Smad3 and PKCdelta mediate TGF-beta1-induced collagen I expression in human mesangial cells.
Transforming growth factor (TGF)-beta has been associated with fibrogenesis in clinical studies and animal models. We previously showed that Smad3 promotes COL1A2 gene activation by TGF-beta1 in human mesangial cells. In addition to the Smad pathway, it has been suggested that TGF-beta1 could also activate more classical growth factor signaling. Here, we report that protein kinase C (PKC)delta plays a role in TGF-beta1-stimulated collagen I production. In an in vitro kinase assay, TGF-beta1 treatment specifically increased mesangial cell PKCdelta activity in a time-dependent manner. Translocation to the membrane was detected by immunocytochemistry and immunoblot, suggesting activation of PKCdelta by TGF-beta1. Inhibition of PKCdelta by rottlerin decreased basal and TGF-beta1-stimulated collagen I production, mRNA expression, and COL1A2 promoter activity, whereas blockade of conventional PKCs by Gö 6976 had little or no effect. In a Gal4-LUC assay system, inhibition of PKCdelta abolished TGF-beta1-induced transcriptional activity of Gal4-Smad3 and Gal4-Smad4(266-552). Overexpression of Smad3 or Smad3D, in which the three COOH-terminal serine phosphoacceptor residues have been mutated, increased activity of the SBE-LUC construct, containing four DNA binding sites for Smad3 and Smad4. This induction was blocked by PKCdelta inhibition, suggesting that rottlerin decreased Smad3 transcriptional activity independently of COOH-terminal serine phosphorylation. Blockade of PKCdelta abolished ligand-independent and ligand-dependent stimulation of COL1A2 promoter activity by Smad3. These data indicate that PKCdelta is activated by TGF-beta1 in human mesangial cells. TGF-beta1-stimulated PKCdelta activity positively regulates Smad transcriptional activity and is required for COL1A2 gene transcription. Thus cross talk among multiple signaling pathways likely contributes to the pathogenesis of glomerular matrix accumulation.
Runyan
Constance E
CE
Northwestern Univ., Dept. of Pediatrics, Chicago, IL 60611-3008, USA.
Schnaper
H William
HW
Poncelet
Anne-Christine
AC
D024042:Collagen Type I / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051745:Protein Kinase C-delta; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D024042:Collagen Type I; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta