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Kotulkar M, Paine-Cabrera D, Apte U. Role of Hepatocyte Nuclear Factor 4 Alpha in Liver Cancer. Semin Liver Dis 2024; 44:383-393. [PMID: 38901435 DOI: 10.1055/a-2349-7236] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 06/22/2024]
Abstract
Liver cancer is the sixth most common cancer and the fourth leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and the incidence of HCC is on the rise. Liver cancers in general and HCC in particular do not respond to chemotherapy. Radiological ablation, surgical resection, and liver transplantation are the only medical therapies currently available. Hepatocyte nuclear factor 4 α (HNF4α) is an orphan nuclear receptor expressed only in hepatocytes in the liver. HNF4α is considered the master regulator of hepatic differentiation because it regulates a significant number of genes involved in various liver-specific functions. In addition to maintaining hepatic differentiation, HNF4α also acts as a tumor suppressor by inhibiting hepatocyte proliferation by suppressing the expression of promitogenic genes and inhibiting epithelial to mesenchymal transition in hepatocytes. Loss of HNF4α expression and function is associated with rapid progression of chronic liver diseases that ultimately lead to liver cirrhosis and HCC, including metabolism-associated steatohepatitis, alcohol-associated liver disease, and hepatitis virus infection. This review summarizes the role of HNF4α in liver cancer pathogenesis and highlights its potential as a potential therapeutic target for HCC.
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Affiliation(s)
- Manasi Kotulkar
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas
| | - Diego Paine-Cabrera
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas
| | - Udayan Apte
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas
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2
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Thakur A, Park K, Cullum R, Fuglerud BM, Khoshnoodi M, Drissler S, Stephan TL, Lotto J, Kim D, Gonzalez FJ, Hoodless PA. HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements. Commun Biol 2024; 7:144. [PMID: 38297077 PMCID: PMC10830483 DOI: 10.1038/s42003-024-05835-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Accepted: 01/18/2024] [Indexed: 02/02/2024] Open
Abstract
Hepatocyte nuclear factor 4A (HNF4A/NR2a1), a transcriptional regulator of hepatocyte identity, controls genes that are crucial for liver functions, primarily through binding to enhancers. In mammalian cells, active and primed enhancers are marked by monomethylation of histone 3 (H3) at lysine 4 (K4) (H3K4me1) in a cell type-specific manner. How this modification is established and maintained at enhancers in connection with transcription factors (TFs) remains unknown. Using analysis of genome-wide histone modifications, TF binding, chromatin accessibility and gene expression, we show that HNF4A is essential for an active chromatin state. Using HNF4A loss and gain of function experiments in vivo and in cell lines in vitro, we show that HNF4A affects H3K4me1, H3K27ac and chromatin accessibility, highlighting its contribution to the establishment and maintenance of a transcriptionally permissive epigenetic state. Mechanistically, HNF4A interacts with the mixed-lineage leukaemia 4 (MLL4) complex facilitating recruitment to HNF4A-bound regions. Our findings indicate that HNF4A enriches H3K4me1, H3K27ac and establishes chromatin opening at transcriptional regulatory regions.
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Affiliation(s)
- Avinash Thakur
- Terry Fox Laboratory, BC Cancer, Vancouver, V5Z 1L3, Canada
- Department of Medical Genetics, University of British Columbia, Vancouver, V6T 1Z4, Canada
| | - Kwangjin Park
- Terry Fox Laboratory, BC Cancer, Vancouver, V5Z 1L3, Canada
- Department of Medical Genetics, University of British Columbia, Vancouver, V6T 1Z4, Canada
| | - Rebecca Cullum
- Terry Fox Laboratory, BC Cancer, Vancouver, V5Z 1L3, Canada
| | - Bettina M Fuglerud
- Terry Fox Laboratory, BC Cancer, Vancouver, V5Z 1L3, Canada
- Department of Medical Genetics, University of British Columbia, Vancouver, V6T 1Z4, Canada
| | | | - Sibyl Drissler
- Terry Fox Laboratory, BC Cancer, Vancouver, V5Z 1L3, Canada
- Cell and Developmental Biology Program, University of British Columbia, Vancouver, V6T 1Z4, Canada
| | - Tabea L Stephan
- Terry Fox Laboratory, BC Cancer, Vancouver, V5Z 1L3, Canada
- Cell and Developmental Biology Program, University of British Columbia, Vancouver, V6T 1Z4, Canada
| | - Jeremy Lotto
- Terry Fox Laboratory, BC Cancer, Vancouver, V5Z 1L3, Canada
- Cell and Developmental Biology Program, University of British Columbia, Vancouver, V6T 1Z4, Canada
| | - Donghwan Kim
- Center of Cancer Research, National Cancer Institute, Bethesda, 2089, USA
| | - Frank J Gonzalez
- Center of Cancer Research, National Cancer Institute, Bethesda, 2089, USA
| | - Pamela A Hoodless
- Terry Fox Laboratory, BC Cancer, Vancouver, V5Z 1L3, Canada.
- Department of Medical Genetics, University of British Columbia, Vancouver, V6T 1Z4, Canada.
- Cell and Developmental Biology Program, University of British Columbia, Vancouver, V6T 1Z4, Canada.
- School of Biomedical Engineering, University of British Columbia, Vancouver, V6T 1Z4, Canada.
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Jones C, Avino M, Giroux V, Boudreau F. HNF4α Acts as Upstream Functional Regulator of Intestinal Wnt3 and Paneth Cell Fate. Cell Mol Gastroenterol Hepatol 2023; 15:593-612. [PMID: 36464209 PMCID: PMC9871320 DOI: 10.1016/j.jcmgh.2022.11.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/26/2021] [Revised: 11/24/2022] [Accepted: 11/28/2022] [Indexed: 12/03/2022]
Abstract
BACKGROUND & AIMS The intestinal epithelium intrinsically renews itself ex vivo via the proliferation of Lgr5+ intestinal stem cells, which is sustained by the establishment of an epithelial stem cell niche. Differentiated Paneth cells are the main source of epithelial-derived niche factor supplies and produce Wnt3 as an essential factor in supporting Lgr5+ stem cell activity in the absence of redundant mesenchymal Wnts. Maturation of Paneth cells depends on canonical Wnt signaling, but few transcriptional regulators have been identified to this end. The role of HNF4α in intestinal epithelial cell differentiation is considered redundant with its paralog HNF4γ. However, it is unclear whether HNF4α alone controls intrinsic intestinal epithelial cell growth and fate in the absence of a mesenchymal niche. METHODS We used transcriptomic analyses to dissect the role of HNF4α in the maintenance of jejunal epithelial culture when cultured ex vivo as enteroids in the presence or absence of compensatory mesenchymal cells. RESULTS HNF4α plays a crucial role in supporting the growth and survival of jejunal enteroids. Transcriptomic analyses revealed an autonomous function of HNF4α in Wnt3 transcriptional regulation and Paneth cell differentiation. We showed that Wnt3a supplementation or co-culture with intestinal subepithelial mesenchymal cells reversed cell death and transcriptional changes caused by the deletion of Hnf4a in jejunal enteroids. CONCLUSIONS Our results support the intrinsic epithelial role of HNF4α in regulating Paneth cell homeostasis and intestinal epithelium renewal in the absence of compensatory Wnt signaling.
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Affiliation(s)
- Christine Jones
- Department of Immunology and Cell Biology, Université de Sherbrooke, Sherbrooke, Québec, Canada
| | - Mariano Avino
- Department of Biochemistry and Functional Genomics, Université de Sherbrooke, Sherbrooke, Québec, Canada
| | - Véronique Giroux
- Department of Immunology and Cell Biology, Université de Sherbrooke, Sherbrooke, Québec, Canada
| | - Francois Boudreau
- Department of Immunology and Cell Biology, Université de Sherbrooke, Sherbrooke, Québec, Canada.
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Liver fat storage is controlled by HNF4α through induction of lipophagy and is reversed by a potent HNF4α agonist. Cell Death Dis 2021; 12:603. [PMID: 34117215 PMCID: PMC8193211 DOI: 10.1038/s41419-021-03862-x] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2021] [Revised: 05/18/2021] [Accepted: 05/21/2021] [Indexed: 12/13/2022]
Abstract
We report the discovery of strong HNF4α agonists and their use to uncover a previously unknown pathway by which HNF4α controls the level of fat storage in the liver. This involves the induction of lipophagy by dihydroceramides, the synthesis and secretion of which is controlled by genes induced by HNF4α. The HNF4α activators are N-trans caffeoyltyramine (NCT) and N-trans feruloyltyramine (NFT), which are structurally related to the known drugs alverine and benfluorex, which we previously showed to be weak HNF4α activators. In vitro, NCT and NFT induced fat clearance from palmitate-loaded cells. In DIO mice, NCT led to recovery of hepatic HNF4α expression and reduction of steatosis. Mechanistically, increased dihydroceramide production and action downstream of HNF4α occurred through increased expression of HNF4α downstream genes, including SPNS2 and CYP26A1. NCT was completely nontoxic at the highest dose administered and so is a strong candidate for an NAFLD therapeutic.
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Smith JL, Rodríguez TC, Mou H, Kwan SY, Pratt H, Zhang XO, Cao Y, Liang S, Ozata DM, Yu T, Yin Q, Hazeltine M, Weng Z, Sontheimer EJ, Xue W. YAP1 Withdrawal in Hepatoblastoma Drives Therapeutic Differentiation of Tumor Cells to Functional Hepatocyte-Like Cells. Hepatology 2021; 73:1011-1027. [PMID: 32452550 PMCID: PMC8500588 DOI: 10.1002/hep.31389] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/02/2019] [Revised: 04/16/2020] [Accepted: 04/27/2020] [Indexed: 12/29/2022]
Abstract
BACKGROUND AND AIMS Despite surgical and chemotherapeutic advances, the 5-year survival rate for stage IV hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. Yes-associated protein 1 (YAP1) and β-catenin co-activation occurs in 80% of children's HB; however, a lack of conditional genetic models precludes tumor maintenance exploration. Thus, the need for a targeted therapy remains unmet. Given the predominance of YAP1 and β-catenin activation in HB, we sought to evaluate YAP1 as a therapeutic target in HB. APPROACH AND RESULTS We engineered the conditional HB murine model using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1S127A , constitutive β-cateninDelN90 , and a luciferase reporter to murine liver. Tumor regression was evaluated using bioluminescent imaging, tumor landscape characterized using RNA and ATAC sequencing, and DNA footprinting. Here we show that YAP1S127A withdrawal mediates more than 90% tumor regression with survival for 230+ days in mice. YAP1S127A withdrawal promotes apoptosis in a subset of tumor cells, and in remaining cells induces a cell fate switch that drives therapeutic differentiation of HB tumors into Ki-67-negative hepatocyte-like HB cells ("HbHeps") with hepatocyte-like morphology and mature hepatocyte gene expression. YAP1S127A withdrawal drives the formation of hbHeps by modulating liver differentiation transcription factor occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and rescue liver damage in mice. CONCLUSIONS YAP1S127A withdrawal, without silencing oncogenic β-catenin, significantly regresses hepatoblastoma, providing in vivo data to support YAP1 as a therapeutic target for HB. YAP1S127A withdrawal alone sufficiently drives long-term regression in HB, as it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes.
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Affiliation(s)
- Jordan L Smith
- RNA Therapeutics InstituteUniversity of Massachusetts Medical SchoolWorcesterMA.,Medical Scientist Training ProgramUniversity of Massachusetts Medical SchoolWorcesterMA
| | - Tomás C Rodríguez
- RNA Therapeutics InstituteUniversity of Massachusetts Medical SchoolWorcesterMA.,Medical Scientist Training ProgramUniversity of Massachusetts Medical SchoolWorcesterMA
| | - Haiwei Mou
- Cold Spring Harbor LaboratoryCold Spring HarborNY
| | - Suet-Yan Kwan
- RNA Therapeutics InstituteUniversity of Massachusetts Medical SchoolWorcesterMA
| | - Henry Pratt
- Medical Scientist Training ProgramUniversity of Massachusetts Medical SchoolWorcesterMA.,Program in Bioinformatics and Integrative BiologyUniversity of Massachusetts Medical SchoolWorcesterMA
| | - Xiao-Ou Zhang
- Program in Bioinformatics and Integrative BiologyUniversity of Massachusetts Medical SchoolWorcesterMA
| | - Yueying Cao
- RNA Therapeutics InstituteUniversity of Massachusetts Medical SchoolWorcesterMA
| | - Shunqing Liang
- RNA Therapeutics InstituteUniversity of Massachusetts Medical SchoolWorcesterMA
| | - Deniz M Ozata
- RNA Therapeutics InstituteUniversity of Massachusetts Medical SchoolWorcesterMA
| | - Tianxiong Yu
- Department of BioinformaticsSchool of Life Science and TechnologyTongji UniversityShanghaiChina
| | - Qiangzong Yin
- Graduate School of Biomedical SciencesUniversity of Massachusetts Medical SchoolWorcesterMA
| | - Max Hazeltine
- Department of SurgeryUniversity of Massachusetts Medical SchoolWorcesterMA
| | - Zhiping Weng
- Program in Bioinformatics and Integrative BiologyUniversity of Massachusetts Medical SchoolWorcesterMA
| | - Erik J Sontheimer
- RNA Therapeutics InstituteUniversity of Massachusetts Medical SchoolWorcesterMA.,Program in Molecular MedicineUniversity of Massachusetts Medical SchoolWorcesterMA.,Li Weibo Institute for Rare Diseases ResearchUniversity of Massachusetts Medical SchoolWorcesterMA
| | - Wen Xue
- RNA Therapeutics InstituteUniversity of Massachusetts Medical SchoolWorcesterMA.,Program in Molecular MedicineUniversity of Massachusetts Medical SchoolWorcesterMA.,Li Weibo Institute for Rare Diseases ResearchUniversity of Massachusetts Medical SchoolWorcesterMA.,Department of Molecular, Cell and Cancer BiologyUniversity of Massachusetts Medical SchoolWorcesterMA
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Qiu MJ, Zhang L, Fang XF, Li QT, Zhu LS, Zhang B, Yang SL, Xiong ZF. Research on the circadian clock gene HNF4a in different malignant tumors. Int J Med Sci 2021; 18:1339-1347. [PMID: 33628089 PMCID: PMC7893568 DOI: 10.7150/ijms.49997] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/27/2020] [Accepted: 01/04/2021] [Indexed: 11/28/2022] Open
Abstract
Background: The circadian rhythm is produced by multiple feedback loops formed by the core clock genes after transcription and translation, thus regulating various metabolic and physiological functions of the human body. We have shown previously that the abnormal expression of 14 clock genes is related closely to the occurrence and development of different malignant tumors, and these genes may play an anti-cancer or pro-cancer role in different tumors. HNF4a has many typical properties of clock proteins involved in the clock gene negative feedback loop regulation process. We need to explore the function of HNF4a as a circadian clock gene in malignant tumors further. Methods: We used The Cancer Genome Atlas (TCGA) database to download the clinicopathological information of twenty malignant tumors and the corresponding RNA-seq data. The HNF4a RNA-seq data standardized by R language and clinical information were integrated to reveal the relationship between HNF4a and prognosis of patients. Results: Analysis of TCGA data showed that the prognosis of HNF4a was significantly different in BLCA, KIRC, LUSC, and READ. High HNF4a expression is correlated with good prognosis in BLCA, KIRC, and READ but poor prognosis in LUSC. However, HNF4a was associated with the stages, T stages, and lymph node status only in BLCA. Conclusions: HNF4a plays different roles in different malignancies, and the abnormal expression of HNF4a has a great correlation with the biological characteristics of BLCA. The low expression of HNF4a could be a reference index for the metastasis, recurrence, and prognosis of BLCA.
