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Contextual Regulation of TGF-β Signaling in Liver Cancer. Cells 2019; 8:cells8101235. [PMID: 31614569 PMCID: PMC6829617 DOI: 10.3390/cells8101235] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2019] [Revised: 10/09/2019] [Accepted: 10/10/2019] [Indexed: 02/06/2023] Open
Abstract
Primary liver cancer is one of the leading causes for cancer-related death worldwide. Transforming growth factor beta (TGF-β) is a pleiotropic cytokine that signals through membrane receptors and intracellular Smad proteins, which enter the nucleus upon receptor activation and act as transcription factors. TGF-β inhibits liver tumorigenesis in the early stage by inducing cytostasis and apoptosis, but promotes malignant progression in more advanced stages by enhancing cancer cell survival, EMT, migration, invasion and finally metastasis. Understanding the molecular mechanisms underpinning the multi-faceted roles of TGF-β in liver cancer has become a persistent pursuit during the last two decades. Contextual regulation fine-tunes the robustness, duration and plasticity of TGF-β signaling, yielding versatile albeit specific responses. This involves multiple feedback and feed-forward regulatory loops and also the interplay between Smad signaling and non-Smad pathways. This review summarizes the known regulatory mechanisms of TGF-β signaling in liver cancer, and how they channel, skew and even switch the actions of TGF-β during cancer progression.
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Arboatti AS, Lambertucci F, Sedlmeier MG, Pisani G, Monti J, Álvarez MDL, Francés DEA, Ronco MT, Carnovale CE. Diethylnitrosamine enhances hepatic tumorigenic pathways in mice fed with high fat diet (Hfd). Chem Biol Interact 2019; 303:70-78. [PMID: 30826251 DOI: 10.1016/j.cbi.2019.02.024] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2018] [Revised: 01/28/2019] [Accepted: 02/24/2019] [Indexed: 12/21/2022]
Abstract
Obesity has been implicated in the genesis of metabolic syndromes including insulin resistance and Type 2 Diabetes Mellitus (T2DM). Given the association between T2DM and the risk of hepatocellular carcinoma (HCC), our specific goal was to determine whether the liver of HFD-induced T2DM mice is more sensitive to the carcinogen diethylnitrosamine (DEN), due to a modification of the molecular pathways implicated in the early stages of HCC pathogenesis. C57BL/6 male mice (five-week-old) were divided into 4 groups: C, C + DEN, HFD and HFD + DEN. Mice were euthanized twenty-five weeks after DEN-injection. Livers of HDF-fed mice showed a higher proliferative index than Control groups. In line with this, HFD groups showed an increase of nuclear β-catenin, and interestingly, DEN treatment led to a slight increase in the expression of this protein in HFD group. Based on these results, and to confirm this effect, we analyzed β-catenin target genes, finding that DEN treatment in HFD group led to a significant increase of Vegf, c-myc, c-jun and cyclin D1 expression levels. According to our results, the expression of TCF4 showed to be significantly increased in HFD + DEN vs. HFD. In this regard, the β-catenin/TCF4 complex enhanced its association with pSmads 2/3, as we observed an increase of nuclear Smads expression in HFD + DEN, suggesting a possible role of TGF-β1/Smads signaling pathway in this phenomenon. Our results show that the liver of HFD fed model that resembles early T2DM pathology in mice, is more sensitive to DEN, by inducing both Wnt/β-catenin and TGF β1/Smads tumorigenic pathways.
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Affiliation(s)
- A S Arboatti
- Instituto de Fisiología Experimental (IFISE-CONICET), Cátedra de Fisiología, Facultad de Ciencias Bioquímicas y Farmacéuticas- UNR, Suipacha 570, 2000, Rosario, Argentina
| | - F Lambertucci
- Instituto de Fisiología Experimental (IFISE-CONICET), Cátedra de Fisiología, Facultad de Ciencias Bioquímicas y Farmacéuticas- UNR, Suipacha 570, 2000, Rosario, Argentina
| | - M G Sedlmeier
- Instituto de Fisiología Experimental (IFISE-CONICET), Cátedra de Fisiología, Facultad de Ciencias Bioquímicas y Farmacéuticas- UNR, Suipacha 570, 2000, Rosario, Argentina
| | - G Pisani
- Cátedra de Morfología, Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR, Suipacha 570, 2000, Rosario, Argentina
| | - J Monti
- Instituto de Fisiología Experimental (IFISE-CONICET), Cátedra de Fisiología, Facultad de Ciencias Bioquímicas y Farmacéuticas- UNR, Suipacha 570, 2000, Rosario, Argentina
| | - M de L Álvarez
- Instituto de Fisiología Experimental (IFISE-CONICET), Cátedra de Fisiología, Facultad de Ciencias Bioquímicas y Farmacéuticas- UNR, Suipacha 570, 2000, Rosario, Argentina; Cátedra de Morfología, Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR, Suipacha 570, 2000, Rosario, Argentina
| | - D E A Francés
- Instituto de Fisiología Experimental (IFISE-CONICET), Cátedra de Fisiología, Facultad de Ciencias Bioquímicas y Farmacéuticas- UNR, Suipacha 570, 2000, Rosario, Argentina
| | - M T Ronco
- Instituto de Fisiología Experimental (IFISE-CONICET), Cátedra de Fisiología, Facultad de Ciencias Bioquímicas y Farmacéuticas- UNR, Suipacha 570, 2000, Rosario, Argentina
| | - C E Carnovale
- Instituto de Fisiología Experimental (IFISE-CONICET), Cátedra de Fisiología, Facultad de Ciencias Bioquímicas y Farmacéuticas- UNR, Suipacha 570, 2000, Rosario, Argentina.
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Zhang J, Wang Y, Fu L, Wang B, Ji YL, Wang H, Xu DX. Chronic cadmium exposure induced hepatic cellular stress and inflammation in aged female mice. J Appl Toxicol 2018; 39:498-509. [PMID: 30375035 DOI: 10.1002/jat.3742] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2018] [Revised: 09/11/2018] [Accepted: 09/25/2018] [Indexed: 01/04/2023]
Abstract
Previous studies have revealed that acute cadmium (Cd) exposure led to inflammation in different organs through an oxidative stress mechanism. However, whether chronic Cd exposure induces inflammation in liver and the mechanistic link between inflammation and cell stress remains unclear. In the present study, we investigated the effects of chronic Cd exposure on hepatic cellular stress and inflammatory responses. Female CD1 mice were administrated with CdCl2 (10 and 100 mg/L) in drinking water for 57 weeks. Our results showed that the mRNA levels of Inos and the protein content of HO-1, markers of oxidative stress, were markedly increased in Cd-treated mice. In addition, the protein level of GRP78, the chaperone of endoplasmic reticulum (ER) stress, was significantly increased in Cd-treated mice. The expression of the proteins CHOP and peIF2α, two proteins downstream of ER stress, was also upregulated in the Cd-100 mg/L and Cd-10 mg/L group, respectively. Moreover, there were increased inflammatory cells existing in liver after Cd administration. Besides, there was a significant elevation in the mRNA level of Mip-2, Il-10 and Il-12 in the Cd-100 mg/L group. The mRNA level of Tgf-β was also upregulated in Cd-treated mice. Moreover, we also found that the number of Ki67-positive hepatic cells was increased in the Cd-10 mg/L group. Hence, our results indicated that chronic Cd exposure induced oxidative stress, ER stress, inflammatory responses and proliferation in the liver of aged female mice.
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Affiliation(s)
- Jun Zhang
- Department of Toxicology, School of Public Health, Anhui Medical University, Hefei, China.,Anhui Provincial Key Laboratory of Population Health & Aristogenics, Anhui Medical University, Hefei, China.,Laboratory of Environmental Toxicology, Anhui Medical University, Hefei, China
| | - Yan Wang
- Department of Toxicology, School of Public Health, Anhui Medical University, Hefei, China.,Laboratory of Environmental Toxicology, Anhui Medical University, Hefei, China
| | - Lin Fu
- Department of Toxicology, School of Public Health, Anhui Medical University, Hefei, China.,Anhui Provincial Key Laboratory of Population Health & Aristogenics, Anhui Medical University, Hefei, China.,Laboratory of Environmental Toxicology, Anhui Medical University, Hefei, China
| | - Bo Wang
- Department of Toxicology, School of Public Health, Anhui Medical University, Hefei, China.,Laboratory of Environmental Toxicology, Anhui Medical University, Hefei, China
| | - Yan-Li Ji
- Department of Toxicology, School of Public Health, Anhui Medical University, Hefei, China.,Anhui Provincial Key Laboratory of Population Health & Aristogenics, Anhui Medical University, Hefei, China.,Laboratory of Environmental Toxicology, Anhui Medical University, Hefei, China
| | - Hua Wang
- Department of Toxicology, School of Public Health, Anhui Medical University, Hefei, China.,Anhui Provincial Key Laboratory of Population Health & Aristogenics, Anhui Medical University, Hefei, China.,Laboratory of Environmental Toxicology, Anhui Medical University, Hefei, China
| | - De-Xiang Xu
- Department of Toxicology, School of Public Health, Anhui Medical University, Hefei, China.,Anhui Provincial Key Laboratory of Population Health & Aristogenics, Anhui Medical University, Hefei, China.,Laboratory of Environmental Toxicology, Anhui Medical University, Hefei, China
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Wang Y. The inhibition of microRNA-15a suppresses hepatitis B virus-associated liver cancer cell growth through the Smad/TGF-β pathway. Oncol Rep 2017; 37:3520-3526. [PMID: 28498453 DOI: 10.3892/or.2017.5618] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2017] [Accepted: 03/02/2017] [Indexed: 11/06/2022] Open
Abstract
In the present study, the role of microRNA‑15a (miR‑15a) was investigated in hepatitis B virus (HBV)‑associated liver cancer. The results revealed that the expression levels of miR-15a were increased in HBV-associated liver cancer tissues compared with the levels in normal tumor‑adjacent tissues. Moreover, Smad-7 protein expression in patients with HBV-associated liver cancer was higher than that in normal tumor-adjacent tissues. In addition, miR-15a expression and Smad-7 protein expression were increased in HepG2 hepatocellular carcinoma cells compared with that noted in L-02 normal hepatocytes. In HepG2 cells, miR-15a inhibition suppressed cell proliferation and increased Smad-7 protein expression. The inhibition of miR-15a was also demonstrated to decrease transforming growth factor (TGF)-β1 protein expression and Smad-2, p-Smad-2 and Smad-4 expression levels in HepG2 cells. Furthermore, FSP1 protein expression and caspase-3/-7 activities were enhanced by miR-15a inhibition in HepG2 cells compared with the control group. Treatment with recombinant TGF-β1 was demonstrated to activate Smad‑2/-4 and FSP1 protein expression and increase caspase-3/-7 activity in HepG2 cells. Collectively, these findings demonstrate that the miR-15a/Smad-7/TGF-β pathway is important in HBV-associated liver cancer.
