Elsevier

Experimental Cell Research

Volume 362, Issue 2, 15 January 2018, Pages 343-348
Experimental Cell Research

Osteopontin promotes collagen I synthesis in hepatic stellate cells by miRNA-129-5p inhibition

https://doi.org/10.1016/j.yexcr.2017.11.035Get rights and content

Abstract

Activation of hepatic stellate cells (HSCs) is an essential event in the initiation and progression of liver fibrosis. HSCs are believed to be the major source of collagen-producing myofibroblasts in fibrotic livers. A key feature in the pathogenesis of liver fibrosis is fibrillar Collagen I (Col 1) deposition. Osteopontin (OPN), an extracellular matrix (ECM) cytokine expressed in HSCs, could drive fibrogenesis by modulating the HSC pro-fibrogenic phenotype and Col 1 expression. Here, we aimed to investigate the molecular mechanism of OPN regulating the activation of HSCs. Our results showed that hepatic expression of OPN was increased in patients with liver fibrosis. In addition, hepatic OPN was positively correlated with Col 1 and α-SMA. Recombinant OPN (rOPN) upregulated Col 1 and α-SMA expression in LX-2 cells. However, OPN knockdown downregulated Col 1 expression. The 3′-UTR of the collagen 1 (Col 1) was identified to bind miR-129-5p. Transfection of miR-129-5p mimic in HSC resulted in a marked reduction of Col 1 expression. Conversely, a decrease in miR-129-5p in HSCs transfected by anti-sense miR-129-5p (AS-miR-129-5p) caused Col 1 upregulation. Furthermore, luciferase reporter assay showed that miR-129-5p directly target the 3′-UTR of Col1α1 mRNA via repressing its post-transcriptional activities. Finally, miR-129-5p level was decreased in fibrotic liver of human, and reduced by rOPN treatment. In contrast, miR-129-5p was induced in HSCs transfected by OPN siRNA. These data suggested that OPN induces Col 1 expression via suppression of miR-129-5p in HSCs.

Introduction

Liver fibrosis is a chronic scarring process of the liver and characterized by an increased and altered deposition of extracellular matrix (ECM) components, particular collagen type I [1]. The fibrillar collagen type I, is encoded by two different genes, col1A1 and col1A2, and accounts for 36% of the total collagens in ECM of healthy liver. During liver fibrogenesis, collagen type I is the predominant isoform deposited into perisinusoidal space [2], [3].

In the fibrotic liver, activated hepatic stellate cells (HSCs) are identified as the main producers of ECM in liver and their activation represents the critical event in liver fibrosis. After liver injury, the quiescent HSCs trans-differentiate into myofibroblast-like cells that characterized by the expression of α-smooth muscle actin (α-SMA) [4]. In addition, the ECM contains a complex mix of proteins that promote cell proliferation, migration, and differentiation. One ECM component with such roles is the matricellular glycophosphoprotein, osteopontin (OPN), also known as secreted phosphoprotein 1. Recently, OPN levels have been highlighted as a potential biomarker of liver disease, levels correlating with the severity of disease. Moreover, OPN has been reported to promote the progression of fibrosis in nonalcoholic steatohepatitis [5] and liver fibrosis [6]. OPN emerges as a key cytokine within the ECM protein network, expressed in HSCs, could drive the HSC pro-fibrogenic phenotype and collagen type I protein expression, contributing to scarring and liver fibrosis [7]. However, the molecular mechanism on OPN mediating collagen type I remain elusive.

MicroRNAs (miRNAs) are short 20–22 nucleotides that regulate gene expression by binding to the 3′-untranslated region (3′-UTR) of the target mRNAs [8]. There is an increasing evidence that aberrant miRNAs are involved in liver fibrosis, and miRNAs are HSC regulators which may play roles in the regulation of antifibrotic or profibrotic genes during hepatic fibrosis [9], [10]. For example, collagen type I is predicted as miR-129-5p target gene by TARGETSCAN database in human cell lines. Recently, it was reported that miR-129-5p, one of the downregulated microRNAs in systemic sclerosis fibroblasts, mediated the collagen type I reduction [11]. However, knowledge of the function and role of miR-129-5p in liver fibrosis is still not understood.

In this study, we found that miR-129-5p was decreased in OPN-induced HSC and human liver fibrotic tissues. Upregulation of miR-129-5p decreased the expression levels of collagen type I and α-SMA and promoted HSC proliferation. It is confirmed that collagen type I is a direct target of miR-129-5p. Therefore, the major aim of this work was to determine how miR-129-5p become a antifibrogenic “switch” and to characterize whether OPN mediates collagen type I expression through miR-129-5p.