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Affiliation(s)
- Meng-Jun Qiu
- Division of Gastroenterology, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430077, China
| | - Li Zhang
- Division of Gastroenterology, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430077, China
| | - Xie-Fan Fang
- Charles River Laboratories, Inc., 6995 Longley Lane, Reno NV 89511
| | - Qiu-Ting Li
- Division of Gastroenterology, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430077, China
| | - Li-Sheng Zhu
- Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Bin Zhang
- Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Sheng-Li Yang
- Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Zhi-Fan Xiong
- Division of Gastroenterology, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430077, China
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Hepatic Polarization Accelerated by Mechanical Compaction Involves HNF4 α Activation. BIOMED RESEARCH INTERNATIONAL 2020; 2020:8016306. [PMID: 32802875 PMCID: PMC7426769 DOI: 10.1155/2020/8016306] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/22/2020] [Revised: 06/23/2020] [Accepted: 07/07/2020] [Indexed: 11/25/2022]
Abstract
There remain few data about the role of homeostatic compaction in hepatic polarization. A previous study has found that mechanical compaction can accelerate hepatocyte polarization; however, the cellular mechanism underlying the effect is mostly unclear. Hepatocyte nuclear factor 4 alpha (HNF4α) is crucial for hepatic polarization in liver morphogenesis. Therefore, we sought to identify any possible involvement of HNF4α in the process of hepatocyte polarization accelerated by mechanical compaction. We first verified in the nonhepatic cell model HEK-293T, and the hepatic cell model primary hepatocytes that the mechanical compaction on cell aggregates simulated by using transient centrifugation can directly activate the expression of HNF4α promoters. Moreover, data using primary hepatocytes showed that the HNF4α expression is positively associated with the levels of compaction force: 2.1-folds higher at the mRNA level and 2.1-folds higher at the protein level for 500 g vs. 0 g. Furthermore, activated HNF4α expression is associated with the enhanced biliary canalicular formation and the increased production of albumin and urea. Pretreatment with Latrunculin B, an inhibitor of F-actin, and SHE78-7, an inhibitor of E-cadherin, which both interrupt the pathway of mechanical transduction, partially but significantly reduced the HNF4α expression and production of albumin and urea. In conclusion, HNF4α can be actively involved in the hepatic polarization in the context of environmental mechanical compaction.
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Bolleyn J, Rombaut M, Nair N, Branson S, Heymans A, Chuah M, VandenDriessche T, Rogiers V, De Kock J, Vanhaecke T. Genetic and Epigenetic Modification of Rat Liver Progenitor Cells via HNF4α Transduction and 5' Azacytidine Treatment: An Integrated miRNA and mRNA Expression Profile Analysis. Genes (Basel) 2020; 11:E486. [PMID: 32365562 PMCID: PMC7291069 DOI: 10.3390/genes11050486] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2020] [Revised: 04/27/2020] [Accepted: 04/28/2020] [Indexed: 12/17/2022] Open
Abstract
Neonatal liver-derived rat epithelial cells (rLEC) from biliary origin are liver progenitor cells that acquire a hepatocyte-like phenotype upon sequential exposure to hepatogenic growth factors and cytokines. Undifferentiated rLEC express several liver-enriched transcription factors, including the hepatocyte nuclear factors (HNF) 3β and HNF6, but not the hepatic master regulator HNF4α. In this study, we first investigated the impact of the ectopic expression of HNF4α in rLEC on both mRNA and microRNA (miR) level by means of microarray technology. We found that HNF4α transduction did not induce major changes to the rLEC phenotype. However, we next investigated the influence of DNA methyl transferase (DNMT) inhibition on the phenotype of undifferentiated naïve rLEC by exposure to 5' azacytidine (AZA), which was found to have a significant impact on rLEC gene expression. The transduction of HNF4α or AZA treatment resulted both in significantly downregulated C/EBPα expression levels, while the exposure of the cells to AZA had a significant effect on the expression of HNF3β. Computationally, dysregulated miRNAs were linked to target mRNAs using the microRNA Target Filter function of Ingenuity Pathway Analysis. We found that differentially regulated miRNA-mRNA target associations predict ectopic HNF4α expression in naïve rLEC to interfere with cell viability and cellular maturation (miR-19b-3p/NR4A2, miR30C-5p/P4HA2, miR328-3p/CD44) while it predicts AZA exposure to modulate epithelial/hepatic cell proliferation, apoptosis, cell cycle progression and the differentiation of stem cells (miR-18a-5p/ESR1, miR-503-5p/CCND1). Finally, our computational analysis predicts that the combination of HNF4α transduction with subsequent AZA treatment might cause changes in hepatic cell proliferation and maturation (miR-18a-5p/ESR1, miR-503-5p/CCND1, miR-328-3p/CD44) as well as the apoptosis (miR-16-5p/BCL2, miR-17-5p/BCL2, miR-34a-5p/BCL2 and miR-494-3p/HMOX1) of naïve rLEC.
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Affiliation(s)
- Jennifer Bolleyn
- Department of In Vitro Toxicology and Dermato-cosmetology (IVTD), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; (J.B.); (M.R.); (S.B.); (A.H.); (V.R.); (T.V.)
| | - Matthias Rombaut
- Department of In Vitro Toxicology and Dermato-cosmetology (IVTD), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; (J.B.); (M.R.); (S.B.); (A.H.); (V.R.); (T.V.)
| | - Nisha Nair
- Department of Gene Therapy and Regenerative Medicine (GTRM), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; (N.N.); (M.C.); (T.V.)
| | - Steven Branson
- Department of In Vitro Toxicology and Dermato-cosmetology (IVTD), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; (J.B.); (M.R.); (S.B.); (A.H.); (V.R.); (T.V.)
| | - Anja Heymans
- Department of In Vitro Toxicology and Dermato-cosmetology (IVTD), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; (J.B.); (M.R.); (S.B.); (A.H.); (V.R.); (T.V.)
| | - Marinee Chuah
- Department of Gene Therapy and Regenerative Medicine (GTRM), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; (N.N.); (M.C.); (T.V.)
| | - Thierry VandenDriessche
- Department of Gene Therapy and Regenerative Medicine (GTRM), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; (N.N.); (M.C.); (T.V.)
| | - Vera Rogiers
- Department of In Vitro Toxicology and Dermato-cosmetology (IVTD), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; (J.B.); (M.R.); (S.B.); (A.H.); (V.R.); (T.V.)
| | - Joery De Kock
- Department of In Vitro Toxicology and Dermato-cosmetology (IVTD), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; (J.B.); (M.R.); (S.B.); (A.H.); (V.R.); (T.V.)
| | - Tamara Vanhaecke
- Department of In Vitro Toxicology and Dermato-cosmetology (IVTD), Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; (J.B.); (M.R.); (S.B.); (A.H.); (V.R.); (T.V.)
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Gao Y, Yan Y, Guo J, Zhang Q, Bi D, Wang F, Chang Z, Lu L, Yao X, Wei Q. HNF‑4α downregulation promotes tumor migration and invasion by regulating E‑cadherin in renal cell carcinoma. Oncol Rep 2019; 42:1066-1074. [PMID: 31322246 PMCID: PMC6667891 DOI: 10.3892/or.2019.7214] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2018] [Accepted: 06/18/2019] [Indexed: 12/19/2022] Open
Abstract
Renal cell carcinoma (RCC) is the most common malignant disease of the kidneys in adults. Patients with metastatic RCC have an unusually poor prognosis and exhibit resistance to all current therapies. Therefore, it is necessary to explore novel molecules involved in the progression of RCC and to identify effective therapeutic targets. Hepatocyte nuclear factor‑4α (HNF‑4α) serves an important role in hepatocyte differentiation and is involved in the progression of liver cancer; however, the functional role of HNF‑4α has not been well established in RCC. The present study reported that HNF‑4α expression was markedly downregulated in RCC tissue samples compared with in normal controls by immunohistochemistry and RNA‑sequencing analysis. Statistical analysis demonstrated that HNF‑4α downregulation was significantly associated with tumor stage, recurrence, metastasis and poor prognosis in patients with RCC. Furthermore, wound‑healing and Transwell assays revealed that downregulation of HNF‑4α promoted cell migration and invasion by transcriptionally regulating E‑cadherin in RCC. Finally, a positive correlation was revealed between HNF‑4α expression and E‑cadherin expression, and patients with low E‑cadherin expression also had a poor prognosis. These findings may provide novel insights into the biological effects of HNF‑4α and lay the foundation for the discovery of molecular therapeutic targets in RCC.
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MESH Headings
- Aged
- Antigens, CD/genetics
- Antigens, CD/metabolism
- Apoptosis
- Biomarkers, Tumor/genetics
- Biomarkers, Tumor/metabolism
- Cadherins/genetics
- Cadherins/metabolism
- Carcinoma, Renal Cell/genetics
- Carcinoma, Renal Cell/metabolism
- Carcinoma, Renal Cell/pathology
- Case-Control Studies
- Cell Movement
- Cell Proliferation
- Female
- Follow-Up Studies
- Gene Expression Regulation, Neoplastic
- Hepatocyte Nuclear Factor 4/genetics
- Hepatocyte Nuclear Factor 4/metabolism
- Humans
- Kidney Neoplasms/genetics
- Kidney Neoplasms/metabolism
- Kidney Neoplasms/secondary
- Male
- Middle Aged
- Neoplasm Invasiveness
- Neoplasm Recurrence, Local/genetics
- Neoplasm Recurrence, Local/metabolism
- Neoplasm Recurrence, Local/pathology
- Prognosis
- Survival Rate
- Tumor Cells, Cultured
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Affiliation(s)
- Yaohui Gao
- Department of Pathology, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, P.R. China
| | - Yang Yan
- Department of Urology, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, P.R. China
| | - Jing Guo
- Department of Pathology, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, P.R. China
| | - Qian Zhang
- Department of Pathology, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, P.R. China
| | - Dexi Bi
- Department of Pathology, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, P.R. China
| | - Fen Wang
- Department of Urology, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, P.R. China
- Department of Anesthesiology, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, P.R. China
| | - Zhengyan Chang
- Department of Pathology, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, P.R. China
| | - Ling Lu
- Department of Pathology, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, P.R. China
| | - Xudong Yao
- Department of Urology, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, P.R. China
| | - Qing Wei
- Department of Pathology, Shanghai Tenth People's Hospital, Tongji University, Shanghai 200072, P.R. China
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Blau BJ, Miki T. The role of cellular interactions in the induction of hepatocyte polarity and functional maturation in stem cell-derived hepatic cells. Differentiation 2019; 106:42-48. [PMID: 30878880 DOI: 10.1016/j.diff.2019.02.006] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2019] [Revised: 02/25/2019] [Accepted: 02/28/2019] [Indexed: 02/08/2023]
Abstract
The unique microenvironment found within the liver in vivo plays a key role in the induction of functional maturation in the developing hepatocyte. During organogenesis, hepatocytes acquire a polar phenotype that allows them to perform their functions of bile production and transport, protein synthesis, metabolism, and detoxification simultaneously, independently, and efficiently. It is thought that the induction of polarity and functional maturation in hepatocytes is dependent on the complex interplay of cell-cell and cell-extracellular matrix (ECM) interactions. While this process is highly efficient in the human liver, it has been shown that hepatocytes rapidly lose their functions when placed in cell culture. This poses a challenge for the development of a bioartificial liver (BAL) support system, which utilizes a live cellular source to perform hepatic functions in the event of acute liver failure or primary nonfunction. However, once the molecular mechanisms underlying the induction of hepatocyte polarity are fully identified, it will be possible to develop highly functional hepatic cells from human pluripotent stem cells (hPSCs). This new cell line would be an ideal cellular source for a BAL system, as it would have both the functionality and longevity to support a patient through the entire clinical course of treatment. In this review, we explore the literature that has examined the potential mechanisms that induce polarity in the developing hepatocyte and discuss the future implications of this knowledge in a clinical setting from a bioengineering perspective.
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Affiliation(s)
- Brandon J Blau
- Department of Surgery, Keck School of Medicine, University of Southern California, USA
| | - Toshio Miki
- Department of Surgery, Keck School of Medicine, University of Southern California, USA.
