Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jan;1862(1):135-44.
doi: 10.1016/j.bbadis.2015.10.028. Epub 2015 Oct 31.

Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis (NASH) fibrosis

Jason D Coombes  1 , Steve S Choi  2 , Marzena Swiderska-Syn  3 , Paul Manka  4 , Danielle T Reid  5 , Elena Palma  6 , Marco A Briones-Orta  1 , Guanhua Xie  3 , Rasha Younis  7 , Naoto Kitamura  7 , Marco Della Peruta  8 , Shanna Bitencourt  9 , Laurent Dollé  9 , Ye Htun Oo  10 , Zhiyong Mi  11 , Paul C Kuo  11 , Roger Williams  1 , Shilpa Chokshi  6 , Ali Canbay  12 , Lee C Claridge  13 , Bertus Eksteen  5 , Anna Mae Diehl  3 , Wing-Kin Syn  14
Affiliations

Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis (NASH) fibrosis

Jason D Coombes et al. Biochim Biophys Acta. 2016 Jan.

Abstract

Introduction: Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH.

Methods: Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis.

Results: MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFβ mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice.

Conclusion: OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.

Keywords: Adipokine; Fibrosis; Hepatic; Signaling.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Osteopontin (OPN)-expressing hepatic stellate cells (HSC) accumulate in non-alcoholic steatohepatitis (NASH) fibrosis. Wild type (WT) and ob/ob (leptin deficient) mice were fed control chow or the methionine choline deficient (MCD) diet for 8 weeks. Livers were harvested for IHC and qRTPCR. Representative staining are shown. (A) OPN staining. (B) OPN morphometry. (C) OPN mRNA; results are expressed as fold change relative to WT control mice. (D) OPN (brown) and Desmin (marker of HSC) (green)—double immunostaining (magnification ×400); graph shows number of OPN/Desmin double-positive cells per HPF (quantified by cell counting); black arrows indicate cells which co-express OPN and Desmin. All results are graphed as mean ± SEM; *p < 0.05 vs. WT control mice or otherwise indicated.
Fig. 2
Fig. 2
OPN loss abrogates leptin fibrogenic effects in HSC. HSC (mouse GRX line) with OPN knockdown (shOPN) and control HSC (shScr) were treated with recombinant leptin (or vehicle) for 48 h. Cells were then harvested and RNA analyzed by qRTPCR. (A) OPN mRNA. (B) αSMA mRNA. (C) Collagen 1α1 mRNA. (D) TGFβ mRNA. Results are expressed as fold changes relative to shScr, and graphed as mean ± SEM. Wound-healing and transmigration studies in shOPN or shScr HSC were performed under leptin-treated conditions (E–F). (E) Wound healing; migration was quantified by measuring the distance dividing the two sides of the monolayer. Mean (% wound closure) ± SEM were graphed. (F) Transmigration was evaluated 24 h after seeding of cells, by counting crystal violet-stained cells on the underside membrane in 15 random HPF. Mean cell numbers ± SEM were graphed. *p < 0.05 vs shScr or otherwise indicated.
Fig. 3
Fig. 3
OPN is PI3K regulated and enhances fibrogenesis in GRX HSC line. HSC (mouse GRX line) with OPN knockdown (shOPN) and control HSC (shScr) were treated with recombinant leptin, with or without LY294002 (a PI3K inhibitor). Cells were then harvested and RNA analyzed by qRTPCR. (A) OPN mRNA. (B) αSMA mRNA. (C) Collagen 1α1 mRNA. (D) TGFβ mRNA. Results are expressed as fold changes relative to shScr-Leptin, and graphed as mean ± SEM. *p < 0.05 vs. shScr-Leptin.
Fig. 4
Fig. 4