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Affiliation(s)
- Yan Wang
- Department of Infectious Diseases, Binzhou Tuberculosis Prevention and Control Hospital, Huimin, Binzhou, Shandong 251700, P.R. China
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5
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Herbertz S, Sawyer JS, Stauber AJ, Gueorguieva I, Driscoll KE, Estrem ST, Cleverly AL, Desaiah D, Guba SC, Benhadji KA, Slapak CA, Lahn MM. Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway. Drug Des Devel Ther 2015; 9:4479-99. [PMID: 26309397 PMCID: PMC4539082 DOI: 10.2147/dddt.s86621] [Citation(s) in RCA: 277] [Impact Index Per Article: 27.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Transforming growth factor-beta (TGF-β) signaling regulates a wide range of biological processes. TGF-β plays an important role in tumorigenesis and contributes to the hallmarks of cancer, including tumor proliferation, invasion and metastasis, inflammation, angiogenesis, and escape of immune surveillance. There are several pharmacological approaches to block TGF-β signaling, such as monoclonal antibodies, vaccines, antisense oligonucleotides, and small molecule inhibitors. Galunisertib (LY2157299 monohydrate) is an oral small molecule inhibitor of the TGF-β receptor I kinase that specifically downregulates the phosphorylation of SMAD2, abrogating activation of the canonical pathway. Furthermore, galunisertib has antitumor activity in tumor-bearing animal models such as breast, colon, lung cancers, and hepatocellular carcinoma. Continuous long-term exposure to galunisertib caused cardiac toxicities in animals requiring adoption of a pharmacokinetic/pharmacodynamic-based dosing strategy to allow further development. The use of such a pharmacokinetic/pharmacodynamic model defined a therapeutic window with an appropriate safety profile that enabled the clinical investigation of galunisertib. These efforts resulted in an intermittent dosing regimen (14 days on/14 days off, on a 28-day cycle) of galunisertib for all ongoing trials. Galunisertib is being investigated either as monotherapy or in combination with standard antitumor regimens (including nivolumab) in patients with cancer with high unmet medical needs such as glioblastoma, pancreatic cancer, and hepatocellular carcinoma. The present review summarizes the past and current experiences with different pharmacological treatments that enabled galunisertib to be investigated in patients.
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Affiliation(s)
| | - J Scott Sawyer
- Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA
| | - Anja J Stauber
- Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA
| | | | - Kyla E Driscoll
- Lilly Research Laboratories, Eli Lilly and Company, New York, NY, USA
| | - Shawn T Estrem
- Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA
| | - Ann L Cleverly
- Lilly Research Laboratories, Eli Lilly and Company, Windlesham, Surrey, UK
| | - Durisala Desaiah
- Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA
| | - Susan C Guba
- Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA
| | - Karim A Benhadji
- Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA
| | | | - Michael M Lahn
- Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA
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6
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Feng T, Dzieran J, Gu X, Marhenke S, Vogel A, Machida K, Weiss TS, Ruemmele P, Kollmar O, Hoffmann P, Grässer F, Allgayer H, Fabian J, Weng HL, Teufel A, Maass T, Meyer C, Lehmann U, Zhu C, Mertens PR, Gao CF, Dooley S, Meindl-Beinker NM. Smad7 regulates compensatory hepatocyte proliferation in damaged mouse liver and positively relates to better clinical outcome in human hepatocellular carcinoma. Clin Sci (Lond) 2015; 128:761-74. [PMID: 25602745 PMCID: PMC10618913 DOI: 10.1042/cs20140606] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/29/2023]
Abstract
Transforming growth factor β (TGF-β) is cytostatic towards damage-induced compensatory hepatocyte proliferation. This function is frequently lost during hepatocarcinogenesis, thereby switching the TGF-β role from tumour suppressor to tumour promoter. In the present study, we investigate Smad7 overexpression as a pathophysiological mechanism for cytostatic TGF-β inhibition in liver damage and hepatocellular carcinoma (HCC). Transgenic hepatocyte-specific Smad7 overexpression in damaged liver of fumarylacetoacetate hydrolase (FAH)-deficient mice increased compensatory proliferation of hepatocytes. Similarly, modulation of Smad7 expression changed the sensitivity of Huh7, FLC-4, HLE and HLF HCC cell lines for cytostatic TGF-β effects. In our cohort of 140 HCC patients, Smad7 transcripts were elevated in 41.4% of HCC samples as compared with adjacent tissue, with significant positive correlation to tumour size, whereas low Smad7 expression levels were significantly associated with worse clinical outcome. Univariate and multivariate analyses indicate Smad7 levels as an independent predictor for overall (P<0.001) and disease-free survival (P=0.0123). Delineating a mechanism for Smad7 transcriptional regulation in HCC, we identified cold-shock Y-box protein-1 (YB-1), a multifunctional transcription factor. YB-1 RNAi reduced TGF-β-induced and endogenous Smad7 expression in Huh7 and FLC-4 cells respectively. YB-1 and Smad7 mRNA expression levels correlated positively (P<0.0001). Furthermore, nuclear co-localization of Smad7 and YB-1 proteins was present in cancer cells of those patients. In summary, the present study provides a YB-1/Smad7-mediated mechanism that interferes with anti-proliferative/tumour-suppressive TGF-β actions in a subgroup of HCC cells that may facilitate aspects of tumour progression.
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MESH Headings
- Animals
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/metabolism
- Carcinoma, Hepatocellular/pathology
- Cell Line, Tumor
- Cell Proliferation
- Cells, Cultured
- Female
- Gene Expression Regulation, Neoplastic/drug effects
- Hep G2 Cells
- Hepatocytes/metabolism
- Humans
- Liver Diseases/genetics
- Liver Diseases/metabolism
- Liver Diseases/pathology
- Liver Neoplasms/genetics
- Liver Neoplasms/metabolism
- Liver Neoplasms/pathology
- Male
- Mice, 129 Strain
- Mice, Knockout
- Mice, Transgenic
- Microscopy, Confocal
- Middle Aged
- Multivariate Analysis
- RNA Interference
- Reverse Transcriptase Polymerase Chain Reaction
- Smad7 Protein/genetics
- Smad7 Protein/metabolism
- Survival Analysis
- Transforming Growth Factor beta/pharmacology
- Y-Box-Binding Protein 1/genetics
- Y-Box-Binding Protein 1/metabolism
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Affiliation(s)
- Teng Feng
- *Molecular Hepatology Alcohol Associated Diseases, Dept. of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Johanna Dzieran
- *Molecular Hepatology Alcohol Associated Diseases, Dept. of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Xing Gu
- †Department of Laboratory Medicine, Eastern Hepatobiliary Hospital, Second Military Medical University, Shanghai, China
| | - Silke Marhenke
- ‡Department of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Hannover, Germany
| | - Arndt Vogel
- ‡Department of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Hannover, Germany
| | - Keigo Machida
- §Department of Molecular Microbiology and Immunology and Southern California Research Center for ALPD and Cirrhosis, Los Angeles, CA, U.S.A
| | - Thomas S Weiss
- ║Department of Pediatrics and Juvenile Medicine, Center for Liver Cell Research, University of Regensburg Hospital, Regensburg, Germany
| | - Petra Ruemmele
- ¶Institute of Pathology, University of Regensburg, Regensburg, Germany
| | - Otto Kollmar
- **Department of General, Visceral, Vascular and Pediatric Surgery, University of Saarland, Homburg/Saar, Germany
| | - Patrick Hoffmann
- ††Saarland University Medical School, Institute of Virology, Homburg/Saar, Germany
| | - Friedrich Grässer
- ††Saarland University Medical School, Institute of Virology, Homburg/Saar, Germany
| | - Heike Allgayer
- ‡‡Department of Experimental Surgery, University of Heidelberg, Mannheim and Molecular Oncology of Solid Tumors, DKFZ, Heidelberg, Germany
| | - Jasmin Fabian
- *Molecular Hepatology Alcohol Associated Diseases, Dept. of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Hong Lei Weng
- *Molecular Hepatology Alcohol Associated Diseases, Dept. of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Andreas Teufel
- §§Klinik und Poliklinik für Innere Medizin I, Universitätsklinikum Regensburg, Regensburg, Germany
| | - Thorsten Maass
- §§Klinik und Poliklinik für Innere Medizin I, Universitätsklinikum Regensburg, Regensburg, Germany
| | - Christoph Meyer
- *Molecular Hepatology Alcohol Associated Diseases, Dept. of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Ulrich Lehmann
- ║║Institute of Pathology, Medical School Hannover, Hannover, Germany
| | - Cheng Zhu
- ¶¶Department of Nephrology and Hypertension, Diabetes and Endocrinology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
| | - Peter R Mertens
- ¶¶Department of Nephrology and Hypertension, Diabetes and Endocrinology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
| | - Chun Fang Gao
- †Department of Laboratory Medicine, Eastern Hepatobiliary Hospital, Second Military Medical University, Shanghai, China
| | - Steven Dooley
- *Molecular Hepatology Alcohol Associated Diseases, Dept. of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Nadja M Meindl-Beinker
- *Molecular Hepatology Alcohol Associated Diseases, Dept. of Medicine II, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
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TGF-β signal shifting between tumor suppression and fibro-carcinogenesis in human chronic liver diseases. J Gastroenterol 2014; 49:971-81. [PMID: 24263677 DOI: 10.1007/s00535-013-0910-2] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/25/2013] [Accepted: 11/04/2013] [Indexed: 02/04/2023]
Abstract
Perturbation of transforming growth factor (TGF)-β signaling in hepatocytes persistently infected with hepatitis viruses promotes both fibrogenesis and carcinogenesis (fibro-carcinogenesis). Insights into hepatocytic fibro-carcinogenesis have emerged from recent detailed analyses of context-dependent and cell type-specific TGF-β signaling processes directed by multiple phosphorylated forms (phospho-isoforms) of Smad mediators. In the course of hepatitis virus-related chronic liver diseases, chronic inflammation, ongoing viral infection, and host genetic/epigenetic alterations additively shift hepatocytic Smad phospho-isoform signaling from tumor suppression to fibro-carcinogenesis, accelerating liver fibrosis and increasing risk of hepatocellular carcinoma (HCC). After successful antiviral therapy, patients with chronic hepatitis can experience less risk of HCC occurrence by reversing Smad phospho-isoform signaling from fibro-carcinogenesis to tumor suppression. However, patients with cirrhosis can still develop HCC owing to sustained, intense fibro-carcinogenic signaling. Recent progress in understanding Smad phospho-isoform signaling should permit use of Smad phosphorylation as a tool predicting the likelihood of liver disease progression, and as a biomarker for assessing the effectiveness of interventions aimed at reducing fibrosis and cancer risk.
