Regulation of biglycan gene expression by transforming growth factor-β requires MKK6-p38 mitogen-activated protein kinase signaling downstream of Smad signaling

Abstract

Several signaling pathways have been implicated in mediating TGF-β1-induced extracellular matrix production and fibrosis. We have shown recently that induction of biglycan (BGN) expression by TGF-β1 depended on a functional Smad pathway (Chen, W.-B., Lenschow, W., Tiede, K., Fischer, J. W., Kalthoff, H., and Ungefroren, H. (2002) J. Biol. Chem. 277, 36118-36128). Here, we present evidence that the ability of TGF-β1 to induce BGN mRNA, in addition to Smads, requires p38 MAPK signaling, because 1) pharmacological inhibitors of p38 dose-dependently inhibited the TGF-β effect without significantly affecting the transcriptional activity of a constitutively active mutant of the TGF-β type I receptor or Smad2 phosphorylation at concentrations up to 10 μM, 2) the up-regulation of BGN mRNA was preceded by a delayed increase in the phosphorylation of p38 and its upstream activator MKK6 in TGF-β1-treated PANC-1 cells, 3) inhibition of the p38 pathway by stable retroviral transduction with a dominant negative mutant of either p38 or MKK6 reduced TGF-β1-induced BGN mRNA expression, and 4) overexpression of wild-type p38 or MKK6, but not MKK3, augmented the TGF-β1 effect on BGN mRNA. We further demonstrate that the (delayed) p38 activation by TGF-β1 is downstream of Smads and requires a functional Smad pathway, because blocking TGF-β-induced p38 activity with SB202190 had no effect on Smad2 phosphorylation, but blocking Smad signaling by forced expression of Smad7 abolished TGF-β1 induction of p38 activation and, as shown earlier, BGN mRNA expression; finally, re-expression of Smad4 in Smad4-null CFPAC-1 cells restored TGF-β-induced p38 phosphorylation and, as demonstrated previously, BGN mRNA accumulation. These results clearly show that TGF-β induction of BGN expression in pancreatic cells requires activation of MKK6-p38 MAPK signaling downstream of Smad signaling and provide a mechanistic clue to the up-regulation of BGN seen in inflammatory response-related fibrosis and desmoplasia.