OPN is a downstream target of leptin and is PI3K/Akt regulated in primary HSC. Freshly isolated HSC were treated with leptin (or vehicle), with or without LY294002. Additional HSC were also pre-treated with Ad5dnAkt (or Ad5GFP) prior to treatment with leptin (100 ng/ml) or Ad5mryAkt alone. RNA was isolated and changes in gene expression evaluated by qRT-PCR. (A) OPN mRNA. (B) αSMA mRNA. (C) Collagen 1α1 mRNA. Results are expressed as fold changes relative to vehicle-treated HSC, and graphed as mean ± SEM. *p < 0.05 vs. vehicle.
Fig. 5
Fig. 5
OPN neutralization abrogates fibrogenesis in precision-cut liver slices (PCLS). Precision-cut, viable mouse liver slices (PCLS) (a liver organ culture system) were stimulated with leptin (or vehicle), in the presence of sham- or OPN-specific aptamers for 48 h. At the end of treatment, liver slices were harvested, viability assessed by ATP content, and RNA analyzed by qRTPCR. (A) ATP content (viability) in nmol/mg protein; hashed line denotes the accepted viability threshold. (B) OPN mRNA. (C) αSMA mRNA. (D) Collagen 1α1 mRNA. Results are expressed as fold changes relative to vehicle-treated PCLS, and graphed as mean ± SEM. *p < 0.05 vs. vehicle.
Fig. 6
Fig. 6
OPN neutralization inhibits diet-induced NASH fibrosis in leptin-intact mice. Leptin-intact (WT) mice were fed control chow or the MCD diet for 6 weeks, in the presence of sham or OPN-aptamers. Mice were sacrificed 24 h after final dose of aptamers, and livers analyzed. (A) Representative Sirius-red staining; black arrows indicate Sirius-red stained fibrils. Insert shows Sirius-red staining from a control-fed mouse. (B) Sirius-red morphometry. (C) Collagen 1α1 mRNA. (D) αSMA mRNA. Results are expressed as fold changes relative to sham-aptamer treated mice, and graphed as mean ± SEM. *p < 0.05 vs. sham-aptamer treated mice. (E) Representative OPN staining; black arrows indicated OPN-positive cells. Insert shows OPN staining from control-fed mouse. (F) OPN morphometry.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Singh S, Allen AM, Wang Z, Prokop LJ, Murad MH, Loomba R. Fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: a systematic review and meta-analysis of paired-biopsy studies. Clin. Gastroenterol. Hepatol. 2015 Apr;13(4):643–654. e1–e9. quiz e39–40 http://dx.doi.org/10.1016/j.cgh.2014.04.014 (Epub 2014 Apr 24). - DOI - PMC - PubMed
    1. Pais R, Charlotte F, Fedchuk L, et al. A systematic review of follow-up biopsies reveals disease progression in patients with non-alcoholic fatty liver. J. Hepatol. 2013 Sep;59(3):550–556. http://dx.doi.org/10.1016/j.jhep.2013.04.027 (Epub 2013 May 9). - DOI - PubMed
    1. Bhala N, Angulo P, van der Poorten D, et al. The natural history of nonalcoholic fatty liver disease with advanced fibrosis or cirrhosis: an international collaborative study. Hepatology. 2011 Oct;54(4):1208–1216. http://dx.doi.org/10.1002/hep.24491 (Epub 2011 Aug 9). - DOI - PMC - PubMed
    1. Mittal S, El-Serag HB, Sada YH, et al. Hepatocellular carcinoma in the absence of cirrhosis in US veterans is associated with non-alcoholic fatty liver disease. Clin. Gastroenterol. Hepatol. 2015 Jul;18 http://dx.doi.org/10.1016/j.cgh.2015.07.019 (pii: S1542-3565(15)00978–7 [Epub ahead of print]). - DOI - PMC - PubMed
    1. Michelotti GA, Machado MV, Diehl AM. NAFLD, NASH and liver cancer. Nat. Rev. Gastroenterol. Hepatol. 2013 Nov;10(11):656–665. http://dx.doi.org/10.1038/nrgastro.2013.183 (Epub 2013 Oct 1). - DOI - PubMed

Publication types

MeSH terms

-