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

The precise mechanism by which protein kinase C-delta (PKCdelta) inhibits cell cycle progression is not known. We investigated the regulation of cyclin D-1 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 D-1 promoter, whereas overexpression of a dominant-negative PKCdelta increased promoter activity. A PKCdelta-specific pseudosubstrate increased cyclin D-1 protein abundance. To determine the transcriptional mechanism by which PKCdelta negatively regulates cyclin D-1 expression, we transiently transfected cells with cDNAs encoding cyclin D-1 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 -33 to -30 nuclear factor-kappaB site functions as a basal level suppressor. Further, PDGF and PKCdelta responsiveness of the cyclin D; 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 D-1 promoter activity via the regulation of three distinct cis-acting regulatory elements.