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G?12 overexpression induced by miR-16 dysregulation contributes to liver fibrosis by promoting autophagy in hepatic stellate cells.

ABSTRACT: BACKGROUND & AIMS:Hepatic stellate cells (HSCs) have a role in liver fibrosis. Guanine nucleotide-binding ?-subunit 12 (G?12) converges signals from G-protein-coupled receptors whose ligand levels are elevated in the environment during liver fibrosis; however, information is lacking on the effect of G?12 on HSC trans-differentiation. This study investigated the expression of G?12 in HSCs and the molecular basis of the effects of its expression on liver fibrosis. METHODS:G?12 expression was assessed by immunostaining, and immunoblot analyses of mouse fibrotic liver tissues and primary HSCs. The role of G?12 in liver fibrosis was estimated using a toxicant injury mouse model with G?12 gene knockout and/or HSC-specific G?12 delivery using lentiviral vectors, in addition to primary HSCs and LX-2 cells using microRNA (miR) inhibitors, overexpression vectors, or adenoviruses. miR-16, G?12, and LC3 were also examined in samples from patients with fibrosis. RESULTS:G?12 was overexpressed in activated HSCs and fibrotic liver, and was colocalised with desmin. In a carbon tetrachloride-induced fibrosis mouse model, G?12 ablation prevented increases in fibrosis and liver injury. This effect was attenuated by HSC-specific lentiviral delivery of G?12. Moreover, G?12 activation promoted autophagy accompanying c-Jun N-terminal kinase-dependent ATG12-5 conjugation. In addition, miR-16 was found to be a direct inhibitor of the de novo synthesis of G?12. Modulations of miR-16 altered autophagy in HSCs. In a fibrosis animal model or patients with severe fibrosis, miR-16 levels were lower than in their corresponding controls. Consistently, cirrhotic patient liver tissues showed G?12 and LC3 upregulation in desmin-positive areas. CONCLUSIONS:miR-16 dysregulation in HSCs results in G?12 overexpression, which activates HSCs by facilitating autophagy through ATG12-5 formation. This suggests that G?12 and its regulatory molecules could serve as targets for the amelioration of liver fibrosis. LAY SUMMARY:Guanine nucleotide-binding ?-subunit 12 (G?12) is upregulated in activated hepatic stellate cells (HSCs) as a consequence of the dysregulation of a specific microRNA that is abundant in HSCs, facilitating the progression of liver fibrosis. This event is mediated by c-Jun N-terminal kinase-dependent ATG12-5 formation and the promotion of autophagy. We suggest that G?12 and its associated regulators could serve as new targets in HSCs for the treatment of liver fibrosis.

SUBMITTER: Kim KM

PROVIDER: S-EPMC5818314 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Gα<sub>12</sub> overexpression induced by miR-16 dysregulation contributes to liver fibrosis by promoting autophagy in hepatic stellate cells.

Kim Kyu Min KM Han Chang Yeob CY Kim Ji Young JY Cho Sam Seok SS Kim Yun Seok YS Koo Ja Hyun JH Lee Jung Min JM Lim Sung Chul SC Kang Keon Wook KW Kim Jae-Sung JS Hwang Se Jin SJ Ki Sung Hwan SH Kim Sang Geon SG

Journal of hepatology 20180102 3

<h4>Background & aims</h4>Hepatic stellate cells (HSCs) have a role in liver fibrosis. Guanine nucleotide-binding α-subunit 12 (Gα<sub>12</sub>) converges signals from G-protein-coupled receptors whose ligand levels are elevated in the environment during liver fibrosis; however, information is lacking on the effect of Gα<sub>12</sub> on HSC trans-differentiation. This study investigated the expression of Gα<sub>12</sub> in HSCs and the molecular basis of the effects of its expression on liver fi ...[more]

