c-Myb-dependent inositol 1,4,5-trisphosphate receptor type-1 expression in vascular smooth muscle cells

Objective-The JP3 receptor-1 (IP3R1) mediates Ca2+ signals critical to vascular smooth muscle cell (VSMC) proliferation. The cell cycle-associated transcription factor c-Myb increases Ca2+ at the G(1)/S transition. Here we show the mechanism through which c-Myb regulates expression of IP3R1. Methods & Results-Ribonuclease protection confirmed transcriptional start (TS), and qRT-PCR revealed a 6-fold increase in IP3R1 mRNA as immortalized VSMC progress from G(0) to G(1)/S. A c-Myb neutralizing antibody decreased IP3R1 mRNA expression 3-fold, and abolished the 3.4-fold increase in IP3R1 protein observed at G(1)/S. Primary aortic VSMCs in culture and proliferating carotid VSMCs in vivo showed similar regulation of IP3R1 mRNA and protein. Sequence analysis of a 3.1-Kb mouse IP3R1 promoter revealed 17 putative c-Myb binding sites. Reporter assays demonstrated a 2-fold increase in promoter activity in G(1)/S- versus G(0)-synchronized VSMCs, which was abolished by functional c-Myb knockdown or deletion of promoter sequences upstream and downstream of TS. Point mutations in Myb sites-13 or -15 significantly blunted G(1)/S-specific promoter induction in both immortalized and primary VSMCs. Gel shift and ChlP confirmed binding of c-Myb to sites-13 and -15 in G(1)/S stage VSMCs. Conclusion-c-Myb regulates cell cycle-associated IP3R1 transcription in VSMCs via specific highly conserved Myb-binding sites in the IP3R1 promoter.