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Bertran E, Crosas-Molist E, Sancho P, Caja L, Lopez-Luque J, Navarro E, Egea G, Lastra R, Serrano T, Ramos E, Fabregat I. Overactivation of the TGF-β pathway confers a mesenchymal-like phenotype and CXCR4-dependent migratory properties to liver tumor cells. Hepatology 2013; 58:2032-44. [PMID: 23813475 DOI: 10.1002/hep.26597] [Citation(s) in RCA: 111] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/04/2013] [Accepted: 06/15/2013] [Indexed: 12/11/2022]
Abstract
UNLABELLED Transforming growth factor-beta (TGF-β) is an important regulatory suppressor factor in hepatocytes. However, liver tumor cells develop mechanisms to overcome its suppressor effects and respond to this cytokine by inducing other processes, such as the epithelial-mesenchymal transition (EMT), which contributes to tumor progression and dissemination. Recent studies have placed chemokines and their receptors at the center not only of physiological cell migration but also of pathological processes, such as metastasis in cancer. In particular, CXCR4 and its ligand, stromal cell-derived factor 1α (SDF-1α) / chemokine (C-X-C motif) ligand 12 (CXCL12) have been revealed as regulatory molecules involved in the spreading and progression of a variety of tumors. Here we show that autocrine stimulation of TGF-β in human liver tumor cells correlates with a mesenchymal-like phenotype, resistance to TGF-β-induced suppressor effects, and high expression of CXCR4, which is required for TGF-β-induced cell migration. Silencing of the TGF-β receptor1 (TGFBR1), or its specific inhibition, recovered the epithelial phenotype and attenuated CXCR4 expression, inhibiting cell migratory capacity. In an experimental mouse model of hepatocarcinogenesis (diethylnitrosamine-induced), tumors showed increased activation of the TGF-β pathway and enhanced CXCR4 levels. In human hepatocellular carcinoma tumors, high levels of CXCR4 always correlated with activation of the TGF-β pathway, a less differentiated phenotype, and a cirrhotic background. CXCR4 concentrated at the tumor border and perivascular areas, suggesting its potential involvement in tumor cell dissemination. CONCLUSION A crosstalk exists among the TGF-β and CXCR4 pathways in liver tumors, reflecting a novel molecular mechanism that explains the protumorigenic effects of TGF-β and opens new perspectives for tumor therapy.
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Affiliation(s)
- Esther Bertran
- Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain
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9
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Mu X, Lin S, Yang J, Chen C, Chen Y, Herzig MC, Washburn K, Halff GA, Walter CA, Sun B, Sun LZ. TGF-β signaling is often attenuated during hepatotumorigenesis, but is retained for the malignancy of hepatocellular carcinoma cells. PLoS One 2013; 8:e63436. [PMID: 23704908 PMCID: PMC3660330 DOI: 10.1371/journal.pone.0063436] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2013] [Accepted: 03/29/2013] [Indexed: 01/07/2023] Open
Abstract
The role of transforming growth factor-beta (TGF-β) signaling in hepatocarcinogenesis remains controversial. We aimed to reveal TGF-β signaling status in human and murine tissues of hepatocellular carcinoma (HCC) and the mechanisms that mediate TGF-β’s role in regulating HCC malignancy. Here, TGF-β pathway component expression and activation in human and murine HCC tissues were measured with quantitative RT-PCR and Western blotting assays. The role of TGF-β receptor and Smad signaling in the growth and survival of several HCC cell lines was determined with several in vitro and in vivo approaches. We found that TGF-β receptor II (TβRII) expression was downregulated in two different HCC patient cohorts. Consistently, Smad3 phosphorylation was also downregulated in HCC tissues in comparison to that in adjacent normal tissues. Interestingly, many HCC cell lines were sensitive to TGF-β and growth-inhibited by exogenous TGF-β. However, stable knockdown of TβRII inhibited cell growth on plastic and in soft agar, and induced apoptosis resulting in suppressed subcutaneous tumor growth and metastatic potential in vivo. Furthermore, knockdown of Smad4 also led to a significant inhibition of growth on plastic and in soft agar with concomitant increase of apoptosis, PTEN expression, and reduced nuclear accumulation of linker region-phosphorylated Smad3. Taken together, TGF-β signaling pathway plays a dichotomous role in hepatocellular carcinogenesis. It appears to suppress HCC development, but is retained for HCC cell survival and malignancy. Furthermore, Smad4 can mediate both growth inhibitory activity induced by exogenous TGF-β and the survival activity induced by autocrine TGF-β revealing a delicate selection of the two opposing activities of TGF-β during HCC evolution.
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Affiliation(s)
- Xiaoxin Mu
- Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China
- Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas, United States of America
| | - Shu Lin
- Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas, United States of America
| | - Junhua Yang
- Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas, United States of America
| | - Chen Chen
- Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China
| | - Yun Chen
- Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China
| | - Maryanne C. Herzig
- Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas, United States of America
| | - Kenneth Washburn
- Transplant Center, University of Texas Health Science Center, San Antonio, Texas, United States of America
| | - Glenn A. Halff
- Transplant Center, University of Texas Health Science Center, San Antonio, Texas, United States of America
| | - Christi A. Walter
- Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas, United States of America
- Cancer Therapy and Cancer Center, University of Texas Health Science Center, San Antonio, Texas, United States of America
- South Texas Veteran’s Health Care System, Audie Murphy Hospital, San Antonio, Texas, United States of America
| | - Beicheng Sun
- Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China
- * E-mail: ) (L-ZS; (LS) (BS)
| | - Lu-Zhe Sun
- Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas, United States of America
- Cancer Therapy and Cancer Center, University of Texas Health Science Center, San Antonio, Texas, United States of America
- * E-mail: ) (L-ZS; (LS) (BS)
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10
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Hepatitis B surface antigen could contribute to the immunopathogenesis of hepatitis B virus infection. ISRN GASTROENTEROLOGY 2013; 2013:935295. [PMID: 23401786 PMCID: PMC3562682 DOI: 10.1155/2013/935295] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/11/2012] [Accepted: 12/24/2012] [Indexed: 12/20/2022]
Abstract
Various findings concerning the clinical significance of quantitative changes in hepatitis B surface antigen (HBsAg) during the acute and chronic phase of hepatitis B virus (HBV) infection have been reported. In addition to being a biomarker of HBV-replication activity, it has been reported that HBsAg could contribute to the immunopathogenesis of HBV persistent infection. Moreover, HBsAg could become an attractive target for immune therapy, since the cellular and humeral immune response against HBsAg might be able to control the HBV replication and life cycle. However, several reports have described the immune suppressive function of HBsAg. HBsAg might suppress monocytes, dendritic cells (DCs), natural killer (NK), and natural killer T (NK-T) cells by direct interaction. On the other hand, cytotoxic T lymphocytes (CTLs) and helper T (Th) cells were exhausted by high amounts of HBsAg. In this paper, we focused on the immunological aspects of HBsAg, since better understanding of the interaction between HBsAg and immune cells could contribute to the development of an immune therapy as well as a biomarker of the state of HBV persistent infection.
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11
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Suppression of glypican 3 inhibits growth of hepatocellular carcinoma cells through up-regulation of TGF-β2. Neoplasia 2011; 13:735-47. [PMID: 21847365 DOI: 10.1593/neo.11664] [Citation(s) in RCA: 207] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2011] [Revised: 06/06/2011] [Accepted: 06/07/2011] [Indexed: 12/27/2022] Open
Abstract
Glypican 3 (GPC3) is a valuable diagnostic marker and a potential therapeutic target in hepatocellular carcinoma (HCC). To evaluate the efficacy of targeting GPC3 at the translational level, we used RNA interference to examine the biologic and molecular effects of GPC3 suppression in HCC cells in vitro and in vivo. Transfection of Huh7 and HepG2 cells with GPC3-specific small interfering RNA (siRNA) inhibited cell proliferation (P < .001) together with cell cycle arrest at the G(1) phase, down-regulation of antiapoptotic protein (Bcl-2, Bcl-xL, and Mcl-1), and replicative senescence. Gene expression analysis revealed that GPC3 suppression significantly correlated with transforming growth factor beta receptor (TGFBR) pathway (P = 4.57e-5) and upregulated TGF-β2 at both RNA and protein levels. The effects of GPC3 suppression by siRNA can be recapitulated by addition of human recombinant TGF-β2 to HCC cells in culture, suggesting the possible involvement of TGF-β2 in growth inhibition of HCC cells. Cotransfection of siRNA-GPC3 with siRNA-TGF-β2 partially attenuated the effects of GPC3 suppression on cell proliferation, cell cycle progression, apoptosis, and replicative senescence, confirming the involvement of TGF-β2 in siRNA-GPC3-mediated growth suppression. In vivo, GPC3 suppression significantly inhibited the growth of orthotopic xenografts of Huh7 and HepG2 cells (P < .05), accompanied by increased TGF-β2 expression, reduced cell proliferation (observed by proliferating cell nuclear antigen staining), and enhanced apoptosis (by TUNEL staining). In conclusion, molecular targeting of GPC3 at the translational level offers an effective option for the clinical management of GPC3-positive HCC patients.
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12
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Interferon-α2b and transforming growth factor-β1 treatments on HCC cell lines: Are Wnt/β-catenin pathway and Smads signaling connected in hepatocellular carcinoma? Biochem Pharmacol 2011; 82:1682-91. [PMID: 21843516 DOI: 10.1016/j.bcp.2011.08.001] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2011] [Revised: 08/01/2011] [Accepted: 08/01/2011] [Indexed: 01/04/2023]
Abstract
Wnt/β-catenin pathway is often dysregulated in hepatocellular carcinoma (HCC). Activated β-catenin accumulates in the cytosol and nucleus and forms a nuclear complex with TCF/LEF factors like TCF4. Interferon-α (IFN-α) has recently been recognized to harbor therapeutic potential in prevention and treatment of HCC. Transforming Growth Factor-β1 (TGF-β1) is a mediator of apoptosis, exerting its effects via Smads proteins. One mode of interaction between Wnt/β-catenin and TGF-β1/Smads pathways is the association of Smads with β-catenin/TCF4. In this study we analyzed the effects of IFN-α2b and TGF-β1 treatments on Wnt/β-catenin pathway, Smads proteins levels, β-catenin/TCF4/Smads interaction and proliferation and apoptotic death in HepG2/C3A and Huh7 cell lines. IFN-α2b and TGF-β1 attenuated Wnt/β-catenin signal by decreasing β-catenin and Frizzled7 receptor proteins contents and the interaction of β-catenin with TCF4. Truncated β-catenin form present in C3A cell line also diminished after treatments. Both cytokines declined Smads proteins and their interaction with TCF4. The overall cellular response to cytokines was the decrease in proliferation and increase in apoptotic death. Treatment with Wnt3a, which elevates β-catenin protein levels, also generated the increment of Smads proteins contents when comparing with untreated cells. In conclusion, IFN-α2b and TGF-β1 proved to be effective as modulators of Wnt/β-catenin pathway in HCC cell lines holding both wild-type and truncated β-catenin. Since the inhibition of β-catenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis, IFN-α2b and TGF-β1 could be useful as potential treatments in patients with HCC.