PMID: 29080810

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Project description:Background & aimsAutophagy plays a crucial role in hepatic homeostasis and its deregulation has been associated with chronic liver disease. However, the effect of autophagy on the release of fibrogenic extracellular vesicles (EVs) by platelet-derived growth factor (PDGF)-stimulated hepatic stellate cells (HSCs) remains unknown. Herein, we aimed to elucidate the role of autophagy, specifically relating to fibrogenic EV release, in fibrosis.MethodsIn vitro experiments were conducted in primary human and murine HSCs as well as LX2 cells. Small EVs were purified by differential ultracentrifugation. Carbon tetrachloride (CCl4) or bile duct ligation (BDL) were used to induce fibrosis in our mouse model. Liver lysates from patients with cirrhosis or healthy controls were compared by RNA sequencing.ResultsIn vitro, PDGF and its downstream molecule SHP2 (Src homology 2-containing protein tyrosine phosphatase 2) inhibited autophagy and increased HSC-derived EV release. We used this PDGF/SHP2 model to further investigate how autophagy affects fibrogenic EV release. RNA sequencing identified an mTOR (mammalian target of rapamycin) signaling molecule that was regulated by SHP2 and PDGF. Disruption of mTOR signaling abolished PDGF-dependent EV release. Activation of mTOR signaling induced the release of multivesicular body-derived exosomes (by inhibiting autophagy) and microvesicles (by activating ROCK1 signaling). These mTOR-dependent EVs promoted in vitro HSC migration. To assess the importance of this mechanism in vivo, SHP2 was selectively deleted in HSCs, which attenuated CCl4- or BDL-induced liver fibrosis. Furthermore, in the CCl4 model, mice receiving circulating EVs derived from mice with HSC-specific Shp2 deletion had less fibrosis than mice receiving EVs from control mice. Correspondingly, SHP2 was upregulated in patients with liver cirrhosis.ConclusionThese results demonstrate that autophagy in HSCs attenuates liver fibrosis by inhibiting the release of fibrogenic EVs.Lay summaryDuring liver fibrosis and cirrhosis, activated hepatic stellate cells (HSCs) are the key cell type responsible for fibrotic tissue deposition. Recently, we demonstrated that activated HSCs release nano-sized vesicles enriched with fibrogenic proteins. In the current study, we unveil the mechanism by which these fibrogenic vesicles are released, moving a step closer to the long-term goal of therapeutically targeting this process.

Project description:IntroductionAutophagy is an intracellular catabolic process that removes and recycles unnecessary/dysfunctional cellular components, contributing to cellular health and survival. Autophagy is a highly regulated cellular process that responds to several intracellular signals, many of which are deregulated by human papillomavirus (HPV) infection through the expression of HPV-encoded oncoproteins. This adaptive inhibitory response helps prevent viral clearance. A strong correlation remains between HPV infection and the development of squamous cell carcinoma (SCC) of the anus, particularly in HIV positive and other immunosuppressed patients. We hypothesize that autophagy is inhibited by HPV-encoded oncoproteins thereby promoting anal carcinogenesis (Fig 1).Materials and methodsHPV16 transgenic mice (K14E6/E7) and non-transgenic mice (FVB/N), both of which do not spontaneously develop anal tumors, were treated topically with the chemical carcinogen, 7,12-Dimethylbenz[a]anthracene (DMBA), to induce anal cancer. The anuses at different time points of treatment (5, 10, 15 and 20 weeks) were analyzed using immunofluorescence (IF) for two key autophagy marker proteins (LC3β and p62) in addition to histological grading. The anuses from the K14E6/E7 mice were also analyzed for visual evidence of autophagic activity by electron microscopy (EM). To see if there was a correlation to humans, archival anal specimens were assessed histologically for grade of dysplasia and then analyzed for LC3β and p62 protein content. To more directly examine the effect of autophagic inhibition on anal carcinogenesis, nontransgenic mice that do not develop anal cancer with DMBA treatment were treated with a known pharmacologic inhibitor of autophagy, chloroquine, and examined for tumor development and analyzed by IF for autophagic proteins.ResultsHistologically, we observed the progression of normal anoderm to invasive SCC with DMBA treatment in K14E6/E7 mice but not in nontransgenic, syngeneic FVB/N background control mice. With the development of low-grade dysplasia in the K14E6/E7 mice, there was an increase in both punctate LC3β and p62 expression while EM revealed increased autophagosomes without evidence of autophagolysosomes. These observations are consistent with autophagy being inhibited at a later stage in the autophagic process. In contrast, in high-grade dysplasia and SCC in the DMBA-treated K14E6/E7 mice, there were decreased levels of p62 with a continued increase in punctate LC3β expression by IF, while autophagolysosomes were seen on EM, consistent with the process of autophagy proceeded to completion. Similar findings, including histological grade dependent changes in LC3β and p62 expression, were noted with human samples upon analysis of IF. Finally, with pharmacologic inhibition of autophagy in DMBA-treated, nontrangenic FVB/N mice, there was a significant increase in anal cancer development similar to that observed in DMBA- treated K14E6/E7 mice.ConclusionAutophagic dysregulation is noted early on in HPV-associated anal carcinogenesis (low-grade dysplasia), with normalization of the autophagic process arising in late stages of HPV-associated anal carcinogenesis (high-grade dysplasia and invasive carcinoma).

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G?12 overexpression induced by miR-16 dysregulation contributes to liver fibrosis by promoting autophagy in hepatic stellate cells.

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Gα<sub>12</sub> overexpression induced by miR-16 dysregulation contributes to liver fibrosis by promoting autophagy in hepatic stellate cells.

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