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Danielpour D. Advances and Challenges in Targeting TGF-β Isoforms for Therapeutic Intervention of Cancer: A Mechanism-Based Perspective. Pharmaceuticals (Basel) 2024; 17:533. [PMID: 38675493 PMCID: PMC11054419 DOI: 10.3390/ph17040533] [Citation(s) in RCA: 19] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2024] [Revised: 04/11/2024] [Accepted: 04/16/2024] [Indexed: 04/28/2024] Open
Abstract
The TGF-β family is a group of 25 kDa secretory cytokines, in mammals consisting of three dimeric isoforms (TGF-βs 1, 2, and 3), each encoded on a separate gene with unique regulatory elements. Each isoform plays unique, diverse, and pivotal roles in cell growth, survival, immune response, and differentiation. However, many researchers in the TGF-β field often mistakenly assume a uniform functionality among all three isoforms. Although TGF-βs are essential for normal development and many cellular and physiological processes, their dysregulated expression contributes significantly to various diseases. Notably, they drive conditions like fibrosis and tumor metastasis/progression. To counter these pathologies, extensive efforts have been directed towards targeting TGF-βs, resulting in the development of a range of TGF-β inhibitors. Despite some clinical success, these agents have yet to reach their full potential in the treatment of cancers. A significant challenge rests in effectively targeting TGF-βs' pathological functions while preserving their physiological roles. Many existing approaches collectively target all three isoforms, failing to target just the specific deregulated ones. Additionally, most strategies tackle the entire TGF-β signaling pathway instead of focusing on disease-specific components or preferentially targeting tumors. This review gives a unique historical overview of the TGF-β field often missed in other reviews and provides a current landscape of TGF-β research, emphasizing isoform-specific functions and disease implications. The review then delves into ongoing therapeutic strategies in cancer, stressing the need for more tools that target specific isoforms and disease-related pathway components, advocating mechanism-based and refined approaches to enhance the effectiveness of TGF-β-targeted cancer therapies.
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Affiliation(s)
- David Danielpour
- Case Comprehensive Cancer Center Research Laboratories, The Division of General Medical Sciences-Oncology, Case Western Reserve University, Cleveland, OH 44106, USA; ; Tel.: +1-216-368-5670; Fax: +1-216-368-8919
- Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA
- Institute of Urology, University Hospitals, Cleveland, OH 44106, USA
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2
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Systematic Analysis of Cytostatic TGF-Beta Response in Mesenchymal-Like Hepatocellular Carcinoma Cell Lines. J Gastrointest Cancer 2021; 52:1320-1335. [PMID: 34463913 DOI: 10.1007/s12029-021-00704-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/23/2021] [Indexed: 10/20/2022]
Abstract
BACKGROUND Hepatocellular carcinoma (HCC) is one of the most challenging malignancies, with high morbidity and mortality rates. The transforming growth factor-β (TGF-β) pathway plays a dual role in HCC, acting as both tumor suppressor and promoter. A thorough understanding of the mechanisms underlying its opposing functions is important. The growth suppressive effects of TGF-β remain largely unknown for mesenchymal HCC cells. Using a systematic approach, here we assess the cytostatic TGF-β responses and intracellular transduction of the canonical TGF-β/Smad signaling cascade in mesenchymal-like HCC cell lines. METHODS Nine mesenchymal-like HCC cell lines, including SNU182, SNU387, SNU398, SNU423, SNU449, SNU475, Mahlavu, Focus, and Sk-Hep1, were used in this study. The cytostatic effects of TGF-β were evaluated by cell cycle analysis, BrdU labeling, and SA-β-Gal assay. RT-PCR and western blot analysis were utilized to determine the mRNA and protein expression levels of TGF-β signaling components and cytostatic genes. Immunoperoxidase staining and luciferase reporter assays were performed to comprehend the transduction of the canonical TGF-β pathway. RESULTS We report that mesenchymal-like HCC cell lines are resistant to TGF-β-induced growth suppression. The vast majority of cell lines have an active canonical signaling from the cell membrane to the nucleus. Three cell lines had lost the expression of cytostatic effector genes. CONCLUSION Our findings reveal that cytostatic TGF-β responses have been selectively lost in mesenchymal-like HCC cell lines. Notably, their lack of responsiveness was not associated with a widespread impairment of TGF-β signaling cascade. These cell lines may serve as valuable models for studying the molecular mechanisms underlying the loss of TGF-β-mediated cytostasis during hepatocarcinogenesis.
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Sinha A, Iyengar PV, ten Dijke P. E3 Ubiquitin Ligases: Key Regulators of TGFβ Signaling in Cancer Progression. Int J Mol Sci 2021; 22:E476. [PMID: 33418880 PMCID: PMC7825147 DOI: 10.3390/ijms22020476] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2020] [Revised: 12/25/2020] [Accepted: 12/29/2020] [Indexed: 02/07/2023] Open
Abstract
Transforming growth factor β (TGFβ) is a secreted growth and differentiation factor that influences vital cellular processes like proliferation, adhesion, motility, and apoptosis. Regulation of the TGFβ signaling pathway is of key importance to maintain tissue homeostasis. Perturbation of this signaling pathway has been implicated in a plethora of diseases, including cancer. The effect of TGFβ is dependent on cellular context, and TGFβ can perform both anti- and pro-oncogenic roles. TGFβ acts by binding to specific cell surface TGFβ type I and type II transmembrane receptors that are endowed with serine/threonine kinase activity. Upon ligand-induced receptor phosphorylation, SMAD proteins and other intracellular effectors become activated and mediate biological responses. The levels, localization, and function of TGFβ signaling mediators, regulators, and effectors are highly dynamic and regulated by a myriad of post-translational modifications. One such crucial modification is ubiquitination. The ubiquitin modification is also a mechanism by which crosstalk with other signaling pathways is achieved. Crucial effector components of the ubiquitination cascade include the very diverse family of E3 ubiquitin ligases. This review summarizes the diverse roles of E3 ligases that act on TGFβ receptor and intracellular signaling components. E3 ligases regulate TGFβ signaling both positively and negatively by regulating degradation of receptors and various signaling intermediates. We also highlight the function of E3 ligases in connection with TGFβ's dual role during tumorigenesis. We conclude with a perspective on the emerging possibility of defining E3 ligases as drug targets and how they may be used to selectively target TGFβ-induced pro-oncogenic responses.
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Affiliation(s)
| | | | - Peter ten Dijke
- Department of Cell and Chemical Biology and Oncode Institute, Leiden University Medical Center, 2300 RC Leiden, The Netherlands; (A.S.); (P.V.I.)
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4
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Bellomo C, Caja L, Fabregat I, Mikulits W, Kardassis D, Heldin CH, Moustakas A. Snail mediates crosstalk between TGFβ and LXRα in hepatocellular carcinoma. Cell Death Differ 2017; 25:885-903. [PMID: 29230000 PMCID: PMC5943406 DOI: 10.1038/s41418-017-0021-3] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2017] [Revised: 10/17/2017] [Accepted: 10/20/2017] [Indexed: 12/21/2022] Open
Abstract
Understanding the complexity of changes in differentiation and cell survival in hepatocellular carcinoma (HCC) is essential for the design of new diagnostic tools and therapeutic modalities. In this context, we have analyzed the crosstalk between transforming growth factor β (TGFβ) and liver X receptor α (LXRα) pathways. TGFβ is known to promote cytostatic and pro-apoptotic responses in HCC, and to facilitate mesenchymal differentiation. We here demonstrate that stimulation of the nuclear LXRα receptor system by physiological and clinically useful agonists controls the HCC response to TGFβ. Specifically, LXRα activation antagonizes the mesenchymal, reactive oxygen species and pro-apoptotic responses to TGFβ and the mesenchymal transcription factor Snail mediates this crosstalk. In contrast, LXRα activation and TGFβ cooperate in enforcing cytostasis in HCC, which preserves their epithelial features. LXRα influences Snail expression transcriptionally, acting on the Snail promoter. These findings propose that clinically used LXR agonists may find further application to the treatment of aggressive, mesenchymal HCCs, whose progression is chronically dependent on autocrine or paracrine TGFβ.
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Affiliation(s)
- Claudia Bellomo
- Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Box 582, Biomedical Center, Uppsala University, SE-75123, Uppsala, Sweden.,Ludwig Institute for Cancer Research, Science for Life Laboratory, Box 595, Biomedical Center, Uppsala University, SE-75124, Uppsala, Sweden
| | - Laia Caja
- Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Box 582, Biomedical Center, Uppsala University, SE-75123, Uppsala, Sweden.,Ludwig Institute for Cancer Research, Science for Life Laboratory, Box 595, Biomedical Center, Uppsala University, SE-75124, Uppsala, Sweden
| | - Isabel Fabregat
- Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet, and Department of Physiological Sciences, School of Medicine, University of Barcelona, ES-08908, Barcelona, Spain
| | - Wolfgang Mikulits
- Department of Medicine I, Division: Institute of Cancer Research, Comprehensive Cancer Center Vienna, Medical University of Vienna, A-1090, Vienna, Austria
| | - Dimitris Kardassis
- Division of Basic Medical Sciences, University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, GR-71003, Heraklion, Greece
| | - Carl-Henrik Heldin
- Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Box 582, Biomedical Center, Uppsala University, SE-75123, Uppsala, Sweden.,Ludwig Institute for Cancer Research, Science for Life Laboratory, Box 595, Biomedical Center, Uppsala University, SE-75124, Uppsala, Sweden
| | - Aristidis Moustakas
- Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Box 582, Biomedical Center, Uppsala University, SE-75123, Uppsala, Sweden. .,Ludwig Institute for Cancer Research, Science for Life Laboratory, Box 595, Biomedical Center, Uppsala University, SE-75124, Uppsala, Sweden.
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Leucine-rich α-2-glycoprotein promotes TGFβ1-mediated growth suppression in the Lewis lung carcinoma cell lines. Oncotarget 2016; 6:11009-22. [PMID: 25826092 PMCID: PMC4484435 DOI: 10.18632/oncotarget.3557] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2014] [Accepted: 02/12/2015] [Indexed: 12/19/2022] Open
Abstract
Leucine-rich α2-glycoprotein (LRG) is an approximately 50-kDa glycoprotein that has been found to be elevated in the sera of patients with several types of cancer. LRG directly binds to transforming growth factor beta 1 (TGFβ1) and modulates TGFβ1 signaling in endothelial cells; however, the precise function of LRG in cancer remains unclear. This study aimed to investigate the role of LRG in cancer. Lewis lung carcinoma (LLC) cells hardly expressed LRG. The growth of LLC tumors allografted in the LRG knockout (KO) mice was significantly increased compared with wild-type (WT) mice. Conversely, overexpression of LRG significantly inhibited the growth of LLC tumors in WT mice. In the presence of LRG, TGFβ1 significantly inhibited the proliferation of LLC cells and human hepatocellular carcinoma Hep3B cells in vitro by inducing apoptosis via the potent activation of smad2 and its downstream signaling pathway. Furthermore, administration of a TGFβR1 inhibitor (SB431542) significantly enhanced the growth of LLC tumors in WT mice compared with LRG KO mice via inhibition of apoptosis. We propose that LRG potentiates the effect of TGFβ1 in cancer cells whose growth is suppressed in the presence of TGFβ1.