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13
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Smad phosphoisoform signals in acute and chronic liver injury: similarities and differences between epithelial and mesenchymal cells. Cell Tissue Res 2011; 347:225-43. [PMID: 21626291 PMCID: PMC3250618 DOI: 10.1007/s00441-011-1178-6] [Citation(s) in RCA: 74] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2011] [Accepted: 04/15/2011] [Indexed: 12/17/2022]
Abstract
Hepatocellular carcinoma (HCC) usually arises from hepatic fibrosis caused by chronic inflammation. In chronic liver damage, hepatic stellate cells undergo progressive activation to myofibroblasts (MFB), which are important extracellular-matrix-producing mesenchymal cells. Concomitantly, perturbation of transforming growth factor (TGF)-β signaling by pro-inflammatory cytokines in the epithelial cells of the liver (hepatocytes) promotes both fibrogenesis and carcinogenesis (fibro-carcinogenesis). Insights into fibro-carcinogenic effects on chronically damaged hepatocytes have come from recent detailed analyses of the TGF-β signaling process. Smad proteins, which convey signals from TGF-β receptors to the nucleus, have intermediate linker regions between conserved Mad homology (MH) 1 and MH2 domains. TGF-β type I receptor and pro-inflammatory cytokine-activated kinases differentially phosphorylate Smad2 and Smad3 to create phosphoisoforms phosphorylated at the COOH-terminal, linker, or both (L/C) regions. After acute liver injury, TGF-β-mediated pSmad3C signaling terminates hepatocytic proliferation induced by the pro-inflammatory cytokine-mediated mitogenic pSmad3L pathway; TGF-β and pro-inflammatory cytokines synergistically enhance collagen synthesis by activated hepatic stellate cells via pSmad2L/C and pSmad3L/C pathways. During chronic liver disease progression, pre-neoplastic hepatocytes persistently affected by TGF-β together with pro-inflammatory cytokines come to exhibit the same carcinogenic (mitogenic) pSmad3L and fibrogenic pSmad2L/C signaling as do MFB, thereby accelerating liver fibrosis while increasing risk of HCC. This review of Smad phosphoisoform-mediated signals examines similarities and differences between epithelial and mesenchymal cells in acute and chronic liver injuries and considers Smad linker phosphorylation as a potential target for the chemoprevention of fibro-carcinogenesis.
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14
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Liu X, Yang Y, Zhang X, Xu S, He S, Huang W, Roberts MS. Compound Astragalus and Salvia miltiorrhiza extract inhibits cell invasion by modulating transforming growth factor-beta/Smad in HepG2 cell. J Gastroenterol Hepatol 2010; 25:420-6. [PMID: 19793165 DOI: 10.1111/j.1440-1746.2009.05981.x] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND AND AIMS Compound Astragalus and Salvia miltiorrhiza extract (CASE) is made up of astragalosides, astragalus polysaccharide and salvianolic acids extracted from Astragalus membranaceus Bunge (Leguminosae) and Salvia miltiorhiza Bunge (Lamiaceae) with a standard ratio. Previous reports showed that CASE inhibited hepatic fibrosis by mediating transforming growth factor (TGF)-beta/Smad signaling. This study further investigated the effect of CASE on hepatoma HepG2 cells stimulated by TGF-beta(1) and its potential action mechanisms by TGF-beta/Smad signaling. METHODS Cell proliferation was studied by MTT assay and cell invasion was evaluated by measuring cell migration through Matrigel. Protein expression in hepatoma HepG2 cells stimulated by TGF-beta(1) was analyzed by western blotting and plasminogen activator inhibitor type 1 (PAI-1) transcriptional activity in HepG2 cells was evaluated. RESULTS CASE (40 microg/mL) markedly suppressed cell invasion triggered by TGF-beta(1). Smad3 phosphorylation at the linker region (pSmad3L) and Samd2 phosphorylation at the C-terminal region (pSmad2C) were significantly reduced by CASE. Mild elevated Smad3 phosphorylation at C-terminal (pSmade3C) region was enhanced by CASE at 20 microg/mL. In addition, treatment of CASE decreased the level of Smad2/3/4 complex at 80 microg/mL, but upregulated the expression of Smad7 in a dose-dependent manner. CASE also showed inhibitory effect on PAI-1 transcriptional activity. CONCLUSION All these results suggest that CASE exerts anti-HepG2 cell invasion effect by modulating TGF-beta/Smad signaling.
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Affiliation(s)
- Xin Liu
- Department of Pharmacology, Anhui Medical University, Hefei, Anhui, China
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15
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Sánchez A, Fabregat I. Genetically modified animal models recapitulating molecular events altered in human hepatocarcinogenesis. Clin Transl Oncol 2009; 11:208-14. [DOI: 10.1007/s12094-009-0342-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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16
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Matsuzaki K, Murata M, Yoshida K, Sekimoto G, Uemura Y, Sakaida N, Kaibori M, Kamiyama Y, Nishizawa M, Fujisawa J, Okazaki K, Seki T. Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma. Hepatology 2007; 46:48-57. [PMID: 17596875 DOI: 10.1002/hep.21672] [Citation(s) in RCA: 230] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
UNLABELLED Many patients with chronic hepatitis caused by hepatitis C virus (HCV) infection develop liver fibrosis with high risk for hepatocellular carcinoma (HCC), but the mechanism underling this process is unclear. Conversely, transforming growth factor beta (TGF-beta) activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), which convert the mediator Smad3 into two distinctive phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Whereas the TbetaRI/pSmad3C pathway suppresses epithelial cell growth by upregulating p21(WAF1) transcription, JNK/pSmad3L-mediated signaling promotes extracellular matrix deposition, partly, by upregulating plasminogen activator inhibitor 1 (PAI-1). We studied the domain-specific Smad3 phosphorylation in biopsy specimens representing chronic hepatitis, cirrhosis, or HCC from 100 patients chronically infected with HCV, and correlated Smad3 phosphorylation with clinical course. As HCV-infected livers progressed from chronic hepatitis through cirrhosis to HCC, hepatocytic pSmad3L/PAI-1 increased with fibrotic stage and necroinflammatory grade, and pSmad3C/p21(WAF1) decreased. Of 14 patients with chronic hepatitis C with strong hepatocytic pSmad3L positivity, 8 developed HCC within 12 years; only 1 of 12 showing little pSmad3L positivity developed HCC. We further sought molecular mechanisms in vitro. JNK activation by the pro-inflammatory cytokine interleukin-1beta stimulated the pSmad3L/PAI-1 pathway in facilitating hepatocytic invasion, in the meantime reducing TGF-beta-dependent tumor-suppressive activity by the pSmad3C/p21(WAF1) pathway. CONCLUSION These results indicate that chronic inflammation associated with HCV infection shifts hepatocytic TGF-beta signaling from tumor-suppression to fibrogenesis, accelerating liver fibrosis and increasing risk for HCC.
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Affiliation(s)
- Koichi Matsuzaki
- Department of Gastroenterology and Hepatology, Kansai Medical University, Moriguchi, Osaka, Japan.
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17
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Adesina AM, Nguyen Y, Guanaratne P, Pulliam J, Lopez-Terrada D, Margolin J, Finegold M. FOXG1 is overexpressed in hepatoblastoma. Hum Pathol 2007; 38:400-9. [PMID: 17217994 DOI: 10.1016/j.humpath.2006.09.003] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/03/2006] [Revised: 08/31/2006] [Accepted: 09/05/2006] [Indexed: 11/29/2022]
Abstract
Bacterial artificial chromosome array comparative genomic hybridization analysis of hepatoblastomas reveals a deletion in the 14q12 locus in 12 of 16 cases. A high frequency of copy gain is seen on chromosomes 1q, 2, 5p, 8, and 20. Frequent deletions are also seen at 6q, 17q, and 1p with less frequent gains on 4p, 6p, and 19p. 14q12 deletion locus analyses using quantitative real-time polymerase chain reaction reveals copy number gain/amplification in the region immediately telomeric to the deleted locus, including copy number gain (2- to 4-fold) of FOXG1 in 13 out of 16 tumors. This is associated with up-regulation (approximately 87-fold) of FOXG1 gene transcripts and increased protein expression. Immunostaining reveals an inverse relationship between FOXG1 expression and p21cip1 expression in all histologic subtypes. However, FOXG1 transcript levels were significantly higher (approximately 75-fold) in tumors with embryonal and small cell components when compared with pure fetal hepatoblastomas. FOXG1 has been implicated in the repression of transforming growth factor beta-induced expression of p21cip1 and cytostasis. Our findings are consistent with such a role for FOXG1. We propose that FOXG1 overexpression may contribute to the maintenance of the undifferentiated state in hepatoblastomas and could be a potential target for molecular therapeutics. This is the first report of a possible role for FOXG1 in hepatoblastoma and pediatric neoplasia.
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Affiliation(s)
- Adekunle Michael Adesina
- Department of Pathology, Texas Children's Hospital, Baylor College of Medicine, Houston, TX 77030, USA.
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18
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MIKULA M, PROELL V, FISCHER A, MIKULITS W. Activated hepatic stellate cells induce tumor progression of neoplastic hepatocytes in a TGF-beta dependent fashion. J Cell Physiol 2006; 209:560-7. [PMID: 16883581 PMCID: PMC2900580 DOI: 10.1002/jcp.20772] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The development of hepatocellular carcinomas from malignant hepatocytes is frequently associated with intra- and peritumoral accumulation of connective tissue arising from activated hepatic stellate cells. For both tumorigenesis and hepatic fibrogenesis, transforming growth factor (TGF)-beta signaling executes key roles and therefore is considered as a hallmark of these pathological events. By employing cellular transplantation we show that the interaction of neoplastic MIM-R hepatocytes with the tumor microenvironment, containing either activated hepatic stellate cells (M1-4HSCs) or myofibroblasts derived thereof (M-HTs), induces progression in malignancy. Cotransplantation of MIM-R hepatocytes with M-HTs yielded strongest MIM-R generated tumor formation accompanied by nuclear localization of Smad2/3 as well as of beta-catenin. Genetic interference with TGF-beta signaling by gain of antagonistic Smad7 in MIM-R hepatocytes diminished epithelial dedifferentiation and tumor progression upon interaction with M1-4HSCs or M-HTs. Further analysis showed that tumors harboring disrupted Smad signaling are devoid of nuclear beta-catenin accumulation, indicating a crosstalk between TGF-beta and beta-catenin signaling. Together, these data demonstrate that activated HSCs and myofibroblasts directly govern hepatocarcinogenesis in a TGF-beta dependent fashion by inducing autocrine TGF-beta signaling and nuclear beta-catenin accumulation in neoplastic hepatocytes. These results indicate that intervention with TGF-beta signaling is highly promising in liver cancer therapy.
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Affiliation(s)
| | | | | | - W. MIKULITS
- Correspondence to: W. Mikulits, Department of Medicine I, Division: Institute of Cancer Research, Medical University of Vienna, Borschke-Gasse 8a, A-1090 Vienna, Austria.
