Abstract 725: Protein Kinase C delta Inhibits Type I Collagen Basal Expression through CCAAT-binding Factor and c-Krox in Vascular Smooth Muscle Cells
Protein Kinase C delta (PKCδ) regulates multiple functions in vascular smooth muscle cells (VSMCs) including proliferation, migration, and apoptosis. In previous studies, we have demonstrated that PKCδ activity is necessary for TGFβ-induced fibronectin production in VSMCs. The purpose of the current study is to understand the role of this kinase in regulation of type I collagen synthesis. Using A10 VSMCs derived from rat thoracic aorta, we showed that TGFβ (5ng/ml x 48 hours) increased type I collagen expression. Pretreatment of A10 with Rottlerin (2uM, 1 hour), a selective inhibitor of PKCδ, dramatically increased type I collagen basal expression independent of TGFβ. To understand the mechanism involved with PKCδ, we used a Luciferase reporter that contains the human COL1A2 promoter (−772 to +58). Rottlerin stimulated the Luciferase reporter activity by 3.53±0.13 fold (n=3, p<0.01). Moreover, inhibition of PKCδ with a dominant negative mutant led to upregulation of reporter activity (1.77±0.15 fold induction, n=3, p<0.01), while activation of PKCδ with a constitutive active mutant reduced collagen reporters (0.19±0.02 fold reduction, n=3, p<0.01). Using a series of 5′ deletions of COL1A2/Luciferase constructs (−772, −353, and −108), we narrowed the location of the PKCδ-response element to the -108/+58 region of the COL1A2 promoter, which is distinct from the TGFβ response element that is further upstream. This region contains a CCAAT motif and a c-Krox motif that have been previously implicated in collagen expression. Using a gel shift assay, we found that inhibition of PKCδ with Rottlerin or the dominant negative mutant increased the binding of CCAAT-binding Factor (CBF) to the CCAAT motif but decreased the binding at the c-Krox site. Taken together, our data indicate that PKCδ inhibits type I collagen basal expression by affecting its gene transcription through a mechanism involving CBF and c-Krox in VSMCs. We believe that PKCδ is one of the key players in mediating extracellular matrix production and could contribute to pathogenesis of vascular diseases.