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Tokay E, Kockar F. Identification of intracellular pathways through which TGF-β1 upregulates URG-4/URGCP gene expression in hepatoma cells. Life Sci 2016; 144:121-8. [DOI: 10.1016/j.lfs.2015.12.010] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2015] [Revised: 11/22/2015] [Accepted: 12/04/2015] [Indexed: 10/22/2022]
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7
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He T, Quan T, Fisher GJ. Ultraviolet irradiation represses TGF-β type II receptor transcription through a 38-bp sequence in the proximal promoter in human skin fibroblasts. Exp Dermatol 2015; 23 Suppl 1:2-6. [PMID: 25234828 DOI: 10.1111/exd.12389] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/24/2014] [Indexed: 12/31/2022]
Abstract
Transforming growth factor-β (TGF-β) is a major regulator of collagen gene expression in human skin fibroblasts. Cellular responses to TGF-β are mediated primarily through its cell surface type I (TβRI) and type II (TβRII) receptors. Ultraviolet (UV) irradiation impairs TGF-β signalling largely due to reduced TβRII gene expression, thereby decreasing type I procollagen synthesis, in human skin fibroblasts. UV irradiation does not alter either TβRII mRNA or protein stability, indicating that UV reduction in TβRII expression likely results from transcriptional or translational repression. To understand how UV irradiation regulates TβRII transcription, we used a series of TβRII promoter-luciferase 5'-deletion constructs (covering 2 kb of the TβRII proximal promoter) to determine transcriptional rate in response to UV irradiation. We identified a 137-bp region upstream of the transcriptional start site that exhibited high promoter activity and was repressed 60% by UV irradiation, whereas all other TβRII promoter reporter constructs exhibited either low promoter activities or no regulation by UV irradiation. Mutation of potential transcription factor binding sites within the promoter region revealed that an inverted CCAAT box (-81 bp from transcription start site) is required for promoter activity. Mutation of the CCAAT box completely abolished UV irradiation regulation of the TβRII promoter. Protein-binding assay, as determined by electrophoretic mobility-shift assays (EMSAs) using the inverted CCAAT box as probe (-100/-62), demonstrated significantly enhanced protein binding in response to UV irradiation. Super shift experiments indicated that nuclear factor Y (NFY) is able to binding to this sequence, but NFY binding was not altered in response to UV irradiation, indicating additional protein(s) are capable of binding this sequence in response to UV irradiation. Taken together, these data indicate that UV irradiation reduces TβRII expression, at least partially, through transcriptional repression. This repression is mediated by a 38-bp sequence in TβRII promoter, in human skin fibroblasts.
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Affiliation(s)
- Tianyuan He
- Department of Dermatology, University of Michigan, Ann Arbor, MI, USA
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8
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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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9
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Danielpour D. Transforming Growth Factor-Beta in Prostate Cancer. Prostate Cancer 2013. [DOI: 10.1007/978-1-4614-6828-8_7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022] Open
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10
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Ehrlich M, Gutman O, Knaus P, Henis YI. Oligomeric interactions of TGF-β and BMP receptors. FEBS Lett 2012; 586:1885-96. [PMID: 22293501 DOI: 10.1016/j.febslet.2012.01.040] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2012] [Revised: 01/15/2012] [Accepted: 01/19/2012] [Indexed: 10/14/2022]
Abstract
Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) cytokines participate in a multiplicity of ways in the regulation of numerous physiological and pathological processes. Their wide-ranging biological functions are controlled by several mechanisms, including regulation of transcription, complex formation among the signaling receptors (oligomerization) and with co-receptors, binding of the receptors to scaffolding proteins or their targeting to specific membrane domains. Here, we address the generation of TGF-β and BMP receptor homo- and hetero-oligomers and its roles as a mechanism capable of fast regulation of signaling by these crucial cytokines. We examine the available biochemical, biophysical and structural evidence for the ternary structure of these complexes, and the possible roles of homomeric and heteromeric receptor oligomers in signaling.
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Affiliation(s)
- Marcelo Ehrlich
- Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
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11
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Seth D, Haber PS, Syn WK, Diehl AM, Day CP. Pathogenesis of alcohol-induced liver disease: classical concepts and recent advances. J Gastroenterol Hepatol 2011; 26:1089-105. [PMID: 21545524 DOI: 10.1111/j.1440-1746.2011.06756.x] [Citation(s) in RCA: 112] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Alcoholic liver disease (ALD) is a primary consequence of heavy and prolonged drinking. ALD contributes to the bulk of liver disease burden worldwide. Progression of ALD is a multifactorial and multistep process that includes many genetic and environmental risk factors. The molecular pathogenesis of ALD involves alcohol metabolism and secondary mechanisms such as oxidative stress, endotoxin, cytokines and immune regulators. The histopathological manifestation of ALD occurs as an outcome of complex but controlled interactions between hepatic cell types. Hepatic stellate cells (HSCs) are the key drivers of fibrogenesis, but transformation of hepatocytes to myofibroblastoids also implicate parenchymal cells as playing an active role in hepatic fibrogenesis. Recent discoveries indicate that lipogenesis during the early stages of ALD is a risk for advancement to cirrhosis. Other recently identified novel molecules and physiological/cell signaling pathways include fibrinolysis, osteopontin, transforming growth factor-β-SMAD and hedgehog signaling, and involvement of novel cytokines in hepatic fibrogenesis. The observation that ALD and non-alcoholic steatohepatitis share common pathways and genetic polymorphisms suggests operation of parallel pathogenic mechanisms. Future research involving genomics, epigenomics, deep sequencing and non-coding regulatory elements holds promise to identify novel diagnostic and therapeutic targets for ALD. There is also a need for adequate animal models to study pathogenic mechanisms at the molecular level and targeted therapy.
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Affiliation(s)
- Devanshi Seth
- Drug Health Services, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia.
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Homomeric and heteromeric complexes among TGF-β and BMP receptors and their roles in signaling. Cell Signal 2011; 23:1424-32. [PMID: 21515362 DOI: 10.1016/j.cellsig.2011.04.004] [Citation(s) in RCA: 70] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2011] [Accepted: 04/04/2011] [Indexed: 02/08/2023]
Abstract
Transforming growth factor-β (TGF-β) ligands and bone morphogenetic proteins (BMPs) play myriad roles in many biological processes and diseases. Their pluripotent activities are subject to multiple levels of regulation, including receptor oligomerization, endocytosis, association with co-receptors, cellular scaffolds or membrane domains, as well as transcriptional control. In this review, we focus on TGF-β and BMP receptor homomeric and heteromeric complex formation and their modulation by ligand binding, which regulate signaling on a near-immediate timescale. We discuss the current structural, biochemical and biophysical evidence for the oligomerization of these receptors, and the potential roles of distinct oligomeric interactions in signaling.
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Senturk S, Mumcuoglu M, Gursoy-Yuzugullu O, Cingoz B, Akcali KC, Ozturk M. Transforming growth factor-beta induces senescence in hepatocellular carcinoma cells and inhibits tumor growth. Hepatology 2010; 52:966-74. [PMID: 20583212 DOI: 10.1002/hep.23769] [Citation(s) in RCA: 182] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
UNLABELLED Senescence induction could be used as an effective treatment for hepatocellular carcinoma (HCC). However, major senescence inducers (p53 and p16(Ink4a)) are frequently inactivated in these cancers. We tested whether transforming growth factor-beta (TGF-beta) could serve as a potential senescence inducer in HCC. First, we screened for HCC cell lines with intact TGF-beta signaling that leads to small mothers against decapentaplegic (Smad)-targeted gene activation. Five cell lines met this condition, and all of them displayed a strong senescence response to TGF-beta1 (1-5 ng/mL) treatment. Upon treatment, c-myc was down-regulated, p21(Cip1) and p15(Ink4b) were up-regulated, and cells were arrested at G(1). The expression of p16(Ink4a) was not induced, and the senescence response was independent of p53 status. A short exposure of less than 1 minute was sufficient for a robust senescence response. Forced expression of p21(Cip1) and p15(Ink4b) recapitulated TGF-beta1 effects. Senescence response was associated with reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) induction and intracellular reactive oxygen species (ROS) accumulation. The treatment of cells with the ROS scavenger N-acetyl-L-cysteine, or silencing of the NOX4 gene, rescued p21(Cip1) and p15(Ink4b) accumulation as well as the growth arrest in response to TGF-beta. Human HCC tumors raised in immunodeficient mice also displayed TGF-beta1-induced senescence. More importantly, peritumoral injection of TGF-beta1 (2 ng) at 4-day intervals reduced tumor growth by more than 75%. In contrast, the deletion of TGF-beta receptor 2 abolished in vitro senescence response and greatly accelerated in vivo tumor growth. CONCLUSION TGF-beta induces p53-independent and p16(Ink4a)-independent, but Nox4-dependent, p21(Cip1)-dependent, p15(Ink4b)-dependent, and ROS-dependent senescence arrest in well-differentiated HCC cells. Moreover, TGF-beta-induced senescence in vivo is associated with a strong antitumor response against HCC.
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Affiliation(s)
- Serif Senturk
- BilGen Research Center and Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
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Seth D, D'Souza El-Guindy NB, Apte M, Mari M, Dooley S, Neuman M, Haber PS, Kundu GC, Darwanto A, de Villiers WJ, Vonlaufen A, Xu Z, Phillips P, Yang S, Goldstein D, Pirola RM, Wilson JS, Moles A, Fernández A, Colell A, García-Ruiz C, Fernández-Checa JC, Meyer C, Meindl-Beinker NM. Alcohol, signaling, and ECM turnover. Alcohol Clin Exp Res 2010; 34:4-18. [PMID: 19860812 DOI: 10.1111/j.1530-0277.2009.01060.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2023]
Abstract
Alcohol is recognized as a direct hepatotoxin, but the precise molecular pathways that are important for the initiation and progression of alcohol-induced tissue injury are not completely understood. The current understanding of alcohol toxicity to organs suggests that alcohol initiates injury by generation of oxidative and nonoxidative ethanol metabolites and via translocation of gut-derived endotoxin. These processes lead to cellular injury and stimulation of the inflammatory responses mediated through a variety of molecules. With continuing alcohol abuse, the injury progresses through impairment of tissue regeneration and extracellular matrix (ECM) turnover, leading to fibrogenesis and cirrhosis. Several cell types are involved in this process, the predominant being stellate cells, macrophages, and parenchymal cells. In response to alcohol, growth factors and cytokines activate many signaling cascades that regulate fibrogenesis. This mini-review brings together research focusing on the underlying mechanisms of alcohol-mediated injury in a number of organs. It highlights the various processes and molecules that are likely involved in inflammation, immune modulation, susceptibility to infection, ECM turnover and fibrogenesis in the liver, pancreas, and lung triggered by alcohol abuse.
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Affiliation(s)
- Devanshi Seth
- Drug Health Services & Centenary Institute, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
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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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Zimonjic DB, Zhou X, Lee JS, Ullmannova-Benson V, Tripathi V, Thorgeirsson SS, Popescu NC. Acquired genetic and functional alterations associated with transforming growth factor beta type I resistance in Hep3B human hepatocellular carcinoma cell line. J Cell Mol Med 2009; 13:3985-92. [PMID: 19426152 PMCID: PMC4516545 DOI: 10.1111/j.1582-4934.2009.00769.x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
Abstract
During the neoplastic process tumour cells frequently acquire resistance to the antiproliferative signals of transforming growth factor-β (TGF-β). Here we examined a human hepatocellular carcinoma cell line (Hep3B-TS) sensitive to TGF-β signalling, and a derivative line (Hep3B-TR) rendered resistant to TGF-β by stepwise exposure to TGF-β1. Comprehensive molecular cytogenetic analysis revealed that the acquisition of TGF-β-resistance by Hep3B-TR cells was due to loss of TGF-β receptor 2 (TGFβRII) gene. As demonstrated by spectral karyotyping and array-based comparative genomic hybridization, and in difference to Hep3B-TS cells, which have three rearranged and two normal copies of chromosome 3 that harbour the TGFβRII gene, Hep3B-TR cells have four rearranged and one apparently normal chromosome 3, which nonetheless underwent a critical microdeletion at the site of TGFβRII gene. Gene expression analysis using an oligonucleotide microarray of 21,397 genes showed that Hep3B-TR differentially expressed 307 genes, out of which 197 and 110 were up- and down-regulated, respectively, compared to Hep3B-TS. Six of differentially expressed genes were identified as downstream targets of the tumour necrosis factor (TNF) gene, suggesting that loss of TGFβRII triggered activation of the TNF pathway known to be regulated by TGF-β1 network. On the functional level, the TGF-β-resistant Hep3B-TR cells displayed significantly enhanced capacity for anchorage independent growth and cell migration in vitro, and also increased tumorigenicity in vivo and in vitro and in vivo tumorigenicity compared with parental sensitive cells.
