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Hepatic stellate cell (HSC) activation plays an important role in the pathogenesis of liver fibrosis, and epithelial-mesenchymal transition (EMT) is suggested to potentially promote HSC activation. Superoxide dismutase 3 (SOD3) is an extracellular antioxidant defense against oxidative damage. Here, we found downregulation of SOD3 in a mouse model of liver fibrosis induced by carbon tetrachloride (CCl 4 ). SOD3 deficiency induced spontaneous liver injury and fibrosis with increased collagen deposition, and further aggravated CCl 4 -induced liver injury in mice. Depletion of SOD3 enhanced HSC activation marked by increased α-smooth muscle actin and subsequent collagen synthesis primarily collagen type I in vivo, and promoted transforming growth factor-β1 (TGF-β1)-induced HSC activation in vitro. SOD3 deficiency accelerated EMT process in the liver and TGF-β1-induced EMT of AML12 hepatocytes, as evidenced by loss of E-cadherin and gain of N-cadherin and vimentin. Notably, SOD3 expression and its pro-fibrogenic effect were positively associated with sirtuin 1 (SIRT1) expression. SOD3 deficiency inhibited adenosine monophosphate-activated protein kinase (AMPK) signaling to downregulate SIRT1 expression and thus involving in liver fibrosis. Enforced expression of SIRT1 inhibited SOD3 deficiency-induced HSC activation and EMT, whereas depletion of SIRT1 counteracted the inhibitory effect of SOD3 in vitro. These findings demonstrate that SOD3 deficiency contributes to liver fibrogenesis by promoting HSC activation and EMT process, and suggest a possibility that SOD3 may function through modulating SIRT1 via the AMPK pathway in liver fibrosis., (© 2020 Wiley Periodicals LLC.)

Myofibroblastic trans-differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S-adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATβ proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up-regulation during trans-differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post-translational modifications in the MATα2 and MATβ proteins that stabilize them and favor human HSC trans-differentiation. Culture-activation of human HSCs induced the MATα2 and MATβ proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATβ predicted to be phosphorylated by mitogen-activated protein kinase (MAPK) family members (ERK1/2, V-Raf Murine Sarcoma Viral Oncogene Homolog B1 [B-Raf], MEK). Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B-Raf lowered the phosphorylation and stability of MATβ but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATβ phospho-sites destabilized them and prevented HSC trans-differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans-differentiation., (© 2014 Wiley Periodicals, Inc.)

β-Arrestins are multifunctional adaptor proteins. Recently, some new roles of β-arrestins in regulating intracellular signaling networks have been discovered, which regulate cell growth, proliferation, and apoptosis. Though, the role of β-arrestins expression in the pathology of hepatic fibrosis remains unclear. In this study, the possible relationship between the expression of β-arrestins with the experimental hepatic fibrosis and the proliferation of hepatic stellate cells (HSCs) were investigated. Porcine serum induced liver fibrosis was established in this study. At five time points, the dynamic expression of β-arrestin1, β-arrestin2, and α-smooth muscle actin (α-SMA) in rat liver tissues, was measured by immunohistochemical staining, double immunofluorescent staining, and Western blotting. This study showed that aggravation of hepatic fibrosis with gradually increasing expression of β-arrestin2 in the hepatic tissues, but not β-arrestin1. Further, as hepatic fibrosis worsens, β-arrestin2-expressing activated HSCs accounts for an increasingly larger percentage of all activated HSCs. And the expression of β-arrestin2 had a significant positive correlation with the expression of α-SMA, an activated HSCs marker. In vitro studies, the dynamic expression of β-arrestin1 and β-arrestin2 in platelet derived growth factor-BB (PDGF-BB) stimulated HSCs was assessed by Western blotting. The expression of β-arrestin2 was remarkably increased in PDGF-BB stimulated HSCs. Furthermore, the small interfering RNA (siRNA) technique was used to explore the effect of β-arrestins on the proliferation of HSCs and the activation of ERK1/2. Transfection of siRNA targeting β-arrestin2 mRNA (siβ-arrestin2) into HSCs led to a 68% and 70% reduction of β-arrestin2 mRNA and protein expression, respectively. siβ-arrestin2 abolished the effect of PDGF-BB on the proliferation of HSCs. In addition, siβ-arrestin2 exerted the inhibition of the activation of ERK1/2 in HSCs. The present study provided strong evidence for the participation of the β-arrestin2 in the pathogenesis of hepatic fibrosis. The β-arrestin2 depletion diminishes HSCs ERK1/2 signaling and proliferation stimulated by PDGF-BB. Selective targeting of β-arrestin2 inhibitors to HSCs might present as a novel strategy for the treatment of hepatic fibrosis., (Copyright © 2012 Wiley Periodicals, Inc.)