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19
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Luo JH, Ren B, Keryanov S, Tseng GC, Rao UNM, Monga SP, Strom S, Demetris AJ, Nalesnik M, Yu YP, Ranganathan S, Michalopoulos GK. Transcriptomic and genomic analysis of human hepatocellular carcinomas and hepatoblastomas. Hepatology 2006; 44:1012-24. [PMID: 17006932 PMCID: PMC1769554 DOI: 10.1002/hep.21328] [Citation(s) in RCA: 265] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
This study analyzed gene expression patterns and global genomic alterations in hepatocellular carcinomas (HCC), hepatoblastomas (HPBL), tissue adjacent to HCC and normal liver tissue derived from normal livers and hepatic resections. We found that HCC and adjacent non-neoplastic cirrhotic tissue have considerable overlap in gene expression patterns compared to normal liver. Several genes including Glypican 3, spondin-2, PEG10, EDIL3 and Osteopontin are over-expressed in HCC vs. adjacent tissue whereas Ficolin 3 is the most consistently under-expressed gene. HCC can be subdivided into three clusters based on gene expression patterns. HCC and HPBL have clearly different patterns of gene expression, with genes IGF2, Fibronectin, DLK1, TGFb1, MALAT1 and MIG6 being over-expressed in HPBL versus HCC. In addition, specific areas of the genome appear unstable in HCC, with the same regions undergoing either deletion or increased gene dosage in all HCC. In conclusion, a set of specific genes and areas of genomic instability are found across the board in liver neoplasia.
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Affiliation(s)
- Jian-Hua Luo
- From the Departments of Pathology, School of Medicine, and
| | - Baoguo Ren
- From the Departments of Pathology, School of Medicine, and
| | | | - George C. Tseng
- Biostatistics, Graduate School of Public Health, University of
Pittsburgh, Pittsburgh, PA 15261
| | - Uma N. M. Rao
- From the Departments of Pathology, School of Medicine, and
| | | | - Steven Strom
- From the Departments of Pathology, School of Medicine, and
| | | | | | - Yan P. Yu
- From the Departments of Pathology, School of Medicine, and
| | | | - George K. Michalopoulos
- From the Departments of Pathology, School of Medicine, and
- Address reprint requests to: George K. Michalopoulos, S410 BST,
University of Pittsburgh School of Medicine, Dept. of Pathology, Pittsburgh, PA,
15241. E-mail: ; fax:
412-648-9846
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20
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Mikula M, Proell V, Fischer ANM, Mikulits W. Activated hepatic stellate cells induce tumor progression of neoplastic hepatocytes in a TGF-beta dependent fashion. J Cell Physiol 2006. [PMID: 16883581 DOI: 10.1002/jcp.20772.] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The development of hepatocellular carcinomas from malignant hepatocytes is frequently associated with intra- and peritumoral accumulation of connective tissue arising from activated hepatic stellate cells. For both tumorigenesis and hepatic fibrogenesis, transforming growth factor (TGF)-beta signaling executes key roles and therefore is considered as a hallmark of these pathological events. By employing cellular transplantation we show that the interaction of neoplastic MIM-R hepatocytes with the tumor microenvironment, containing either activated hepatic stellate cells (M1-4HSCs) or myofibroblasts derived thereof (M-HTs), induces progression in malignancy. Cotransplantation of MIM-R hepatocytes with M-HTs yielded strongest MIM-R generated tumor formation accompanied by nuclear localization of Smad2/3 as well as of beta-catenin. Genetic interference with TGF-beta signaling by gain of antagonistic Smad7 in MIM-R hepatocytes diminished epithelial dedifferentiation and tumor progression upon interaction with M1-4HSCs or M-HTs. Further analysis showed that tumors harboring disrupted Smad signaling are devoid of nuclear beta-catenin accumulation, indicating a crosstalk between TGF-beta and beta-catenin signaling. Together, these data demonstrate that activated HSCs and myofibroblasts directly govern hepatocarcinogenesis in a TGF-beta dependent fashion by inducing autocrine TGF-beta signaling and nuclear beta-catenin accumulation in neoplastic hepatocytes. These results indicate that intervention with TGF-beta signaling is highly promising in liver cancer therapy.
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Affiliation(s)
- M Mikula
- Department of Medicine I, Division: Institute of Cancer Research, Medical University of Vienna, Vienna, Austria
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21
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Jin P, Wang E, Provenzano M, Deola S, Selleri S, Ren J, Voiculescu S, Stroncek D, Panelli MC, Marincola FM. Molecular signatures induced by interleukin-2 on peripheral blood mononuclear cells and T cell subsets. J Transl Med 2006; 4:26. [PMID: 16805915 PMCID: PMC1557669 DOI: 10.1186/1479-5876-4-26] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2006] [Accepted: 06/28/2006] [Indexed: 12/03/2022] Open
Abstract
Experimentally, interleukin-2 (IL-2) exerts complex immunological functions promoting the proliferation, survival and activation of T cells on one hand and inducing immune regulatory mechanisms on the other. This complexity results from a cross talk among immune cells which sways the effects of IL-2 according to the experimental or clinical condition tested. Recombinant IL-2 (rIL-2) stimulation of peripheral blood mononuclear cells (PBMC) from 47 donors of different genetic background induced generalized T cell activation and anti-apoptotic effects. Most effects were dependent upon interactions among immune cells. Specialized functions of CD4 and CD8 T cells were less dependent upon and often dampened by the presence of other PBMC populations. In particular, cytotoxic T cell effector function was variably affected with a component strictly dependent upon the direct stimulation of CD8 T cells in the absence of other PBMC. This observation may provide a roadmap for the interpretation of the discrepant biological activities of rIL-2 observed in distinct pathological conditions or treatment modalities.
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Affiliation(s)
- Ping Jin
- Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, 20892, USA
| | - Ena Wang
- Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, 20892, USA
| | - Maurizio Provenzano
- Immune Oncology Section, Department of Surgery, University Hospital ZLF, Hebelstrasse 20, 4031, Basel, Switzerland
| | - Sara Deola
- Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, 20892, USA
| | - Silvia Selleri
- Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, 20892, USA
| | - Jiaqiang Ren
- Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, 20892, USA
| | - Sonia Voiculescu
- Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, 20892, USA
| | - David Stroncek
- Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, 20892, USA
| | - Monica C Panelli
- Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, 20892, USA
| | - Francesco M Marincola
- Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, 20892, USA
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Elsammak MY, Amin GM, Khalil GM, Ragab WS, Abaza MM. Possible contribution of serum activin A and IGF-1 in the development of hepatocellular carcinoma in Egyptian patients suffering from combined hepatitis C virus infection and hepatic schistosomiasis. Clin Biochem 2006; 39:623-9. [PMID: 16624274 DOI: 10.1016/j.clinbiochem.2006.01.022] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2005] [Revised: 12/06/2005] [Accepted: 01/13/2006] [Indexed: 12/15/2022]
Abstract
OBJECTIVES The present study evaluated the role of activin A, insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) in Egyptian patients suffering from combined hepatitis C virus (HCV) infection and hepatic schistosomiasis. DESIGN AND METHODS Four groups were included in the present study. Group I: 30 healthy subjects were included as controls; Group II (HCV): 30 patients with chronic liver disease due to HCV infection without evidence of schistosomiasis; Group III (SHF + HCV): 30 patients with combined disease, chronic schistosomal hepatic fibrosis (SHF) and chronic hepatitis C infection; Group IV (HCC): 30 patients with hepatocellular carcinoma associated with chronic hepatitis C virus and schistosomal infection. RESULTS Patients with HCV, HCV + SHF and those with HCC had a significantly higher serum activin A compared with the control group (P < 0.001). Serum activin A level (mean +/- SD) was 5.7 +/- 2.76, 10.59 +/- 3.59, 15.39 +/- 4.61 and 19.93 +/- 5.43 ng/mL in controls, HCV patients, HCV + SHF patients and HCC patients, respectively. Serum IGF-1 was significantly lower in HCV patients, HCV + SHF patients and HCC patients compared to the control group (P < 0.001). Serum IGF-1 was 121.7 +/- 73.4, 76.7 +/- 23.5, 35.7 +/- 17.6 and 39.9 +/- 25.9 ng/mL in controls, HCV patients, HCV + SHF patients and HCC patients, respectively. Similarly, serum IGFBP-3 was significantly lower in HCV patients, HCV + SHF patients and HCC patients compared to the control group (P < 0.001). Furthermore, serum insulin-like growth factor binding protein 3 (IGFBP-3) was significantly lower in patients with HCC compared to patients with HCV or those with HCV + SHF (P < 0.01 and P = 0.024, respectively). The median (range) of serum IGFBP-3 was 4452 (352.2-8965), 3457 (1114-6000), 2114 (867-5901) and 1202 (576-3994) ng/mL in controls, HCV patients, HCV + SHF patients and HCC patients, respectively. Serum activin A correlated positively with Child-Pugh scoring in patients with HCV, HCV + SHF and those with HCC. The correlation coefficient was significant, at 0.001, in total cases. CONCLUSIONS We conclude that patients with HCV, HCV + SHF and those with HCC have a significantly higher serum activin A when compared with controls. Serum activin A level was significantly higher in patients with HCV + SHF compared to those with HCV alone (P < 0.01) with a significant positive correlation between the serum activin A level and Child-Pugh scoring in patients with HCV, HCV + SHF and those with HCC. Furthermore, serum IGF-1 and IGFBP-3 levels were significantly reduced in patients with HCV, HCV + SHF and those with HCC compared to the control group. We suggest that this pattern (high activin A and low IGF-1 and its binding protein 3) may play a role in development of HCC in Egyptian patients suffering from combined hepatitis C virus infection and hepatic schistosomiasis.
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Affiliation(s)
- Mohamed Yousry Elsammak
- Department of Chemical Pathology, Medical Research Instituitre, Alexandria University, Alexandria, Egypt.
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23
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Liu Y, Wang LF, Zou HF, Song XY, Xu HF, Lin P, Zheng HH, Yu XG. Expression and location of Smad2, 4 mRNAs during and after liver fibrogenesis of rats. World J Gastroenterol 2006; 12:1577-82. [PMID: 16570350 PMCID: PMC4124290 DOI: 10.3748/wjg.v12.i10.1577] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the location alteration of Smad2 and Smad4 mRNAs in the liver during and after fibrogenesis in rats.
METHODS: Eighty male Wistar rats weighing approximately 200 g each were used. The rat models of experimental hepatic fibrosis were established by injection with carbon tetrachloride (CCl4), normal rats and rats were injected with olive oil and served as control groups. In situ hybridization(ISH) was used to detect the Smad2 and Smad4 mRNA in liver.
RESULTS: In situ hybridization showed Smad2 and Smad4 mRNA expressions in the cytoplasm of hepatic stellate cells (HSC), fibroblasts and myofibroblasts around the central vein and hepatic sinus during and after fibrogenesis. Expression of Smad2, 4 mRNA was higher than that in normal and control rats.
