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The TSP-1 domain of the matricellular protein CCN5 is essential for its nuclear localization and anti-fibrotic function.

ABSTRACT: The matricellular protein CCN5 exerts anti-fibrotic activity in hearts partly by inducing reverse trans-differentiation of myofibroblasts (MyoFBs) to fibroblasts (FBs). CCN5 consists of three structural domains: an insulin-like growth factor binding protein (IGFBP), a von Willebrand factor type C (VWC), and a thrombospondin type 1 (TSP-1). In this study, we set out to elucidate the roles of these domains in the context of the reverse trans-differentiation of MyoFBs to FBs. First, human cardiac FBs were trans-differentiated to MyoFBs by treatment with TGF-β; this was then reversed by treatment with recombinant human CCN5 protein or various recombinant proteins comprising individual or paired CCN5 domains. Subcellular localization of these recombinant proteins was analyzed by immunocytochemistry, cellular fractionation, and western blotting. Anti-fibrotic activity was also evaluated by examining expression of MyoFB-specific markers, α-SMA and fibronectin. Our data show that CCN5 is taken up by FBs and MyoFBs mainly via clathrin-mediated endocytosis, which is essential for the function of CCN5 during the reverse trans-differentiation of MyoFBs. Furthermore, we showed that the TSP-1 domain is essential and sufficient for endocytosis and nuclear localization of CCN5. However, the TSP-1 domain alone is not sufficient for the anti-fibrotic function of CCN5; either the IGFBP or VWC domain is needed in addition to the TSP-1 domain.

SUBMITTER: Song MH

PROVIDER: S-EPMC9045603 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Cardiac fibrosis (CF) is associated with increased ventricular stiffness and diastolic dysfunction and is an independent predictor of long-term clinical outcomes of patients with heart failure (HF). We previously showed that the matricellular CCN5 protein is cardioprotective via its ability to inhibit CF and preserve cardiac contractility. OBJECTIVES:This study examined the role of CCN5 in human heart failure and tested whether CCN5 can reverse established CF in an experimental model of HF induced by pressure overload. METHODS:Human hearts were obtained from patients with end-stage heart failure. Extensive CF was induced by applying transverse aortic constriction for 8 weeks, which was followed by adeno-associated virus-mediated transfer of CCN5 to the heart. Eight weeks following gene transfer, cellular and molecular effects were examined. RESULTS:Expression of CCN5 was significantly decreased in failing hearts from patients with end-stage heart failure compared to nonfailing hearts. Trichrome staining and myofibroblast content measurements revealed that the established CF had been reversed by CCN5 gene transfer. Anti-CF effects of CCN5 were associated with inhibition of the transforming growth factor beta signaling pathway. CCN5 significantly inhibited endothelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation, which are 2 critical processes for CF progression, both in vivo and in vitro. In addition, CCN5 induced apoptosis in myofibroblasts, but not in cardiomyocytes or fibroblasts, both in vivo and in vitro. CCN5 provoked the intrinsic apoptotic pathway specifically in myofibroblasts, which may have been due the ability of CCN5 to inhibit the activity of NF?B, an antiapoptotic molecule. CONCLUSIONS:CCN5 can reverse established CF by inhibiting the generation of and enhancing apoptosis of myofibroblasts in the myocardium. CCN5 may provide a novel platform for the development of targeted anti-CF therapies.

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The TSP-1 domain of the matricellular protein CCN5 is essential for its nuclear localization and anti-fibrotic function.

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