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Affiliation(s)
- Drazen B Zimonjic
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-4262, USA
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Ding W, Mouzaki M, You H, Laird JC, Mato J, Lu SC, Rountree CB. CD133+ liver cancer stem cells from methionine adenosyl transferase 1A-deficient mice demonstrate resistance to transforming growth factor (TGF)-beta-induced apoptosis. Hepatology 2009; 49:1277-86. [PMID: 19115422 PMCID: PMC2853874 DOI: 10.1002/hep.22743] [Citation(s) in RCA: 80] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
UNLABELLED Methionine adenosyltransferase (MAT) is an essential enzyme required for S-adenosylmethionine biosynthesis. Hepatic MAT activity falls during chronic liver injury, and mice lacking Mat1a develop spontaneous hepatocellular carcinoma by 18 months. We have previously demonstrated that CD133(+)CD45(-) oval cells isolated from 16-month-old Mat1a(-/-) mice represent a liver cancer stem cell population. The transforming growth factor beta (TGF-beta) pathway constitutes a central signaling network in proliferation, apoptosis, and tumorigenesis. In this study, we tested the response of tumorigenic liver stem cells to TGF-beta. CD133(+)CD45(-) oval cells were isolated from premalignant 16-month-old Mat1a(-/-) mice by flow cytometry and expanded as five clone lines derived from a single cell. All clone lines demonstrated expression of both hepatocyte and cholangiocyte markers and maintained a small population (0.5% to 2%) of CD133(+) cells in vitro, and three of five clone lines produced tumors. Although TGF-beta1 inhibited cell growth equally in CD133(-) and CD133(+) cells from each clone line, the CD133(+) population demonstrated significant resistance to TGF-beta-induced apoptosis compared with CD133(-) cells. Furthermore, CD133(+) cells demonstrated a substantial increase in mitogen-activated protein kinase (MAPK) pathway activation, as demonstrated by phosphorylated extracellular signal-regulated kinase levels before and after TGF-beta stimulation. MAPK inhibition using mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD98059 led to a significant increase in TGF-beta-induced apoptosis in CD133(+) cells. Conversely, a constitutively active form of MEK1 blocked the apoptotic effects of TGF-beta in CD133(-) cells. CONCLUSION CD133(+) liver cancer stem cells exhibit relative resistance to TGF-beta-induced apoptosis. One mechanism of resistance to TGF-beta-induced apoptosis in CD133(+) cancer stem cells is an activated mitogen-activated protein kinase/extracellular signal-regulated kinase pathway.
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Affiliation(s)
- Wei Ding
- Department of Pediatrics, The Pennsylvania State University College of Medicine, Penn State Children’s Hospital, Hershey, PA
- Department of Pharmacology, The Pennsylvania State University College of Medicine, Penn State Children’s Hospital, Hershey, PA
| | - Marialena Mouzaki
- Department of Pediatrics, The Pennsylvania State University College of Medicine, Penn State Children’s Hospital, Hershey, PA
- Department of Pharmacology, The Pennsylvania State University College of Medicine, Penn State Children’s Hospital, Hershey, PA
| | - Hanning You
- Department of Pediatrics, The Pennsylvania State University College of Medicine, Penn State Children’s Hospital, Hershey, PA
- Department of Pharmacology, The Pennsylvania State University College of Medicine, Penn State Children’s Hospital, Hershey, PA
| | - Joshua C. Laird
- Department of Pediatrics, The Pennsylvania State University College of Medicine, Penn State Children’s Hospital, Hershey, PA
- Department of Pharmacology, The Pennsylvania State University College of Medicine, Penn State Children’s Hospital, Hershey, PA
| | - Jose Mato
- CIC bioGUNE, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Technology, Park of Bizkaia, Bizkaia, Spain
| | - Shelly C. Lu
- Division of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA
| | - C. Bart Rountree
- Department of Pediatrics, The Pennsylvania State University College of Medicine, Penn State Children’s Hospital, Hershey, PA
- Department of Pharmacology, The Pennsylvania State University College of Medicine, Penn State Children’s Hospital, Hershey, PA
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18
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Rechtman MM, Nakaryakov A, Shapira KE, Ehrlich M, Henis YI. Different domains regulate homomeric and heteromeric complex formation among type I and type II transforming growth factor-beta receptors. J Biol Chem 2009; 284:7843-52. [PMID: 19147499 DOI: 10.1074/jbc.m809215200] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Transforming growth factor-beta (TGF-beta) binds to and signals via two serine-threonine kinase receptors, type I (TbetaRI) and type II (TbetaRII). The oligomerization of TGF-beta receptors modulates ligand binding and receptor trafficking and may contribute to signal diversification. However, numerous features of the molecular domains that determine the homo- and hetero-oligomerization of full-length receptors at the cell surface and the mode of these interactions remain unclear. Here, we address these questions through computerized immunofluorescence co-patching and patch/fluorescence recovery after photobleaching measurements of different combinations of epitope-tagged receptors and their mutants in live cells. We show that TbetaRI and TbetaRII are present on the plasma membrane both as monomers and homo- and hetero-oligomers. The homodimerization of TbetaRII depends on a cytoplasmic juxtamembrane region (amino acid residues 200-220). In contrast, the cytoplasmic domain of TbetaRI is dispensable for its homodimerization. TbetaRI.TbetaRII hetero-oligomerization depends on the cytoplasmic domain of TbetaRI and on a C-terminal region of TbetaRII (residues 419-565). TGF-beta1 elevates TbetaRII homodimerization to some degree and strongly enhances TbetaRI.TbetaRII heteromeric complex formation. Both ligand-induced effects depend on the region encompassed between residues 200-242 of TbetaRII. Furthermore, the kinase activity of TbetaRI is also necessary for the latter effect. All forms of the homo- and hetero-oligomers, whether constitutively present on the membrane or formed upon TGF-beta1 stimulation, were stable in the time-scale of our patch/FRAP measurements. We suggest that the different forms of receptor oligomerization may serve as a basis for the heterogeneity of TGF-beta signaling responses.
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Affiliation(s)
- Maya Mouler Rechtman
- Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
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19
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Dancea HC, Shareef MM, Ahmed MM. Role of Radiation-induced TGF-beta Signaling in Cancer Therapy. MOLECULAR AND CELLULAR PHARMACOLOGY 2009; 1:44-56. [PMID: 20336170 PMCID: PMC2844640 DOI: 10.4255/mcpharmacol.09.06] [Citation(s) in RCA: 101] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
TGF-β signaling regulates several different biological processes involving cell-growth, differentiation, apoptosis, motility, angiogenesis, epithelial mesenchymal transition and extracellular matrix production that affects embryonic development and pathogenesis of various diseases, including cancer, its effects depending on the cellular context and physiological environment. Growth suppression mediated by TGF-β signaling often associated with inhibition of c-myc, cdks and induction of p15, p27, Bax and p21. Despite its growth inhibitory effect, in certain conditions TGF-β may act as a promoter of cell proliferation and invasion. Loss of responsiveness to growth suppression by TGF-β due to mutation or loss of TGF-beta type II receptor (TβRII) and Smad4 in several different cancer cells are reported. In addition, TGF-β binding to its receptor activates many non-canonical signaling pathways. Radiation induced TGF-β is primarily involved in normal tissue injury and fibrosis. Seminal studies from our group have used radio-adjuvant therapies, involving classical components of the pathway such as TβRII and SMAD4 to overcome the growth promoting effects of TGF-β. The main impediment in the radiation-induced TGF-β signaling is the induction of SMAD7 that blocks TGF-β signaling in a negative feedback manner. It is well demonstrated from our studies that the use of neutralizing antibodies against TGF- β can render a robust radio-resistant effect. Thus, understanding the functional interactions of TGF-β signaling components of the pathway with other molecules may help tailor appropriate adjuvant radio-therapeutic strategies for treatment of solid tumors.
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Affiliation(s)
- Horatiu C Dancea
- Department of General Surgery, Geisinger Clinic, Danville, Pennsylvania
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20
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Meindl-Beinker NM, Dooley S. Transforming growth factor-beta and hepatocyte transdifferentiation in liver fibrogenesis. J Gastroenterol Hepatol 2008; 23 Suppl 1:S122-7. [PMID: 18336655 DOI: 10.1111/j.1440-1746.2007.05297.x] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Currently, hepatic stellate cells (HSC) are thought to be the major fibrotic precursor cells that transdifferentiate to fibrogenic, extracellular matrix producing myofibroblasts in inflammatory liver tissue upon transforming growth factor-beta (TGF-beta) signaling, whereas hepatocytes are thought to respond with apoptosis to this cytokine. Starting out from in vitro experiments with primary hepatocyte cultures and immortalized AML-12 cells, TGF-beta signaling in this cell type was assessed and apoptosis was found to be only a minor effect. Instead, hepatocytes undergo epithelial mesenchymal transition (EMT), a physiological process in embryogenesis and of relevance for cancerous cell transformation. In injured liver, however, this process contributes to the promotion of fibrosis. Already after a few days of culture, hepatocytes lose their epithelial honeycomb-like shape towards a fibroblast-like phenotype. We could demonstrate by microarray analysis that stimulation of hepatocytes with TGF-beta regulates the expression of genes involved in EMT and fibrosis. Among these were, for example, Snail, a known mediator of EMT, and connective tissue growth factor (CTGF), a strong inducer of fibrosis. In a mouse model, hepatocyte-specific overexpression of Smad7 was able to blunt a fibrogenic response after CCl(4) intoxication. These results emphasize the dynamic nature of liver fibrosis, challenge the paradigm of HSC as a crucial source of liver myofibroblasts and hint towards a prominent role for hepatocytes in liver fibrogenesis.
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Affiliation(s)
- Nadja M Meindl-Beinker
- Molecular Alcohol Research in Gastroenterology, II, Medical Clinic, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany
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21
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Lin SJ, Chang C, Ng AK, Wang SH, Li JJ, Hu CP. Prevention of TGF-beta-induced apoptosis by interlukin-4 through Akt activation and p70S6K survival signaling pathways. Apoptosis 2007; 12:1659-70. [PMID: 17624592 DOI: 10.1007/s10495-007-0085-5] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-beta (TGF-beta). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-beta-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-beta-induced apoptosis.
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Affiliation(s)
- Sue-Jane Lin
- Institute of Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan, Republic of China
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22
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Celikkaya H, Ciraci C, Oztas E, Avci ME, Ozturk M, Yagci T. Immunization with UV-Induced Apoptotic Cells Generates Monoclonal Antibodies Against Proteins Differentially Expressed in Hepatocellular Carcinoma Cell Lines. Hybridoma (Larchmt) 2007; 26:55-61. [PMID: 17451351 DOI: 10.1089/hyb.2006.047] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Early and differential diagnosis of hepatocellular carcinoma (HCC) requires sensitive and specific tissue and serum markers. On the other hand, proteins involved in tumorigenesis are extensively modulated on exposure to apoptotic stimuli, including ultraviolet (UVC) irradiation. Hence, we generated monoclonal antibodies by using UVC-irradiated apoptotic cells of an HCC cell line, HUH7, aiming to explore proteins differentially expressed in tumors and apoptosis. We obtained 18 hybridoma clones recognizing protein targets in apoptotic HUH7 cells, and clone 6D5 was chosen for characterization studies because of its strong reactivity in cell-ELISA assay. Subtype of the antibody was IgG3 (kappa). Targets of 6D5 antibody were found to be abundantly expressed in all HCC cell lines except FLC4, which resembles normal hepatocytes. We also observed the secretion of 6D5 ligands by some of the HCC cell lines. Moreover, cellular proteins recognized by the antibody displayed a late upregulation in UVC-induced apoptotic cells. We concluded that 6D5 target proteins are modulated in liver tumorigenesis and apoptotic processes. We therefore propose the validation of our antibody in tissue and serum samples of HCC patients to assess its potential use for the early diagnosis of HCC and to understand the role of 6D5 ligands in liver carcinogenesis.