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11
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Krivtsova O, Makarova A, Lazarevich N. Aberrant expression of alternative isoforms of transcription factors in hepatocellular carcinoma. World J Hepatol 2018; 10:645-661. [PMID: 30386458 PMCID: PMC6206146 DOI: 10.4254/wjh.v10.i10.645] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/20/2018] [Revised: 06/08/2018] [Accepted: 06/28/2018] [Indexed: 02/06/2023] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide and the second leading cause of death among all cancer types. Deregulation of the networks of tissue-specific transcription factors (TFs) observed in HCC leads to profound changes in the hepatic transcriptional program that facilitates tumor progression. In addition, recent reports suggest that substantial aberrations in the production of TF isoforms occur in HCC. In vitro experiments have identified distinct isoform-specific regulatory functions and related biological effects of liver-specific TFs that are implicated in carcinogenesis, which may be relevant for tumor progression and clinical outcome. This study reviews available data on the expression of isoforms of liver-specific and ubiquitous TFs in the liver and HCC and their effects, including HNF4α, C/EBPs, p73 and TCF7L2, and indicates that assessment of the ratio of isoforms and targeting specific TF variants may be beneficial for the prognosis and treatment of HCC.
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Affiliation(s)
- Olga Krivtsova
- Federal State Budgetary Institution, “N. N. Blokhin Medical Research Center of Oncology” of the Ministry of Health of the Russian Federation, Moscow 115478, Russian
- M. V. Lomonosov Moscow State University, Moscow 119991, Russian
| | - Anna Makarova
- Federal State Budgetary Institution, “N. N. Blokhin Medical Research Center of Oncology” of the Ministry of Health of the Russian Federation, Moscow 115478, Russian
| | - Natalia Lazarevich
- Federal State Budgetary Institution, “N. N. Blokhin Medical Research Center of Oncology” of the Ministry of Health of the Russian Federation, Moscow 115478, Russian
- M. V. Lomonosov Moscow State University, Moscow 119991, Russian
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12
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Ott M, Ma Q, Li B, Gagandeep S, Rogler LE, Gupta S. Regulation of hepatitis B virus expression in progenitor and differentiated cell types: evidence for negative transcriptional control in nonpermissive cells. Gene Expr 2018; 8:175-86. [PMID: 10634319 PMCID: PMC6157367] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/15/2023]
Abstract
Mechanisms regulating cell type-specific gene expression are not completely understood. We utilized hepatitis B virus (HBV) enhancer I and preS1 promoter sequences, which exhibit cell type specificity, to analyze transcriptional control in pluripotential murine embryonic stem (ES) cells, bipotential HBC-3 progenitor liver cells, mature hepatocytes, and fibroblasts. In transient transfection assays, HBV sequences were most active in primary hepatocytes, followed by HBC-3 and ES cells, and became inactive in fibroblasts. Cotransfections with HNF-3 or C/EBP plasmids increased expression of HBV sequences in hepatocytes and HBC-3 cells. However, increased HBV expression was not observed in ES cells and HBV remained inactive in fibroblasts, suggesting different transcriptional controls, which was compatible with alterations in the abundance of endogenous transcription factors. Analysis in somatic hybrid cells created from NIH 3T3 fibroblasts and Hepa1-6 mouse hepatocytes with expression of albumin and selected hepatic transcription factors showed that HBV sequences were expressed weakly but without increased expression following transfection of HNF-1, HNF-3, and C/EBP plasmids. These findings indicated that repression of HBV in nonpermissive cells involved inactivation of transcription factor activity. Expression of HBV in stem cells is relevant for mechanisms concerning viral persistence and oncogenesis, as well as analysis of hepatocytic differentiation in progenitor cells.
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Affiliation(s)
- Michael Ott
- Marion Bessin Liver Research Center, Cancer Research Center and Department of Medicine, Jack and Pearl Resnick Campus, Albert Einstein College of Medicine, Bronx, NY
| | - Qiangzhong Ma
- Marion Bessin Liver Research Center, Cancer Research Center and Department of Medicine, Jack and Pearl Resnick Campus, Albert Einstein College of Medicine, Bronx, NY
| | - Baiquan Li
- Marion Bessin Liver Research Center, Cancer Research Center and Department of Medicine, Jack and Pearl Resnick Campus, Albert Einstein College of Medicine, Bronx, NY
| | - S. Gagandeep
- Marion Bessin Liver Research Center, Cancer Research Center and Department of Medicine, Jack and Pearl Resnick Campus, Albert Einstein College of Medicine, Bronx, NY
| | - Leslie E. Rogler
- Marion Bessin Liver Research Center, Cancer Research Center and Department of Medicine, Jack and Pearl Resnick Campus, Albert Einstein College of Medicine, Bronx, NY
| | - Sanjeev Gupta
- Marion Bessin Liver Research Center, Cancer Research Center and Department of Medicine, Jack and Pearl Resnick Campus, Albert Einstein College of Medicine, Bronx, NY
- Address correspondence to Sanjeev Gupta, M.D., Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Ullmann 625, 1300 Morris Park Avenue, Bronx, NY 10461. Tel: (718) 430-2098; Fax: (718) 430-8975; E-mail:
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13
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Guzman‐Lepe J, Cervantes‐Alvarez E, Collin de l'Hortet A, Wang Y, Mars WM, Oda Y, Bekki Y, Shimokawa M, Wang H, Yoshizumi T, Maehara Y, Bell A, Fox IJ, Takeishi K, Soto‐Gutierrez A. Liver-enriched transcription factor expression relates to chronic hepatic failure in humans. Hepatol Commun 2018; 2:582-594. [PMID: 29761173 PMCID: PMC5944584 DOI: 10.1002/hep4.1172] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/03/2017] [Revised: 01/15/2018] [Accepted: 02/21/2018] [Indexed: 12/22/2022] Open
Abstract
The mechanisms by which the liver fails in end-stage liver disease remain elusive. Disruption of the transcription factor network in hepatocytes has been suggested to mediate terminal liver failure in animals. However, this hypothesis remains unexplored in human subjects. To study the relevance of transcription factor expression in terminal stages of chronic liver failure in humans, we analyzed the expression of liver-enriched transcription factors (LETFs) hepatocyte nuclear factor (HNF)4α, HNF1α, forkhead box protein A2 (FOXA2), CCAAT/enhancer-binding protein (CEBP)α, and CEBPβ. We then selected downstream genes responsible for some hepatic functions (ornithine transcarbamylase [OTC], cytochrome P450 3A4 [CYP3A4], coagulation factor VII [F7], cadherin 1 [CDH1], phospho-ezrin (Thr567)/radixin (Thr564)/moesin (Thr558) [p-ERM], phospho-myosin light chain [p-MLC], low-density lipoprotein receptor-related protein 1 [LRP1]) in liver tissue from patients at different stages of decompensated liver function based upon Child-Pugh classification, Model for End-Stage Liver Disease score, and degree of inflammatory activity/fibrosis. We first examined differential expression of LETF and determined whether a relationship exists between transcript and protein expression, and liver function. We found HNF4α expression was down-regulated and correlated well with the extent of liver dysfunction (P = 0.001), stage of fibrosis (P = 0.0005), and serum levels of total bilirubin (P = 0.009; r = 0.35), albumin (P < 0.001; r = 0.52), and prothrombin time activity (P = 0.002; r = 0.41). HNF4α expression also correlated with CYP3A4, OTC, and F7 as well as CDH1 RNA levels. The Rho/Rho-associated protein kinase pathways, which have been implicated in the regulation of HNF4α, were also differentially expressed, in concert with LRP1, a reported upstream regulator of RhoA function. Conclusion: HNF4α and other members of the LETFs appear to be important regulators of hepatocyte function in patients with chronic hepatic failure. (Hepatology Communications 2018;2:582-594).
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Affiliation(s)
| | - Eduardo Cervantes‐Alvarez
- Department of PathologyUniversity of PittsburghPittsburghPA
- PECEM, Facultad de MedicinaUniversidad Nacional Autónoma de MéxicoMexico CityMexico
| | | | - Yang Wang
- Department of PathologyUniversity of PittsburghPittsburghPA
- Department of Hepatobiliary SurgeryPeking University People's HospitalBeijingChina
| | - Wendy M. Mars
- Department of PathologyUniversity of PittsburghPittsburghPA
| | - Yoshinao Oda
- Department of Anatomic Pathology, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan
| | - Yuki Bekki
- Department of Surgery and Science, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan
| | - Masahiro Shimokawa
- Department of Surgery and Science, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan
| | - Huanlin Wang
- Department of Surgery and Science, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan
| | - Tomoharu Yoshizumi
- Department of Surgery and Science, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan
| | - Yoshihiko Maehara
- Department of Surgery and Science, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan
| | - Aaron Bell
- Department of PathologyUniversity of PittsburghPittsburghPA
| | - Ira J. Fox
- Department of SurgeryChildren's Hospital of Pittsburgh of the University of Pittsburgh Medical CenterPittsburghPA
- McGowan Institute for Regenerative MedicineUniversity of PittsburghPittsburghPA
| | - Kazuki Takeishi
- Department of PathologyUniversity of PittsburghPittsburghPA
- Department of Surgery and Science, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan
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14
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Herrera B, Addante A, Sánchez A. BMP Signalling at the Crossroad of Liver Fibrosis and Regeneration. Int J Mol Sci 2017; 19:ijms19010039. [PMID: 29295498 PMCID: PMC5795989 DOI: 10.3390/ijms19010039] [Citation(s) in RCA: 50] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2017] [Revised: 12/15/2017] [Accepted: 12/18/2017] [Indexed: 12/16/2022] Open
Abstract
Bone Morphogenetic Proteins (BMPs) belong to the Transforming Growth Factor-β (TGF-β) family. Initially identified due to their ability to induce bone formation, they are now known to have multiple functions in a variety of tissues, being critical not only during development for tissue morphogenesis and organogenesis but also during adult tissue homeostasis. This review focus on the liver as a target tissue for BMPs actions, devoting most efforts to summarize our knowledge on their recently recognized and/or emerging roles on regulation of the liver regenerative response to various insults, either acute or chronic and their effects on development and progression of liver fibrosis in different pathological conditions. In an attempt to provide the basis for guiding research efforts in this field both the more solid and more controversial areas of research were highlighted.
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Affiliation(s)
- Blanca Herrera
- Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Complutense University of Madrid (UCM), Health Research Institute of the Hospital Clínico San Carlos (IdISSC), 28040 Madrid, Spain.
| | - Annalisa Addante
- Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Complutense University of Madrid (UCM), Health Research Institute of the Hospital Clínico San Carlos (IdISSC), 28040 Madrid, Spain.
| | - Aránzazu Sánchez
- Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Complutense University of Madrid (UCM), Health Research Institute of the Hospital Clínico San Carlos (IdISSC), 28040 Madrid, Spain.
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15
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Gao YH, Wu ZX, Xie LQ, Li CX, Mao YQ, Duan YT, Han B, Han SF, Yu Y, Lu HJ, Yang PY, Xu TR, Xia JL, Chen GQ, Wang LS. VHL deficiency augments anthracycline sensitivity of clear cell renal cell carcinomas by down-regulating ALDH2. Nat Commun 2017; 8:15337. [PMID: 28643803 PMCID: PMC5481740 DOI: 10.1038/ncomms15337] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2015] [Accepted: 03/20/2017] [Indexed: 12/12/2022] Open
Abstract
The von Hippel-Lindau (VHL) is deficient in ∼70% of clear-cell renal cell carcinomas (ccRCC), which contributes to the carcinogenesis and drug resistance of ccRCC. Here we show that VHL-deficient ccRCC cells present enhanced cytotoxicity of anthracyclines in a hypoxia-inducible factor-independent manner. By subtractive proteomic analysis coupling with RNAi or overexpression verification, aldehyde dehydrogenase 2 (ALDH2) is found to be transcriptionally regulated by VHL and contributes to enhanced anthracyclines cytotoxicity in ccRCC cells. Furthermore, VHL regulates ALDH2 expression by directly binding the promoter of -130 bp to -160 bp to activate the transcription of hepatocyte nuclear factor 4 alpha (HNF-4α). In addition, a positive correlation is found among the protein expressions of VHL, HNF-4α and ALDH2 in ccRCC samples. These findings will deepen our understanding of VHL function and shed light on precise treatment for ccRCC patients.
