Research ArticleGα12 overexpression induced by miR-16 dysregulation contributes to liver fibrosis by promoting autophagy in hepatic stellate cells
Graphical abstract
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,9 Gα12 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 Gα12 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
Gα12 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.
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