Stimulation of pro-α1(I) collagen by TGF-β1 in mesangial cells:: role of the p38 MAPK pathway

Transforming growth factor-beta (1) (TGF-beta (1)) is a potent inducer of extracellular matrix protein synthesis and a key mediator of renal fibrosis. However, the intracellular signaling mechanisms by which TGF-beta (1) stimulates this process remain incompletely understood. In this report, we examined the role of a major stress-activated intracellular signaling cascade, belonging to the mitogen-activated protein kinase (MAPK) superfamily, in mediating TGF-beta (1) responses in rat glomerular mesangial cells, using dominant-negative inhibition of TGF-beta (1) signaling receptors. We first stably transfected rat glomerular mesangial cells with a kinase-deleted mutant TGF-beta type II receptor (T betaR-IIM) designed to inhibit TGF-beta (1) signaling in a dominant-negative fashion. Next, expression of T betaR-IIM mRNA was confirmed by Northern analysis. Cell surface expression and ligand binding of T betaR-IIM protein were demonstrated by affinity cross-linking with I-125-labeled-TGF-beta (1). TGF-beta (1) rapidly induced p38 MAPK phosphorylation in wild-type and empty vector (pcDNA3) transfected control mesangial cells. Interestingly, transfection with dominant-negative T betaR-IIM failed to block TGF-beta (1) induced p38 MAPK phosphorylation. Moreover, dominant-negative T betaR-IIM failed to block TGF-beta (1)-stimulated pro-alpha (1) (I) collagen mRNA expression and cellular protein synthesis, whereas TGF-beta (1)-induced extracellular signal-regulated kinase (ERK) 1/ERK2 activation and antiproliferative responses were blocked by T betaR-IIM. In the presence of a specific inhibitor of p38 MAPK, SB-203580, TGF-beta (1) was unable to stimulate pro-alpha (1) (I) collagen mRNA expression in the control and T betaR-IIM-transfected mesangial cells. Finally, we confirmed that both p38 MAPK activation and pro-alpha (1) (I) collagen stimulation were TGF-beta (1) effects that were abrogated by dominant-negative inhibition of TGF-beta type I receptor. Thus we show first demonstration of p38 MAPK activation by TGF-beta (1) in mesangial cells, and, given the rapid kinetics, this TGF-beta (1) effect is likely a direct one. Furthermore, our findings suggest that the p38 MAPK pathway functions as a component in the signaling of pro-alpha (1) (I) collagen induction by TGF-beta (1) in mesangial cells.