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MESH Headings
- Aldehyde Dehydrogenase, Mitochondrial/genetics
- Aldehyde Dehydrogenase, Mitochondrial/metabolism
- Animals
- Anthracyclines/pharmacology
- Anthracyclines/therapeutic use
- Anthracyclines/toxicity
- Carcinoma, Renal Cell/drug therapy
- Carcinoma, Renal Cell/genetics
- Carcinoma, Renal Cell/pathology
- Cell Death/drug effects
- Cell Line, Tumor
- Down-Regulation/drug effects
- Down-Regulation/genetics
- Gene Expression Regulation, Neoplastic/drug effects
- Hepatocyte Nuclear Factor 4/metabolism
- Hypoxia-Inducible Factor 1, alpha Subunit/metabolism
- Kidney Neoplasms/drug therapy
- Kidney Neoplasms/genetics
- Kidney Neoplasms/pathology
- Male
- Mice, Nude
- Neoplasm Proteins/metabolism
- Proteomics
- Transcription, Genetic/drug effects
- Von Hippel-Lindau Tumor Suppressor Protein/metabolism
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Affiliation(s)
- Yao-Hui Gao
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, 170 Xinsong Road, Shanghai 201199, China
| | - Zhao-Xia Wu
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, 170 Xinsong Road, Shanghai 201199, China
| | - Li-Qi Xie
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, 170 Xinsong Road, Shanghai 201199, China
| | - Cai-Xia Li
- Shanghai Universities E-Institute for Chemical Biology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
| | - Yu-Qin Mao
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, 170 Xinsong Road, Shanghai 201199, China
| | - Yan-Tao Duan
- Shanghai Universities E-Institute for Chemical Biology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
| | - Bing Han
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, 170 Xinsong Road, Shanghai 201199, China
| | - San-Feng Han
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, 170 Xinsong Road, Shanghai 201199, China
| | - Yun Yu
- Shanghai Universities E-Institute for Chemical Biology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
| | - Hao-Jie Lu
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, 170 Xinsong Road, Shanghai 201199, China
| | - Peng-Yuan Yang
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, 170 Xinsong Road, Shanghai 201199, China
| | - Tian-Rui Xu
- Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
| | - Jing-Lin Xia
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, 170 Xinsong Road, Shanghai 201199, China
| | - Guo-Qiang Chen
- Shanghai Universities E-Institute for Chemical Biology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
| | - Li-Shun Wang
- Institute of Fudan-Minhang Academic Health System, Minhang Hospital, Fudan University, 170 Xinsong Road, Shanghai 201199, China
- Shanghai Universities E-Institute for Chemical Biology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
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16
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Zhang B, Madden P, Gu J, Xing X, Sankar S, Flynn J, Kroll K, Wang T. Uncovering the transcriptomic and epigenomic landscape of nicotinic receptor genes in non-neuronal tissues. BMC Genomics 2017; 18:439. [PMID: 28583088 PMCID: PMC5460472 DOI: 10.1186/s12864-017-3813-4] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2016] [Accepted: 05/23/2017] [Indexed: 02/17/2023] Open
Abstract
Background Nicotinic acetylcholine receptors (nAChRs) play an important role in cellular physiology and human nicotine dependence, and are closely associated with many human diseases including cancer. For example, previous studies suggest that nAChRs can re-wire gene regulatory networks in lung cancer cell lines. However, the tissue specificity of nAChRs genes and their regulation remain unexplored. Result In this study, we integrated data from multiple large genomic consortiums, including ENCODE, Roadmap Epigenomics, GTEx, and FANTOM, to define the transcriptomic and epigenomic landscape of all nicotinic receptor genes across many different human tissues and cell types. We found that many important nAChRs, including CHRNA3, CHRNA4, CHRNA5, and CHRNB4, exhibited strong non-neuronal tissue-specific expression patterns. CHRNA3, CHRNA5, and CHRNB4 were highly expressed in human colon and small intestine, and CHRNA4 was highly expressed in human liver. By comparing the epigenetic marks of CHRNA4 in human liver and hippocampus, we identified a novel liver-specific transcription start site (TSS) of CHRNA4. We further demonstrated that CHRNA4 was specifically transcribed in hepatocytes but not transcribed in hepatic sinusoids and stellate cells, and that transcription factors HNF4A and RXRA were likely upstream regulators of CHRNA4. Our findings suggest that CHRNA4 has distinct transcriptional regulatory mechanisms in human liver and brain, and that this tissue-specific expression pattern is evolutionarily conserved in mouse. Finally, we found that liver-specific CHRNA4 transcription was highly correlated with genes involved in the nicotine metabolism, including CYP2A6, UGT2B7, and FMO3. These genes were significantly down-regulated in liver cancer patients, whereas CHRNA4 is also significantly down-regulated in cancer-matched normal livers. Conclusions Our results suggest important non-neuronally expressed nicotinic acetylcholine receptors in the human body. These non-neuronal expression patterns are highly tissue-specific, and are epigenetically conserved during evolution in the context of non-conserved DNA sequence. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3813-4) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Bo Zhang
- Center of Regenerative Medicine, Department of Developmental Biology, Washington University School of Medicine, Room 3212, 4515 McKinley Research Building, 4515 McKinley Ave, St. Louis, MO, 63110, USA. .,Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, Room 5211, 4515 McKinley Research Building, 4515 McKinley Ave, St. Louis, MO, 63110, USA.
| | - Pamela Madden
- Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Junchen Gu
- Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, Room 5211, 4515 McKinley Research Building, 4515 McKinley Ave, St. Louis, MO, 63110, USA
| | - Xiaoyun Xing
- Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, Room 5211, 4515 McKinley Research Building, 4515 McKinley Ave, St. Louis, MO, 63110, USA
| | - Savita Sankar
- Center of Regenerative Medicine, Department of Developmental Biology, Washington University School of Medicine, Room 3212, 4515 McKinley Research Building, 4515 McKinley Ave, St. Louis, MO, 63110, USA
| | - Jennifer Flynn
- Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, Room 5211, 4515 McKinley Research Building, 4515 McKinley Ave, St. Louis, MO, 63110, USA
| | - Kristen Kroll
- Center of Regenerative Medicine, Department of Developmental Biology, Washington University School of Medicine, Room 3212, 4515 McKinley Research Building, 4515 McKinley Ave, St. Louis, MO, 63110, USA
| | - Ting Wang
- Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, Room 5211, 4515 McKinley Research Building, 4515 McKinley Ave, St. Louis, MO, 63110, USA.
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17
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Guo H, Novozhilova NM, Bandini G, Turco SJ, Ferguson MAJ, Beverley SM. Genetic metabolic complementation establishes a requirement for GDP-fucose in Leishmania. J Biol Chem 2017; 292:10696-10708. [PMID: 28465349 PMCID: PMC5481574 DOI: 10.1074/jbc.m117.778480] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2017] [Revised: 05/01/2017] [Indexed: 01/10/2023] Open
Abstract
To survive in its sand fly vector, the trypanosomatid protozoan parasite Leishmania first attaches to the midgut to avoid excretion, but eventually it must detach for transmission by the next bite. In Leishmania major strain Friedlin, this is controlled by modifications of the stage-specific adhesin lipophosphoglycan (LPG). During differentiation to infective metacyclics, d-arabinopyranose (d-Arap) caps the LPG side-chain galactose residues, blocking interaction with the midgut lectin PpGalec, thereby leading to parasite detachment and transmission. Previously, we characterized two closely related L. major genes (FKP40 and AFKP80) encoding bifunctional proteins with kinase/pyrophosphorylase activities required for salvage and conversion of l-fucose and/or d-Arap into the nucleotide-sugar substrates required by glycosyltransferases. Whereas only AFKP80 yielded GDP-d-Arap from exogenous d-Arap, both proteins were able to salvage l-fucose to GDP-fucose. We now show that Δafkp80− null mutants ablated d-Arap modifications of LPG as predicted, whereas Δfkp40− null mutants resembled wild type (WT). Fucoconjugates had not been reported previously in L. major, but unexpectedly, we were unable to generate fkp40−/afkp80− double mutants, unless one of the A/FKPs was expressed ectopically. To test whether GDP-fucose itself was essential for Leishmania viability, we employed “genetic metabolite complementation.” First, the trypanosome de novo pathway enzymes GDP-mannose dehydratase (GMD) and GDP-fucose synthetase (GMER) were expressed ectopically; from these cells, the Δfkp40−/Δafkp80− double mutant was now readily obtained. As expected, the Δfkp40−/Δafkp80−/+TbGMD-GMER line lacked the capacity to generate GDP-Arap, while synthesizing abundant GDP-fucose. These results establish a requirement for GDP-fucose for L. major viability and predict the existence of an essential fucoconjugate(s).
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Affiliation(s)
- Hongjie Guo
- From the Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110
| | - Natalia M Novozhilova
- the Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536, and
| | - Giulia Bandini
- the Division of Biological Chemistry and Drug Discovery, School of Life Science, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom
| | - Salvatore J Turco
- the Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536, and
| | - Michael A J Ferguson
- the Division of Biological Chemistry and Drug Discovery, School of Life Science, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom
| | - Stephen M Beverley
- From the Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110,
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18
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Morimoto A, Kannari M, Tsuchida Y, Sasaki S, Saito C, Matsuta T, Maeda T, Akiyama M, Nakamura T, Sakaguchi M, Nameki N, Gonzalez FJ, Inoue Y. An HNF4α-microRNA-194/192 signaling axis maintains hepatic cell function. J Biol Chem 2017; 292:10574-10585. [PMID: 28465351 DOI: 10.1074/jbc.m117.785592] [Citation(s) in RCA: 60] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2017] [Revised: 04/28/2017] [Indexed: 12/14/2022] Open
Abstract
Hepatocyte nuclear factor 4α (HNF4α) controls the expression of liver-specific protein-coding genes. However, some microRNAs are also modulated by HNF4α, and it is not known whether they are direct targets of HNF4α and whether they influence hepatic function. In this study, we found that HNF4α regulates microRNAs, indicated by marked down-regulation of miR-194 and miR-192 (miR-194/192) in liver-specific Hnf4a-null (Hnf4aΔH) mice. Transactivation of the shared miR-194/192 promoter was dependent on HNF4α expression, indicating that miR-194/192 is a target gene of HNF4α. Screening of potential mRNAs targeted by miR-194/192 revealed that expression of genes involved in glucose metabolism (glycogenin 1 (Gyg1)), cell adhesion and migration (activated leukocyte cell adhesion molecule (Alcam)), tumorigenesis and tumor progression (Rap2b and epiregulin (Ereg)), protein SUMOylation (Sumo2), epigenetic regulation (Setd5 and Cullin 4B (Cln4b)), and the epithelial-mesenchymal transition (moesin (Msn)) was up-regulated in Hnf4aΔH mice. Moreover, we also found that miR-194/192 binds the 3'-UTR of these mRNAs. siRNA knockdown of HNF4α suppressed miR-194/192 expression in human hepatocellular carcinoma (HCC) cells and resulted in up-regulation of their mRNA targets. Inhibition and overexpression experiments with miR-194/192 revealed that Gyg1, Setd5, Sumo2, Cln4b, and Rap2b are miR-194 targets, whereas Ereg, Alcam, and Msn are miR-192 targets. These findings reveal a novel HNF4α network controlled by miR-194/192 that may play a critical role in maintaining the hepatocyte-differentiated state by inhibiting expression of genes involved in dedifferentiation and tumorigenesis. These insights may contribute to the development of diagnostic markers for early HCC detection, and targeting of the miR-194/192 pathway could be useful for managing HCC.
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Affiliation(s)
- Aoi Morimoto
- From the Laboratory of Molecular Life Science, Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan
| | - Mana Kannari
- From the Laboratory of Molecular Life Science, Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan
| | - Yuichi Tsuchida
- From the Laboratory of Molecular Life Science, Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan
| | - Shota Sasaki
- From the Laboratory of Molecular Life Science, Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan
| | - Chinatsu Saito
- From the Laboratory of Molecular Life Science, Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan
| | - Tsuyoshi Matsuta
- From the Laboratory of Molecular Life Science, Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan
| | - Tsukasa Maeda
- From the Laboratory of Molecular Life Science, Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan
| | - Megumi Akiyama
- From the Laboratory of Molecular Life Science, Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan
| | - Takahiro Nakamura
- From the Laboratory of Molecular Life Science, Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan
| | - Masakiyo Sakaguchi
- the Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Kita-ku, Okayama 700-8558, Japan, and
| | - Nobukazu Nameki
- From the Laboratory of Molecular Life Science, Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan
| | - Frank J Gonzalez
- the Laboratory of Metabolism, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20852
| | - Yusuke Inoue
- From the Laboratory of Molecular Life Science, Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu, Gunma 376-8515, Japan,
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19
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Cai WY, Lin LY, Hao H, Zhang SM, Ma F, Hong XX, Zhang H, Liu QF, Ye GD, Sun GB, Liu YJ, Li SN, Xie YY, Cai JC, Li BA. Yes-associated protein/TEA domain family member and hepatocyte nuclear factor 4-alpha (HNF4α) repress reciprocally to regulate hepatocarcinogenesis in rats and mice. Hepatology 2017; 65:1206-1221. [PMID: 27809333 DOI: 10.1002/hep.28911] [Citation(s) in RCA: 54] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/17/2015] [Revised: 10/15/2016] [Accepted: 10/19/2016] [Indexed: 12/11/2022]
Abstract
UNLABELLED Great progress has been achieved in the study of Hippo signaling in regulating tumorigenesis; however, the downstream molecular events that mediate this process have not been completely defined. Moreover, regulation of Hippo signaling during tumorigenesis in hepatocellular carcinoma (HCC) remains largely unknown. In the present study, we systematically investigated the relationship between Yes-associated protein/TEA domain family member (YAP-TEAD) and hepatocyte nuclear factor 4-alpha (HNF4α) in the hepatocarcinogenesis of HCC cells. Our results indicated that HNF4α expression was negatively regulated by YAP1 in HCC cells by a ubiquitin proteasome pathway. By contrast, HNF4α was found to directly associate with TEAD4 to compete with YAP1 for binding to TEAD4, thus inhibiting the transcriptional activity of YAP-TEAD and expression of their target genes. Moreover, overexpression of HNF4α was found to significantly compromise YAP-TEAD-induced HCC cell proliferation and stem cell expansion. Finally, we documented the regulatory mechanism between YAP-TEAD and HNF4α in rat and mouse tumor models, which confirmed our in vitro results. CONCLUSION There is a double-negative feedback mechanism that controls TEAD-YAP and HNF4α expression in vitro and in vivo, thereby regulating cellular proliferation and differentiation. Given that YAP acts as a dominant oncogene in HCC and plays a crucial role in stem cell homeostasis and tissue regeneration, manipulating the interaction between YAP, TEADs, and HNF4α may provide a new approach for HCC treatment and regenerative medicine. (Hepatology 2017;65:1206-1221).
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Affiliation(s)
- Wang-Yu Cai
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.,Zhongshan Hospital of Xiamen University, Xiamen, Fujian, China
| | - Ling-Yun Lin
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.,Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Han Hao
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.,Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Sai-Man Zhang
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Fei Ma
- Zhongshan Hospital of Xiamen University, Xiamen, Fujian, China
| | - Xin-Xin Hong
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Hui Zhang
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Qing-Feng Liu
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Guo-Dong Ye
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Guang-Bin Sun
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Yun-Jia Liu
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Sheng-Nan Li
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Yuan-Yuan Xie
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
| | - Jian-Chun Cai
- Zhongshan Hospital of Xiamen University, Xiamen, Fujian, China
| | - Bo-An Li
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.,Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, China
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20
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Chang TT, Thakar D, Weaver VM. Force-dependent breaching of the basement membrane. Matrix Biol 2016; 57-58:178-189. [PMID: 28025167 DOI: 10.1016/j.matbio.2016.12.005] [Citation(s) in RCA: 57] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2016] [Revised: 10/20/2016] [Accepted: 12/12/2016] [Indexed: 12/24/2022]
Abstract
Clinically, non-invasive carcinomas are confined to the epithelial side of the basement membrane and are classified as benign, whereas invasive cancers invade through the basement membrane and thereby acquire the potential to metastasize. Recent findings suggest that, in addition to protease-mediated degradation and chemotaxis-stimulated migration, basement membrane invasion by malignant cells is significantly influenced by the stiffness of the associated interstitial extracellular matrix and the contractility of the tumor cells that is dictated in part by their oncogenic genotype. In this review, we highlight recent findings that illustrate unifying molecular mechanisms whereby these physical cues contribute to tissue fibrosis and malignancy in three epithelial organs: breast, pancreas, and liver. We also discuss the clinical implications of these findings and the biological properties and clinical challenges linked to the unique biology of each of these organs.
