C-Myb–Dependent Inositol 1,4,5-Trisphosphate Receptor Type-1 Expression in Vascular Smooth Muscle Cells

Abstract

The IP3 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 G1/S transition. Here we show the mechanism through which c-Myb regulates expression of IP3R1. Ribonuclease protection confirmed transcriptional start (TS), and qRT-PCR revealed a 6-fold increase in IP3R1 mRNA as immortalized VSMC progress from G0 to G1/S. A c-Myb neutralizing antibody decreased IP3R1 mRNA expression 3-fold, and abolished the 3.4-fold increase in IP3R1 protein observed at G1/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 G1/S- versus G0-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 G1/S-specific promoter induction in both immortalized and primary VSMCs. Gel shift and ChIP confirmed binding of c-Myb to sites-13 and -15 in G1/S stage VSMCs. c-Myb regulates cell cycle-associated IP3R1 transcription in VSMCs via specific highly conserved Myb-binding sites in the IP3R1 promoter.