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.

Clitoria ternatea Flower Extract-Based Gel Elevates TGF-β1 Gene Expression and Collagen Density in UVB-Induced Collagen Loss Rat Skin

“…Repeated exposure to UVB stimulates the cytokine receptors by increasing the transcription of AP-1, which results in TGF-β1 inactivation and collagen degradation (3,9,33,36). UVB irradiation modulates the TGF-β pathway at several levels; it decreases type II TGF-β receptor in the dermal fibroblast-cell membrane in vivo, stimulates the intracellular inhibitor of TGF-β1 signaling Smad-7, and reduces levels of connective tissue growth factor (CCN2), an essential mediator of TGF-β1 effects on collagen synthesis (37)(38)(39)(40)(41). At the intracellular level, UVB can cause DNA damage by cross-linking adjacent pyrimidine bases and generating free radicals or reactive oxygen species (ROS), consequent, resulting in transforming growth factor (TGF-β) inhibition and AP-1 (activator protein) activation (23,31,42).…”

The matricellular protein CCN5 prevents anti-VEGF drug-induced epithelial-mesenchymal transition of retinal pigment epithelium

“…Recombinant CCN5 protein was purified as previous described 51 . The CCN5 proteins was treated at a concentration of 500 ng/mL in ARPE-19 cells or intravitreally injected into mouse eyes (40 ng/eye).…”

Structural insights into regulation of CCN protein activities and functions

“…Aided by either N‐ or C‐terminally appended fusion partners, both expression and solubility increased many‐fold and the TSP1‐fusion protein was able to convey activities previously reported for full‐length CCN5. It was recently published that TSP1 from CCN5 with either the IGFBP or vWC domain appended N‐terminally was required for the TSP1 domain to reduce the expression of the fibrotic markers fibronectin and alpha smooth muscle actin (αSMA) in fibroblasts, in a similar manner to full‐length CCN5 (Song et al 2022). Considering the poor solubility of TSP1 alone, it is possible that the IGFBP and vWC domains may function as chaperones to ensure proper folding of the TSP1 domain in the Song study, similarly as the role of the fusion partners in our study.…”