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Affiliation(s)
- Tammy T Chang
- Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA; Liver Center, University of California, San Francisco, San Francisco, CA 94143, USA.
| | - Dhruv Thakar
- Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA; Center for Bioengineering and Tissue Regeneration, University of California San Francisco, San Francisco, CA 94143, USA.
| | - Valerie M Weaver
- Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA; Center for Bioengineering and Tissue Regeneration, University of California San Francisco, San Francisco, CA 94143, USA; Department of Anatomy, UCSF, San Francisco, CA 94143, USA; Department of Bioengineering and Therapeutic Sciences, UCSF, San Francisco, CA 94143, USA; Department of Radiation Oncology, UCSF, San Francisco, CA 94143, USA; Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, CA 94143, USA; The Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, CA 94143, USA.
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21
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Colleypriest BJ, Burke ZD, Griffiths LP, Chen Y, Yu WY, Jover R, Bock M, Biddlestone L, Quinlan JM, Ward SG, Mark Farrant J, Slack JMW, Tosh D. Hnf4α is a key gene that can generate columnar metaplasia in oesophageal epithelium. Differentiation 2016; 93:39-49. [PMID: 27875772 PMCID: PMC5293356 DOI: 10.1016/j.diff.2016.11.001] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2016] [Revised: 11/02/2016] [Accepted: 11/07/2016] [Indexed: 12/20/2022]
Abstract
Barrett's metaplasia is the only known morphological precursor to oesophageal adenocarcinoma and is characterized by replacement of stratified squamous epithelium by columnar epithelium. The cell of origin is uncertain and the molecular mechanisms responsible for the change in cellular phenotype are poorly understood. We therefore explored the role of two transcription factors, Cdx2 and HNF4α in the conversion using primary organ cultures. Biopsy samples from cases of human Barrett's metaplasia were analysed for the presence of CDX2 and HNF4α. A new organ culture system for adult murine oesophagus is described. Using this, Cdx2 and HNF4α were ectopically expressed by adenoviral infection. The phenotype following infection was determined by a combination of PCR, immunohistochemical and morphological analyses. We demonstrate the expression of CDX2 and HNF4α in human biopsy samples. Our oesophageal organ culture system expressed markers characteristic of the normal SSQE: p63, K14, K4 and loricrin. Ectopic expression of HNF4α, but not of Cdx2 induced expression of Tff3, villin, K8 and E-cadherin. HNF4α is sufficient to induce a columnar-like phenotype in adult mouse oesophageal epithelium and is present in the human condition. These data suggest that induction of HNF4α is a key early step in the formation of Barrett's metaplasia and are consistent with an origin of Barrett's metaplasia from the oesophageal epithelium.
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Affiliation(s)
- Benjamin J Colleypriest
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK; Department of Gastroenterology, Royal United Hospital, Combe Park, Bath BA1 3NG, UK
| | - Zoë D Burke
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
| | - Leonard P Griffiths
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK; Department of Gastroenterology, Royal United Hospital, Combe Park, Bath BA1 3NG, UK
| | - Yu Chen
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
| | - Wei-Yuan Yu
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
| | - Ramiro Jover
- Unidad Mixta Hepatologia Experimental & CIBERehd, Departamento de Bioquimica y Biologia Molecular, Universidad de Valencia, Spain
| | - Michael Bock
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
| | - Leigh Biddlestone
- Department of Gastroenterology, Royal United Hospital, Combe Park, Bath BA1 3NG, UK
| | - Jonathan M Quinlan
- Department of Gastroenterology, Royal United Hospital, Combe Park, Bath BA1 3NG, UK
| | - Stephen G Ward
- Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
| | - J Mark Farrant
- Department of Gastroenterology, Royal United Hospital, Combe Park, Bath BA1 3NG, UK
| | - Jonathan M W Slack
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK; Stem Cell Institute, University of Minnesota, Minneapolis 55455, USA
| | - David Tosh
- Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.
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22
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Vasconcellos R, Alvarenga ÉC, Parreira RC, Lima SS, Resende RR. Exploring the cell signalling in hepatocyte differentiation. Cell Signal 2016; 28:1773-88. [DOI: 10.1016/j.cellsig.2016.08.011] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2016] [Revised: 08/18/2016] [Accepted: 08/18/2016] [Indexed: 02/08/2023]
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23
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Berasain C, Avila MA. Regulation of hepatocyte identity and quiescence. Cell Mol Life Sci 2015; 72:3831-51. [PMID: 26089250 PMCID: PMC11114060 DOI: 10.1007/s00018-015-1970-7] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2015] [Revised: 05/23/2015] [Accepted: 06/12/2015] [Indexed: 12/11/2022]
Abstract
The liver is a highly differentiated organ with a central role in metabolism, detoxification and systemic homeostasis. To perform its multiple tasks, liver parenchymal cells, the hepatocytes, express a large complement of enabling genes defining their complex phenotype. This phenotype is progressively acquired during fetal development and needs to be maintained in adulthood to guarantee the individual's survival. Upon injury or loss of functional mass, the liver displays an extraordinary regenerative response, mainly based on the proliferation of hepatocytes which otherwise are long-lived quiescent cells. Increasing observations suggest that loss of hepatocellular differentiation and quiescence underlie liver malfunction in chronic liver disease and pave the way for hepatocellular carcinoma development. Here, we briefly review the essential mechanisms leading to the acquisition of liver maturity. We also identify the key molecular factors involved in the preservation of hepatocellular homeostasis and finally discuss potential strategies to preserve liver identity and function.
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Affiliation(s)
- Carmen Berasain
- Division of Hepatology, CIMA, University of Navarra, CIBEREHD, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Avda. Pio XII, n55, 31008, Pamplona, Spain.
| | - Matías A Avila
- Division of Hepatology, CIMA, University of Navarra, CIBEREHD, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Avda. Pio XII, n55, 31008, Pamplona, Spain.
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24
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Sahu SK, Garding A, Tiwari N, Thakurela S, Toedling J, Gebhard S, Ortega F, Schmarowski N, Berninger B, Nitsch R, Schmidt M, Tiwari VK. JNK-dependent gene regulatory circuitry governs mesenchymal fate. EMBO J 2015; 34:2162-81. [PMID: 26157010 PMCID: PMC4557668 DOI: 10.15252/embj.201490693] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2014] [Accepted: 06/05/2015] [Indexed: 12/14/2022] Open
Abstract
The epithelial to mesenchymal transition (EMT) is a biological process in which cells lose cell–cell contacts and become motile. EMT is used during development, for example, in triggering neural crest migration, and in cancer metastasis. Despite progress, the dynamics of JNK signaling, its role in genomewide transcriptional reprogramming, and involved downstream effectors during EMT remain largely unknown. Here, we show that JNK is not required for initiation, but progression of phenotypic changes associated with EMT. Such dependency resulted from JNK-driven transcriptional reprogramming of critical EMT genes and involved changes in their chromatin state. Furthermore, we identified eight novel JNK-induced transcription factors that were required for proper EMT. Three of these factors were also highly expressed in invasive cancer cells where they function in gene regulation to maintain mesenchymal identity. These factors were also induced during neuronal development and function in neuronal migration in vivo. These comprehensive findings uncovered a kinetically distinct role for the JNK pathway in defining the transcriptome that underlies mesenchymal identity and revealed novel transcription factors that mediate these responses during development and disease.
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Affiliation(s)
| | | | - Neha Tiwari
- Institute of Physiological Chemistry University Medical Center Johannes Gutenberg University, Mainz, Germany
| | | | | | - Susanne Gebhard
- Department of Obstetrics and Gynecology, Johannes Gutenberg University, Mainz, Germany
| | - Felipe Ortega
- Institute of Physiological Chemistry University Medical Center Johannes Gutenberg University, Mainz, Germany
| | - Nikolai Schmarowski
- Institute for Microscopic Anatomy and Neurobiology University Medical Center Johannes Gutenberg University, Mainz, Germany
| | - Benedikt Berninger
- Institute of Physiological Chemistry University Medical Center Johannes Gutenberg University, Mainz, Germany
| | - Robert Nitsch
- Institute for Microscopic Anatomy and Neurobiology University Medical Center Johannes Gutenberg University, Mainz, Germany
| | - Marcus Schmidt
- Department of Obstetrics and Gynecology, Johannes Gutenberg University, Mainz, Germany
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25
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Epigenetic control of EMT/MET dynamics: HNF4α impacts DNMT3s through miRs-29. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2015; 1849:919-29. [PMID: 26003733 DOI: 10.1016/j.bbagrm.2015.05.005] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/17/2014] [Revised: 04/17/2015] [Accepted: 05/13/2015] [Indexed: 01/11/2023]
Abstract
BACKGROUND AND AIMS Epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial transition (MET) are manifestations of cellular plasticity that imply a dynamic and profound gene expression reprogramming. While a major epigenetic code controlling the coordinated regulation of a whole transcriptional profile is guaranteed by DNA methylation, DNA methyltransferase (DNMT) activities in EMT/MET dynamics are still largely unexplored. Here, we investigated the molecular mechanisms directly linking HNF4α, the master effector of MET, to the regulation of both de novo of DNMT 3A and 3B. METHODS Correlation among EMT/MET markers, microRNA29 and DNMT3s expression was evaluated by RT-qPCR, Western blotting and immunocytochemical analysis. Functional roles of microRNAs and DNMT3s were tested by anti-miRs, microRNA precursors and chemical inhibitors. ChIP was utilized for investigating HNF4α DNA binding activity. RESULTS HNF4α silencing was sufficient to induce positive modulation of DNMT3B, in in vitro differentiated hepatocytes as well as in vivo hepatocyte-specific Hnf4α knockout mice, and DNMT3A, in vitro, but not DNMT1. In exploring the molecular mechanisms underlying these observations, evidence have been gathered for (i) the inverse correlation between DNMT3 levels and the expression of their regulators miR-29a and miR-29b and (ii) the role of HNF4α as a direct regulator of miR-29a-b transcription. Notably, during TGFβ-induced EMT, DNMT3s' pivotal function has been proved, thus suggesting the need for the repression of these DNMTs in the maintenance of a differentiated phenotype. CONCLUSIONS HNF4α maintains hepatocyte identity by regulating miR-29a and -29b expression, which in turn control epigenetic modifications by limiting DNMT3A and DNMT3B levels.
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26
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Kwon SM, Jung YY, Hwang CJ, Park MH, Yoon NY, Kim TM, Yu JM, Kim DH, Seo DW, Youn HS, Seo HO, Chung IS, Han SB, Hwang BY, Yoo HS, Jung JK, Lee H, Hong JT. Anti-cancer effect of N-(3,5-bis(trifluoromethyl)phenyl)-5-chloro-2,3-dihydronaphtho[1,2-b]furan-2-carboxamide, a novel synthetic compound. Mol Carcinog 2015; 55:659-70. [DOI: 10.1002/mc.22311] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2014] [Revised: 12/27/2014] [Accepted: 02/18/2015] [Indexed: 01/05/2023]
Affiliation(s)
- Sun Mi Kwon
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Yu Yeon Jung
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Chul Ju Hwang
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Mi Hee Park
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Na Young Yoon
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Tae Myung Kim
- College of Veterinary Medicine; Chungbuk National University; Chungbuk Korea
| | - Ji Myung Yu
- College of Veterinary Medicine; Chungbuk National University; Chungbuk Korea
| | - Dae Hwan Kim
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Doo Won Seo
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Hyu Seok Youn
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Hyun Ok Seo
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - In Sung Chung
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Sang Bae Han
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Bang Yeon Hwang
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Hwan-Soo Yoo
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Jae-Kyung Jung
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Heesoon Lee
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
| | - Jin Tae Hong
- College of Pharmacy and Medical Research Center; Chungbuk National University; Chungbuk Korea
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27
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Cicchini C, Amicone L, Alonzi T, Marchetti A, Mancone C, Tripodi M. Molecular mechanisms controlling the phenotype and the EMT/MET dynamics of hepatocyte. Liver Int 2015; 35:302-10. [PMID: 24766136 PMCID: PMC4344819 DOI: 10.1111/liv.12577] [Citation(s) in RCA: 74] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/06/2013] [Accepted: 04/19/2014] [Indexed: 02/06/2023]
Abstract
The complex spatial and paracrine relationships between the various liver histotypes are essential for proper functioning of the hepatic parenchymal cells. Only within a correct tissue organization, in fact, they stably maintain their identity and differentiated phenotype. The loss of histotype identity, which invariably occurs in the primary hepatocytes in culture, or in vivo in particular pathological conditions (fibrosis and tumours), is mainly because of the phenomenon of epithelial-to-mesenchymal transition (EMT). The EMT process, that occurs in the many epithelial cells, appears to be driven by a number of general, non-tissue-specific, master transcriptional regulators. The reverse process, the mesenchymal-to-epithelial transition (MET), as yet much less characterized at a molecular level, restores specific epithelial identities, and thus must include tissue-specific master elements. In this review, we will summarize the so far unveiled events of EMT/MET occurring in liver cells. In particular, we will focus on hepatocyte and describe the pivotal role in the control of EMT/MET dynamics exerted by a tissue-specific molecular mini-circuitry. Recent evidence, indeed, highlighted as two transcriptional factors, the master gene of EMT Snail, and the master gene of hepatocyte differentiation HNF4α, exhorting a direct reciprocal repression, act as pivotal elements in determining opposite cellular outcomes. The different balances between these two master regulators, further integrated by specific microRNAs, in fact, were found responsible for the EMT/METs dynamics as well as for the preservation of both hepatocyte and stem/precursor cells identity and differentiation. Overall, these findings impact the maintenance of stem cells and differentiated cells both in in vivo EMT/MET physio-pathological processes as well as in culture.
