Sirtuin 3 (SIRT3) Regulates α-Smooth Muscle Actin (α-SMA) Production through the Succinate Dehydrogenase-G Protein-coupled Receptor 91 (GPR91) Pathway in Hepatic Stellate Cells

Abstract BackgroundEchinococcus multilocularis is a causative agent of human alveolar echinococcosis (AE). AE leads to cirrhosis in several organs, such as the liver, triggering severe conditions, including hepatic failure and encephalopathy. The main purpose of this study is to explore the interaction between treated hepatic stellate cells and AE-protoscoleces (AE-PSCs). The results of this study will be provided experimental basis for revealing the mechanisms of hepatic fibrosis after AE infection.MethodsWe investigated the role of alveolar echinococcosis-protoscoleces (AE-PSCs) in liver fibrosis and structural changes and liver fibrosis-related protein expression in a coculture of PSCs and human hepatic stellate cells (HSCs). Structural changes were detected by transmission electron microscopy, whereas liver fibrosis-related proteins, collagen I, alpha-smooth muscle actin, and osteopontin levels were measured by western blotting and enzyme-linked immunosorbent assay. ResultsPSCs exhibited morphological changes, specifically changes in shape, and showed slight changes in the cytoplasmic membrane, whereas structural modifications were observed in HSCs. Additionally, western blotting and enzyme-linked immunosorbent assay revealed that PSCs treated in vitro with HSC-LX2 showed significantly increased collagen-Ⅰ, α-smooth muscle actin, and osteopontin expression levels after 3–4 days of incubation in a coculture system. AE-PSCs induced liver fibrosis by inducing extracellular matrix expression and HSC activation.ConclusionsThese results provide insight into the pathogenesis of echinococcosis- induced hepatic fibrosis and introduce therapeutic targets and diagnostic criteria for managing echinococcosis-dependent liver fibrosis.

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c-Myb modulates transcription of the α-smooth muscle actin gene in activated hepatic stellate cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2000.278.2.g321 ◽

2000 ◽

Vol 278 (2) ◽

pp. G321-G328 ◽

Cited By ~ 27

Author(s):

Martina Buck ◽

Dong Joon Kim ◽

Karl Houglum ◽

Tarek Hassanein ◽

Mario Chojkier

Keyword(s):

Smooth Muscle ◽

Hepatic Stellate Cells ◽

Cell Activation ◽

Stellate Cell ◽

Smooth Muscle Actin ◽

Stellate Cells ◽

Dominant Negative ◽

Muscle Actin ◽

Muscle Actin Gene ◽

High Level

Expression of α-smooth muscle actin (α-SMA) defines the phenotype of activated (myofibroblastic) hepatic stellate cells. These cells, but not quiescent stellate cells, have a high level of α-SMA and c-Myb expression, as well as increased c-Myb-binding activities to the proximal α-SMA E box. Therefore, we analyzed the role of c-Myb in α-SMA transcription and stellate cell activation. Activated primary rat stellate cells displayed a high expression of the −724 and −271 α-SMA/luciferase (LUC) chimeric genes, which contain c-Myb binding sites (−223/−216 bp). α-SMA/LUC minigenes with mutation (−219/−217 bp), truncation (−224 bp), or deletion (−191 bp) of the c-Myb binding site were not efficiently transcribed. Transfection of wild-type c-Myb into quiescent stellate cells, which do not express endogenous c-Myb, induced a ∼10-fold stimulation of −724 α-SMA/LUC expression. Conversely, expression of either a dominant-negative c-Myb basic domain mutant (Cys43 → Asp) or a c-Myb antisense RNA blocked transcription from the −724 α-SMA/LUC or −271 α-SMA/LUC in activated cells. Moreover, transfection of c- myb antisense, but not sense, RNA inhibited both expression of the endogenous α-SMA gene and stellate cell activation, whereas transfection of c- myb stimulated α-SMA expression in quiescent stellate cells. These findings suggest that c-Myb modulates the activation of stellate cells and that integrity of the redox sensor Cys43in c-Myb is required for this effect.

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De novo expression of glutamine synthetase during transformation of hepatic stellate cells into myofibroblast-like cells

Biochemical Journal ◽

10.1042/bj3350697 ◽

1998 ◽

Vol 335 (3) ◽

pp. 697-700 ◽

Cited By ~ 19

Author(s):

Johannes G. BODE ◽

Thorsten PETERS-REGEHR ◽

Axel M. GRESSNER ◽

Dieter HÄUSSINGER

Keyword(s):

Enzyme Activity ◽

Smooth Muscle ◽

Glutamine Synthetase ◽

Western Blot ◽

Hepatic Stellate Cells ◽

De Novo ◽

Smooth Muscle Actin ◽

Stellate Cells ◽

Cell Protein ◽

Muscle Actin

The expression of glutamine synthetase (GS) was studied in cultured quiescent hepatic stellate cells (HSC) and during their transformation into myofibroblast-like cells. GS mRNA was detectable in quiescent HSC (1-day culture); however, the enzyme protein was not expressed, as assessed by Western blot analysis, immunocytochemistry and the absence of detectable enzyme activity. Similar findings were obtained after 2 days of culture; in addition, the mRNA levels had dropped by about 70%, but they increased again thereafter during the process of HSC transformation in culture, as indicated by the expression of α-smooth-muscle actin. In parallel with the accumulation of α-smooth-muscle actin, GS was expressed, as shown by Western blot analysis and immunocytochemistry, and enzyme activity increased from undetectable levels in quiescent cells to 0.13±0.01 µmol/h per mg of cell protein within 7–14 days. This value compares with GS activity in liver parenchymal cells of 0.57±0.03 µmol/h per mg of cell protein. The findings suggest that activation of HSC results in the de novo expression of GS protein and activity, and this may serve as another marker of HSC transformation.

