Regulation of Biglycan Gene Expression by Transforming Growth Factor-β Requires MKK6-p38 Mitogen-activated Protein Kinase Signaling Downstream of Smad Signaling

Hendrik Ungefroren,Wolfgang Lenschow,Wen-Bin Chen,Fred Faendrich,Holger Kalthoff

doi:10.1074/jbc.m300035200

Abstract

Several signaling pathways have been implicated in mediating TGF-beta1-induced extracellular matrix production and fibrosis. We have shown recently that induction of biglycan (BGN) expression by TGF-beta1 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-beta 1 to induce BGN mRNA, in addition to Smads, requires p38 MAPK signaling, because 1) pharmacological inhibitors of p38 dose-dependently inhibited the TGF-beta effect without significantly affecting the transcriptional activity of a constitutively active mutant of the TGF-beta type I receptor or Smad2 phosphorylation at concentrations up to 10 microm, 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-beta 1-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-beta 1-induced BGN mRNA expression, and 4) overexpression of wild-type p38 or MKK6, but not MKK3, augmented the TGF-beta 1 effect on BGN mRNA. We further demonstrate that the (delayed) p38 activation by TGF-beta 1 is downstream of Smads and requires a functional Smad pathway, because blocking TGF-beta-induced p38 activity with SB202190 had no effect on Smad2 phosphorylation, but blocking Smad signaling by forced expression of Smad7 abolished TGF-beta1 induction of p38 activation and, as shown earlier, BGN mRNA expression; finally, re-expression of Smad4 in Smad4-null CFPAC-1 cells restored TGF-beta-induced p38 phosphorylation and, as demonstrated previously, BGN mRNA accumulation. These results clearly show that TGF-beta 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.

Highlights

§ Present address: The First Affiliated Hospital, Medical College, Zhejiang University, Zhejiang, 310003 Hangzhou, People’s Republic of China
We further demonstrate that the p38 activation by TGF-␤1 is downstream of Smads and requires a functional Smad pathway, because blocking TGF-␤-induced p38 activity with SB202190 had no effect on Smad2 phosphorylation, but blocking Smad signaling by forced expression of Smad7 abolished TGF-␤1 induction of p38 activation and, as shown earlier, BGN mRNA expression; reexpression of Smad4 in Smad4-null CFPAC-1 cells restored TGF-␤-induced p38 phosphorylation and, as demonstrated previously, BGN mRNA accumulation
Effect of p38 MAPK Blockade on TGF-␤ Induction of BGN Expression—Several observations suggested that in PANC-1 cells an additional pathway is activated by TGF-␤ that, together with the Smad pathway, functions to amplify the TGF-␤ signal for the BGN mRNA up-regulation

Summary

Introduction

§ Present address: The First Affiliated Hospital, Medical College, Zhejiang University, Zhejiang, 310003 Hangzhou, People’s Republic of China. Effect of p38 MAPK Blockade on TGF-␤ Induction of BGN Expression—Several observations suggested that in PANC-1 cells an additional pathway is activated by TGF-␤ that, together with the Smad pathway, functions to amplify the TGF-␤ signal for the BGN mRNA up-regulation (see Introduction).

Results

Conclusion