Stimulation of pro-α1(I) collagen by TGF-β1in mesangial cells: role of the p38 MAPK pathway

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Abstract
Transforming growth factor-β1(TGF-β1) is a potent inducer of extracellular matrix protein synthesis and a key mediator of renal fibrosis. However, the intracellular signaling mechanisms by which TGF-β1stimulates 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-β1responses in rat glomerular mesangial cells, using dominant-negative inhibition of TGF-β1signaling receptors. We first stably transfected rat glomerular mesangial cells with a kinase-deleted mutant TGF-β type II receptor (TβR-IIM) designed to inhibit TGF-β1signaling in a dominant-negative fashion. Next, expression of TβR-IIMmRNA was confirmed by Northern analysis. Cell surface expression and ligand binding of TβR-IIMprotein were demonstrated by affinity cross-linking with125I-labeled-TGF-β1. TGF-β1rapidly induced p38 MAPK phosphorylation in wild-type and empty vector (pcDNA3)-transfected control mesangial cells. Interestingly, transfection with dominant-negative TβR-IIMfailed to block TGF-β1-induced p38 MAPK phosphorylation. Moreover, dominant-negative TβR-IIMfailed to block TGF-β1-stimulated pro-α1(I) collagen mRNA expression and cellular protein synthesis, whereas TGF-β1-induced extracellular signal-regulated kinase (ERK) 1/ERK2 activation and antiproliferative responses were blocked by TβR-IIM. In the presence of a specific inhibitor of p38 MAPK, SB-203580, TGF-β1was unable to stimulate pro-α1(I) collagen mRNA expression in the control and TβR-IIM-transfected mesangial cells. Finally, we confirmed that both p38 MAPK activation and pro-α1(I) collagen stimulation were TGF-β1effects that were abrogated by dominant-negative inhibition of TGF-β type I receptor. Thus we show first demonstration of p38 MAPK activation by TGF-β1in mesangial cells, and, given the rapid kinetics, this TGF-β1effect is likely a direct one. Furthermore, our findings suggest that the p38 MAPK pathway functions as a component in the signaling of pro-α1(I) collagen induction by TGF-β1in mesangial cells.