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.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation/physiology , Mitogen-Activated Protein Kinases/metabolism , Proteoglycans/genetics , Signal Transduction , Trans-Activators/metabolism , Transforming Growth Factor beta/physiology , Base Sequence , Biglycan , DNA Primers , Enzyme Activation , Extracellular Matrix Proteins , Humans , MAP Kinase Kinase 6 , Smad Proteins , Tumor Cells, Cultured , p38 Mitogen-Activated Protein KinasesABSTRACT
Overexpression of the small leucine-rich proteoglycan biglycan (BGN) in fibrosis and desmoplasia results from enhanced activity of transforming growth factor-beta (TGF-beta). In pancreatic adenocarcinoma, the tumor cells themselves may contribute to BGN synthesis in vivo, since 8 of 18 different pancreatic carcinoma cell lines constitutively expressed BGN mRNA, as shown by reverse transcription-PCR analysis. In PANC-1 cells, TGF-beta1 dramatically stimulated BGN mRNA accumulation through a BGN transcription-independent, cycloheximide-sensitive mechanism and strongly increased the synthesis and release of the proteoglycan form of BGN. The ability of TGF-beta1 to induce BGN mRNA was critically dependent on Smad signaling, since 1) the up-regulation of BGN mRNA was preceded by a marked increase in Smad2 phosphorylation in TGF-beta1-treated PANC-1 cells, 2) TGF-beta1 was unable to induce BGN mRNA in pancreatic carcinoma cell lines that carry homozygous deletions of the Smad4/DPC4 gene, 3) inhibition of the Smad pathway in PANC-1 cells by transfection with a dominant negative Smad4/DPC4 mutant significantly reduced TGF-beta1-induced BGN mRNA expression, 4) stable reintroduction of wild type Smad4/DPC4 into Smad4-null CFPAC-1 cells restored the TGF-beta1 effect, and 5) overexpression of Smad2 and Smad3 in PANC-1 cells augmented TGF-beta1 induction of BGN mRNA, whereas forced expression of Smad7, an inhibitory Smad, effectively blocked it. These results clearly show that a functional Smad pathway is crucial for TGF-beta regulation of BGN mRNA expression. Since BGN has been shown to inhibit growth of pancreatic cancer cells, the Smad4/DPC4 mediation of the TGF-beta effect may represent a novel tumor suppressor function for Smad4/DPC4: antiproliferation via expression of autoinhibitory BGN.