CONCLUSION: In the process of and after hepatic fibrosis formation, HSC, fibroblasts and myofibroblasts are the major cells that express Smad2 and Smad4. The more serious the hepatic fibrosis is in the injured liver, the higher the level of Smad2 and Smad4 gene expression is during and after fibrogenesis respectively.
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Affiliation(s)
- Yang Liu
- Department of Biochemistry and Molecular Biology, Harbin Medical University, Harbin 150086, Heilongjiang Province, China
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Choi SH, Hwang SB. Modulation of the transforming growth factor-beta signal transduction pathway by hepatitis C virus nonstructural 5A protein. J Biol Chem 2006; 281:7468-78. [PMID: 16407286 DOI: 10.1074/jbc.m512438200] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Transforming growth factor-beta (TGF-beta) is implicated in the pathogenesis of liver disease. TGF-beta is involved both in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis C virus (HCV) infection often leads to cirrhosis and hepatocellular carcinoma. HCV nonstructural 5A (NS5A) protein is a multifunctional protein that modulates cytokine-mediated signal transduction pathways. To elucidate the molecular mechanism of HCV pathogenesis, we examined the effect of NS5A protein on TGF-beta-stimulated signaling cascades. We show that NS5A protein inhibited the TGF-beta-mediated signaling pathway in hepatoma cell lines as determined by reporter gene assay. To further investigate the role of NS5A, we examined the protein/protein interaction between NS5A and TGF-beta signal transducers. Both in vitro and in vivo binding data showed that NS5A protein directly interacted with TGF-beta receptor I (TbetaR-I) in hepatoma cell lines. This interaction was mapped to amino acids 148-238 of NS5A. We also found that NS5A protein co-localized with TbetaR-I in the cytoplasm of Huh7 cells and inhibited TGF-beta-mediated nuclear translocation of Smad2. Furthermore, we demonstrate that NS5A protein abrogated the phosphorylation of Smad2 and the heterodimerization of Smad3 and Smad4. To further explore the relevance to viral infection, we examined the effect of the HCV subgenomic replicon on the TGF-beta signaling pathway. We show that the HCV subgenomic replicon also inhibited TGF-beta-induced signaling cascades. These results indicate that HCV NS5A modulates TGF-beta signaling through interaction with TbetaR-I and that NS5A may be an important risk factor in HCV-associated liver pathogenesis.
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Affiliation(s)
- Soo-Ho Choi
- Ilsong Institute of Life Science, Hallym University, 1 Ockcheon-dong, Chuncheon 200-702, Korea
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Sebestyén A, Barna G, Nagy K, Jánosi J, Paku S, Kohut E, Berczi L, Mihalik R, Kopper L. Smad signal and TGFβ induced apoptosis in human lymphoma cells. Cytokine 2005; 30:228-35. [PMID: 15927846 DOI: 10.1016/j.cyto.2005.01.013] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2004] [Revised: 10/26/2004] [Accepted: 01/25/2005] [Indexed: 11/17/2022]
Abstract
Transforming growth factor beta1 (TGF beta1) has antiproliferative and/or apoptotic effect on lymphoid cells. In certain lymphomas exogenous TGF beta1 is able to induce apoptosis, however many lymphoid malignancies are resistant to the endogenous TGF beta1 production. We studied the expression and the activity of TGF beta1 signalling components in B cell lymphoma cell lines (e.g. HT 58 cells) and in isolated human peripheral mononuclear cells (PBMCs) from healthy individual's and B-CLL patient's blood. We found that all signal transducer Smads (Smad2,-3; Smad4) and at least one of the inhibitory Smads (Smad6,-7) were expressed in non-treated lymphoma cells, but the inhibitory Smads did not in normal/control PBMCs. However, after TGF beta1 treatment Smad6 disappeared, while the expression of Smad7 increased in HT 58 cells. The activity of Smad signals was proved by phosphorylation of Smad2, nuclear translocation of Smad2/3, and the increased expression of Smad-dependent gene, TIEG in TGF beta1 treated lymphoma cells. These results showed that Smad signaling is available in certain different human lymphoma cells, however ISmads expression could inhibit the signal transmission. This findings indicates that the lost sensitivity of lymphoma cells toward a physiological regulatory factor could be reversed.
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Affiliation(s)
- Anna Sebestyén
- Ist Department of Pathology and Experimental Cancer Research, Faculty of Medicine, Semmelweis University, 1085 Budapest, Hungary.
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Liao D, Luo GH, Wu JZ, Jiang JN, Huang YQ. Expression of TGF-b1 and smad4 mRNA in chronic hepatitis, liver cirrhosis, hepatocellular carcinoma and para-cancerous tissues, and their significance. Shijie Huaren Xiaohua Zazhi 2004; 12:2091-2094. [DOI: 10.11569/wcjd.v12.i9.2091] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: Transforming growth factor b1 (TGF-b1) signaling pathway is involved in a variety of important cellular regulative functions. including cell growth, differentiation, adhesion migration, extracellular matrix formation and immune regulation. The study was aimed to evaluate the relationship and mechanism of transforming growth factor b1 protein and smad4 mRNA in formation and development of fibrosis by detecting the expression of transforming growth factor b1 protein and smad4 mRNA in liver biopsy of chronic virus hepatitis, hepatocirrhosis and para-cancerous tissues.
METHODS: In situ hybridization and immunohistochemistry were used in 10 cases of normal liver tissue, 17 cases of para-cancerous tissues, 70 cases of chronic virus hepatitis and hepatocirrhosis.
RESULTS: Increased expression of TGF-b1 and smad4 mRNA in the intermediate degree of chronic virus hepatitis and hepatocirrhosis; the positive rates of TGF-b1 and smad4 mRNA were obviously higher in the chronic virus hepatitis and hepatocirrhosis (83.3%, 87.0% and 87.5%, 87.0%) than those in the normal liver (20.0%, 20.0%), the expression of TGF-b1 and smad4 mRNA had a significant relationship between the intermediate degree of chronic virus hepatitis, hepatocirrhosis and the normal liver (b3P <0.01, χ2 = 12.3980; b4P <0.01, χ2 = 14.0 609; b1P <0.01, χ2 = 14.6 953; b2P <0.01, χ2 = 14.0 609); the expression of TGF-b1 and smad4 mRNA in pare-cancerous tissues were significantly lower than that in the chronic virus hepatitis and hepatocirrhosis, as comparied with with the normal liver; the expression of TGF-b1 and smad4 mRNA reached the highest level at the stage S3, and decreased slightly at the stage S4, with positive relevance (χ2 = 4.5 064, P = 0.0 336, r = 0.2 668), in which most of the positive cells were distributed surroundings of portal tract, central vein, and the sinus.
CONCLUSION: TGF-b/smad pathway plays a significant role in formation and development of hepatic fibrosis, and the localization of TGF-b1 and smad4mRNA in hepatic tissue section can be used to evaluate accurately situation and tendency of hepatic fibrosis.
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de Luján Alvarez M, Ronco MT, Ochoa JE, Monti JA, Carnovale CE, Pisani GB, Lugano MC, Carrillo MC. Interferon alpha-induced apoptosis on rat preneoplastic liver is mediated by hepatocytic transforming growth factor beta(1). Hepatology 2004; 40:394-402. [PMID: 15368444 DOI: 10.1002/hep.20307] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
In previous work we showed that interferon alfa-2b (IFN-alpha2b) increases apoptosis on rat hepatic preneoplastic foci. The aim of this study was to determine if transforming growth factor beta1 (TGF-beta1) was involved in the programmed cell death on the foci. Animals were divided into 6 groups: subjected to a 2-phase model (diethylnitrosamine plus 2-acetylaminofluorene) of preneoplasia development (group 1); treated with IFN-alpha2b during the 2 phases (group 2); treated with IFN-alpha2b during initiation with diethylnitrosamine (group 3); treated with IFN-alpha2b during 2-acetylaminofluorene administration (group 4); subjected only to an initiation stage (group 5); and treated with IFN-alpha2b during the initiation period (group 6). Serum TGF-beta1 levels were increased in IFN-alpha2b-treated rats. Immunohistochemical studies showed that IFN-alpha2b significantly increased the quantity of TGF-beta1-positive hepatocytes in groups 2 to 4. Phosphorylated-Smads-2/3 (p-Smads-2/3) proteins in liver nuclear extracts were significantly elevated. To determine the source of TGF-beta1, isolated hepatocytes, Kupffer cells, and peritoneal macrophages from animals in groups 1 and 5 were cultured with or without IFN-alpha2b. IFN-alpha2b stimulus induced several-fold increases of TGF-beta1 secretion from hepatocytes. Neither Kupffer cells nor peritoneal macrophages secreted detectable TGF-beta1 levels when they were treated with IFN-alpha2b. IFN-alpha2b-stimulated cultured hepatocytes from preneoplastic livers showed enhanced apoptosis, measured by fluorescence microscopy and caspase-3 activity. They presented higher nuclear accumulation of p-Smads-2/3, indicating increased TGF-beta1 signaling. When anti-TGF-beta1 was added to the culture media, TGF-beta1 activation and apoptosis induced by IFN-alpha2b were blocked. In conclusion, IFN-alpha2b-induced production of TGF-beta1 by hepatocytes from preneoplastic liver is involved in the apoptotic elimination of altered hepatic foci.
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Affiliation(s)
- María de Luján Alvarez
- Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Rosario, Rosario, Argentina
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Abstract
AIM: To analyze the genetic and epigenetic alterations of RUNX3 gene, a potential putative tumor suppressor gene, in hepatocellular carcinoma (HCC).
METHODS: PCR-based loss of heterozygosity (LOH) detection, analysis of mutation with PCR-single strand conformational polymorphism (SSCP) and sequencing, and methylation study with methylation specific PCR (MSP) were performed on RUNX3 gene in a series of 62 HCCs along with their matched normal tissues.
RESULTS: Mutation of RUNX3 gene was not found, but one single nucleotide polymorphism with T to A transversion at the second nucleotide of the 18th condon was found. Nine of 26 informative cases (34.6%) showed allelic loss on the polymorphic site and 30 cases (48.4%) revealed hypermethylation of RUNX3 gene in promoter CpG islands. Furthermore, of the 9 cases with LOH, 8 (88.9%) also had hypermethylation.
CONCLUSION: Our findings indicate that inactivation of RUNX3 gene through allelic loss and promoter hypermethylation might be one of the major mechanisms in hepatocellualr carcinogenesis.
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Affiliation(s)
- Wen-Hua Xiao
- Department of Oncology, 304th Hospital of PLA, Beijing 100037, China.
