- Authors: Song MH. et al.
- Year: 2022
- Journal: PLoS One 17 e0267629
- Applications: in vitro / DNA / FectoPRO
- Cell type: HEK-293F
Cell culture HEK 293-F cells (Gibco, #R79007) were suspension-cultured in Freestyle 293 medium (Gibco, #12338018) at 37°C in a 8% CO2 incubator with shaking. Purification of recombinant proteins cDNAs encoding the full-length human CCN5 protein, and individual domains (IGFBP, VWC, and TSP-1), and pairs of domains (VWC-TSP-1, IGFBP-TSP-1, and IGFBP-VWC) of human CCN5, were subcloned into a pcDNA3.1-myc-his plasmid and then transfected into HEK 293-F cells. One day before transfection, 1.5 × 107 cells were seeded into 25 mL of Freestyle 293 medium. On the day of transfection, 20 μg of plasmid DNA was diluted in 3 mL of Opti-MEM medium (Gibco, #51985034). Next, 25 μl FectoPRO transfection reagent (Polyplus, #116–001) was added to the diluted plasmid DNA and the mixture was incubated for 10 mins at room temperature before being added gently to cultured HEK 293-F cells. Three days after transfection, the culture medium was collected and centrifuged at 2,000 × g for 3 mins. The proteins contained in culture medium were purified using Capturem His-Tagged Purification Maxiprep Columns (Takara, 635715). The purified proteins were stored in a buffer (20 mM NaHPO4, 150 mM NaCl and 250 mM imidazole, and 10% (v/v) glycerol) at -70°C.
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.