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Affiliation(s)
- Hilal Celikkaya
- Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
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23
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Xu W, Song S, Huang Y, Gong Z. Effects of perindopril and valsartan on expression of transforming growth factor-beta-Smads in experimental hepatic fibrosis in rats. J Gastroenterol Hepatol 2006; 21:1250-6. [PMID: 16872305 DOI: 10.1111/j.1440-1746.2006.04331.x] [Citation(s) in RCA: 20] [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
BACKGROUND Previous studies have shown that the renin-angiotensin system (RAS) plays an important role in the pathogenesis of hepatic fibrosis, and blockers of the RAS may be active as an antifibrogenic goal. However, the potential role of RAS inhibition on expression transforming growth factor (TGF)-beta-Smads in hepatic fibrosis remains unknown. The aim of this study was to investigate the effect and mechanism of an angiotensin-converting enzyme inhibitor (perindopril) and an angiotensin II receptor blocker (valsartan) on TGF-beta1 and TGF receptor II (TRII) mRNA, Smad3 and Smad7 in fibrotic hepatic livers in rats. METHODS Sixty Wistar rats were randomly divided into four study groups (n = 15 for each group), including normal controls, hepatic fibrosis models, and two treated groups with either perindopril or valsartan, starting from the fourth week after being exposed to carbon tetrachloride (CCl(4)) for 4 weeks. The levels of TGF-beta and TRII mRNA in liver tissue were analyzed by RT-PCR. The expressions of TGF-beta1, Smad3 and Smad7 in liver tissues were evaluated by immunohistochemistry. The liver histopathology was examined by hematoxylin and eosin (HE) staining and by electron microscopy, respectively. The liver function and serum hyaluronic acid were also assayed by biochemistry and radioimmunoassay. RESULTS Compared with the hepatic fibrosis models, the levels of TGF-beta1, TRII mRNA and the expression Smad3 significantly decreased in the two treated groups, and the expression of Smad7 was significantly increased in the liver of rats treated with perindopril or valsartan (P < 0.05 or P < 0.01). The histological changes and ultrastructure of fibrotic liver, liver function and hyaluronic acid also remarkably improved in the treated rats. CONCLUSIONS The angiotensin-converting enzyme inhibitors perindopril and valsartan have a protective effect on liver injury and can ameliorate hepatic fibrosis in rats induced by CCl(4). The mechanisms may be associated with their effects of down-regulating TGF-beta1, TRII mRNA and smad3, and up-regulating Smad7.
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Affiliation(s)
- Wei Xu
- Department of Infectious Diseases, Renmin Hospital, State Key Laboratory of Virology, Wuhan University, Wuhan, China
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24
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Song SL, Gong ZJ, Zhang QR, Huang TX. Effects of Chinese traditional compound, JinSanE, on expression of TGF-β1 and TGF-β1 type II receptor mRNA, Smad3 and Smad7 on experimental hepatic fibrosis in vivo. World J Gastroenterol 2005; 11:2269-76. [PMID: 15818738 PMCID: PMC4305811 DOI: 10.3748/wjg.v11.i15.2269] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [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: The transforming growth factor-beta (TGF-β)/Smad signaling pathway system plays a prominent role in the control of cell growth and extracellular matrix formation in the progression of liver fibrogenesis. Smad proteins can either positively or negatively regulate TGF-β responses. In this study, the therapeutic effects of Chinese traditional compound decoction, JinSanE, and the changes of TGF-β/Smad signaling pathway system in carbon tetrachloride (CCl4)-induced rat experimental liver fibrosis were examined.
METHODS: Seventy-two healthy Wistar rats were assigned to groups including normal control group, CCl4 model group, JinSanE treatment group I and JinSanE treatment group II. Each group contained 18 rats. All groups, except the normal control group, received CCl4 subcutaneous injection for 8 wk. Rats in JinSanE groups I and II were orally treated with JinSanE daily at the 1st and 5th wk, respectively, after exposure to CCl4. The expression of TGF-β1 and TGF-β1 type II receptor (TRII) mRNA in the liver was determined by reverse transcription polymerase chain reaction, and the expression of TGF-β1, Smad3 and Smad7 by immunohistochemistry. The liver histopathology was also examined by HE staining and observed under electron microscope. The activities of several serum fibrosis-associated enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), the levels of serum hyaluronic acid (HA) were assayed.
RESULTS: Hepatic fibrosis caused by CCl4 was significantly inhibited in the JinSanE-treated groups. The degrees of necrosis/degeneration and fibrosis scores were significantly lower in the JinSanE-treated groups than in the model control group. The expression of TGF-β1, TRII and Smad3 was significantly higher in the model group than that in the JinSanE-treated groups, and the active/total TGF-β1 ratio in the JinSanE groups was suppressed. Expression of TRII mRNA and Smad3 proteins showed a distribution pattern similar to that of TGF-β1 with a direct correlation in terms of the degree of hepatic fibrosis. The amount of positive staining Smad7 cells was significantly less in the model group than in the JinSanE-treated groups and the normal group. The contents of ALT, AST and HA were significantly lower in the JinSanE-treated groups than those in the model group.
CONCLUSION: Traditional Chinese medicine, JinSanE, prevents the progression of hepatic damage and fibrosis through the inhibition of TGF-β1, TRII and Smad3 signal proteins, and increases expression of Smad7 signal protein in vivo.
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Affiliation(s)
- Shi-Ling Song
- Department of Infectious Diseases, Renmin Hospital, Key Laboratory of Virology for Ministry of Education, Wuhan University, Wuhan 430060, Hubei Province, China
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25
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Abstract
AIM: To investigate the effects of cyclosporine A (CsA) on thioacetamide (TAA)-induced liver injury.
METHODS: CsA was co-administrated (7.5 μg/kg body weight per day, i.p.) into rat to investigate the role of CsA on TAA-(200 mg/kg body weight per 3 d for 30 d, i.p.)induced liver injury.
RESULTS: The data show that TAA caused liver fibrosis in rat after 30 d of treatment. CsA alleviates the morphological changes of TAA-induced fibrosis in rat liver. The blood glutamyl oxaloacetic transaminase (GOT)/glutamyl pyruvic transaminase (GPT) in the TAA-injury group is elevated compared to that of the normal rat. Compared with the TAA-injury group, the blood GOT/GPT and TGFβ1 (by RT-PCR analysis) are reduced in the CsA plus TAA-treated rat. The level of the transforming growth factor receptor I (TGFβ-R1) in the CsA plus TAA-treated group shows higher than that in the TAA only group, but shows a lower level of the fibroblast growth factor receptor 4 (FGFR4) in the CsA plus TAA-treated group, when using the Western blot analysis. After immunostaining of the frozen section, TGFβ-R1 and FGFR4 are more concentrated in rat liver after CsA plus TAA injury.
CONCLUSION: This result suggests that CsA has an alleviated effect on TAA-induced liver injury by increasing the multidrug resistance P-glycoprotein and could be through the regulation of TGFβ-R1 and FGFR4.
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Affiliation(s)
- Sabrina Fan
- Institute of Biotechnology, National Dong Hwa University, Hualien 974, Taiwan, China
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26
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Xu Z, Ma DZ, Wang LY, Su JM, Zha XL. Transforming growth factor-β1 stimulated protein kinase B serine-473 and focal adhesion kinase tyrosine phosphorylation dependent on cell adhesion in human hepatocellular carcinoma SMMC-7721 cells. Biochem Biophys Res Commun 2003; 312:388-96. [PMID: 14637150 DOI: 10.1016/j.bbrc.2003.10.130] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Transforming growth factor-beta1 (TGF-beta1) is a potent growth inhibitor and apoptosis inducer for most normal cells. However, tumor cells are commonly nevertheless sensitive to the tumor-suppressing effects of TGF-beta1. In this paper, we focus on the effects of TGF-beta1 on two important anti-apoptotic protein kinases, protein kinase B (PKB), and focal adhesion kinase (FAK), in SMMC-7721 cells. We found that PKB-Ser-473 phosphorylation was apparently up-regulated by TGF-beta1. In the meantime, PKB-Thr-308 phosphorylation was slightly up-regulated by TGF-beta1. TGF-beta1 could also enhance FAK-Tyr phosphorylation. We observed that integrin-linked kinase (ILK) was also up-regulated by TGF-beta1 in good accordance with PKB-Ser-473 phosphorylation. We first found that TGF-beta1 could stimulate PKB-Ser-473 phosphorylation possibly via up-regulating ILK expression. Furthermore, we also failed to detect PKB-Ser-473 and FAK-Tyr phosphorylation with various concentrations of TGF-beta1 treatment when cells were kept in suspension. The above results indicate that PKB-Ser-473 and FAK-Tyr phosphorylation stimulated by TGF-beta1 are both dependent on cell adhesion.
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Affiliation(s)
- Zhen Xu
- Department of Biochemistry and Molecular Biology, Ministry of Education, Shanghai Medical College, Fudan University, Shanghai, 200032, China
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27
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Kracklauer MP, Schmidt C, Sclabas GM. TGFbeta1 signaling via alphaVbeta6 integrin. Mol Cancer 2003; 2:28. [PMID: 12935295 PMCID: PMC184456 DOI: 10.1186/1476-4598-2-28] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2003] [Accepted: 08/07/2003] [Indexed: 11/25/2022] Open
Abstract
BACKGROUND Transforming growth factor beta1 (TGFbeta1) is a potent inhibitor of epithelial cell growth, thus playing an important role in tissue homeostasis. Most carcinoma cells exhibit a reduced sensitivity for TGFbeta1 mediated growth inhibition, suggesting TGFbeta1 participation in the development of these cancers. The tumor suppressor gene DPC4/SMAD4, which is frequently inactivated in carcinoma cells, has been described as a key player in TGFbeta1 mediated growth inhibition. However, some carcinoma cells lacking functional SMAD4 are sensitive to TGFbeta1 induced growth inhibition, thus requiring a SMAD4 independent TGFbeta1 pathway. RESULTS Here we report that mature TGFbeta1 is a ligand for the integrin alphaVbeta6, independent of the common integrin binding sequence motif RGD. After TGFbeta1 binds to alphaVbeta6 integrin, different signaling proteins are activated in TGFbeta1-sensitive carcinoma cells, but not in cells that are insensitive to TGFbeta1. Among others, interaction of TGFbeta1 with the alphaVbeta6 integrin resulted in an upregulation of the cell cycle inhibitors p21/WAF1 and p27 leading to growth inhibition in SMAD4 deleted as well as in SMAD4 wildtype carcinoma cells. CONCLUSIONS Our data provide support for the existence of an alternate TGFbeta1 signaling pathway that is independent of the known SMAD pathway. This alternate pathway involves alphaVbeta6 integrin and the Ras/MAP kinase pathway and does not employ an RGD motif in TGFbeta1-sensitive tumor cells. The combined action of these two pathways seems to be necessary to elicit a complete TGFbeta1 signal.
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Affiliation(s)
- Martin P Kracklauer
- Section of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station, A4800, 78712, Austin, TX, USA
| | - Christian Schmidt
- Department of Surgical Oncology and Molecular Oncology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA
| | - Guido M Sclabas
- Department of Surgical Oncology and Molecular Oncology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA
- Department of Visceral and Transplantation Surgery, The University of Bern, Inselspital, Bern, 3010, Switzerland
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28
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Wilder PJ, Bernadt CT, Kim JH, Rizzino A. Stimulation of the murine type II transforming growth factor-beta receptor promoter by the transcription factor Egr-1. Mol Reprod Dev 2002; 63:282-90. [PMID: 12237943 DOI: 10.1002/mrd.10165] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Previous studies have demonstrated that differentiation of murine embryonal carcinoma (EC) cells leads to the appearance of high affinity receptors for transforming growth factor-beta (TGF-beta). Subsequently, it was demonstrated that differentiation of F9 EC cells leads to increases in the transcription of the type II TGF-beta-receptor gene (TbetaR-II) and leads to significant increases in the steady-state levels of TbetaR-II mRNA. Analysis of the human TbetaR-II promoter in F9-differentiated cells identified several cis-regulatory elements that influence the activity of the promoter, including a CRE/ATF site and a CCAAT box motif. In the work described in this report, we focused on the effect of the transcription factor Egr-1 on the murine TbetaR-II promoter. We have identified an Egr-1 response-element approximately 150 bp upstream of the major transcription start site of the murine TbetaR-II gene. We demonstrate by electrophoretic mobility shift analysis (EMSA) that this cis-regulatory element binds Egr-1, and we demonstrate that disruption of this site eliminates the response to Egr-1. As part of this analysis, we also examined the effect of Egr-1 on human TbetaR-II promoter. In contrast to a previous report, which reported that Egr-1 inhibits expression of human TbetaR-II promoter/reporter gene constructs, we did not observe an inhibitory effect of Egr-1 that was specific for the human TbetaR-II promoter. Taken together, the findings described in this report identify important differences between the human and the murine TbetaR-II promoter, and our findings identify an Egr-1 cis-regulatory element that is capable of stimulating the activity of the murine TbetaR-II promoter.
