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

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-β₁ 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-β₁ responses in rat glomerular mesangial cells, using dominant-negative inhibition of TGF-β₁ signaling receptors. We first stably transfected rat glomerular mesangial cells with a kinase-deleted mutant TGF-β₁ type II receptor (TβR-II_M) designed to inhibit TGF-β₁ signaling in a dominant-negative fashion. Next, expression of TβR-II_M mRNA was confirmed by Northern analysis. Cell surface expression and ligand binding of TβR-II_M protein were demonstrated by affinity cross-linking with ¹²⁵I-labeled-TGF-β₁. TGF-β₁ rapidly induced p38 MAPK phosphorylation in wild-type and empty vector (pcDNA3)-transfected control mesangial cells. Interestingly, transfection with dominant-negative TβR-II_M failed to block TGF-β₁-induced p38 MAPK phosphorylation. Moreover, dominant-negative TβR-II_M failed 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-II_M. In the presence of a specific inhibitor of p38 MAPK, SB-203580, TGF-β₁ was unable to stimulate pro-α₂I) collagen mRNA expression in the control and TβR-II_M-transfected mesangial cells. Finally, we confirmed that both p38 MAPK activation and pro-α₁(I) collagen stimulation were TGF-β₁ effects that were abrogated by dominant-negative inhibition of TGF-β type I receptor. Thus we show first demonstration of p38 MAPK activation by TGF-β₂ in mesangial cells, and, given the rapid kinetics, this TGF-β₁ effect 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-β₂ in mesangial cells.