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Cutroneo KR, Phan SH. TGF-beta1-induced Smad 3 binding to the Smad 7 gene: knockout of Smad 7 gene transcription by sense phosphorothioate oligos, autoregulation, and effect on TGF-beta1 secretion: bleomycin acts through TGF-beta1. J Cell Biochem 2003; 89:474-83. [PMID: 12761881 DOI: 10.1002/jcb.10528] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Bleomycin produces its fibrogenic effect, at least in part, by TGF-beta1 secretion. Treatment of IMR-90 human embryonic lung fibroblasts with bleomycin at 0.5 microg/ml results in a 1.6-fold increase of TGF-beta1 as determined by a specific ELISA assay for TGF-beta1 after acidification of the conditioned media. This elevation of TGF-beta1 secretion is furthermore enhanced in vivo by TGF-beta1 autoinduction of the TGF-beta1 gene. To demonstrate TGF-beta1 autoinduction, the fibroblasts were pretreated with 12.5 ng/ml TGF-beta1, washed extensively to remove any residual TGF-beta1, and then allowed to incubate for 24 h in AIM V synthetic serum-free media. The media when assayed using the ELISA assay contained a 1.6-fold increase of TGF-beta1. The distal promoter of the human TGF-beta1 gene contains a Smad 3 element (CAGGACA), which is homologous to the Smad 3 binding element motif (CAGA). The nuclear extracts of human embryonic lung fibroblasts treated for either 15 min or 24 h with TGF-beta1 did not demonstrate specificity of binding of a protein(s) to the homologous Smad 3 element as determined by cold wild-type oligodeoxynucleotide competition experiments. However, specific Smad 3 binding to the Smad 3 element (GTCTAGAC) found in proximal promoter of the Smad 7 gene was observed by cold oligo competition and supershift assays using a goat polyclonal Smad 3 antibody in the presence and absence of an N-terminal Smad 3 peptide. To determine the functionality of this Smad 3 binding to the Smad 3 element in the proximal promoter of the Smad 7 inhibitory gene to TGF-beta1 secretion, fibroblasts were transiently pretransfected with double-stranded phosphorothioate oligo "decoys" containing the Smad 7/Smad 3 element in the presence of plasmin to convert latent TGF-beta1 to active TGF-beta1. Under these conditions, which simulate the in vivo situation of 2.2-fold increase of total active TGF-beta1 was observed. Fibroblasts were also pretransfected with these double-stranded oligo "decoys," washed, then treated with TGF-beta1, washed and incubated in AIM V for an additional 24 h. In this latter experiment, a superinduction of TGF-beta1 secretion was observed. We propose that these oligo "decoys" bind Smad 3 preventing this initiation factor from binding to the Smad 7/Smad 3 element thereby decreasing the transcription of the Smad 7 gene. The decrease of the inhibitory Smad 7 would result in less binding of this Smad inhibitor to the Type I TGF-beta receptor and less antagonism of active TGF-beta1, more autoinduction of the TGF-beta1 gene, and more of the fibrogenic effects of TGF-beta1.
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Affiliation(s)
- Kenneth R Cutroneo
- Department of Biochemistry, College of Medicine, University of Vermont, Burlington, Vermont 05405-0068, USA.
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30
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Sugano Y, Matsuzaki K, Tahashi Y, Furukawa F, Mori S, Yamagata H, Yoshida K, Matsushita M, Nishizawa M, Fujisawa J, Inoue K. Distortion of autocrine transforming growth factor beta signal accelerates malignant potential by enhancing cell growth as well as PAI-1 and VEGF production in human hepatocellular carcinoma cells. Oncogene 2003; 22:2309-21. [PMID: 12700666 DOI: 10.1038/sj.onc.1206305] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Resistance to growth inhibitory effects of transforming growth factor (TGF)-beta is a frequent consequence of malignant transformation. On the other hand, serum concentrations of TGF-beta, plasminogen activator inhibitor type 1 (PAI-1), and vascular endothelial growth factor (VEGF) are elevated as tumor progresses. The molecular mechanism of autocrine TGF-beta signaling and its effects on PAI-1 and VEGF production in human hepatocellular carcinoma (HCC) is unknown. TGF-beta signaling involves TGF-beta type I receptor-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. To investigate the involvement of autocrine TGF-beta signal in cell growth, PAI-1 and VEGF production of HCC, we made stable transfectants of human HCC line (HuH-7 cells) to express a mutant Smad2(3S-A), in which serine residues of SSXS motif were changed to alanine. The transfectants demonstrated an impaired Smad2 signaling. Along with the resistance to growth inhibition by TGF-beta, forced expression of Smad2(3S-A) induced endogenous TGF-beta secretion. Moreover, this increased TGF-beta enhanced ligand-dependent signaling through the activated Smad3 and Smad4 complex, and transcriptional activities of PAI-1 and VEGF genes. In conclusion, distortion of autocrine TGF-beta signals in human HCC accelerates their malignant potential by enhancing cell growth as well as PAI-1 and VEGF production.
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Affiliation(s)
- Yasushi Sugano
- Third Department of Internal Medicine, 10-15 Fumizonocho, Mariguchi, Osaka 570-8507, Japan
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Kitamura Y, Ninomiya H. Smad expression of hepatic stellate cells in liver cirrhosis in vivo and hepatic stellate cell line in vitro. Pathol Int 2003; 53:18-26. [PMID: 12558865 DOI: 10.1046/j.1440-1827.2003.01431.x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Smad expressions, signaling mediators of transforming growth factor-beta (TGF-beta) superfamily of cytokines, were investigated in paraffin-embedded tissue sections of liver cirrhosis due to the hepatitis C virus infection and in the hepatic stellate cell (HSC) line in vitro. Smad 2/3, 4 and 7 was expressed in the nucleus of the HSC in the cirrhotic liver, while the expression was weak in the non-cirrhotic liver. TGF-beta1 expression in the HSC of the cirrhotic liver was strong, while the expression was weak in the non-cirrhotic liver. In situ hybridization also demonstrated the Smad signalings in the HSC of the cirrhotic liver, which confirmed the results of the Smad expressions by immunohistochemistry. The HSC line showed a cytoplasmic and a weak nuclear expression of Smads without TGF-beta1 stimulation, while these cells showed a strong Smad expression in the nucleus by TGF-beta1 stimulation. Immunocytochemical assay demonstrated that the TGF-beta1 stimulation induced the increase of the Smad expressions and the decrease of the autocrine TGF-beta1 in the HSC line. In situ hybridization assay also demonstrated an increase of the Smad mRNA signalings by TGF-beta1 stimulation in vitro. These observations suggest that the Smad expressions increase in the nucleus of the HSC in the cirrhotic liver and that the TGF-beta1 stimulation induces the Smad expression.
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Affiliation(s)
- Yukisato Kitamura
- Second Department of Pathology, School of Life Science, Faculty of Medicine, Tottori University, Yonago, Japan.
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33
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Torbenson M, Marinopoulos S, Dang DT, Choti M, Ashfaq R, Maitra A, Boitnott J, Wilentz RE. Smad4 overexpression in hepatocellular carcinoma is strongly associated with transforming growth factor beta II receptor immunolabeling. Hum Pathol 2002; 33:871-6. [PMID: 12378510 DOI: 10.1053/hupa.2002.128061] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
In the normal liver, the transforming growth factor beta (TGF-beta) signaling pathway plays an important role in inhibiting hepatocyte growth. This effect is mediated through Smad4 (or Dpc4), a tumor-suppressor gene that affects gene transcription and controls cell growth. A loss of Smad4 is associated with carcinoma in a number of other organs, including the pancreas and colon. Despite these facts, several recent studies using cDNA microarrays have surprisingly shown overexpression of Smad4 in hepatocellular carcinoma (HCC). Because Smad4 plays a central role in the TGF-beta signaling pathway, we hypothesized that activation of the TGF-beta signaling pathway may explain Smad4 overexpression. To investigate this, 21 surgically resected HCCs were immunostained with antibodies to Smad4 and TGF-beta receptor II. Tumor and normal liver tissues were stained in all cases, and expression in the tumor was scored in comparison to the nonneoplastic liver. Thirteen hepatic adenomas were also immunostained as a control group. The average age at resection was 58 +/- 16 years for the 17 men and 4 women with HCC. TGF-beta receptor II was weakly expressed in the hepatocyte cytoplasm of all normal livers and was overexpressed in 10 of 21 HCCs. Of these 10 HCCs increased Smad4 immunolabeling was also present in 10 of 10 cases. In contrast, of the 11 of HCCs that did not show TGF-beta overexpression, only 1 showed increased Smad4 immunolabeling. Increased TGF-beta receptor II and Smad4 labeling was associated with a worse nuclear grade and increased mitotic activity. For the hepatic adenomas, the 13 women had an average age at resection of 36 +/- 10 years. Whereas 2 adenomas showed over expression of TGF-beta receptor II, there was no Smad4 overexpression in any case. In conclusion, increased Smad4 protein expression in HCC is tightly linked to overexpression of TGF-beta II receptors and is associated with increased mitoses and a worse nuclear grade. Hepatic adenomas only rarely show overexpression of TGF-beta II receptors and did not show increased Smad4 labeling. The results from this study indicate that Smad4 protein overexpression is present in a subset of HCCs and is strongly correlated with immunostaining for TGF-beta II receptor, findings that may represent activation or dysregulation of the TGF-beta signaling pathway.
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Affiliation(s)
- Michael Torbenson
- Department of Pathology, The Johns Hopkins Hospital, Baltimore, MD 21231, USA
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Lala PK, Lee BP, Xu G, Chakraborty C. Human placental trophoblast as an in vitro model for tumor progression. Can J Physiol Pharmacol 2002; 80:142-9. [PMID: 11934257 DOI: 10.1139/y02-006] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The human placenta is a highly invasive tumor-like structure in which a subpopulation of placental trophoblast cells known as the "extravillous trophoblast" (EVT) invades the uterine decidua and its vasculature to establish adequate fetal-maternal exchange of molecules. By utilizing in vitro-propagated short-lived EVT cell lines we found that molecular mechanisms responsible for their invasiveness are identical to those of cancer cells; however, unlike cancer cells, their proliferation, migration, and invasiveness in situ are stringently controlled by decidua-derived transforming growth factor (TGF)-beta. By SV40T antigen transfection of normal EVT cells followed by a forced crisis regimen in culture we produced an immortalized premalignant derivative that is hyperproliferative, hyperinvasive, and deficient in gap-junctional intercellular communication. Both premalignant and malignant EVT (JAR and JEG-3 choriocarcinoma) cell lines were found to be TGF-beta-resistant. Using these cell lines, we investigated genetic changes responsible for transition of the normal EVT cells to premalignant and malignant phenotype. Hyperinvasiveness in both cases resulted from a downregulation of tissue inhibitor of metalloprotease (TIMP)-1 and plasminogen activator inhibitor (PAI)-1 genes. In contrast to normal EVT cells, both cell types failed to upregulate these genes in response to TGF-beta. Loss of TGF-beta response in malignant EVT cells was explained by the loss of expression of Smad3 gene. Differential mRNA display of normal and premalignant EVT cells identified up- and down-regulation of numerous known or novel genes in premalignant EVT cells, with potential oncogenic and (or) tumor-suppressor functions, e.g., loss of fibronectin and insulin-like growth factor binding protein (IGFBP-5). Premalignant EVT cells also lost IGF receptor type 2 (IGFR-II). IGFBP-5 was shown to be a negative regulator of IGF-1-induced proliferation of premalignant EVT cells, so that loss of IGFBP-5 as well as IGFR-II permitted their unrestricted proliferation in an IGF-I-rich microenvironment of the fetal-maternal interface. The present model may be a good prototype for identifying genetic changes underlying epithelial tumor progression.