Section snippets

Human liver samples

Liver tissues were taken from 10 patients with clinically diagnosed liver cirrhosis suffered from HBV who underwent liver transplantation. Liver tissues with normal histology (n = 8) obtained from patients with various benign liver conditions or transplant donors were used as controls. All clinical samples are from the First Affiliated Hospital of the Guangdong Pharmaceutical University, Guangzhou, China. Written informed consent was obtained from each patients and volunteers, and the study was

OPN is induced in liver fibrosis

The expression of OPN was significantly increased in fibrotic liver, compared to control liver (Fig. 1A), and presented quantitatively in Fig. 1B. Furthermore, increased mRNA levels of OPN was also observed in the fibrotic liver tissues (Fig. 1C). Consistently, the expression of Col1α1 and α-SMA was also increased in fibrotic livers (Fig. 1D and E). These data suggest that OPN signaling is activated in human fibrotic livers and might be involved in the pathogenesis of liver fibrosis.

OPN regulates collagen expression in HSCs

Next, we

Discussion

Liver fibrosis is characterized by accumulation of ECM, mainly produced by activated HSCs. Our results has been for the first time demonstrated that the expression of miR-129-5p was significantly correlated with Col 1 synthesis and HSC activation, and miR-129-5p is the central mediator of induction of Col 1 induced by OPN.

It is well-known that OPN is significantly induced during liver injury, both in humans and in rodents [5], [16], [17]. In recent years, it has been demonstrated that the

Acknowledgements

This work was supported by the research grants from the National Natural Science Foundation of China (81300331); the Science and Technology Planning Project of Guangdong Province (2014A020212460), China; the Key Project of Natural Science Foundation of Guangdong Province (2016A030311014), China; Natural Science Foundation of Guangdong Province (2015A030313582), China; and Guangdong Provincial Medical Science and Technology Research Foundation (A2015092), China. We also thank the support from

Potential conflict of interest

None to report.

References (28)

  • S.L. Friedman

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

    Physiol. Rev.

    (2008)
  • W.K. Syn et al.

    Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis

    Hepatology

    (2011)
  • J. Pritchett et al.

    Osteopontin is a novel downstream target of SOX9 with diagnostic implications for progression of liver fibrosis in humans

    Hepatology

    (2012)
  • R. Urtasun et al.

    Osteopontin, an oxidant stress sensitive cytokine, up-regulates collagen-I via integrin alpha(V)beta(3) engagement and PI3K/pAkt/NFkappaB signaling

    Hepatology

    (2012)
  • Cited by (41)

    • The critical role of osteopontin (OPN) in fibrotic diseases

      2023, Cytokine and Growth Factor Reviews
    • Pathogenesis of Alcohol-Associated Liver Disease

      2022, Journal of Clinical and Experimental Hepatology
      Citation Excerpt :

      Investigations into how/whether OPN induces liver fibrosis indicated that OPN activates HSCs by upregulating the hedgehog pathway218 and Akt and Erk phosphorylations.217 OPN also increases collagen type I expression by reducing miR-129–5p, an inhibitor of collagen expression.219 90 kDa ribosomal S6 kinase (p90RSK): p90RSK is a serine/threonine kinase of the S6 ribosomal kinase (RSK) family, whose members are downstream effectors of the Ras/Raf/MEK/ERK signaling pathway, which controls cell growth, proliferation, survival, and apoptosis, cytokine production, and collagen synthesis.220,221

    • Is human umbilical cord mesenchymal stem cell-derived conditioned medium effective against oxidative and inflammatory status in CCl<inf>4</inf>-induced acute liver injury?

      2022, Life Sciences
      Citation Excerpt :

      This effect had also mentioned in the previous study [16]. TGF-β activates HSCs as a major source of collagen [34]. Therefore, hMSC-CM probably prevents the activation of HSCs by reducing TGF-β levels, and this event could lead to decreased collagen production.

    • C[sbnd]C motif chemokine ligand 16 inhibits the progression of liver cirrhosis via inactivating hepatic stellate cells

      2020, Hepatobiliary and Pancreatic Diseases International
      Citation Excerpt :

      Our IHC in the 7 liver cirrhotic tissues showed that CCL16 expression was negatively related to HIF-1α (P = 0.018, Fig. 4C, D). Collagen is deposited in the progression of cirrhotic liver, due to activation of hepatic stellate cells (HSC) [14]. Overexpression of CCL16 in LX2 resulted in downregulation of collagen-related markers, Col1 and Col4 (Fig. 5A).

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text