ALK5 inhibition blocks TGFβ-induced CCN1 expression in human foreskin fibroblasts

Abstract

The potent profibrotic cytokine TGFβ induces connective tissue growth factor (CCN2/CTGF) is induced in fibroblasts in a fashion sensitive to SB-431542, a specific pharmacological inhibitor of TGFβ type I receptor (ALK5). In several cell types, TGFβ induces CCN1 but suppresses CCN3, which opposes CCN1/CCN2 activities. However, whether SB-431542 alters TGFβ-induced CCN1 or CCN3 in human foreskin fibroblasts in unclear. Here we show that TGFβ induces CCN1 but suppresses CCN3 expression in human foreskin fibroblasts in a SB-431542-sensitive fashion. These results emphasize that CCN1/CCN2 and CCN3 are reciprocally regulated and support the notion that blocking ALK5 or addition of CCN3 may be useful anti-fibrotic approaches.

Fig. 1

Inhibiting ALK5 decreased TGFβ-induced CCN1 and increases CCN3 mRNA expression. Human foreskin dermal fibroblasts were serum-starved overnight, incubated for 45 min with DMSO or SB-431542 (10 μM) prior to treatment for 6 h with or without TGFβ1 (1 ng/ml). Total RNAs were harvested and subjected to real time RT-PCR. Each sample was conducted in triplicate. CCN2, CCN1 and CCN3 expression were detected and normalized to that of 18S using the ΔΔCt method. Columns, Average (mRNA fold change compared to DMSO control) of triplicate samples performed on cells derived from each of three different experiments (N = 3; Averages +/-SEM are shown; One way ANOVA * p < 0.05, ** p < 0.001, *** p < 0.0001)

Fig. 2

Inhibiting ALK5 decreased TGFβ-induced CCN1 protein expression: Western blot analysis Human foreskin dermal fibroblasts were serum-starved overnight, incubated for 45 min with DMSO or SB-431542 (10 μM) prior to treatment for 24 h with or without TGFβ1 (1 ng/ml). rCCN3 = recombinant CCN3 control. Protein was harvested and subjected to western blot analyses with anti-CCN1, anti-CCN2 and anti-CCN3 and anti-βactin antibodies. Experiments were conducted thrice, representative Western blots are shown