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Affiliation(s)
- Phillip J Wilder
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA
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29
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Zhou Q, Dong Wang L, Du F, Zhou Y, Rui Zhang Y, Liu B, Wei Feng C, Gao SS, Fan ZM, Yang CS, Zheng S. Changes of TGFbeta1 and TGFbetaRII expression in esophageal precancerous and cancerous lesions: a study of a high-risk population in Henan, northern China. Dis Esophagus 2002; 15:74-9. [PMID: 12060047 DOI: 10.1046/j.1442-2050.2002.00227.x] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The level of transforming growth factor beta1 (TGFbeta1) and transforming growth factor betaII receptor (TGFbetaRII) was determined immunohistochemically in normal tissues and tissues with different severities of lesions (basal cell hyperplasia, BCH; dysplasia, DYS; carcinoma in situ, CIS; and squamous cell carcinoma, SCC) from surgically resected human esophagi and esophageal biopsies of symptom-free subjects. The samples were from an area with high esophageal cancer incidence in northern China (Linzhou, formerly Linxian, and nearby county Huixian in Henan Province). Peroxidase immunostain (ABC) and conventional hematoxylin and eosin stain were used. The tissue sections were incubated with antibodies of TGFbeta1 and TGFbetaRII overnight. The immunoreactivity was observed in cytoplasm of the esophageal specimen. From normal to BCH to DYS to CIS and to SCC, the positive immunostaining rates for TGFbeta1 increased significantly (P < 0.05). A linear correlation between the positive immunostaining rates of TGFbeta1 and the different lesions was observed (P < 0.05). From well- to moderately- and poorly differentiated SCC, the positive immunostaining rates for TGFbeta1 decreased gradually, but the difference was not significant (P > 0.05). In contrast, with the lesions progressing from normal to BCH to DYS to CIS and to SCC, the positive immunostaining rates for TGFbetaRII decreased significantly (P < 0.05). From well- to moderately- and poorly differentiated SCC, the positive immunostaining rates for TGFbetaRII decreased significantly (P < 0.05). There was a linear correlation between the positive rates of TGFbetaRII and different lesions and SCC differentiation (P < 0.05). The present results indicated that the alterations of TGFbeta1 and TGFbetaRII is a frequent event in esophageal multistage carcinogenesis, the absent or lower expression of TGFbetaRII may lead to the loss of cell proliferation control by TGFbeta1 and the overexpression of TGFbeta1 may be a negative feedback response caused by the lower expression of TGFbetaRII protein.
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Affiliation(s)
- Q Zhou
- Laboratory for Cancer Research, College of Medicine, Zhengzhou University, Henan, People's Republic of China
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30
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Uchiyama-Tanaka Y, Matsubara H, Mori Y, Kosaki A, Kishimoto N, Amano K, Higashiyama S, Iwasaka T. Involvement of HB-EGF and EGF receptor transactivation in TGF-beta-mediated fibronectin expression in mesangial cells. Kidney Int 2002; 62:799-808. [PMID: 12164862 DOI: 10.1046/j.1523-1755.2002.00537.x] [Citation(s) in RCA: 76] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
BACKGROUND Gq-coupled receptors are known to transactivate epidermal growth factor receptor (EGFR) via the Ca2+ and PKC pathways to phosphorylate extracellular signal-regulated kinase (ERK). METHODS We studied the involvement of EGFR in transforming growth factor-beta (TGF-beta)-mediated fibronectin (FN) expression using glomerular mesangial cells. RESULTS TGF-beta up-regulated FN mRNA accumulation in a time- and dose-dependent manner, which was completely inhibited by phosphatidylcholine-phospholipase C (PC-PLC) inhibitor and PKC inhibitors (calphostin-C and staurosporin). The EGFR inhibitor AG1478 completely abolished TGF-beta-mediated FN expression. ERK inactivation by PD98059, and p38MAPK inhibitor SB203580 also showed significant inhibitory effects. Addition of neutralizing anti-heparin-binding EGF-like growth factor (HB-EGF) antibody, pretreatment with heparin and the metalloproteinase (MMP) inhibitor batimastat blocked FN expression. In mesangial cells stably transfected with a chimera containing HB-EGF and alkaline phosphatase (ALP) genes, ALP activity in incubation medium was rapidly increased by TGF-beta (2.1-fold at 0.5 min) and reached a 3.7-fold increase at two minutes, which was abolished by calphostin-C or batimastat. TGF-beta phosphorylated EGFR, ERK and p38MAPK in a PKC- and MMP-dependent manner. Smad2 phosphorylation by TGF-beta was not affected by AG1478, and HB-EGF did not activate Smad2. FN mRNA stability was not affected by TGF-beta. Cycloheximde did not interfere with TGF-beta-mediated FN expression. CONCLUSIONS The present study demonstrated that HB-EGF processed and released via PC-PLC-PKC signaling is an intermediate molecule for TGF-beta-mediated EGFR transactivation, and subsequent activation of ERK and p38MAPK is involved in FN expression via transcriptional regulation without requiring new protein synthesis.
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Affiliation(s)
- Yoko Uchiyama-Tanaka
- Department of Medicine II, Kansai Medical University, Moriguchi, Osaka 570-8507, Japan
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31
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Kishibe K, Yamada Y, Ogawa K. Production of nerve growth factor by mouse hepatocellular carcinoma cells and expression of TrkA in tumor-associated arteries in mice. Gastroenterology 2002; 122:1978-86. [PMID: 12055603 DOI: 10.1053/gast.2002.33581] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
BACKGROUND & AIMS Nerve growth factor (NGF) has been suggested to play a role in cancer progression. We found that NGF is specifically elevated in mouse hepatocellular carcinomas (HCCs) by cDNA array analysis. The present study aimed to elucidate expression of NGF and its receptors during hepatocarcinogenesis and under other conditions. METHODS Expression of NGF, TrkA, and p75NTR was investigated in HCCs developing and regenerating livers, and primary hepatocyte cultures in B6C3F(1) mice by reverse-transcription polymerase chain reaction, Northern blotting, and/or immunohistochemistry. The biological activity of NGF produced by the HCC cells was studied by using PC12 cells. Nerve fibers in hepatic tumors were immunohistochemically examined. RESULTS Although NGF was negative in adult and developing livers, it was markedly elevated in focal hepatocytic lesions from early stages of carcinogenesis. Appreciable levels were also detected in regenerating livers and hepatocytes in culture. The conditioned medium of HCC cells caused PC12 neurite outgrowth, but this was reduced on pretreatment of the conditioned medium with an anti-NGF antibody or NGF antisense expression in HCC cells. Although neither TrkA nor p75NTR was detectable in either HCC or normal hepatic cells, TrkA was shown in the walls of tumor-associated arteries that contain abundant nerve fibers. CONCLUSIONS NGF is expressed by hepatocytes during carcinogenesis, regeneration, and primary culture but may have cells other than hepatocytes as the target. TrkA expression and the abundance of nerve fibers in the walls of tumor-associated arteries suggest a possible role for NGF in angiogenesis.
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Affiliation(s)
- Kan Kishibe
- Department of Pathology, Asahikawa Medical College, Midorigaoka, Japan
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32
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Fernandez T, Amoroso S, Sharpe S, Jones GM, Bliskovski V, Kovalchuk A, Wakefield LM, Kim SJ, Potter M, Letterio JJ. Disruption of transforming growth factor beta signaling by a novel ligand-dependent mechanism. J Exp Med 2002; 195:1247-55. [PMID: 12021305 PMCID: PMC2193757 DOI: 10.1084/jem.20011521] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Transforming growth factor (TGF)-beta is the prototype in a family of secreted proteins that act in autocrine and paracrine pathways to regulate cell development and function. Normal cells typically coexpress TGF-beta receptors and one or more isoforms of TGF-beta, thus the synthesis and secretion of TGF-beta as an inactive latent complex is considered an essential step in regula-ting the activity of this pathway. To determine whether intracellular activation of TGF-beta results in TGF-beta ligand-receptor interactions within the cell, we studied pristane-induced plasma cell tumors (PCTs). We now demonstrate that active TGF-beta1 in the PCT binds to intracellular TGF-beta type II receptor (TbetaRII). Disruption of the expression of TGF-beta1 by antisense TGF-beta1 mRNA restores localization of TbetaRII at the PCT cell surface, indicating a ligand-induced impediment in receptor trafficking. We also show that retroviral expression of a truncated, dominant-negative TbetaRII (dnTbetaRII) effectively competes for intracellular binding of active ligand in the PCT and restores cell surface expression of the endogenous TbetaRII. Analysis of TGF-beta receptor-activated Smad2 suggests the intracellular ligand-receptor complex is not capable of signaling. These data are the first to demonstrate the formation of an intracellular TGF-beta-receptor complex, and define a novel mechanism for modulating the TGF-beta signaling pathway.
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Affiliation(s)
- Tania Fernandez
- Laboratory of Cell Regulation and Carcinogenesis, The National Cancer Institute, The National Institutes of Health, Bethesda, MD 20892, USA
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33
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Kim JH, Wilder PJ, Hou J, Nowling T, Rizzino A. Activation of the murine type II transforming growth factor-beta receptor gene: up-regulation and function of the transcription factor Elf-3/Ert/Esx/Ese-1. J Biol Chem 2002; 277:17520-30. [PMID: 11893733 DOI: 10.1074/jbc.m110434200] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Previous studies demonstrated that differentiation of mouse embryonal carcinoma cells leads to transcriptional up-regulation of the mouse type II transforming growth factor-beta receptor (mTbetaR-II) gene. To elucidate the molecular mechanisms regulating transcription of this gene, we isolated the 5'-flanking region of the mTbetaR-II gene and characterized its expression in F9-differentiated cells. Analysis of mTbetaR-II promoter/reporter gene constructs demonstrates that two conserved Ets-binding sites play an important role in the activity of the mTbetaR-II promoter. Importantly, we present evidence that mElf-3, a member of the Ets family, plays a key role in the activation of the mTbetaR-II promoter. Northern blot analysis reveals that the steady-state levels of mTbetaR-II mRNA increase in parallel with those of mElf-3 mRNA during the differentiation of F9 embryonal carcinoma cells. We also demonstrate that mElf-3 contains one or more domains that influence its binding to DNA. Finally, we report that a single amino acid substitution in the transactivation domain of mElf-3 reduces its ability to transactivate and elevates its steady-state levels of expression. In conclusion, our data argue that mElf-3 plays a key role in the regulation of the mTbetaR-II gene, and Elf-3 itself is regulated at multiple levels.
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Affiliation(s)
- Jae-Hwan Kim
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA
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34
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Lee JS, Thorgeirsson SS. Functional and genomic implications of global gene expression profiles in cell lines from human hepatocellular cancer. Hepatology 2002; 35:1134-43. [PMID: 11981763 DOI: 10.1053/jhep.2002.33165] [Citation(s) in RCA: 89] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Global gene expression profiles in cancer have impacted both classification of tumors and definition of molecular pathways in neoplasia. To explore the possibility of employing human tumor cell lines to obtain information on the functional genomics of the early stages of tumorigenesis, we have characterized variation in gene-expression patterns in a cytogenetically well-defined series of cell lines derived from human hepatocellular carcinoma (HCC). Microarrays containing 6,720 sequence-verified human cDNAs were used in this study. Nineteen well-characterized HCC cell lines were analyzed, and a nontumorigenic liver-derived epithelial cell line (Chang) was used as a reference. Each sample was examined at least twice by switching fluorescent dyes, Cy-5 and Cy-3, and average values of 2 experiments on each sample were used for further analysis. Analysis of the clustered data revealed 2 distinctive subtypes of gene-expression patterns among the 19 cell lines, suggesting a degree of heterogeneity among the gene-expression profiles of cell lines. Remarkably, expression of alpha-fetoprotein (AFP) was highly correlated with the molecular subtypes of HCC. Although the 3 most distinctive gene-expression modules represented the signatures of 2 different subgroups of HCC, most of the cell lines shared many coexpressed genes. However, sets of coexpressed genes that are specific for the subtypes of HCC were identified. Furthermore, our results indicate that the comparison between gene-expression patterns and structural alterations in chromosomes is potentially useful in identifying genes critical in early stages of tumorigenesis. In conclusion, these results not only identified unrecognized subtypes of HCC, but also provided potential molecular markers for each subtype that can be useful for diagnostic and/or therapeutic purposes.
