Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

et al. (2011) Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction. PLoS ONE 6(9): e24568. doi:10.1371/journal.pone.0024568 Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction Monika Kwiecinski 0 Andrea Noetel 0 Natalia Elfimova 0 Jonel Trebicka 0 Stephanie Schievenbusch 0 Ingo Strack 0 Levente Molnar 0 Melanie von Brandenstein 0 Ulrich To x 0 Roswitha Nischt 0 Oliver 0 Coutelle 0 Hans Peter Dienes 0 Margarete Odenthal 0 Costanza Emanueli, University of Bristol, United Kingdom 0 1 Institute for Pathology, University Hospital Cologne , Cologne, Germany , 2 Department of Internal Medicine I, University of Bonn , Bonn, Germany , 3 Department of Gastroenterology and Hepatology, University Hospital of Cologne , Cologne, Germany , 4 Department of Dermatology, University Hospital of Cologne , Cologne, Germany , 5 Department of Internal Medicine, University Hospital Cologne , Cologne , Germany Background: In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-b (TGF-b) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-b on the miR-29 collagen axis in HSC. Methodology: HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-b, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-b stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 39-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting. Principal Findings: The 39-UTR of the collagen-1 and 24 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-b stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-b exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-b stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis. Conclusions: Upregulation of miRNA-29 by HGF and downregulation by TGF-b take part in the anti- or profibrogenic response of HSC, respectively. - Funding: This study was partly supported by the Research and Education program of the Medical Faculty of the University Cologne and by the German Competence Network for Viral Hepatitis (HepNet), funded by the German Ministry of Education and Research (BMBF), Grant No 01KI0601 (to HPD / MO) and the German Liver Foundation. No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Progressive liver fibrosis due to chronic viral hepatitis, autoimmune, metabolic or hereditary disorders is a leading cause of morbidity and mortality in the Western world (reviewed in [1,2,3]). Regardless of the underlying etiology, liver fibrosis is characterized by an excessive deposition and reorganization of extracellular matrix (ECM) with a dramatic increase in noncollagenous and collagenous ECM proteins. The fibrillar collagen type I, is encoded by two different genes, col1A1 and col1A2, and accounts for 36% of the total collagens in ECM of healthy liver. During liver fibrogenesis, collagen type I is the predominant isoform deposited into the perisinusoidal space. However, collagen type IV, that constitutes less than 10% of total collagen in the normal liver, is most dramatically upregulated in fibrosis [4,5,6]. In the fibrotic liver, hepatic stellate cells (HSC) undergo myofibroblastic transdifferentiation. These myofibroblastic HSC are regarded as the main source of ECM production [1,3,7,8] although portal myofibroblasts, infiltrating fibroblasts and fibrocytes may also participate in the synthesis and restructuring of the connective tissue [9,10]. HSC get activated in response to chronic liver injury by proinflammatory and profibrogenic mediators such as transforming growth factor-b (TGF-b) [11,12] and plateletderived growth factor b [13,14]. TGF-b is recognized as the main profibrogenic mediator, triggering the myofibroblastic transition of HSC. Furthermore, it promotes the synthesis of ECM proteins, and inhibits expression and activity of matrix degrading enzymes in HSC [15]. TGF-b stimulated matrix production and deposition has been shown in a wide range of models of experimental fibrosis [16,17] and in patients with chronic hepatitis and cirrhosis [18,19,20]. Interestingly, there is good evidence for hepatic growth factor (HGF) opposing TGF-b signalling by reducing TGF-b mRNA levels [21]. HGF is a multifunctional cytokine that elicits mitogenic, motogenic, and morphogenic properties [22,23] by activation of the tyrosine kinase receptor Met, a product of the proto-oncogene c-met [24,25]. In addition, HGF is known to inhibit accumulation of extracellular matrix and development of hepatic fibrosis in vivo [26,27,28]. TGF-b can in turn dramatically suppress HGF mRNA expression in HSC, demonstrating the reciprocal effects of these cytokines on ECM accumulation [29]. The synthesis of extracellular matrix proteins is modulated by microRNA-29 (miR-29) in extrahepatic tissue [30,31,32,33]. Recent reports suggest that miR-29 is also involved in the synthesis of collagen type I in liver fibrosis [34,35]. The miR-29 family consists of miR-29a, miR-29b (b1, b2), and miR-29c, which differ in only two or three nucleotides, respectively. The genes for miR-29a and miR-29b1 are both located on chromosome 7, whereas the genes for miR-29c and miR-29b2 are located on chromosome 1. Each gene pair is transcribed in tandem resulting in a common pri-miRNA from which the mature miR-29 members are released after further processing [36,37]. In the present study, we investigate the role of the members of the miR-29 family in HGF mediated repression of collagen synthesis. We demonstrat (...truncated)