Elsevier

Journal of Hepatology

Volume 68, Issue 3, March 2018, Pages 493-504
Journal of Hepatology

Research Article
12 overexpression induced by miR-16 dysregulation contributes to liver fibrosis by promoting autophagy in hepatic stellate cells

https://doi.org/10.1016/j.jhep.2017.10.011Get rights and content

Highlights

  • 12 expression was enhanced during HSC activation due to miR-16 dysregulation.

  • 12 signaling facilitates autophagy via JNK-dependent ATG12-5 formation.

  • 12 associated molecules in HSCs may serve as targets for fibrosis treatment.

Background & Aims

Hepatic stellate cells (HSCs) have a role in liver fibrosis. Guanine nucleotide-binding α-subunit 12 (Gα12) 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 Gα12 on HSC trans-differentiation. This study investigated the expression of Gα12 in HSCs and the molecular basis of the effects of its expression on liver fibrosis.

Methods

12 expression was assessed by immunostaining, and immunoblot analyses of mouse fibrotic liver tissues and primary HSCs. The role of Gα12 in liver fibrosis was estimated using a toxicant injury mouse model with 12 gene knockout and/or HSC-specific Gα12 delivery using lentiviral vectors, in addition to primary HSCs and LX-2 cells using microRNA (miR) inhibitors, overexpression vectors, or adenoviruses. miR-16, Gα12, and LC3 were also examined in samples from patients with fibrosis.

Results

12 was overexpressed in activated HSCs and fibrotic liver, and was colocalised with desmin. In a carbon tetrachloride-induced fibrosis mouse model, Gα12 ablation prevented increases in fibrosis and liver injury. This effect was attenuated by HSC-specific lentiviral delivery of Gα12. Moreover, Gα12 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 Gα12. 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 Gα12 and LC3 upregulation in desmin-positive areas.

Conclusions

miR-16 dysregulation in HSCs results in Gα12 overexpression, which activates HSCs by facilitating autophagy through ATG12-5 formation. This suggests that Gα12 and its regulatory molecules could serve as targets for the amelioration of liver fibrosis.

Lay summary

Guanine nucleotide-binding α-subunit 12 (Gα12) 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 Gα12 and its associated regulators could serve as new targets in HSCs for the treatment of liver fibrosis.

Introduction

Liver fibrosis is a scarring response promoting the deposition of extracellular matrix (ECM) components against hepatocyte injury.1 ECM deposition disturbs normal liver function and architecture and can progress to liver cirrhosis when left untreated. When exposed to various external stimuli during chronic injury, hepatic stellate cells (HSCs) are primarily responsible for the disproportionate fibre accumulation that leads to liver fibrosis. Additionally, HSCs cause ECM deposition through trans-differentiation from a quiescent phenotype to a myofibroblastic phenotype.2 However, there is currently no clinically effective way to inhibit HSC activation and fibrosis progression;3 therefore, identification of the regulatory molecules and mechanisms involved in this process could assist in the implementation of new diagnostic and therapeutic targets related to liver fibrosis.

Given their roles in complex biological processes, increased attention has been paid to G-protein-coupled receptors (GPCRs), which recognise extracellular ligands, and their related downstream molecules and pathways.4 GPCR activation and subsequent downstream activity is promoted by guanine nucleotide-binding α-subunits (Gα) proteins. Among these, Gα12 has attracted particular interest owing to its potent transforming capacity. Specifically, Gα12 transduces signals to control cell proliferation, migration, and inflammation.[5], [6] Additionally, ligands that promote specific GPCR coupling to Gα12 [i.e. sphingosine-1-phosphate (S1P), lysophosphatidic acid, angiotensin 2 (Ang II), thrombin, and endothelin-1] are implicated in liver fibrosis.[7], [8] Given that Gα12 converges signals from multiple GPCRs and coordinates GPCR-specific signalling cascades to common downstream molecules,912 levels could have a profound effect on the blunting or amplification of biochemical and physiological activities.

Autophagy is an essential catabolic process involved in lysosomal degradation and recycling of intracellular organelles and proteins to maintain cellular homeostasis. In HSCs, autophagy increases the fibrogenic response through the breakdown of lipid droplets.10 Autophagy is orchestrated by events and proteins, including microtubule-associated protein 1 light chain 3 (LC3), and autophagy-related gene (ATG)12-5 and ATG16, with LC3-II being necessary for membrane extension and closure to form vesicles.11 In HSCs, autophagy is activated by many ligands of GPCRs that interact with Gα12;[12], [13] therefore, we hypothesised that Gα12 could affect autophagy.

