Regulation of airway smooth muscle cyclin D1 transcription by protein kinase C-delta.

Abstract

The precise mechanism by which protein kinase C-delta (PKCdelta) inhibits cell cycle progression is not known. We investigated the regulation of cyclin D1 transcription by PKCdelta in primary bovine airway smooth muscle cells. Overexpression of the active catalytic subunit of PKCdelta attenuated platelet-derived growth factor (PDGF)-mediated transcription from the cyclin D1 promoter, whereas overexpression of a dominant-negative PKCdelta increased promoter activity. A PKCdelta-specific pseudosubstrate increased cyclin D1 protein abundance. To determine the transcriptional mechanism by which PKCdelta negatively regulates cyclin D1 expression, we transiently transfected cells with cDNAs encoding cyclin D1 promoter 5' deletions and site mutations in the context of a -66 promoter fragment. We found that the -57 to -52 CRE/ATF2 site functions as a basal level and PDGF enhancer, whereas the -39 to -30 nuclear factor-kappaB site functions as a basal level suppressor. Further, PDGF and PKCdelta responsiveness of the cyclin D1 promoter was maintained following 5' deletion to the Ets-containing -22 minimal promoter. Finally, using electrophoretic mobility gel shift and reporter assays, we determined that PKCdelta inhibits CRE/ATF2 binding and transactivation, activates nuclear factor-kappaB binding and transactivation, and attenuates Ets transactivation. These data suggest that PKCdelta attenuates cyclin D1 promoter activity via the regulation of three distinct cis-acting regulatory elements.