2002/08/01 by Kristen Page, Jing Li, Kevin C. Corbit +7
paper · doi:10.1165/ajrcmb.27.2.20010016oc
Abstract The precise mechanism by which protein kinase C-δ (PKCδ) inhibits cell cycle progression is not known. We investigated the regulation of cyclin D1 transcription by PKCδ in primary bovine airway smooth muscle cells. Overexpression of the active catalytic subunit of PKCδ attenuated platelet-derived growth factor (PDGF)-mediated transcription from the cyclin D1 promoter, whereas overexpression of a dominant-negative PKCδ increased promoter activity. A PKCδ-specific pseudosubstrate increased cyclin D1 protein abundance. To determine the transcriptional mechanism by which PKCδ 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-κB site functions as a basal level suppressor. Further, PDGF and PKCδ 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 PKCδ inhibits CRE/ATF2 binding and transactivation, activates nuclear factor-κB binding and transactivation, and attenuates Ets transactivation. These data suggest that PKCδ attenuates cyclin D1 promoter activity via the regulation of three distinct cis-acting regulatory elements.