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Affiliation(s)
- Carla Cicchini
- Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of RomeRome, Italy
| | - Laura Amicone
- Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of RomeRome, Italy
| | - Tonino Alonzi
- National Institute for Infectious Diseases L. Spallanzani, IRCCSRome, Italy
| | - Alessandra Marchetti
- Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of RomeRome, Italy
| | - Carmine Mancone
- National Institute for Infectious Diseases L. Spallanzani, IRCCSRome, Italy
| | - Marco Tripodi
- Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of RomeRome, Italy,National Institute for Infectious Diseases L. Spallanzani, IRCCSRome, Italy
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28
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Abstract
Hepatocyte nuclear factor 4α (HNF4α) is an orphan nuclear receptor commonly known as the master regulator of hepatic differentiation, owing to the large number of hepatocyte-specific genes it regulates. Whereas the role of HNF4α in hepatocyte differentiation is well recognized and extensively studied, its role in regulation of cell proliferation is relatively less known. Recent studies have revealed that HNF4α inhibits proliferation not only of hepatocytes but also cells in colon and kidney. Further, a growing number of studies have demonstrated that inhibition or loss of HNF4α promotes tumorigenesis in the liver and colon, and reexpression of HNF4α results in decreased cancer growth. Studies using tissue-specific conditional knockout mice, knock-in studies, and combinatorial bioinformatics of RNA/ChIP-sequencing data indicate that the mechanisms of HNF4α-mediated inhibition of cell proliferation are multifold, involving epigenetic repression of promitogenic genes, significant cross talk with other cell cycle regulators including c-Myc and cyclin D1, and regulation of miRNAs. Furthermore, studies indicate that posttranslational modifications of HNF4α may change its activity and may be at the core of its dual role as a differentiation factor and repressor of proliferation. This review summarizes recent findings on the role of HNF4α in cell proliferation and highlights the newly understood function of this old receptor.
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Affiliation(s)
- Chad Walesky
- *Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA
- †Department of Medicine – Genetics Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Udayan Apte
- *Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, USA
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29
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Yao R, Wang J, Li X, Jung Jung D, Qi H, Kee KK, Du Y. Hepatic differentiation of human embryonic stem cells as microscaled multilayered colonies leading to enhanced homogeneity and maturation. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2014; 10:4311-4323. [PMID: 25059765 DOI: 10.1002/smll.201401040] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/14/2014] [Revised: 05/28/2014] [Indexed: 06/03/2023]
Abstract
Directed differentiation of human embryonic stem cells (hESCs) towards hepatocyte-like cells on planar tissue culture plates has been extensively investigated with great promise to provide alternative cell sources for drug metabolism/toxicity testing. Recently, hepatic differentiation of hESCs in 3D configuration with better mimicry of embryonic liver development represents incremental efforts to improve the differentiation efficiency and cellular maturation. However, most of the present 3D differentiation configurations involved interruptive operations during the multi-staged differentiation process, which might impose unwanted influence on cellular differentiation. Most of the current researches resulted in generation of hepatocytes with high expression of AFP, which is minimally expressed in primary hepatocytes. Here, off-the-shelf micro-stencil arrays are developed to generate adherent multilayered colonies composed of hESCs-derived cells. Uninterrupted cellular differentiation and proliferation is achieved to recapitulate the continuous and multi-stage liver development. Compared with conventional 2D format, the micro-scaled multilayered colonies with uniform and defined sizes constrained within the microwells are composed of more homogenous and mature hepatocyte-like cells with significantly lowered AFP expression and elevated hepatic functions. The multilayered colonies as novel 3D configuration for hepatic differentiation of hESCs represent a significant step toward efficient generation of functional hepatocytes for regenerative medicine and drug discovery.
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Affiliation(s)
- Rui Yao
- Biofabrication Center, Department of Mechanical Engineering, Tsinghua University, Beijing, People's Republic of China; Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing, 100084, China
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Glucocorticoid receptor regulates organic cation transporter 1 (OCT1, SLC22A1) expression via HNF4α upregulation in primary human hepatocytes. Pharmacol Rep 2014; 65:1322-35. [PMID: 24399729 DOI: 10.1016/s1734-1140(13)71491-9] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2013] [Revised: 06/07/2013] [Indexed: 01/11/2023]
Abstract
BACKGROUND Organic cation transporter 1 (OCT1, SLC22A1) is a membrane transporter that is important for therapeutic effect of the antidiabetic drug metformin. Its liver-specific expression in hepatocytes is strongly controlled by hepatocyte nuclear factor-4α (HNF4α). HNF4α expression and transcriptional activity have been demonstrated to be augmented by glucocorticoid receptor (GR) in human hepatocytes and rodent livers. METHODS It was examined whether GR activation indirectly induces OCT1 gene expression via HNF4α up-regulation in primary human hepatocytes. We also examined which other transcription factors are involved in OCT1 gene expression and whether they are regulated by dexamethasone using qRT-PCR and gene reporter assays. RESULTS We found that dexamethasone significantly up-regulates OCT1 mRNA and protein in normal primary human hepatocytes, but not in hepatocyte-derived tumor cell lines HepG2 and MZ-Hep1. Consistently, we observed that HNF4α is induced by dexamethasone in primary human hepatocytes, but not in hepatocyte tumor-derived cell lines. Viral transduction of MZ-Hep1 cells with the expression constructs for HNF4α, CCAAT/enhancer binding proteins β (C/EBPβ) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) demonstrated significant roles of the transcription factors in OCT1 gene regulation. We found that expression of OCT1 mRNA in human livers significantly correlates with C/EBPβ and HNF4α mRNAs expression and that C/EBPβ co-transfection stimulates OCT1 gene reporter construct in HepG2 cells. Nevertheless, neither C/EBPβ nor PGC1α were upregulated in human hepatocytes by dexamethasone. CONCLUSION We can conclude that GR-induced expression of HNF4α may contribute to indirect OCT1 gene up-regulation by dexamethasone in primary human hepatocytes, but not in hepatocyte-derived tumor cell lines.
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Let-7f microRNA negatively regulates hepatic differentiation of human adipose tissue-derived stem cells. J Physiol Biochem 2014; 70:781-9. [DOI: 10.1007/s13105-014-0346-z] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2014] [Accepted: 07/08/2014] [Indexed: 12/21/2022]
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Simeone P, Trerotola M, Urbanella A, Lattanzio R, Ciavardelli D, Di Giuseppe F, Eleuterio E, Sulpizio M, Eusebi V, Pession A, Piantelli M, Alberti S. A unique four-hub protein cluster associates to glioblastoma progression. PLoS One 2014; 9:e103030. [PMID: 25050814 PMCID: PMC4106866 DOI: 10.1371/journal.pone.0103030] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2014] [Accepted: 06/25/2014] [Indexed: 01/09/2023] Open
Abstract
Gliomas are the most frequent brain tumors. Among them, glioblastomas are malignant and largely resistant to available treatments. Histopathology is the gold standard for classification and grading of brain tumors. However, brain tumor heterogeneity is remarkable and histopathology procedures for glioma classification remain unsatisfactory for predicting disease course as well as response to treatment. Proteins that tightly associate with cancer differentiation and progression, can bear important prognostic information. Here, we describe the identification of protein clusters differentially expressed in high-grade versus low-grade gliomas. Tissue samples from 25 high-grade tumors, 10 low-grade tumors and 5 normal brain cortices were analyzed by 2D-PAGE and proteomic profiling by mass spectrometry. This led to identify 48 differentially expressed protein markers between tumors and normal samples. Protein clustering by multivariate analyses (PCA and PLS-DA) provided discrimination between pathological samples to an unprecedented extent, and revealed a unique network of deranged proteins. We discovered a novel glioblastoma control module centered on four major network hubs: Huntingtin, HNF4α, c-Myc and 14-3-3ζ. Immunohistochemistry, western blotting and unbiased proteome-wide meta-analysis revealed altered expression of this glioblastoma control module in human glioma samples as compared with normal controls. Moreover, the four-hub network was found to cross-talk with both p53 and EGFR pathways. In summary, the findings of this study indicate the existence of a unifying signaling module controlling glioblastoma pathogenesis and malignant progression, and suggest novel targets for development of diagnostic and therapeutic procedures.
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Affiliation(s)
- Pasquale Simeone
- Unit of Cancer Pathology, Ce.S.I., Foundation University “G. d'Annunzio,” Chieti, Italy
| | - Marco Trerotola
- Unit of Cancer Pathology, Ce.S.I., Foundation University “G. d'Annunzio,” Chieti, Italy
| | - Andrea Urbanella
- Unit of Cancer Pathology, Ce.S.I., Foundation University “G. d'Annunzio,” Chieti, Italy
| | - Rossano Lattanzio
- Unit of Cancer Pathology, Ce.S.I., Foundation University “G. d'Annunzio,” Chieti, Italy
- Department of Experimental and Clinical Sciences, School of Medicine and Health Science, University “G. d'Annunzio,” Chieti, Italy
| | - Domenico Ciavardelli
- School of Human and Social Science, University “Kore” of Enna, Enna, Italy
- Molecular Neurology Unit, Ce.S.I., University “G. d'Annunzio,” Chieti, Italy
| | - Fabrizio Di Giuseppe
- Aging Research Center, Ce.S.I., University “G. d'Annunzio” Foundation, Chieti, Italy
- Department of Experimental and Clinical Sciences, School of Medicine and Health Science, University “G. d'Annunzio,” Chieti, Italy
- StemTeCh Group, Chieti, Italy
| | - Enrica Eleuterio
- Aging Research Center, Ce.S.I., University “G. d'Annunzio” Foundation, Chieti, Italy
- Department of Experimental and Clinical Sciences, School of Medicine and Health Science, University “G. d'Annunzio,” Chieti, Italy
- StemTeCh Group, Chieti, Italy
| | - Marilisa Sulpizio
- Aging Research Center, Ce.S.I., University “G. d'Annunzio” Foundation, Chieti, Italy
- Department of Experimental and Clinical Sciences, School of Medicine and Health Science, University “G. d'Annunzio,” Chieti, Italy
- StemTeCh Group, Chieti, Italy
| | - Vincenzo Eusebi
- Department of “Tutela Salute Donna, Vita nascente, Bambino e Adolescente,” Catholic University of the Sacred Heart, Policlinico Universitario “Agostino Gemelli,” Roma, Italy
| | - Annalisa Pession
- Section of Surgical Pathology, “M. Malpighi,” Bellaria Hospital, Bologna, Italy
| | - Mauro Piantelli
- Unit of Cancer Pathology, Ce.S.I., Foundation University “G. d'Annunzio,” Chieti, Italy
- Department of Experimental and Clinical Sciences, School of Medicine and Health Science, University “G. d'Annunzio,” Chieti, Italy
| | - Saverio Alberti
- Unit of Cancer Pathology, Ce.S.I., Foundation University “G. d'Annunzio,” Chieti, Italy
- Department of Neuroscience, Imaging and Clinical Sciences, University “G. d'Annunzio,” Chieti, Italy
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MicroRNA-122 triggers mesenchymal-epithelial transition and suppresses hepatocellular carcinoma cell motility and invasion by targeting RhoA. PLoS One 2014; 9:e101330. [PMID: 24992599 PMCID: PMC4081555 DOI: 10.1371/journal.pone.0101330] [Citation(s) in RCA: 94] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2014] [Accepted: 06/04/2014] [Indexed: 12/15/2022] Open
Abstract
The loss of microRNA-122 (miR-122) expression is strongly associated with increased invasion and metastasis, and poor prognosis of hepatocellular carcinoma (HCC), however, the underlying mechanisms remain poorly understood. In the present study, we observed that miR-122 over-expression in HCC cell lines Sk-hep-1 and Bel-7402 triggered the mesenchymal-epithelial transition (MET), as demonstrated by epithelial-like morphological changes, up-regulated epithelial proteins (E-cadherin, ZO-1, α-catenin, occludin, BVES, and MST4), and down-regulated mesenchymal proteins (vimentin and fibronectin). The over-expression of miRNA-122 also caused cytoskeleton disruption, RhoA/Rock pathway inactivation, enhanced cell adhesion, and suppression of migration and invasion of Sk-hep-1 and Bel-7402 cells, whereas, these effects could be reversed through miR-122 inhibition. Additional studies demonstrated that the inhibition of wild-type RhoA function induced MET and inhibited cell migration and invasion, while RhoA over-expression reversed miR-122-induced MET and inhibition of migration and invasion of HCC cells, suggesting that miR-122 induced MET and suppressed the migration and invasion of HCC cells by targeting RhoA. Moreover, our results demonstrated that HNF4α up-regulated its target gene miR-122 that subsequently induced MET and inhibited cell migration and invasion, whereas miR-122 inhibition reversed these HNF4α-induced phenotypes. These results revealed functional and mechanistic links among the tumor suppressors HNF4α, miR-122, and RhoA in EMT and invasive and metastatic phenotypes of HCC. Taken together, our study provides the first evidence that the HNF4α/miR-122/RhoA axis negatively regulates EMT and the migration and invasion of HCC cells.