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Affiliation(s)
- Ju-Seog Lee
- Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4258, USA
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35
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Bollard CM, Rössig C, Calonge MJ, Huls MH, Wagner HJ, Massague J, Brenner MK, Heslop HE, Rooney CM. Adapting a transforming growth factor beta-related tumor protection strategy to enhance antitumor immunity. Blood 2002; 99:3179-87. [PMID: 11964281 DOI: 10.1182/blood.v99.9.3179] [Citation(s) in RCA: 258] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Transforming growth factor beta (TGF-beta), a pleiotropic cytokine that regulates cell growth and differentiation, is secreted by many human tumors and markedly inhibits tumor-specific cellular immunity. Tumors can avoid the differentiating and apoptotic effects of TGF-beta by expressing a nonfunctional TGF-beta receptor. We have determined whether this immune evasion strategy can be manipulated to shield tumor-specific cytotoxic T lymphocytes (CTLs) from the inhibitory effects of tumor-derived TGF-beta. As our model we used Epstein-Barr virus (EBV)-specific CTLs that are infused as treatment for EBV-positive Hodgkin disease but that are vulnerable to the TGF-beta produced by this tumor. CTLs were transduced with a retrovirus vector expressing the dominant-negative TGF-beta type II receptor HATGF-betaRII-Deltacyt. HATGF-betaRII-Deltacyt- but not green fluorescence protein (eGFP)-transduced CTLs was resistant to the antiproliferative and anticytotoxic effects of exogenous TGF-beta. Additionally, receptor-transduced cells continued to secrete cytokines in response to antigenic stimulation. TGF-beta receptor ligation results in phosphorylation of Smad2, and this pathway was disrupted in HATGF-betaRII-Deltacyt-transduced CTLs, confirming blockade of the signal transduction pathway. Long-term expression of TGF-betaRII-Deltacyt did not affect CTL function, phenotype, or growth characteristics. Tumor-specific CTLs expressing HATGF-betaRII-Deltacyt should have a selective functional and survival advantage over unmodified CTLs in the presence of TGF-beta-secreting tumors and may be of value in treatment of these diseases.
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MESH Headings
- Adjuvants, Immunologic/genetics
- Adjuvants, Immunologic/metabolism
- Adjuvants, Immunologic/therapeutic use
- DNA-Binding Proteins/metabolism
- Genetic Therapy/methods
- Herpesvirus 4, Human
- Hodgkin Disease/immunology
- Hodgkin Disease/therapy
- Hodgkin Disease/virology
- Humans
- Immunotherapy/methods
- Mutation
- Protein Serine-Threonine Kinases
- Receptor, Transforming Growth Factor-beta Type II
- Receptors, Transforming Growth Factor beta/genetics
- Receptors, Transforming Growth Factor beta/metabolism
- Receptors, Transforming Growth Factor beta/therapeutic use
- Smad2 Protein
- T-Lymphocytes, Cytotoxic/drug effects
- T-Lymphocytes, Cytotoxic/immunology
- T-Lymphocytes, Cytotoxic/metabolism
- Trans-Activators/metabolism
- Transduction, Genetic
- Transforming Growth Factor beta/pharmacology
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Affiliation(s)
- Catherine M Bollard
- Center for Cell and Gene Therapy, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA
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36
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Cowin AJ, Hatzirodos N, Holding CA, Dunaiski V, Harries RH, Rayner TE, Fitridge R, Cooter RD, Schultz GS, Belford DA. Effect of healing on the expression of transforming growth factor beta(s) and their receptors in chronic venous leg ulcers. J Invest Dermatol 2001; 117:1282-9. [PMID: 11710945 DOI: 10.1046/j.0022-202x.2001.01501.x] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
The transforming growth factor betas are of major importance in the wound repair process; however, no studies to date have investigated the role of the transforming growth factor beta receptors in chronic venous leg ulcers or what effect healing has on these proteins. To determine whether the transforming growth factor beta peptides and their receptors are expressed in chronic venous wounds, we used immunofluorescent analysis and quantitative competitive reverse transcription polymerase chain reaction to identify the protein and mRNA expression, respectively. Biopsy samples from wounds and normal skin were collected from 12 patients with chronic venous leg ulcers and three patients undergoing reconstructive surgery, respectively. Additionally four of the chronic venous leg ulcer patients were re-biopsied between 2 and 8 wk after the first biopsy when the wounds had entered the healing phase. The tissue excised from the ulcers included the surrounding intact skin, the ulcer edge, and the ulcer base. Immunofluorescent staining for transforming growth factors beta1, beta2, and beta3 was observed within the epidermis of the skin surrounding the chronic venous ulcers and in fibroblasts and inflammatory cells of the dermis, although this staining was not as strong as that seen in normal unwounded skin. Very little staining could be seen within the ulcers for any of the ligands, however. In contrast the transforming growth factor beta type I receptor was observed throughout the ulcers and the normal unwounded skin biopsies, particularly in the basal epidermal cells. No immunofluorescence for the type II transforming growth factor beta receptor was observed in any of the ulcer biopsies investigated, although it was observed throughout the epidermis and in fibroblasts and inflammatory cells in the surrounding skin. Quantitative, competitive reverse transcription polymerase chain reaction was used to analyze mRNA expression for transforming growth factor beta1 and the type II receptor in the nonhealing ulcers and normal unwounded skin biopsies. These studies revealed that transforming growth factor beta1 and transforming growth factor beta receptor II mRNA was expressed in all the chronic nonhealing ulcers albeit at very low levels for the type II receptor. In marked contrast to the staining observed in nonhealing chronic ulcers, positive immunostaining was observed for the transforming growth factor betas and both the type I and type II receptors in healing ulcers. These results suggest that the absence of a viable receptor complex for the transforming growth factor betas in nonhealing chronic venous ulcers may contribute to wound chronicity.
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Affiliation(s)
- A J Cowin
- Cooperative Research Center for Tissue Growth and Repair, Child Health Research Institute, Women's and Children's Hospital, North Adelaide, South Australia, Australia.
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37
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Park DY, Hwang SY, Suh KS. Expression of transforming growth factor (TGF)-beta1 and TGF-beta type II receptor in preneoplastic lesions during chemical hepatocarcinogenesis of rats. Toxicol Pathol 2001; 29:541-9. [PMID: 11695571 DOI: 10.1080/019262301317226348] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Transforming growth factor (TGF)-beta1 is an important apoptotic growth inhibitor of hepatocyte proliferation, and the expression of TGF-beta1, which regulates cell proliferation, is closely associated with the expression level of TGF-beta type II receptor (TGR2). Moreover, TGF-beta1 expression has been regarded to be an important change in hepatocarcinogenesis, We undertook this study to investigate the gene expression and protein localization of TGF-beta1 and TGR2 and their relationship with apoptosis in the chemically induced hepatocarcinogenesis of the rat, as produced using Solt and Farber's method, during the promotion stage (up to 56 days after partial hepatectomy). Northern blot analysis showed a slight, but not a significant, increase in TGF-beta1 transcripts, and a significant decrease in the TGR2 transcripts during the later stage of our experiments (42 days after partial hepatectomy). Immunohistochemical study showed that TGF-beta1-positive preneoplastic hepatocytes increased with time, and this correlated with an increase of TGR2 negative or reduced TGR2 expressed preneoplastic lesions. The TUNEL method revealed that apoptotic cells increased with time and were more numerous in the adjacent liver parenchyme than preneoplastic lesions. Our data suggest that the expressions of TGF-beta1 and TGR2 are significantly altered during the promotion stage of hepatocarcinogenesis of rat and that these changes might contribute to the development and progression of preneoplastic lesions.
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MESH Headings
- Animals
- Apoptosis/drug effects
- Carcinogens/toxicity
- Cell Count
- Disease Models, Animal
- Drug Synergism
- Gene Expression Regulation, Neoplastic
- Hepatectomy
- Immunoenzyme Techniques
- In Situ Nick-End Labeling
- Liver Neoplasms, Experimental/genetics
- Liver Neoplasms, Experimental/metabolism
- Liver Neoplasms, Experimental/pathology
- Male
- Precancerous Conditions/genetics
- Precancerous Conditions/metabolism
- Precancerous Conditions/pathology
- Protein Serine-Threonine Kinases
- RNA, Messenger/analysis
- RNA, Messenger/metabolism
- Rats
- Rats, Sprague-Dawley
- Receptor, Transforming Growth Factor-beta Type II
- Receptors, Transforming Growth Factor beta/biosynthesis
- Receptors, Transforming Growth Factor beta/genetics
- Transforming Growth Factor beta/biosynthesis
- Transforming Growth Factor beta/genetics
- Transforming Growth Factor beta1
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Affiliation(s)
- D Y Park
- Department of Pathology, Pusan National University College of Medicine, Korea
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38
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Ammanamanchi S, Brattain MG. 5-azaC treatment enhances expression of transforming growth factor-beta receptors through down-regulation of Sp3. J Biol Chem 2001; 276:32854-9. [PMID: 11443124 DOI: 10.1074/jbc.m103951200] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
We have previously reported that Sp3 acts as a transcriptional repressor of transforming growth factor-beta receptors type I (RI) and type II (RII). We now present data suggesting that treatment of MCF-7L breast and GEO colon cancer cells with 5-aza cytidine (5-azaC) leads to down-regulation of Sp3 and the concomitant induction of RI and RII. Western blot and gel shift analyses on 5-azaC-treated MCF-7L and GEO nuclear extracts indicated reduced Sp3 protein levels and decreased binding of Sp3 protein to radiolabeled consensus Sp1 oligonucleotide. Southwestern analysis detected decreased binding of Sp3 to RI and RII promoters in 5-azaC-treated MCF-7L and GEO cells, suggesting a correlation between decreased Sp3 binding and enhanced RI and RII expression in these cells. Reverse transcription-polymerase chain reaction and nuclear run-on data from 5-azaC-treated MCF-7L and GEO cells indicated down-regulation of Sp3 mRNA as a result of decreased transcription of Sp3. We reported earlier that 5-azaC treatment induces RI and RII expression through increased Sp1 protein levels/activities in these cells. These studies demonstrate that the effect of 5-azaC involves a combination of effects on Sp1 and Sp3.
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Affiliation(s)
- S Ammanamanchi
- Department of Surgery, The University of Texas Health Science Center, San Antonio, Texas 78229, USA
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39
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Kanzler S, Meyer E, Lohse AW, Schirmacher P, Henninger J, Galle PR, Blessing M. Hepatocellular expression of a dominant-negative mutant TGF-beta type II receptor accelerates chemically induced hepatocarcinogenesis. Oncogene 2001; 20:5015-24. [PMID: 11526486 DOI: 10.1038/sj.onc.1204544] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2001] [Revised: 04/11/2001] [Accepted: 04/12/2001] [Indexed: 12/17/2022]
Abstract
The potent growth-inhibitory activity of cytokines of the transforming growth factor-beta (TGF-beta) superfamily and their widespread expression in epithelia suggest that they may play an important role in the maintenance of epithelial homeostasis. To analyse TGF-beta mediated tumor suppressor activity in the liver, we generated transgenic mice overexpressing a dominant negative type II TGF-beta receptor in hepatocytes under control of the regulatory elements of the human C-reactive protein gene promoter. Transgenic animals exhibited constitutive and liver-specific transgene expression. The functional inactivation of the TGF-beta signaling pathway in transgenic hepatocytes was shown by reduced TGF-beta induced inhibition of DNA synthesis in primary hepatocyte cultures. Liver morphology and spontaneous tumorigenesis were unchanged in transgenic mice suggesting that interruption of the signaling of all three isoforms of TGF-beta in hepatocytes does not disturb tissue homeostasis in the liver under physiological conditions. However, following initiation with the carcinogen diethylnitrosamine and tumor-promotion with phenobarbital transgenic mice exhibited a moderate albeit significant increase in the incidence, size and multiplicity of both preneoplastic tissue lesions in the liver and of hepatocellular carcinomas. These results give in vivo evidence for a tumor suppressor activity of the endogenous TGF-beta system in the liver during chemical hepatocarcinogenesis.
