HGF regulates the activation of TGF-β1 in rat hepatocytes and hepatic stellate cells.

Hepatocyte growth factor (HGF) ameliorates experimental liver fibrosis through many mechanisms, including degradation of accumulated collagen and decreased expression of fibrotic genes. Investigating an upstream mechanism in which HGF could decrease many fibrotic effectors, we asked whether HGF regulates activation of the fibrotic cytokine transforming growth factor-beta 1 (TGF-β1). Specifically, we tested whether HGF decreases the levels of active TGF-β1, and whether such decrease depends on the predominantly hepatocyte-secreted protease plasmin, and whether it depends on the TGF-β1 activator thrombospondin-1 (TSP-1). With hepatocyte monocultures, we found HGF-induced hepatocyte proliferation did increase total levels of plasmin, while decreasing gene expression of fibrotic markers (PAI-1, TGF-β1, and TIMP-2). With in vitro models of fibrotic liver (HSC-T6 hepatic stellate cells, or co-cultures of HSC-T6 and hepatocytes), we found high levels of fibrosis-associated proteins such as TSP-1, active TGF-β1, and Collagen I. HGF treatment on these fibrotic cultures stimulated plasmin levels; increased TSP-1 protein cleavage; and decreased the levels of active TGF-β1 and Collagen I. When plasmin was blocked by the inhibitor aprotinin, HGF could no longer decrease TGF-β1 activation and Collagen I. Meanwhile, the TSP-1-specific peptide inhibitor, LSKL, reduced TGF-β1 to the same level as in the HGF-treated cultures; combining LSKL and HGF treatments caused no further decrease, suggesting that HGF affects the TSP-1 dependent pathway of TGF-β1 activation. Therefore, HGF can decrease TGF-β1 activation and TGF-β1-dependent fibrotic markers, by stimulating hepatocytes to produce plasmin, and by antagonizing TSP-1-dependent activation of TGF-β1.

Narmada BC ，Chia SM ，Tucker-Kellogg L ，Yu H ... -  《-》

Puerarin, isolated from Pueraria lobata (Willd.), protects against hepatotoxicity via specific inhibition of the TGF-β1/Smad signaling pathway, thereby leading to anti-fibrotic effect.

Recently, the TGF-β1/Smad signaling pathway has been investigated in the pathogenesis of hepatofibrosis, and pharmacological treatment of liver fibrosis targeted this pathway to determine its contribution to the inhibition of fibrotic development. Importantly, ethnopharmacology-derived Pueraria lobata has been reported to effectively reverse the fibrotic process in the liver. In the present study, we performed dimethylnitrosamine (DMN)-induced liver fibrosis in rats to assess the benefits of puerarin (PR), which was isolated from Pueraria lobata (Willd.), on ECM-derived hepatocytes associated with the TGF-β1/Smad pathway. Our results showed that the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN), type III precollagen (PCIII) and type IV collagen (CIV) were significantly reduced by PR treatment, while hepatic homogenates showed decreased levels of hydroxyproline (Hyp) and collagen I (Col I). Masson's trichrome staining indicated that the DMN-induced liver fibrosis was alleviated. In addition, the protein expression levels of transforming growth factor-β l (TGF-β l), smad2, smad3, α-SMA and TIMP-1 were downregulated specifically by PR treatment, whereas the protein expression levels of smad7 and MMP-1 were upregulated. Furthermore, we evaluated the PR-mediated inhibitory effect on TGF-β1-treated proliferation and activation in a rat liver stellate cell line (HSC-T6). These data resulted in inhibition of the cell growth of HSC-T6 in a dose-dependent manner and a reduction in TβRI, smad2 and smad3 expressed proteins in the presence of PR on TGF-β1-treated HSC-T6 cells, while smad7 levels were downregulated. Taken together, these findings identify a unique effect for PR-regulation of the TGF-β1/Smad pathway in blocking fibrotic development and provide a promising strategy for hepatofibrosis treatment.

Xu L ，Zheng N ，He Q ，Li R ，Zhang K ，Liang T ... -  《-》

TGF-β1-elevated TRPM7 channel regulates collagen expression in hepatic stellate cells via TGF-β1/Smad pathway.

Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts plays a critical role in the development of liver fibrosis, since myofibroblasts are the key cells responsible for excessive deposition of ECM proteins. Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel with protein serine/threonine kinase activity, has been demonstrated to function in the proliferation of activated HSCs. Here, we investigated the functional role of TRPM7 in collagen deposition in activated HSC-T6 cells (a rat hepatic stellate cell line). TRPM7 mRNA and protein were measured by Real-time PCR and Western blot in TGF-β1-activated HSC-T6 cells in vitro. Results demonstrated that TRPM7 protein was dramatically increased in fibrotic human livers. Stimulation of HSC-T6 cells with TGF-β1 increased TRPM7 mRNA and protein level in a time-dependent manner. Nevertheless, TGF-β1-elicited upregulation of TRPM7 in HSC-T6 cells was abrogated by SB431542 (TGF-β1 receptor blocker) or SIS3 (inhibitor of Smad3 phosphorylation). Additionally, blockade of TRPM7 channels with non-specific TRPM7 blocker 2-APB or synthetic siRNA targeting TRPM7 attenuated TGF-β1-induced expression of myofibroblast markers, as measured by the induction of α-SMA and Col1α1. Silencing TRPM7 also increased the ratio of MMPs/TIMPs by increasing MMP-13 expression and decreasing TIMP-1 and TIMP-2 levels. Strikingly, phosphorylation of p-Smad2 and p-Smad3, associated with collagen production, was decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TGF-β1 elevates TRPM7 expression in HSCs via Smad3-dependant mechanisms, which in turn contributes Smad protein phosphorylation, and subsequently increases fibrous collagen expression. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis.

Fang L ，Huang C ，Meng X ，Wu B ，Ma T ，Liu X ，Zhu Q ，Zhan S ，Li J ... -  《-》

[Effects of bone morphogenic proteins-7 on production of collagen from hepatocytes and hepatic stellate cells during liver fibrosis].

Yang L ，Yang CQ ，Yuan M ，Wang SL ，Chang YZ ，Xu LJ ，Zhang ZJ ... -  《-》

A novel mechanism by which hepatocyte growth factor blocks tubular epithelial to mesenchymal transition.

Yang J ，Dai C ，Liu Y 《JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY》