The effects of molecular target agents on hepatic fibrogenesis
Date Issued
2015
Date
2015
Author(s)
Su, Tung-Hung
Abstract
Chronic hepatitis B and C and their subsequent liver fibrosis progression, cirrhosis, hepatocellular carcinoma (HCC) and death are important health challenges. Repeated liver inflammation and hepatocyte damage are major pathogenesis for liver fibrosis progression and hepatocellular carcinogenesis. Currently, there are several antiviral agents against viral hepatitis B and C, and there is also one molecular target agent against HCC. However, effective antifibrotic therapy is unavailable, which remains an important unmet clinical need. The proliferation and activation of hepatic stellate cells (HSC) is the major contributor for hepatic fibrogenesis. A comprehensive understanding of the molecular mechanisms involved in HSC activation, perpetuation and collagen gene expression, will provide insight to develop antifibrotic therapy. The transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) are key mitogens for HSC proliferation and activation. The signal transducer and activator of transcription 3 (STAT3) is a transcription factor associated with liver injury, inflammation and regeneration. Src-homology protein tyrosine phosphatase-1 (SHP-1) regulates STAT3 and can directly interact with the PDGF receptor. These molecules are potential targets for antifibrotic therapy. Sorafenib is the only FDA-approved molecular target agent against advanced HCC by inhibition of the Raf kinase, the receptor tyrosine kinase, vascular endothelial growth factor receptor, and PDGF receptor. Sorafenib is found to have antifibrotic effect by reducing HSC proliferation and inducing apoptosis. In addition, sorafenib increases the ratio of matrix metalloproteinases to tissue inhibitor of matrix metalloproteinases and reduces collagen synthesis in HSC. Recent studies found sorafenib, and its derivative (SC-1), which lacks Raf kinase activity, all inhibit the STAT3 pathway. Another sorafenib derivative, SC-43, a SHP-1 agonist, can also inhibit the STAT3 pathway. The aim of this study is to investigate the role of SHP-1/STAT3 signaling pathway in fibrogenesis. The antifibrotic activity of sorafenib, SC-1 and SC-43 are examined and the molecular mechanisms are explored. Furthermore, the clinical implications of SHP-1/STAT3 in chronic hepatitis B (CHB) patients with liver fibrosis are studied. In this study, the antifibrotic activities of sorafenib, SC-1, and SC-43 are investigated in experimental murine liver fibrosis models by the induction of thioacetamide or carbon tetrachloride, or by bile duct ligation. We use the rat (HSC-T6) and human (LX2) HSC cell lines and mouse primary HSC for mechanistical investigation, including cell proliferation and apoptosis assays. The expression of STAT3 pathway related proteins are evaluated by western blot. The STAT3, or SHP-1 with its N-SH2 domain mutants are transfected to investigate their role for HSC proliferation and the mechanisms of sorafenib, SC-1 or SC-43. Among CHB patients who received liver biopsies at National Taiwan University Hospital, we randomly select 40 patients with various stage of liver fibrosis. Their hepatic phospho-STAT3 (p-STAT3), α-SMA, and SHP-1 expressions are correlated with the severity of fibrosis. In the first part of this study, after thioacetamide induction or bile duct ligation, sorafenib or SC-1 treatment significantly reduce hepatic fibrosis. In vitro studies show that both sorafenib and SC-1 have dose and time-dependent effects against cell viability and promote the apoptosis of HSCs. Both sorafenib and SC-1 downregulate p-STAT3 signaling pathway. The sorafenib- and SC-1-induced apoptosis are rescued in STAT3-overexpressing HSC cells. SHP-1 phosphatase is involved in downregulation of p-STAT3, and SHP-1 phosphatase inhibition can upregulate p-STAT3 and reverse sorafenib- or SC-1-induced HSC apoptosis. Both sorafenib and SC-1 may increase SHP-1 activity. CHB patients with advanced fibrosis have overexpressed p-STAT3. Finally, interleukin-6 (IL-6) is the main activating signal of STAT3 and we demonstrate IL-6 stimulation activates the STAT3 pathway in HSCs. We further find plasma IL-6 concentration significantly correlates with the severity of liver fibrosis and hepatic p-STAT3 level. In the second part of this study, we further investigate the role of SHP-1 phosphatase in fibrogenesis and the antifibrotic activity of SC-43, a SHP-1 agonist. In vitro studies show that SC-43 may promote the apoptosis of HSC and inhibit the TGF-β and PDGF receptor pathways. In addition, the inhibition of STAT3 is independent to the PDGF receptor inhibition. The enhanced SHP-1 activity significantly inhibits the proliferation of HSC; while knockdown SHP-1 may rescue the SC-43-induced apoptosis. After transfection of the N-SH2 domain mutants of SHP-1, the SC-43 cannot further increase SHP-1 activity or downregulate p-STAT3, and the pro-apoptotic effect of SC-43 is significantly abolished, indicating that SC-43 interacts with the N-SH2 domain of SHP-1 to enhance its SHP-1 phosphatase activity. In the carbon tetrachloride or the bile duct ligation fibrotic murine models, SC-43 effectively prevents and regresses the fibrogenesis, and promotes the mouse survival. Finally, the SHP-1 is overexpressed in the area of significant fibrosis, indicating that SHP-1 agonist may ameliorate fibrosis with tissue specificity. According to our study, we confirm that SHP-1/STAT3 is an important signaling pathway in fibrogenesis. Both SC-1 and SC-43 can regulate the SHP-1/STAT3 pathway to inhibit liver fibrogenesis effectively. STAT3 may be a fibrotic biomarker in CHB patients with active fibrogenesis, and SHP-1 phosphatase-directed antifibrotic therapy may represent a novel strategy for antifibrotic drug discovery.
Subjects
liver fibrosis
stellate cell
STAT3
sorafenib
SDGs
Type
thesis
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