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MESH Headings
- Animals
- C-Reactive Protein/genetics
- Carcinoma, Hepatocellular/chemically induced
- Carcinoma, Hepatocellular/etiology
- Carcinoma, Hepatocellular/metabolism
- Cells, Cultured
- Hepatocytes/drug effects
- Hepatocytes/metabolism
- Liver Neoplasms, Experimental/chemically induced
- Liver Neoplasms, Experimental/metabolism
- Liver Neoplasms, Experimental/pathology
- Male
- Mice
- Mice, Transgenic
- Mutation
- Protein Serine-Threonine Kinases
- RNA, Messenger/biosynthesis
- Receptor, Transforming Growth Factor-beta Type II
- Receptors, Transforming Growth Factor beta/genetics
- Receptors, Transforming Growth Factor beta/metabolism
- Transforming Growth Factor beta/pharmacology
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Affiliation(s)
- S Kanzler
- Department of Medicine, University of Mainz, 55131 Mainz, Germany
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40
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Warren SM, Steinbrech DS, Mehrara BJ, Saadeh PB, Greenwald JA, Spector JA, Bouletreau PJ, Longaker MT. Hypoxia regulates osteoblast gene expression. J Surg Res 2001; 99:147-55. [PMID: 11421617 DOI: 10.1006/jsre.2001.6128] [Citation(s) in RCA: 65] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Vascular disruption secondary to fracture creates a hypoxic gradient of injury wherein the oxygen tension at the center of the wound is very low. In vivo this hypoxic microenvironment stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and osteoblasts. In order to begin to dissect this complex system, we have examined the effects of hypoxia on isolated osteoblast gene expression in vitro. Understanding gene expression in this system may facilitate the development of targeted therapeutic modalities designed to accelerate fracture repair and reduce complications. Using an established model of in vitro hypoxia, we have analyzed the expression of genes involved in bone matrix production and turnover. Subconfluent neonatal rat calvarial osteoblasts were exposed to hypoxia (pO(2) = 35-40 mm Hg) and total cellular RNA was collected at 0, 3, 6, 24, and 48 h. Northern analysis was used to analyze the expression patterns of (1) transforming growth factors (TGFs)-beta1, -beta2, and -beta3 and their type I receptor; (2) collagens I and III; and (3) tissue inhibitor of metalloproteinase-1. We have demonstrated a marked elevation of TGF-beta1 gene expression within 3 h of hypoxia. Although neither TGF-beta2 nor TGF-beta3 expression was affected by hypoxia, the TGF-beta type I receptor was substantially upregulated within 6 h. In addition, extracellular matrix scaffolding molecules (collagens I and III) were markedly, but differentially, upregulated. Finally, we have demonstrated that the expression of an inhibitor of extracellular matrix turnover, the tissue inhibitor of metalloproteinase-1, was strikingly decreased in response to hypoxia. These results imply that hypoxia can affect osseous healing by altering the expression of cytokines, bone-specific extracellular matrix molecules, and their regulators.
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Affiliation(s)
- S M Warren
- Laboratory of Developmental Biology and Repair, Institute of Reconstructive Plastic Surgery, New York, New York 10016, USA
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41
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Auersperg N, Wong AS, Choi KC, Kang SK, Leung PC. Ovarian surface epithelium: biology, endocrinology, and pathology. Endocr Rev 2001; 22:255-88. [PMID: 11294827 DOI: 10.1210/edrv.22.2.0422] [Citation(s) in RCA: 382] [Impact Index Per Article: 15.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
The epithelial ovarian carcinomas, which make up more than 85% of human ovarian cancer, arise in the ovarian surface epithelium (OSE). The etiology and early events in the progression of these carcinomas are among the least understood of all major human malignancies because there are no appropriate animal models, and because methods to culture OSE have become available only recently. The objective of this article is to review the cellular and molecular mechanisms that underlie the control of normal and neoplastic OSE cell growth, differentiation, and expression of indicators of neoplastic progression. We begin with a brief discussion of the development of OSE, from embryonic to the adult. The pathological and genetic changes of OSE during neoplastic progression are next summarized. The histological characteristics of OSE cells in culture are also described. Finally, the potential involvement of hormones, growth factors, and cytokines is discussed in terms of their contribution to our understanding of the physiology of normal OSE and ovarian cancer development.
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Affiliation(s)
- N Auersperg
- Department of Obstetrics and Gynaecology, British Columbia Women's Hospital, University of British Columbia, Vancouver, British Columbia, V6H 3V5, Canada
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Ammanamanchi S, Brattain MG. Sp3 is a transcriptional repressor of transforming growth factor-beta receptors. J Biol Chem 2001; 276:3348-52. [PMID: 11027677 DOI: 10.1074/jbc.m002462200] [Citation(s) in RCA: 46] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
MCF-7E breast cancer cells express transforming growth factor-beta (TGF-beta) receptors RI and RII in comparison to MCF-7L cells. We present data showing that Sp3 acts as a transcriptional repressor of RI and RII in MCF-7L cells and GEO colon cancer cells. MCF-7L and GEO cells express high levels of Sp3 protein. Gel shift analysis indicated enhanced binding of Sp3 from MCF-7L cells to a consensus Sp1 oligonucleotide. Southwestern data indicated increased binding of Sp3 to RI and RII promoters in MCF-7L cells, suggesting a correlation between Sp3 binding and reduced expression of TGF-beta receptors in MCF-7L cells. Cotransfection of CMV-Sp3 cDNA with RI and RII promoter-luciferase reporter constructs decreased RI and RII promoter activities by 70% in MCF-7E and GEO cells. Southwestern analysis detected the binding of transiently expressed Sp3 to RI and RII promoters in MCF-7E cells. Significantly, ectopic Sp3 expression led to repression of RI and RII transcripts in MCF-7E cells. This report demonstrates that inappropriate overexpression of Sp3 is a mechanism that contributes to repression of TGF-beta receptors.
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Affiliation(s)
- S Ammanamanchi
- Department of Surgery, University of Texas Health Science Center, San Antonio, Texas 78229, USA
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43
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Kim TK, Mo EK, Yoo CG, Lee CT, Han SK, Shim YS, Kim YW. Alteration of cell growth and morphology by overexpression of transforming growth factor beta type II receptor in human lung adenocarcinoma cells. Lung Cancer 2001; 31:181-91. [PMID: 11165397 DOI: 10.1016/s0169-5002(00)00169-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
TGF-beta is a potent inhibitory regulator of cell growth, which is transduced through interaction between type I (RI) and type II (RII) receptors that form heteromeric kinase complexes. Abnormal expression of these receptors has been identified in several human epithelial cancers and has been shown to be highly associated with resistance to TGF-beta. In this study, we investigated the expression of RI and RII in 13 human non-small cell lung cancer cell lines (NSCLCs) and demonstrated decreased or loss of RII expression in five lung cancer cell lines, but not of RI. Of these cell lines, the role of RII in NCI-H358 adenocarcinoma, which lacks RII and is insensitive to TGF-beta, was investigated by transducing this cell line with a recombinant retrovirus expressing full-length TGF-beta RII. Stably transfected cells showed significant increase in RII mRNA and protein expression. These cells responded to exogenous TGF-beta1 with suppressed proliferation in a dose-dependent manner and G1 arrest accompanied by morphological change distinct from control cells. We also investigated whether overexpression of dominant-negative RII (dnRII) in NCI-H441 adenocarcinoma, which is sensitive but expresses low levels of RII, could block signaling through the receptor complex. The overexpression of this kinase-domain-truncated RII by expressing the retroviral dnRII construct led to loss of the ability to respond to TGF-beta1 and an exhibition of uncontrolled growth. These results suggest a close association between the loss of the expression of wild-type TGF-beta RII and carcinogenesis in human lung cancer cells.
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Affiliation(s)
- T K Kim
- Department of Internal Medicine, College of Medicine and Lung Institute, SNUMRC, Seoul National University and Clinical Research Institute, Seoul National University Hospital, Seoul 110-744, South Korea
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44
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Lynch MA, Petrel TA, Song H, Knobloch TJ, Casto BC, Ramljak D, Anderson LM, DeGroff V, Stoner GD, Brueggemeier RW, Weghorst CM. Responsiveness to transforming growth factor-beta (TGF-beta)-mediated growth inhibition is a function of membrane-bound TGF-beta type II receptor in human breast cancer cells. Gene Expr 2001; 9:157-71. [PMID: 11444526 PMCID: PMC5964939 DOI: 10.3727/000000001783992560] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/23/2001] [Indexed: 11/24/2022]
Abstract
Transforming growth factor-beta (TGF-beta) is a potent inhibitor of growth and proliferation of breast epithelial cells, and loss of sensitivity to its effects has been associated with malignant transformation and tumorigenesis. The biological effects of TGF-beta are mediated by the TGF-beta receptor complex, a multimer composed of TGF-beta receptor type I (TbetaR-I) and TGF-beta receptor type II (TbetaR-II) subunits. Evidence suggests that loss of expression of Tbeta3R-II is implicated in the loss of sensitivity of tumorigenic breast cell lines to TGF-beta-mediated growth inhibition. A panel of human breast cell lines, including the immortalized MCF-10F and tumorigenic MCF-7, ZR75-1, BT474, T47-D, MDA-MB231, BT20, and SKBR-3 cell lines, was characterized for responsiveness to TGF-beta-induced G1 growth arrest. Only the nontumorigenic MCF-10F and the tumorigenic MDA-MB231 cell lines demonstrated a significant inhibitory response to TGF-beta1 and a significant binding of 125I-labeled TGF-beta ligand. While expression of TbetaR-I mRNA was similar across the panel of cell lines, TbetaR-II mRNA expression was decreased significantly in all seven tumorigenic cell lines in comparison with the nontumorigenic MCF- 10F cell line. When total cellular protein was fractionated by centrifugation, TbetaR-I protein was observed in both the cytosolic and membrane fractions at similar levels in all cell lines; however, TbetaR-II protein was present in the cytosolic fraction in all cell lines, but was observed in the membrane fraction of only the TGF-beta-responsive MCF-10F and MDA-MB231 cells. Thus, lack of membrane-bound TbetaR-II protein appears to be an important determinant of resistance to TGF-beta-mediated growth inhibition in this group of breast cell lines.
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MESH Headings
- Blotting, Western
- Breast Neoplasms/metabolism
- Breast Neoplasms/pathology
- Cell Division/drug effects
- DNA Mutational Analysis
- Dose-Response Relationship, Drug
- Drug Resistance, Neoplasm
- Female
- G1 Phase/drug effects
- Humans
- Mutation/genetics
- Protein Serine-Threonine Kinases
- Protein Subunits
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Receptor, Transforming Growth Factor-beta Type II
- Receptors, Transforming Growth Factor beta/chemistry
- Receptors, Transforming Growth Factor beta/genetics
- Receptors, Transforming Growth Factor beta/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Signal Transduction/drug effects
- Transforming Growth Factor beta/metabolism
- Transforming Growth Factor beta/pharmacology
- Tumor Cells, Cultured
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Affiliation(s)
- Melanie A. Lynch
- *Division of Environmental Health Sciences, School of Public Health, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210
| | - Trevor A. Petrel
- †Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210
| | - Huijuan Song
- *Division of Environmental Health Sciences, School of Public Health, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210
| | - Thomas J. Knobloch
- *Division of Environmental Health Sciences, School of Public Health, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210
| | - Bruce C. Casto
- *Division of Environmental Health Sciences, School of Public Health, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210
| | - Danica Ramljak
- ‡Laboratory of Comparative Carcinogenesis, National Cancer Institute-FCRDC, Frederick, MD 21702
| | - Lucy M. Anderson
- ‡Laboratory of Comparative Carcinogenesis, National Cancer Institute-FCRDC, Frederick, MD 21702
| | - Valerie DeGroff
- *Division of Environmental Health Sciences, School of Public Health, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210
- §Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210
| | - Gary D. Stoner
- *Division of Environmental Health Sciences, School of Public Health, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210
- §Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210
| | - Robert W. Brueggemeier
- †Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210
- §Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210
| | - Christopher M. Weghorst
- *Division of Environmental Health Sciences, School of Public Health, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210
- §Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210
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45
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Oklü R, Hesketh R. The latent transforming growth factor beta binding protein (LTBP) family. Biochem J 2000; 352 Pt 3:601-10. [PMID: 11104663 PMCID: PMC1221494] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/18/2023]
Abstract
The transforming growth factor beta (TGFbeta) cytokines are a multi-functional family that exert a wide variety of effects on both normal and transformed mammalian cells. The secretion and activation of TGFbetas is regulated by their association with latency-associated proteins and latent TGFbeta binding proteins (LTBPs). Over the past few years, three members of the LTBP family have been identified, in addition to the protoype LTBP1 first sequenced in 1990. Three of the LTBP family are expressed in a variety of isoforms as a consequence of alternative splicing. This review summarizes the differences between the isoforms in terms of the effects on domain structure and hence possible function. The close identity between LTBPs and members of the fibrillin family, mutations in which have been linked directly to Marfan's syndrome, suggests that anomalous expression of LTBPs may be associated with disease. Recent data indicating that differential expression of LTBP1 isoforms occurs during the development of coronary heart disease is considered, together with evidence that modulation of LTBP function, and hence of TGFbeta activity, is associated with a variety of cancers.
