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. 2018 Dec 17;8(1):17905.
doi: 10.1038/s41598-018-36037-4.

SOX9 regulated matrix proteins are increased in patients serum and correlate with severity of liver fibrosis

Varinder S Athwal  1   2   3 , James Pritchett  1   2   4 , Katherine Martin  1   2   3 , Jessica Llewellyn  2 , Jennifer Scott  1   2   3 , Emma Harvey  2 , Abed M Zaitoun  5 , Aoibheann F Mullan  1   2   3 , Leo A H Zeef  6 , Scott L Friedman  7 , William L Irving  8   9 , Neil A Hanley  2   3 , Indra N Guha  8 , Karen Piper Hanley  10   11   12
Affiliations

SOX9 regulated matrix proteins are increased in patients serum and correlate with severity of liver fibrosis

Varinder S Athwal et al. Sci Rep. .

Erratum in

Abstract

Extracellular matrix (ECM) deposition and resultant scar play a major role in the pathogenesis and progression of liver fibrosis. Identifying core regulators of ECM deposition may lead to urgently needed diagnostic and therapetic strategies for the disease. The transcription factor Sex determining region Y box 9 (SOX9) is actively involved in scar formation and its prevalence in patients with liver fibrosis predicts progression. In this study, transcriptomic approaches of Sox9-abrogated myofibroblasts identified >30% of genes regulated by SOX9 relate to the ECM. Further scrutiny of these data identified a panel of highly expressed ECM proteins, including Osteopontin (OPN), Osteoactivin (GPNMB), Fibronectin (FN1), Osteonectin (SPARC) and Vimentin (VIM) as SOX9 targets amenable to assay in patient serum. In vivo all SOX-regulated targets were increased in human disease and mouse models of fibrosis and decreased following Sox9-loss in mice with parenchymal and biliary fibrosis. In patient serum samples, SOX9-regulated ECM proteins were altered in response to fibrosis severity, whereas comparison with established clinical biomarkers demonstrated superiority for OPN and VIM at detecting early stages of fibrosis. These data support SOX9 in the mechanisms underlying fibrosis and highlight SOX9 and its downstream targets as new measures to stratify patients with liver fibrosis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
SOX9 regulates multiple ECM proteins in HSCs. (A) Function annotation for gene ontology of all Sox9-regulated genes (±1.2 fold, p < 0.05) represented as proportion of individual categories outlined and listed in Supplementary Figure 2. (B) Top 5 canonical pathways represented and Sox9-regulated genes listed. Down-regulated genes (purple) and up-regulated (red) are highlighted following Sox9-loss.
Figure 2
Figure 2
Identification of a panel of Sox9-regulated genes in HSCs. (A) Comparative levels and representation of highly expressed factors after knockdown of Sox9 in activated rat HSCs (*in A signifies statistically significant change). Relative mRNA levels by qRT-PCR analysis and protein levels quantified by immunoblotting in rHSCs following Sox9 abrogation using siRNA (B) and (C), representative immunoblot shown in (D) or quiescent and activated rHSCs (E) and (F, representative immunoblot shown in (G). All experiments are n ≥3. Data are shown as means ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
SOX9 is localized and directly binds to its ECM targets in HSCs. (A) Immunofluorescence in activated HSCs from rat (rHSCs; top panel) and human (hHSCs; bottom panel). Nuclear SOX9 shown in red with OPN, GPNMB, SPARC, VIM and FN1 shown in green. Co-localisation is shown where antibody compatibility allows. DAPI nuclear stain (blue). Scale bar 10 μm. (B) ChIP assay for SOX9 binding element in primary rat HSCs with negative controls (IgG and ChIP negative primers) and positive control (Input diluted 1:10). n = 3. SOX9 enrichment (IP) is shown for Fn1, Gpnmb, Sparc and Spp1 (Opn).
Figure 4
Figure 4
Localization and quantification of SOX9-regulated proteins in fibrotic liver following Sox9-loss in mice with BDL-induced fibrosis. (A) Immunohistochemistry for VIM, OPN, SPARC, GPNMB and FN1 (brown) counterstained with toludine blue. Images shown for control (Sox9fl/fl; RosaCreER−/−) and Sox9-null (Sox9fl/fl; RosaCreER+/−) livers following CCl4 induced fibrosis. Scale bar 100 μm. (B) Quantification of surface area covered by individual protein staining in control (Sox9fl/fl; RosaCreER−/−) and Sox9-null (Sox9fl/fl; RosaCreER+/−) livers in (A). All experiments are n = 5. Data are shown as means ± s.e.m. ***p < 0.001.
Figure 5
Figure 5
Localization and quantification of SOX9-regulated proteins in fibrotic liver following Sox9-loss in mice with CCl4 induced fibrosis. (A) Immunohistochemistry for VIM, OPN, SPARC, GPNMB and FN1 (brown) counterstained with toludine blue. Images shown for control (Sox9fl/fl; RosaCreER−/−) and Sox9-null (Sox9fl/fl; RosaCreER+/−) livers following BDL induced fibrosis. Scale bar 100 μm. (B) Quantification of surface area covered by individual protein staining in control (Sox9fl/fl; RosaCreER−/−) and Sox9-null (Sox9fl/fl; RosaCreER+/−) livers in (A). All experiments are n = 5. Data are shown as means ± s.e.m. *p < 0.05.
Figure 6
Figure 6
Localization of SOX9-regulated proteins in fibrotic liver from human. Immunohistochemistry for VIM, OPN, SPARC, GPNMB and FN1 (brown) counterstained with toludine blue. Images shown for advanced fibrotic/cirrhotic human liver secondary to CHC infection. Scale bar 100 μm.
Figure 7
Figure 7
SOX9-regulated proteins are present and increased in serum from patients with liver fibrosis. (A–E) Serum concentration of SOX9-regulated targets quantified by ELISA and grouped by stage of fibrosis (Metavir). Data are shown as means ± s.e.m.
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References

    1. Byass P. The global burden of liver disease: a challenge for methods and for public health. BMC medicine. 2014;12:159. doi: 10.1186/s12916-014-0159-5. - DOI - PMC - PubMed
    1. Williams R, et al. Disease burden and costs from excess alcohol consumption, obesity, and viral hepatitis: fourth report of the Lancet Standing Commission on Liver Disease in the UK. Lancet. 2018;391:1097–1107. doi: 10.1016/S0140-6736(17)32866-0. - DOI - PubMed
    1. Williams R, et al. New metrics for the Lancet Standing Commission on Liver Disease in the UK. Lancet. 2017;389:2053–2080. doi: 10.1016/S0140-6736(16)32234-6. - DOI - PubMed
    1. Tsuchida T, Friedman SL. Mechanisms of hepatic stellate cell activation. Nature reviews. Gastroenterology & hepatology. 2017;14:397–411. doi: 10.1038/nrgastro.2017.38. - DOI - PubMed
    1. Ellis EL, Mann DA. Clinical evidence for the regression of liver fibrosis. Journal of hepatology. 2012;56:1171–1180. doi: 10.1016/j.jhep.2011.09.024. - DOI - PubMed

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