CCN2 Is Required for the TGF-β Induced Activation of Smad1 - Erk1/2 Signaling Network
Figure 6
CCN2 induced up-regulation of collagen gene expression is dose dependent.
(A) Foreskin fibroblasts were transduced with increasing doses of CCN2 (5, 10, 25, 50 MOI) overexpressing virus. Collagen mRNA levels were determined by quantitative RT-PCR (A, left panel) and collagen protein levels were determined by western blot (A, right panel). The values represent mean ± S.E. of three independent experiments. *, significant values at p<0.05. (B) Foreskin fibroblasts were stimulated with 2.5 ng/ml of TGF-β or transduced with AdCCN2 (10 MOI) for 48 hours. Collagen and CCN2 protein levels were determined by western blot. (C) COL1A1 mRNA levels was determined by qPCR in foreskin fibroblasts stimulated with TGF-β (2.5 ng/ml) or indicated doses of rhCCN2 for 24 hours. The values represent mean ± S.E. (n = 4; *, significant values at p<0.05). (D) Foreskin fibroblasts were transfected with COL1A2 (−2 Kb) luciferase promoter plasmid construct. Next day after transfection TGF-β or rCCN2 were added for 24 hours and the promoter activity was determined. The values represent mean ± S.E. (n = 4; *, significant values at at p<0.05). (E) Foreskin fibroblasts were treated with rCCN2 at various concentrations and analyzed for collagen levels in the culture medium. (F) Foreskin fibroblasts were treated with rCCN2 (20 ng/ml) at the indicated time periods and with TGF-β (2.5 ng/ml) for 3 hours and analyzed for phosphorylated and total Fli1 by western blot from nuclear extracts.