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Affiliation(s)
- R Oklü
- Northwestern University Medical School, Lake Shore Center, Room 1707, 850 North Lake Shore Drive, Chicago, IL 60611, USA
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46
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Liu X, Sun Y, Ehrlich M, Lu T, Kloog Y, Weinberg RA, Lodish HF, Henis YI. Disruption of TGF-beta growth inhibition by oncogenic ras is linked to p27Kip1 mislocalization. Oncogene 2000; 19:5926-35. [PMID: 11127824 DOI: 10.1038/sj.onc.1203991] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Expression of oncogenic Ras in epithelial tumor cells is linked to the loss of transforming growth factor-beta (TGF-beta) anti-proliferative activity, and was proposed to involve inhibition of Smad2/3 nuclear translocation. Here we studied several epithelial cell lines expressing oncogenic N-RasK61 and show that TGF-beta-induced nuclear translocation of and transcriptional activation by Smad2/3 were unaffected. In contrast, oncogenic Ras mediated nuclearto-cytoplasmic mislocalization of p27KiP1 (p27) and of the cyclin-dependent kinase (CDK) CDK6, but not CDK2. Concomitantly, oncogenic Ras abrogated the ability of TGF-beta to release p27 from CDK6, to enhance its binding to CDK2 and to inhibit CDK2 activity. Inactivation of Ras by a specific antagonist restored the growth inhibitory response to TGF-beta with concurrent normalization of p27 and CDK6 localization. Therefore, the disruption of TGF-beta-mediated growth inhibition by oncogenic Ras appears to be due to lack of inhibition of CDK2, caused by the sequestration of p27 and CDK2 in different subcellular compartments and by the loss of TGF-beta-induced partner switching of p27 from CDK6 to CDK2.
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Affiliation(s)
- X Liu
- Whitehead Institute for Biomedical Research, Nine Cambridge Center, Massachusetts 02142, USA
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47
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Kang MJ, Ingram A, Ly H, Thai K, Scholey JW. Effects of diabetes and hypertension on glomerular transforming growth factor-beta receptor expression. Kidney Int 2000; 58:1677-85. [PMID: 11012901 DOI: 10.1046/j.1523-1755.2000.00328.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
BACKGROUND Several studies have suggested that transforming growth factor-beta1 (TGF-beta1) is an important determinant of diabetic glomerular injury. TGF-beta1 forms a heteromeric complex with two cellular receptor subtypes, designated TGF-beta RII and TGF-beta RI, but the effects of diabetes mellitus on glomerular TGF-beta receptor expression have not been completely elucidated. We first compared the effect of experimental type I diabetes mellitus and uninephrectomy on glomerular TGF-beta receptor expression in spontaneously hypertensive rats (SHRs), and then sought to determine whether changes in TGF-beta receptor expression were strain specific by studying normotensive Wistar-Kyoto (WKY) rats. METHODS Five groups of male SHRs were studied. The first group received streptozotocin (60 mg/kg IV) and was studied after one week. The second group received streptozotocin and was studied after two weeks. The third group received streptozotocin (60 mg/kg IV) but received insulin to maintain euglycemia. The fourth group of age-matched SHRs served as the control group, while a fifth group of SHRs underwent uninephrectomy. Four groups of male WKY rats were also studied. The first group of WKY rats served as the age-matched control group. The second group of WKY rats received streptozotocin, while a third group of WKY rats underwent uninephrectomy. The fourth group underwent uninephrectomy and received streptozotocin. At each time point, glomeruli were isolated for protein extraction, and the protein was subjected to Western blot analysis of TGF-beta RII and TGF-beta RI expression. RESULTS Basal expression of both TGF-beta receptors per microgram of glomerular protein was similar in normotensive WKY rats and hypertensive SHRs. Hyperglycemia (blood glucose level, 17.8 +/- 2.9 mmol/L) led to an early twofold increase in TGF-beta RII protein expression and a fourfold increase in TGF-beta RI protein expression in the glomeruli of hypertensive diabetic SHRs compared with euglycemic SHRs (blood glucose level, 5.8 +/- 0.8 mmol/L), which was sustained after two weeks. Insulin treatment (blood glucose level, 5. 2 +/- 0.9 mmol/L) normalized both TGF-beta RII and TGF-beta RI expression in the glomeruli of SHRs that received streptozotocin. Glomerular capillary hypertension in the uninephrectomized SHRs led to a twofold increase in glomerular TGF-beta RII protein expression, but did not reproduce the effect of diabetes mellitus on TGF-beta RI expression. In contrast to the findings in SHRs, neither hyperglycemia (blood glucose level, 15.5 +/- 2.1 mmol/L), uninephrectomy, nor hyperglycemia (blood glucose level, 16.8 +/- 3.0 mmol/L) and uninephrectomy altered TGF-beta receptor expression in the glomeruli of normotensive WKY rats. CONCLUSION These studies support the hypothesis that hemodynamic factors and metabolic factors influence glomerular TGF-beta receptor in vivo in the SHRs.
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Affiliation(s)
- M J Kang
- Department of Medicine, University of Toronto, Toronto, Ontario, Canada
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48
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West J, Munoz-Antonia T, Johnson JG, Klotch D, Muro-Cacho CA. Transforming growth factor-beta type II receptors and smad proteins in follicular thyroid tumors. Laryngoscope 2000; 110:1323-7. [PMID: 10942134 DOI: 10.1097/00005537-200008000-00019] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
OBJECTIVE Resistance to transforming growth factor (TGF)-beta-mediated cell growth inhibition is a well-known pathogenic mechanism in epithelial neoplasia. TGF-beta signaling requires normal function of downstream mediators such as TGF-beta receptors (TbetaRs) and Smad proteins. The goal of this study is to investigate the expression of components of the TGF-beta signaling pathway in follicular tumors of the thyroid. STUDY DESIGN Twenty follicular thyroid neoplasms were classified as adenomas (11) or minimally invasive follicular carcinomas (9) according to current pathological criteria. Protein expression was evaluated to identify differences between benign and malignant tumors that could be used as an adjunct to histopathological analysis. METHODS Paraffin-embedded tissue sections containing tumor and adjacent nonneoplastic parenchyma were analyzed by immunohistochemistry for the expression of TbetaR type II (TbetaR-II) and Smad2, Smad4, Smad6, and Smad7. Expression of each protein in the tumor was compared with that of the corresponding adjacent nonneoplastic thyroid parenchyma. RESULTS TbetaR-II expression was lost in 78% of the carcinomas. In the remaining 22%, TbetaR-II was preserved but Smad2 expression was lost. In all conventional adenomas, however, TbetaR-II expression was maintained. Furthermore, all tumors with normal expression of all proteins were adenomas. CONCLUSIONS Downregulation of TbetaR-II is a consistent abnormality in follicular carcinomas and can be used to differentiate minimally invasive carcinomas from adenomas. Also, downregulation of Smad proteins is another mechanism by which carcinomas can become independent from TGF-beta-mediated growth inhibition.
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Affiliation(s)
- J West
- Department of Otolaryngology and Head and Neck Surgery, Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612-9297, USA
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49
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Morel AP, Unsal K, Cagatay T, Ponchel F, Carr B, Ozturk M. P53 but not p16INK4a induces growth arrest in retinoblastoma-deficient hepatocellular carcinoma cells. J Hepatol 2000; 33:254-65. [PMID: 10952243 DOI: 10.1016/s0168-8278(00)80366-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIM Both p16INK4a and p53 proteins are negative regulators of the cell cycle. In human hepatocellular carcinomas (HCC), the loss of function of p53, retinoblastoma (pRb) and pl6INK4a genes by different mechanisms has been largely documented, but their hepatocellular effects are poorly known. We compared the growth-inhibitory effects of p16INK4a and p53 proteins in Hep3B cell line-derived clones. METHODS Cells were transfected with inducible p16INK4a and p53 expression vectors, and stable clones were analyzed for transgene expression by Western blotting and immunoperoxidase staining. Effects on cell growth were analyzed by in vitro growth assay, thymidine incorporation and flow cytometry. Biochemical effects of p53 were tested by Northern blotting of p21Cip1 transcripts and by Western blotting of p21Cip1, mdm-2, bax, cyclin-dependent kinase 2 and cyclin E proteins. The pRb protein was studied by Western blotting and immunoprecipitation assays. RESULTS The induction of p16INK4a protein expression did not affect in vitro growth of cells. In contrast, p53 protein in its wild-type conformation provoked a growth arrest accompanied by transactivation of p21Cip1 gene and accumulation of p21Cip1, bax and mdm-2 proteins. p53-induced growth arrest was due to a cell cycle arrest at the G1/S transition, probably mediated by p21Cip1 protein, which inhibits cyclin-dependent kinase 2/cyclin E complexes. CONCLUSIONS The lack of detectable pRb protein and resistance of cells to p16TNK4a strongly suggest that p53 is able to arrest the growth of HCC cells by a mechanism independent of "p53-retinoblastoma pathway". These findings are applicable to HCC with abberrations of both p53 and pRb genes, and may not represent the universal effects of p53 in hepatic cells.
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Affiliation(s)
- A P Morel
- INSERM U453, Centre Leon Berard, Lyon, France
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50
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Fortunel N, Hatzfeld J, Kisselev S, Monier MN, Ducos K, Cardoso A, Batard P, Hatzfeld A. Release from quiescence of primitive human hematopoietic stem/progenitor cells by blocking their cell-surface TGF-beta type II receptor in a short-term in vitro assay. Stem Cells 2000; 18:102-11. [PMID: 10742382 DOI: 10.1634/stemcells.18-2-102] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Genetic alterations of the signaling cascade of transforming growth factor-beta (TGF-beta) are often associated with neoplastic transformation of primitive cells. This demonstrates the key role for this pleiotropic factor in the control of quiescence and cell proliferation in vivo. In the high proliferative potential-quiescent cell (HPP-Q) in vitro assay, the use of TGF-beta1 blocking antibodies (anti-TGF-beta1) allows the detection within two to three weeks of primitive hematopoietic cells called HPP-Q, which otherwise would not grow. However, the possibility of triggering cell proliferation by blocking the cell-surface TGF-beta receptors has not been investigated until now. We have tested here the efficiency of a blocking antibody against TGF-betaRII (anti-TGF-betaRII) on CD34(+)CD38(-) hematopoietic cells, a subpopulation enriched in primitive stem/progenitor cells, and compared its effect with that of anti-TGF-beta1. About twice as many HPP colony-forming cells were detected in the presence of anti-TGF-beta1 or anti-TGF-betaRII, compared to the control (p < 0.02). Moreover, anti-TGF-betaRII was as efficient as anti-TGF-beta1 for activating multipotent HPP-granulocyte erythroid macrophage megakaryocyte and HPP-Mix, bipotent HPP-granulocyte-macrophage (GM) and unipotent HPP-G, HPP-M and HPP-BFU-E. We therefore propose the use of anti-TGF-betaRII to release primitive cells from quiescence in the HPP-Q assay. This strategy could be extended to nonhematopoietic tissues, as TGF-beta1 may be a pleiotropic regulator of somatic stem cell quiescence.
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MESH Headings
- ADP-ribosyl Cyclase
- ADP-ribosyl Cyclase 1
- Activin Receptors, Type I
- Antigens, CD
- Antigens, CD34
- Antigens, Differentiation
- Cell Differentiation
- Cell Division
- Cloning, Molecular
- Hematopoietic Stem Cells/cytology
- Hematopoietic Stem Cells/metabolism
- Humans
- Membrane Glycoproteins
- NAD+ Nucleosidase
- Protein Serine-Threonine Kinases/genetics
- RNA, Messenger
- Receptor, Transforming Growth Factor-beta Type I
- Receptor, Transforming Growth Factor-beta Type II
- Receptors, Transforming Growth Factor beta/genetics
- Receptors, Transforming Growth Factor beta/immunology
- Receptors, Transforming Growth Factor beta/metabolism
- Time Factors
- Transforming Growth Factor beta/genetics
- Transforming Growth Factor beta/immunology
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Affiliation(s)
- N Fortunel
- Laboratoire de Biologie des Cellules Souches Somatiques Humaines, Centre National de la Recherche Scientifique, Villejuif, France
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