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Shim JH, Kang HJ, Han S, Lee YJ, Lee SG, Yu E, Lee HC. Prognostic value of hepatocyte nuclear factors 4α and 1α identified by tissue microarray in resectable hepatocellular carcinoma. J Gastroenterol Hepatol 2014; 29:524-32. [PMID: 23981200 DOI: 10.1111/jgh.12371] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 08/06/2013] [Indexed: 01/11/2023]
Abstract
BACKGROUND AND AIM This study aimed to investigate the prognostic value of expression of hepatocyte nuclear factors (HNFs) involved in hepatic gene transcription in patients undergoing curative resection for hepatocellular carcinoma (HCC). METHODS We performed immunohistochemical analyses on microarrays of the tumors and matched adjacent tissue using antibodies against HNF1α, HNF1β, HNF4α, and α-fetoprotein (AFP). We evaluated the prognostic value of biomarker expression using Cox regression and the Kaplan-Meier method in a training cohort of 220 patients and conducted an independent validation in 232 patients. We also determined whether measurement of HNFs improved risk prediction beyond the use of established factors, using net reclassification improvement (NRI). RESULTS Post-surgical recurrence and hepatic death were predicted by intratumoral HNF4α underexpression in both cohorts. In the training cohort they were also predicted by peritumoral HNF1α positivity. A pooled cohort analysis showed that these predictors were independently associated with early but not late-phase recurrence, and resultant mortality. Intratumoral expression levels of HNF4α were correlated with those of HNF1α, HNF1β, and AFP (P < 0.05). Similarly, HNF1α expression in peritumoral tissue was correlated with that of other markers (P < 0.05). There was no significant correlation between expression of HNF4α in tumors and HNF1α in peritumoral tissue. Adding combinations of intratumoral HNF4α and peritumoral HNF1α to 2-year recurrence and 5-year mortality models including known clinicopathological prognostic factors significantly improved the NRI indexes (39% and 44%, respectively; P < 0.05). CONCLUSIONS Immunohistological activation of intratumoral HNF4α and depletion of peritumoral HNF1α have prognostic significance for delayed recurrence and death after HCC resection.
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Affiliation(s)
- Ju Hyun Shim
- Department of Gastroenterology, Asan Medical Center, Seoul, Korea
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Abstract
Hepatocytes, like other epithelia, are situated at the interface between the organism's exterior and the underlying internal milieu and organize the vectorial exchange of macromolecules between these two spaces. To mediate this function, epithelial cells, including hepatocytes, are polarized with distinct luminal domains that are separated by tight junctions from lateral domains engaged in cell-cell adhesion and from basal domains that interact with the underlying extracellular matrix. Despite these universal principles, hepatocytes distinguish themselves from other nonstriated epithelia by their multipolar organization. Each hepatocyte participates in multiple, narrow lumina, the bile canaliculi, and has multiple basal surfaces that face the endothelial lining. Hepatocytes also differ in the mechanism of luminal protein trafficking from other epithelia studied. They lack polarized protein secretion to the luminal domain and target single-spanning and glycosylphosphatidylinositol-anchored bile canalicular membrane proteins via transcytosis from the basolateral domain. We compare this unique hepatic polarity phenotype with that of the more common columnar epithelial organization and review our current knowledge of the signaling mechanisms and the organization of polarized protein trafficking that govern the establishment and maintenance of hepatic polarity. The serine/threonine kinase LKB1, which is activated by the bile acid taurocholate and, in turn, activates adenosine monophosphate kinase-related kinases including AMPK1/2 and Par1 paralogues has emerged as a key determinant of hepatic polarity. We propose that the absence of a hepatocyte basal lamina and differences in cell-cell adhesion signaling that determine the positioning of tight junctions are two crucial determinants for the distinct hepatic and columnar polarity phenotypes.
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Affiliation(s)
- Aleksandr Treyer
- Albert Einstein College of Medicine, Department of Developmental and Molecular Biology, Bronx, New York, USA
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Yang M, Li SN, Anjum KM, Gui LX, Zhu SS, Liu J, Chen JK, Liu QF, Ye GD, Wang WJ, Wu JF, Cai WY, Sun GB, Liu YJ, Liu RF, Zhang ZM, Li BA. A double-negative feedback loop between Wnt-β-catenin signaling and HNF4α regulates epithelial-mesenchymal transition in hepatocellular carcinoma. J Cell Sci 2013; 126:5692-703. [PMID: 24101726 DOI: 10.1242/jcs.135053] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Wnt-β-catenin signaling participates in the epithelial-mesenchymal transition (EMT) in a variety of cancers; however, its involvement in hepatocellular carcinoma (HCC) and downstream molecular events is largely undefined. HNF4α is the most prominent and specific factor maintaining the differentiation of hepatic lineage cells and a potential EMT regulator in HCC cells. However, the molecular mechanisms by which HNF4α maintains the differentiated liver epithelium and inhibits EMT have not been completely defined. In this study, we systematically explored the relationship between Wnt-β-catenin signaling and HNF4α in the EMT process of HCC cells. Our results indicated that HNF4α expression was negatively regulated during Wnt-β-catenin signaling-induced EMT through Snail and Slug in HCC cells. In contrast, HNF4α was found to directly associate with TCF4 to compete with β-catenin but facilitate transcription co-repressor activities, thus inhibiting expression of EMT-related Wnt-β-catenin targets. Moreover, HNF4α may control the switch between the transcriptional and adhesion functions of β-catenin. Overexpression of HNF4α was found to completely compromise the Wnt-β-catenin-signaling-induced EMT phenotype. Finally, we determined the regulation pattern between Wnt-β-catenin signaling and HNF4α in rat tumor models. Our studies have identified a double-negative feedback mechanism controlling Wnt-β-catenin signaling and HNF4α expression in vitro and in vivo, which sheds new light on the regulation of EMT in HCC. The modulation of these molecular processes may be a method of inhibiting HCC invasion by blocking Wnt-β-catenin signaling or restoring HNF4α expression to prevent EMT.
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Affiliation(s)
- Meng Yang
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology Research, School of Life Sciences, Xiamen University, Xiamen, 361102, China
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Yao D, Peng S, Dai C. The role of hepatocyte nuclear factor 4alpha in metastatic tumor formation of hepatocellular carcinoma and its close relationship with the mesenchymal-epithelial transition markers. BMC Cancer 2013; 13:432. [PMID: 24059685 PMCID: PMC3852538 DOI: 10.1186/1471-2407-13-432] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2013] [Accepted: 09/17/2013] [Indexed: 12/27/2022] Open
Abstract
Background Mesenchymal–epithelial transition (MET) is now suggested to participate in the process of metastatic tumor formation. However, in hepatocellular carcinoma (HCC) the process is still not well revealed. Methods Paraffin-embedded tissue samples were obtained from 13 patients with HCC in Shengjing Hospital of China Medical University. The expression of E-cadherin, Fibronectin, N-cadherin, Vimentin, Hepatocyte nuclear factor 4alpha (HNF4alpha), Snail and Slug was assessed in primary tumors and their corresponding metastases by immunohistochemical staining. Next, the expression of HNF4alpha and E-cadherin in four HCC cell lines was examined. Furthermore, SK-Hep-1 cells were transfected with human HNF4alpha expression vector, and the change of E-cadherin expression was assessed. Results 45.2% (14/31) of the lesions in the metastases showed increased E-cadherin expression compared with the primaries, suggesting the possible occurrence of MET in metastatic tumor formation of HCC, as re-expression of E-cadherin is proposed to be the important hallmark of MET. The occurrence of MET was also confirmed by the reduced expression of Fibronectin (54.8%, 17/31), N-cadherin (38.7%, 12/31) and Vimentin (61.3%, 19/31) in the metastases. 45.2% (14/31) of the lesions in the metastases also showed increased HNF4alpha expression, and 67.7% (21/31) and 48.4% (15/31) of metastases showed decreased Snail and Slug expression respectively. Statistical results showed that the expression of HNF4alpha was positively related with that of E-cadherin, and negatively correlated with that of Snail, Slug and Fibronectin, suggesting that the expression change of the MET markers in the metastatic lesions might be associated with HNF4alpha. Among the four HCC cell lines, both HNF4alpha and E-cadherin expressed high in Hep3B and Huh-7 cells, but low in SK-Hep-1 and Bel-7402 cells. Furthermore, the expression of E-cadherin increased accordingly when SK-Hep-1 cells were transfected with human HNF4alpha expression vector, further confirming the role of HNF4alpha in the regulation of E-cadherin expression. Conclusions Our clinical observations and experimental data indicate that HNF4alpha might play a crucial role in the metastatic tumor formation of HCC, and the mechanism may be related with the process of phenotype transition.
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Affiliation(s)
- Dianbo Yao
- Department of Hepatobiliary and Splenic Surgery, Shengjing Hospital, China Medical University, Shenyang 110004, Liaoning Province, China.
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Abstract
Liver is a prime organ responsible for synthesis, metabolism, and detoxification. The organ is endodermal in origin and its development is regulated by temporal, complex, and finely balanced cellular and molecular interactions that dictate its origin, growth, and maturation. We discuss the relevance of endoderm patterning, which truly is the first step toward mapping of domains that will give rise to specific organs. Once foregut patterning is completed, certain cells within the foregut endoderm gain competence in the form of expression of certain transcription factors that allow them to respond to certain inductive signals. Hepatic specification is then a result of such inductive signals, which often emanate from the surrounding mesenchyme. During hepatic specification bipotential hepatic stem cells or hepatoblasts become apparent and undergo expansion, which results in a visible liver primordium during the stage of hepatic morphogenesis. Hepatoblasts next differentiate into either hepatocytes or cholangiocytes. The expansion and differentiation is regulated by cellular and molecular interactions between hepatoblasts and mesenchymal cells including sinusoidal endothelial cells, stellate cells, and also innate hematopoietic elements. Further maturation of hepatocytes and cholangiocytes continues during late hepatic development as a function of various growth factors. At this time, liver gains architectural novelty in the form of zonality and at cellular level acquires polarity. A comprehensive elucidation of such finely tuned developmental cues have been the basis of transdifferentiation of various types of stem cells to hepatocyte-like cells for purposes of understanding health and disease and for therapeutic applications.
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Affiliation(s)
- Donghun Shin
- Department of Developmental Biology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA.
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Fraczek J, Bolleyn J, Vanhaecke T, Rogiers V, Vinken M. Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies. Arch Toxicol 2012; 87:577-610. [PMID: 23242478 DOI: 10.1007/s00204-012-0983-3] [Citation(s) in RCA: 91] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2012] [Accepted: 11/19/2012] [Indexed: 01/24/2023]
Abstract
Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs. However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates. Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development. In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations. Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry. Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed.
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Affiliation(s)
- J Fraczek
- Department of Toxicology, Faculty of Medicine and Pharmacy, Centre for Pharmaceutical Research, Vrije Universiteit Brussel, Belgium.
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Sheaffer KL, Kaestner KH. Transcriptional networks in liver and intestinal development. Cold Spring Harb Perspect Biol 2012; 4:a008284. [PMID: 22952394 DOI: 10.1101/cshperspect.a008284] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The development of the gastrointestinal tract is a complex process that integrates signaling processes with downstream transcriptional responses. Here, we discuss the regionalization of the primitive gut and formation of the intestine and liver. Anterior-posterior position in the primitive gut is important for establishing regions that will become functional organs. Coordination of signaling between the epithelium and mesenchyme and downstream transcriptional responses is required for intestinal development and homeostasis. Liver development uses a complex transcriptional network that controls the establishment of organ domains, cell differentiation, and adult function. Discussion of these transcriptional mechanisms gives us insight into how the primitive gut, composed of simple endodermal cells, develops into multiple diverse cell types that are organized into complex mature organs.
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Affiliation(s)
- Karyn L Sheaffer
- Department of Genetics, Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA
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Basu A. WITHDRAWN: Expression and functional characterization of guanylyl cyclase C receptor in HepG2 cells: Two-step regulation by dexamethasone and hepatocyte nuclear factor 4 (HNF4). J Steroid Biochem Mol Biol 2012:S0960-0760(12)00123-9. [PMID: 22750460 DOI: 10.1016/j.jsbmb.2012.06.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/06/2012] [Revised: 05/24/2012] [Accepted: 06/21/2012] [Indexed: 11/22/2022]
Abstract
This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
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Affiliation(s)
- Arindam Basu
- University of Pennsylvania, School of Veterinary Medicine, Department of Animal Biology, 3800 Spruce Street, Philadelphia, PA 19104, United States.
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Transcriptional regulation of cell adhesion at the blood-testis barrier and spermatogenesis in the testis. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2012; 763:281-94. [PMID: 23397630 PMCID: PMC4108166 DOI: 10.1007/978-1-4614-4711-5_14] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Spermatogenesis involves precise co-ordination of multiple cellular events that take place in the seminiferous epithelium composed of Sertoli cells and developing germ cells during the seminiferous epithelial cycle. Given the cyclic and co-ordinated nature of spermatogenesis, temporal and spatial expression of certain genes pertinent to a specific cellular event are essential. As such, transcriptional regulation is one of the major regulatory machineries in controlling the cell type- and stage-specific gene expression, some of which are under the influence of gonadotropins (e.g., FSH and LH) and sex steroids (e.g., testosterone and estradiol-17beta). Recent findings regarding transcriptional control of spermatogenesis, most notably target genes at the Sertoli-Sertoli and Sertoli-spermatid interface at the site of the blood-testis barrier (BTB) and apical ectoplasmic specialization (apical ES), respectively, involving in cell adhesion are reviewed and discussed herein. This is a much neglected area of research and a concerted effort by investigators is needed to understand transcriptional regulation of cell adhesion function in the testis particularly at the BTB during spermatogenesis.