Given the lack of understanding of the role of Gα12 in HSC trans-differentiation and the potential link between Gα12 and autophagy, this study investigated whether Gα12 is upregulated in HSCs and, if so, what its impact is on liver fibrosis and how it is regulated in conjunction with autophagy. Our results showed that Gα12 is overexpressed in activated HSCs because of the dysregulation of a specific microRNA (miR) that is abundant in HSCs, facilitating liver fibrosis. In addition, HSC-specific delivery of Gα12 to whole-body 12 knockout (KO) mice or wild-type (WT) mice using two different sets of constructs confirmed the role of Gα12 overexpression in HSCs in the progression of fibrosis. Moreover, our findings indicated that this event was mediated by c-Jun N-terminal kinase (JNK)-dependent ATG12-5 formation and the induction of autophagy.

Section snippets

Materials

Information on the materials used in this study is provided in the Supplementary Material online.

Animal treatment

The protocol for the animal experiments was approved by the Institutional Animal Use and Care Committee at Seoul National University, and followed ARRIVE guidelines. Detailed information is provided in the Supplementary material online.

Targeted gene delivery

Lentiviral SMA-Gα12 and SMA-Gα12-enhanced green fluorescence protein (EGFP) were cloned, and the viruses produced were administered to C57BL/6 mice via tail vein for

12 is overexpressed in HSCs and fibrotic liver

We analysed Gα-isoform levels in different types of liver-resident cell using the proteome data from whole liver and individual hepatic cell types.14 Of the proteins detected in whole-liver lysates, Gα12 showed the highest signal intensities specifically in HSCs (Fig. 1A, left). We compared Gα12 levels in primary cell types isolated from the liver of healthy mice, and found that Gα12 was more abundant in HSCs than in hepatocytes. In addition, hepatocytes expressed Gα12 to a greater extent than

Discussion

HSC activation is mediated by extracellular ligands.[21], [22] In addition, GPCR-signalling pathways have a profound effect on cell behaviour during fibrogenesis. Levels of GPCR ligands are elevated in fibrotic livers compared with healthy controls, and most GPCRs activated by ligands are coupled with Gα12.[7], [8] Despite the known effects of Gα12 on cell proliferation and migration,5 the basis for Gα12 signalling in liver fibrosis has remained unknown. Our results demonstrated that Gα12 was

Financial support

This work was supported by National Research Foundation (NRF) grants funded by the Government of South Korea (MSIP) (NRF-2015R1A2A1A10052663) and the NRF-JSPS program.

Conflict of interest

The authors declare that they have no conflict of interest to report. Please refer to the accompanying ICMJE disclosure forms for further details.

Authors’ contributions

KMK, CYH, JYK, SSC, YSK, JHK, and JML acquired experimental data. SCL, KWK, JSK, and SJH provided administrative, technical, or material support. KMK, CYH, SHK, and SGK were involved in the study concept and design. KMK and SGK drafted the manuscript. SGK contributed to writing the manuscript, study supervision, and obtaining funding.

References (42)

  • O. Vinas et al.

    Human hepatic stellate cells show features of antigen-presenting cells and stimulate lymphocyte proliferation

    Hepatology

    (2003)
  • T. Hanada et al.

    The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy

    J Biol Chem

    (2007)
  • J. Kluwe et al.

    Modulation of hepatic fibrosis by c-Jun-N-terminal kinase inhibition

    Gastroenterology

    (2010)
  • A. Grimson et al.

    MicroRNA targeting specificity in mammals: determinants beyond seed pairing

    Mol Cell

    (2007)
  • T. Ogawa et al.

    Suppression of type I collagen production by microRNA-29b in cultured human stellate cells

    Biochem Biophys Res Commun

    (2010)
  • M.S. Shafiei et al.

    The function of integrin-linked kinase in normal and activated stellate cells: implications for fibrogenesis in wound healing

    Lab Invest

    (2012)
  • T. Fujii et al.

    12/13-mediated production of reactive oxygen species is critical for angiotensin receptor-induced NFAT activation in cardiac fibroblasts

    J Biol Chem

    (2005)
  • C. Yin et al.

    Hepatic stellate cells in liver development, regeneration, and cancer

    J Clin Invest

    (2013)
  • S.L. Friedman

    Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver

    Physiol Rev

    (2008)
  • F. Tacke et al.

    Update on hepatic stellate cells: pathogenic role in liver fibrosis and novel isolation techniques

    Expert Rev Gastroenterol Hepatol

    (2012)
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