Liver fibrosis is a common consequence of various chronic liver injuries, including virus infection and ethanol. Activated hepatic stellate cells (HSCs) contribute to liver fibrosis through the accumulation of extracellular matrix proteins, including type I alpha collagen (COL1A). The activation of adenosine monophosphate-activated protein kinase (AMPK) modulates HSCs activation, but its underlying mechanism remains unclear. Here, we report that AMPK inhibits transforming growth factor (TGF)-β-induced fibrogenic property of HSCs by regulating transcriptional coactivator p300. We treated human (LX-2) and rat (CFSC-2G) HSC lines with TGF-β to induce fibrogenic activation of HSCs. Pharmacological activation of AMPK by treatment with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), metformin, or adiponectin lowered TGF-β-induced expression of COL1A and myofibroblast marker alpha-smooth muscle actin (α-SMA). Transient transduction of constitutively active AMPKα (caAMPKα) was sufficient to attenuate COL1A and α-SMA expression, whereas an AMPK inhibitor considerably abrogated the inhibitory effect of AICAR on fibrogenic gene expression. Although AMPK significantly suppressed Smad-dependent transcription, it did not affect TGF-β-stimulated phosphorylation, nuclear localization, or DNA-binding activity of Smad2/3. AICAR rather attenuated TGF-β-induced Smad3 interaction with transcriptional coactivator p300 accompanying with reduction of Smad3 acetylation. Moreover, AICAR induced not only physical interaction between AMPK and p300 but also proteasomal degradation of p300 protein. Our data provide substantial evidence that AMPK could be a novel therapeutic target for treatment of liver fibrosis, by demonstrating the underlying mechanism of AMPK-induced antifibrotic function in HSCs., (Copyright © 2011 Wiley Periodicals, Inc.)

The biological roles of sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) have been broadly investigated. However, at present pathophysiological roles of S1P/S1PRs axis in liver fibrosis are not well defined. Here, we investigated the functions of S1P/S1PRs axis in human hepatic stellate cells (HSC) line, LX-2 cells. We found that S1PR types 1, 2 and 3 (S1PR1-3) are clearly detected in LX-2 cells, as determined by RT-PCR, Western blot and immunocytochemistry analysis. S1P exerted a powerful migratory action on LX-2 cells, as determined in Boyden chambers, and stimulated fibrogenic activity of LX-2 cells, as demonstrated by increase of expression of smooth muscle α-actin, procollagen α1(I) and α1(III) and total hydroxyproline content. Moreover, the effects of S1P were mimicked by S1PR1 agonist SEW2871, and abrogated by W146 (S1PR1 antagonist) and/or silencing S1PR1, three expression with small interfering RNA, suggesting the main roles of S1PR1 and 3. However, studies with S1PR2 antagonist JTE-013 and silencing S1PR2 expression indicated that S1PR2 negatively regulated S1P-induced cell migration. Interestingly, exogenously added S1P induced significant up-regulation of sphingosine kinase-1 and the synthesis of additional S1P, and expression of S1PR1,3, but not S1PR2. In conclusion, our data have identified an additional function regulated by S1P/S1PR1,3 axis involving migration and fibrogenic activation of HSCs. These results suggest that selective modulation of S1PR activity may represent a new antifibrotic strategy., (Copyright © 2010 Wiley-Liss, Inc.)