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Pelletier L, Rebouissou S, Vignjevic D, Bioulac-Sage P, Zucman-Rossi J. HNF1α inhibition triggers epithelial-mesenchymal transition in human liver cancer cell lines. BMC Cancer 2011; 11:427. [PMID: 21975049 PMCID: PMC3203860 DOI: 10.1186/1471-2407-11-427] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2011] [Accepted: 10/05/2011] [Indexed: 12/31/2022] Open
Abstract
Background Hepatocyte Nuclear Factor 1α (HNF1α) is an atypical homeodomain-containing transcription factor that transactivates liver-specific genes including albumin, α-1-antitrypsin and α- and β-fibrinogen. Biallelic inactivating mutations of HNF1A have been frequently identified in hepatocellular adenomas (HCA), rare benign liver tumors usually developed in women under oral contraceptives, and in rare cases of hepatocellular carcinomas developed in non-cirrhotic liver. HNF1α-mutated HCA (H-HCA) are characterized by a marked steatosis and show activation of glycolysis, lipogenesis, translational machinery and mTOR pathway. We studied the consequences of HNF1α silencing in hepatic cell lines, HepG2 and Hep3B and we reproduced most of the deregulations identified in H-HCA. Methods We transfected hepatoma cell lines HepG2 and Hep3B with siRNA targeting HNF1α and obtained a strong inhibition of HNF1α expression. We then looked at the phenotypic changes by microscopy and studied changes in gene expression using qRT-PCR and Western Blot. Results Hepatocytes transfected with HNF1α siRNA underwent severe phenotypic changes with loss of cell-cell contacts and development of migration structures. In HNF1α-inhibited cells, hepatocyte and epithelial markers were diminished and mesenchymal markers were over-expressed. This epithelial-mesenchymal transition (EMT) was related to the up regulation of several EMT transcription factors, in particular SNAIL and SLUG. We also found an overexpression of TGFβ1, an EMT initiator, in both cells transfected with HNF1α siRNA and H-HCA. Moreover, TGFβ1 expression is strongly correlated to HNF1α expression in cell models, suggesting regulation of TGFβ1 expression by HNF1α. Conclusion Our results suggest that HNF1α is not only important for hepatocyte differentiation, but has also a role in the maintenance of epithelial phenotype in hepatocytes.
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Affiliation(s)
- Laura Pelletier
- Inserm U674 Génomique fonctionnelle des tumeurs solides, Paris, France
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Mizutani A, Koinuma D, Tsutsumi S, Kamimura N, Morikawa M, Suzuki HI, Imamura T, Miyazono K, Aburatani H. Cell type-specific target selection by combinatorial binding of Smad2/3 proteins and hepatocyte nuclear factor 4alpha in HepG2 cells. J Biol Chem 2011; 286:29848-60. [PMID: 21646355 PMCID: PMC3191026 DOI: 10.1074/jbc.m110.217745] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Specific regulation of target genes by transforming growth factor-β (TGF-β) in a given cellular context is determined in part by transcription factors and cofactors that interact with the Smad complex. In this study, we determined Smad2 and Smad3 (Smad2/3) binding regions in the promoters of known genes in HepG2 hepatoblastoma cells, and we compared them with those in HaCaT epidermal keratinocytes to elucidate the mechanisms of cell type- and context-dependent regulation of transcription induced by TGF-β. Our results show that 81% of the Smad2/3 binding regions in HepG2 cells were not shared with those found in HaCaT cells. Hepatocyte nuclear factor 4α (HNF4α) is expressed in HepG2 cells but not in HaCaT cells, and the HNF4α-binding motif was identified as an enriched motif in the HepG2-specific Smad2/3 binding regions. Chromatin immunoprecipitation sequencing analysis of HNF4α binding regions under TGF-β stimulation revealed that 32.5% of the Smad2/3 binding regions overlapped HNF4α bindings. MIXL1 was identified as a new combinatorial target of HNF4α and Smad2/3, and both the HNF4α protein and its binding motif were required for the induction of MIXL1 by TGF-β in HepG2 cells. These findings generalize the importance of binding of HNF4α on Smad2/3 binding genomic regions for HepG2-specific regulation of transcription by TGF-β and suggest that certain transcription factors expressed in a cell type-specific manner play important roles in the transcription regulated by the TGF-β-Smad signaling pathway.
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Affiliation(s)
- Anna Mizutani
- From the Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033
| | - Daizo Koinuma
- From the Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033
| | - Shuichi Tsutsumi
- the Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, Meguro-ku, Tokyo 153-8904, and
| | - Naoko Kamimura
- the Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, Meguro-ku, Tokyo 153-8904, and
| | - Masato Morikawa
- From the Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033
| | - Hiroshi I. Suzuki
- From the Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033
| | - Takeshi Imamura
- the Division of Biochemistry, Cancer Institute of the Japanese Foundation for Cancer Research, Koto-ku, Tokyo 135-8550, Japan
| | - Kohei Miyazono
- From the Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033
- To whom correspondence should be addressed. Tel.: 81-3-5841-3356; Fax: 81-3-5841-3354; E-mail:
| | - Hiroyuki Aburatani
- the Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, Meguro-ku, Tokyo 153-8904, and
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Nagaoka M, Duncan SA. Transcriptional control of hepatocyte differentiation. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2011; 97:79-101. [PMID: 21074730 DOI: 10.1016/b978-0-12-385233-5.00003-9] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The liver is the largest glandular organ in the body and plays a central role in controlling metabolism. During hepatogenesis, complex developmental processes must generate an array of cell types that are spatially arranged to generate a hepatic architecture that is essential to support liver function. The processes that control the ultimate formation of the liver are diverse and complex and in many cases poorly defined. Much of the focus of research during the past three decades has been on understanding how hepatocytes, which are the predominant liver parenchymal cells, differentiate during embryogenesis. Through a combination of mouse molecular genetics, embryology, and molecular biochemistry, investigators have defined a myriad of transcription factors that combine to control formation and function of hepatocytes. Here, we will review the major discoveries that underlie our current understanding of transcriptional regulation of hepatocyte differentiation.
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Affiliation(s)
- Masato Nagaoka
- Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
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Santangelo L, Marchetti A, Cicchini C, Conigliaro A, Conti B, Mancone C, Bonzo JA, Gonzalez FJ, Alonzi T, Amicone L, Tripodi M. The stable repression of mesenchymal program is required for hepatocyte identity: a novel role for hepatocyte nuclear factor 4α. Hepatology 2011; 53:2063-74. [PMID: 21384409 PMCID: PMC6624426 DOI: 10.1002/hep.24280] [Citation(s) in RCA: 97] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
UNLABELLED The concept that cellular terminal differentiation is stably maintained once development is complete has been questioned by numerous observations showing that differentiated epithelium may undergo an epithelial-to-mesenchymal transition (EMT) program. EMT and the reverse process, mesenchymal-to-epithelial transition (MET), are typical events of development, tissue repair, and tumor progression. In this study, we aimed to clarify the molecular mechanisms underlying these phenotypic conversions in hepatocytes. Hepatocyte nuclear factor 4α (HNF4α) was overexpressed in different hepatocyte cell lines and the resulting gene expression profile was determined by real-time quantitative polymerase chain reaction. HNF4α recruitment on promoters of both mesenchymal and EMT regulator genes was determined by way of electrophoretic mobility shift assay and chromatin immunoprecipitation. The effect of HNF4α depletion was assessed in silenced cells and in the context of the whole liver of HNF4 knockout animals. Our results identified key EMT regulators and mesenchymal genes as new targets of HNF4α. HNF4α, in cooperation with its target HNF1α, directly inhibits transcription of the EMT master regulatory genes Snail, Slug, and HMGA2 and of several mesenchymal markers. HNF4α-mediated repression of EMT genes induces MET in hepatomas, and its silencing triggers the mesenchymal program in differentiated hepatocytes both in cell culture and in the whole liver. CONCLUSION The pivotal role of HNF4α in the induction and maintenance of hepatocyte differentiation should also be ascribed to its capacity to continuously repress the mesenchymal program; thus, both HNF4α activator and repressor functions are necessary for the identity of hepatocytes.
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Affiliation(s)
- Laura Santangelo
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - Alessandra Marchetti
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - Carla Cicchini
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - Alice Conigliaro
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - Beatrice Conti
- National Institute for Infectious Diseases L. Spallanzani, Institute of Research and Cure of Scientific Character, Rome, Italy
| | - Carmine Mancone
- National Institute for Infectious Diseases L. Spallanzani, Institute of Research and Cure of Scientific Character, Rome, Italy
| | - Jessica A. Bonzo
- Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD
| | - Frank J Gonzalez
- Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD
| | - Tonino Alonzi
- National Institute for Infectious Diseases L. Spallanzani, Institute of Research and Cure of Scientific Character, Rome, Italy
| | - Laura Amicone
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy
| | - Marco Tripodi
- Department of Cellular Biotechnologies and Hematology, Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy,National Institute for Infectious Diseases L. Spallanzani, Institute of Research and Cure of Scientific Character, Rome, Italy
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Reiner JE, Datta PK. TGF-beta-dependent and -independent roles of STRAP in cancer. Front Biosci (Landmark Ed) 2011; 16:105-15. [PMID: 21196161 DOI: 10.2741/3678] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
The serine-threonine kinase receptor-associated protein (STRAP) was initially identified as a putative inhibitor of the canonical TGF-beta signaling pathway. Because the Smad-dependent TGF-beta pathway negatively regulates cellular growth, early functional studies suggested that STRAP behaves as an oncogene. Indeed, a correlation between STRAP overexpression and various cancers has been identified. With the emergence of new studies on the biological function of STRAP, it is becoming clear that STRAP regulates several distinct cellular processes and modulates multiple signaling pathways. While STRAP itself does not possess enzymatic activity, it appears that STRAP influences biological processes through associations with cellular proteins. In this review, we will describe the TGF-beta-dependent and -independent functions of STRAP and provide a context for the significance of STRAP activity in the development of cancer.
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Affiliation(s)
- Jennifer Elisabeth Reiner
- Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
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Darsigny M, Babeu JP, Seidman EG, Gendron FP, Levy E, Carrier J, Perreault N, Boudreau F. Hepatocyte nuclear factor-4alpha promotes gut neoplasia in mice and protects against the production of reactive oxygen species. Cancer Res 2010; 70:9423-33. [PMID: 21062980 DOI: 10.1158/0008-5472.can-10-1697] [Citation(s) in RCA: 76] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Hepatocyte nuclear factor-4α (Hnf4α) is a transcription factor that controls epithelial cell polarity and morphogenesis. Hnf4α conditional deletion during postnatal development has minor effects on intestinal epithelium integrity but promotes activation of the Wnt/β-catenin pathway without causing tumorigenesis. Here, we show that Hnf4α does not act as a tumor-suppressor gene but is crucial in promoting gut tumorigenesis in mice. Polyp multiplicity in ApcMin mice lacking Hnf4α is suppressed compared with littermate ApcMin controls. Analysis of microarray gene expression profiles from mice lacking Hnf4α in the intestinal epithelium identifies novel functions of this transcription factor in targeting oxidoreductase-related genes involved in the regulation of reactive oxygen species (ROS) levels. This role is supported with the demonstration that HNF4α is functionally involved in the protection against spontaneous and 5-fluorouracil chemotherapy-induced production of ROS in colorectal cancer cell lines. Analysis of a colorectal cancer patient cohort establishes that HNF4α is significantly upregulated compared with adjacent normal epithelial resections. Several genes involved in ROS neutralization are also induced in correlation with HNF4A expression. Altogether, the findings point to the nuclear receptor HNF4α as a potential therapeutic target to eradicate aberrant epithelial cell resistance to ROS production during intestinal tumorigenesis.
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Affiliation(s)
- Mathieu Darsigny
- Canadian Institute of Health Research Team on Digestive Epithelium, Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada
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Kanazawa T, Konno A, Hashimoto Y, Kon Y. Hepatocyte nuclear factor 4 alpha is related to survival of the condensed mesenchyme in the developing mouse kidney. Dev Dyn 2010; 239:1145-54. [DOI: 10.1002/dvdy.22276] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
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Koh W, Sheng CT, Tan B, Lee QY, Kuznetsov V, Kiang LS, Tanavde V. Analysis of deep sequencing microRNA expression profile from human embryonic stem cells derived mesenchymal stem cells reveals possible role of let-7 microRNA family in downstream targeting of hepatic nuclear factor 4 alpha. BMC Genomics 2010; 11 Suppl 1:S6. [PMID: 20158877 PMCID: PMC2822534 DOI: 10.1186/1471-2164-11-s1-s6] [Citation(s) in RCA: 105] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Background Recent literature has revealed that genetic exchange of microRNA between cells can be essential for cell-cell communication, tissue-specificity and developmental processes. In stem cells, as in other cells, this can be accomplished through microvesicles or exosome mediated transfer. However, molecular profiles and functions of microRNAs within the cells and in their exosomes are poorly studied. Next generation sequencing technologies could provide a broad-spectrum of microRNAs and their expression and identify possible microRNA targets. In this work, we performed deep sequencing of microRNAs to understand the profile and expression of the microRNAs in microvesicles and intracellular environment of human embryonic stem cells derived mesenchymal stem cells (hES-MSC). We outline a workflow pertaining to visualizing, statistical analysis and interpreting deep sequencing data of known intracellular and extracellular microRNAs from hES-MSC). We utilized these results of which directed our attention towards establishing hepatic nuclear factor 4 alpha (HNF4A) as a downstream target of let-7 family of microRNAs. Results In our study, significant differences in expression profile of microRNAs were found in the intracellular and extracellular environment of hES-MSC. However, a high level of let-7 family of microRNAs is predominant in both intra- and extra- cellular samples of hES-MSC. Further results derived from visualization of our alignment data and network analysis showed that let-7 family microRNAs could affect the downstream target HNF4A, which is a known endodermal differentiation marker. The elevated presence of let-7 microRNA in both intracellular and extra cellular environment further suggests a possible intercellular signalling mechanism through microvesicles transfer. We suggest that let-7 family microRNAs might play a signalling role via such a mechanism amongst populations of stem cells in maintaining self renewal property by suppressing HNF4A expression. This is in line with recent paradigm where microRNAs regulate self-renewal and differentiation pathways of embryonic stem cells by forming an integral biological network with transcription factors. Conclusion In summary, our study using a combination of alignment, statistical and network analysis tools to examine deep sequencing data of microRNAs in hES-MSC has led to a result that (i) identifies intracellular and exosome microRNA expression profiles of hES-MSCwith a possible mechanism of miRNA mediated intercellular regulation by these cells and (ii) placed HNF4A within the cross roads of regulation by the let-7 family of microRNAs.
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Affiliation(s)
- Winston Koh
- Bioinformatics Institute (BII), Agency of Science Technology and Research (A*STAR), Matrix, Singapore.
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