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Affiliation(s)
- P K Lala
- Department of Anatomy and Cell Biology, University of Western Ontario, London, Canada.
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35
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Uehara K, Ichida T, Sugahara S, Ishikawa T, Yamagiwa S, Yoshida Y, Nomoto M, Katoh M, Satoh H, Watanabe H, Abo T, Asakura H. Systemic administration of liposome-encapsulated OK-432 prolongs the survival of rats with hepatocellular carcinoma through the induction of IFN-gamma-producing hepatic lymphocytes. J Gastroenterol Hepatol 2002; 17:81-90. [PMID: 11895558 DOI: 10.1046/j.1440-1746.2002.02675.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
BACKGROUND OK-432 is known to increase the host antitumor response. We previously reported that systemic administration of OK-432 (OK-Lipo) specifically induced hepatic lymphocytes in mice. Here we aimed to investigate the antitumor effect of OK-Lipo on hepatocellular carcinomas (HCC) in experimental rats. METHODS Diethylnitrosamine was administered for 12 weeks to all rats (n = 36). Rats were divided into three groups of 12 rats each. One group was injected with OK-Lipo from week 5 (OK-5w group) and another from week 9 (OK-9w group). A control group was injected with saline from week 5 (Non-OK group). At week 13, five rats from each group were used for histological analysis and immunofluorescence assays (surface phenotypic and intracellular cytokine analysis of the mononuclear cells in the liver, spleen and peripheral blood). The remaining rats were observed for the remainder of their survival period. RESULTS The mean survival times of Non-OK, OK-5w, and OK-9w groups differed significantly (98.0 +/- 5.3 days, 116.0 +/- 5.8 days, and 106.0 +/- 5.4 days, respectively, P < 0.01). Histological examination revealed many apoptotic tumor cells, infiltration of lymphocytes and macrophages in the OK-5w group. The two-color immunofluorescence assay showed that the proportion of natural killer (NK) cells and IFN-gamma-producing cells in the liver were significantly higher in the OK-5w group. CONCLUSIONS These findings showed that systemic administration of OK-Lipo contributed to prolonging the survival of rats with HCC. OK-Lipo induced NK cells and IFN-gamma-producing cells specifically in the liver and these cells seemed to reduce hepatocarcinogenesis and tumor growth.
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Affiliation(s)
- Kazuhiro Uehara
- Department of Internal Medicine III, Niigata University School of Medicine, Japan
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36
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Tahashi Y, Matsuzaki K, Date M, Yoshida K, Furukawa F, Sugano Y, Matsushita M, Himeno Y, Inagaki Y, Inoue K. Differential regulation of TGF-beta signal in hepatic stellate cells between acute and chronic rat liver injury. Hepatology 2002; 35:49-61. [PMID: 11786959 DOI: 10.1053/jhep.2002.30083] [Citation(s) in RCA: 155] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
During chronic liver injury, transforming growth factor beta (TGF-beta) plays a prominent role in stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells (HSCs). On the other hand, Smad 7 was recently shown to antagonize the TGF-beta-induced activation of signal-transducing Smads (2 and 3). In this study, we investigated the regulatory mechanisms of the TGF-beta signals in rat HSCs during acute liver injury and myofibroblasts (MFBs) during chronic liver injury, focusing on the roles of Smad 2 and antagonistic Smad 7. In acute liver injury, HSC-derived TGF-beta increased plasminogen activator inhibitor type 1 (PAI-1) and alpha2(I) procollagen (COL1A2) transcripts. Smad 2 in HSCs during liver injury and primary cultured HSCs were activated by an autocrine mechanism, because high levels of Smad 2 phosphorylation and induction of PAI-1 transcript by TGF-beta were observed in HSCs. Thereafter, Smad 7 induced by TGF-beta negatively regulated the Smad 2 action. These results indicated that endogenous TGFbeta-mediated Smad 7 in HSCs terminated the fibrotic signals mediated by signal-transducing Smads, and might be involved in the transient response to autocrine TGF-beta signal after acute liver injury. By contrast, Smad 7 was not induced by the autocrine TGF-beta signal, and constitutive Smad 2 activation was observed in MFBs throughout chronic liver injury, although Smad 7 could inhibit the TGF-beta signal requiring Smad 2 phosphorylation by activated TGF-beta receptor in cultured MFBs. This constitutive phosphorylation of Smad 2 by endogenous TGF-beta under a low level of Smad 7 could be involved in the progression of liver fibrosis.
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Affiliation(s)
- Yoshiya Tahashi
- Third Department of Internal Medicine, Kansai Medical University, Osaka, Japan
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Kanamaru C, Yasuda H, Takeda M, Ueda N, Suzuki J, Tsuchida T, Mashima H, Ohnishi H, Fujita T. Smad7 is induced by norepinephrine and protects rat hepatocytes from activin A-induced growth inhibition. J Biol Chem 2001; 276:45636-41. [PMID: 11551920 DOI: 10.1074/jbc.m105302200] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Activin A induces growth arrest of rat hepatocytes in vitro and in vivo. The alpha(1)-adrenergic agonist, norepinephrine (NE), enhances epidermal growth factor-stimulated DNA synthesis and inhibits activin A-induced growth inhibition, but the mechanisms of these actions are unclear. Smad proteins have recently been identified as intracellular signaling mediators of transforming growth factor-beta family members. In the present study, we explored how NE modulates the Smad signaling pathway in rat cultured hepatocytes. We demonstrate that NE inhibits activin A-induced nuclear accumulation of Smad2/3 and that NE rapidly induces inhibitory Smad7 mRNA expression. Infection of Smad7 adenovirus into rat hepatocytes inhibited activin A-induced nuclear accumulation of Smad2/3, enhanced epidermal growth factor-stimulated DNA synthesis, and abolished the growth inhibitory effect of activin A. We also demonstrated that the induction of Smad7 by NE is dependent on nuclear factor-kappa B (NF-kappa B). The amount of active NF-kappa B complex rapidly increased after NE treatment. Preincubation of the cells with an NF-kappa B pathway inhibitor N-tosyl-l-phenylalanine chloromethyl ketone or infection of the cells with an adenovirus expressing an I kappa B super-repressor (Ad5I kappa B) abolished the NE-induced Smad7 expression. These results indicate a mechanism of transmodulation between the Smad and trimeric G protein signaling pathways in rat hepatocytes.
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Affiliation(s)
- C Kanamaru
- Department of Medicine, University of Tokyo School of Medicine, Tokyo 113-8655, Japan
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Hsieh CS, Huang CC, Wu JJ, Chaung HC, Wu CL, Chang NK, Chang YM, Chou MH, Chuang JH. Ascending cholangitis provokes IL-8 and MCP-1 expression and promotes inflammatory cell infiltration in the cholestatic rat liver. J Pediatr Surg 2001; 36:1623-8. [PMID: 11685687 DOI: 10.1053/jpsu.2001.27933] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
BACKGROUND/PURPOSE Postoperative cholangitis is one of the most common complications after bile duct reconstruction. The pathogenesis and early consequences of ascending cholangitis still are unidentified. METHODS Male Sprague-Dawley rats were divided into 5 treatment groups: control (n = 4), blood sampling and liver biopsy only; group I, [BDL/Eschericha coli; n = 6], ligation of common bile duct (BDL) for a week, followed by Roux-en-Y choledochojejunostomy (RYCJ) and injection of E coli (ATCC 25922) into Roux limb after 24 hours; group II, [BDL/NS; n = 5], same procedures as in group I, with injection of normal saline (NS) into Roux limb; group III, [SBDL/E coli; n = 6], primary RYCJ was constructed 1 week after sham ligation of common bile duct (SBDL) followed by the same treatment as group I; Group IV, [SBDL/N.S; n = 6], same procedures as in group III, but injecting NS into Roux limb. All animals were killed after 24 hours of treatment. Blood was sampled for culture and serum cytokine levels. The liver was harvested for quantitative bacterial culture, as well as for MCP-1, interleukin (IL)-8 (CINC in the rat) and transforming growth factor beta1 mRNA expression by reverse transcriptase polymerase chain reaction (RT-PCR) and for immunohistochemistry. The choledochojejunostomy was resected for culture. Serum cytokine levels were detected by ELISA kits. RESULTS A significant increase of E coli ATCC 25922, occurred in the livers of group I rats, compared with group IV (P =.037). MCP-1 expression increased in all groups, compared with control (P =.000). The IL-8 mRNA expression was significantly higher in group I than in control (P =.021). The expression of TGF-beta1 mRNA was similar among the groups (P =.361), consistent with the immunohistochemistry results. The serum MCP-1 and IL-8 levels were higher in the 4 groups than in the control (P =.000) and were significantly higher in group I than in group IV (P =.001). CONCLUSIONS This study found that a significant colonization of E coli of the same strain was present in the cholestatic rat liver injected into the Roux limb, which was associated with a higher expression of liver MCP-1 and IL-8 mRNA, a significant increase of serum MCP-1 and IL-8, and a more evident inflammatory cell infiltration into the porta hepatis.
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Affiliation(s)
- C S Hsieh
- Department of Pediatric Surgery, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
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Xu G, Chakraborty C, Lala PK. Expression of TGF-beta signaling genes in the normal, premalignant, and malignant human trophoblast: loss of smad3 in choriocarcinoma cells. Biochem Biophys Res Commun 2001; 287:47-55. [PMID: 11549251 DOI: 10.1006/bbrc.2001.5533] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We had earlier shown that TGF-beta controls proliferation, migration, and invasiveness of normal human trophoblast cells, whereas premalignant and malignant trophoblast cells are resistant to TGF-beta. To identify signaling defects responsible for TGF-beta resistance in premalignant and malignant trophoblasts, we have compared the expression of TGF-beta signaling molecules in a normal trophoblast cell line (HTR-8), its premalignant derivative (RSVT2/C), and two choriocarcinoma cell lines (JAR and JEG-3). RT-PCR analysis revealed that all these cell lines expressed the mRNA of TGF-beta1, -beta2, and -beta3, TGF-beta receptors type I, II, and III, and post-receptor signaling genes smad2, smad3, smad4, smad6, and smad7 with the exception that TGF-beta2 and smad3 were undetectable in JAR and JEG-3 cells. Immunoblot analysis confirmed the absence of smad3 protein in choriocarcinoma cells. Treatment with TGF-beta1 induced smad3 phosphorylation and smad3 translocation to the nucleus in the normal and premalignant trophoblast cells. These results suggest that loss of smad3 may account for a functional disruption in the TGF-beta signaling pathway in choriocarcinomas, but not in the premalignant trophoblast.
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Affiliation(s)
- G Xu
- Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario, Canada N6A 5C1
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