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Dynamics of Sept4 expression in fibrotic livers of mice infected with Schistosoma japonicum

Parasitology ◽

10.1017/s0031182011000667 ◽

2011 ◽

Vol 138 (8) ◽

pp. 1003-1010 ◽

Cited By ~ 10

Author(s):

Y. N. DUAN ◽

H. Y. QIAN ◽

Y. W. QIN ◽

D. D. ZHU ◽

X. X. HE ◽

...

Keyword(s):

Smooth Muscle ◽

Liver Fibrosis ◽

Mouse Model ◽

Schistosoma Japonicum ◽

Hepatic Stellate Cells ◽

Immunohistochemical Staining ◽

Smooth Muscle Actin ◽

Stellate Cells ◽

Muscle Actin ◽

Qrt Pcr

SUMMARYIn order to investigate the dynamics of Septin4 (Sept4) expression and its function in the formation of fibrotic livers in mice infected with Schistosoma japonicum, we constructed the mouse model of S. japonicum egg-induced liver fibrosis for 24 weeks. Immunohistochemical staining, qRT-PCR and Western blot were used to detect the expression of Sept4 and α-smooth muscle actin (α-SMA). We found Sept4 localized in the perisinusoidal space where hepatic stellate cells (HSCs) distribute in the periphery of circumoval granulomas and the portal venule. The expression of Sept4 and α-SMA had a similar significant tendency of an up-regulation to a peak at 12 weeks post-infection (p.i.) followed by a down-regulation. At 24 weeks p.i. both were at a low level. These results suggest that Sept4 and α-SMA may interact together in HSCs. Based on this evidence, we hypothesize that Sept4 seems to be involved in the formation of inflammatory granulomata and subsequent liver fibrosis by regulating HSCs activation.

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Increased contractility of hepatic stellate cells in cirrhosis is mediated by enhanced Ca2+-dependent and Ca2+-sensitization pathways

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00350.2010 ◽

2011 ◽

Vol 300 (6) ◽

pp. G1010-G1021 ◽

Cited By ~ 27

Author(s):

Masateru Iizuka ◽

Takahisa Murata ◽

Masatoshi Hori ◽

Hiroshi Ozaki

Keyword(s):

Smooth Muscle ◽

Hepatic Stellate Cells ◽

Smooth Muscle Actin ◽

Stellate Cells ◽

Regulatory Proteins ◽

Muscle Actin ◽

Rock Inhibitor ◽

Duct Ligation

Activation of hepatic stellate cells (HSCs) results in cirrhosis and portal hypertension due to intrahepatic resistance. Activated HSCs increase their contraction after receptor agonist stimulation; however, the signaling pathways for the regulation of contraction are not fully understood. The aim of this study was to elucidate the change in contractile mechanisms of HSCs after cirrhotic activation. The expression pattern of contractile regulatory proteins was analyzed with quantitative RT-PCR and Western blotting. The phosphorylation levels of myosin light chain (MLC), 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), and MLC phosphatase targeting subunit 1 (MYPT1) after endothelin-1 (ET-1) stimulation in culture-activated HSCs were measured using phosphorylation-specific antibodies. In vivo-activated HSCs were isolated from rats subjected to bile duct ligation and repeated dimethylnitrosoamine injections. HSCs showed increased expression of not only α-smooth muscle actin, but also the contractile regulatory proteins MLC kinase (MLCK), Rho kinase 2 (ROCK2), and CPI-17 during HSC activation in vitro. In culture-activated HSCs, ET-1 increased phosphorylation of CPI-17 at Thr18, which was markedly inhibited by the PKC inhibitor Ro-31–8425. ET-1 induced phosphorylation of MYPT1 at Thr853, which was suppressed by the ROCK inhibitor Y-27632. ET-1 induced sustained phosphorylation of MLC at Thr18/Ser19, which was inhibited by both Ro-31–8425 and Y-27632. Consistent with the data obtained from the in vitro study, HSCs isolated from cirrhotic rats showed increased expression of α-smooth muscle actin, MLCK, CPI-17, and ROCK2 compared with HSCs from nontreated rats. Furthermore, MLC phosphorylation in in vivo-activated HSCs was increased, according to enhanced phosphorylation of CPI-17 and MYPT1 in the presence of ET-1. These results suggest that activated HSCs may participate in constriction of hepatic sinusoids in the cirrhotic liver through both Ca2+-dependent (MLCK pathway) and Ca2+-sensitization mechanism (CPI-17 and MYPT1 pathways).

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