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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:1305.)
© 2007 American Heart Association, Inc.

Vascular Biology

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

Talat Afroze; Al Muktafi Sadi; M. Abdul Momen; Steven Gu; Scott Heximer; Mansoor Husain

From the Division of Cell and Molecular Biology (T.A., A.M.S., M.A.M., M.H.), Toronto General Hospital Research Institute; Heart & Stroke Richard Lewar Centre of Excellence in Cardiovascular Research (S.H., M.H.) and the Departments of Medicine (M.H.) and Physiology (S.G., S.H., M.H.), University of Toronto, Ontario, Canada.

Correspondence to Mansoor Husain, TMDT 3-909, MaRS East, University Health Network, University of Toronto, 200 Elizabeth St, Toronto, Ontario M5G 2C4, Canada. E-mail mansoor.husain{at}utoronto.ca

Objective— 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.

Methods & Results— 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.

Conclusion— c-Myb regulates cell cycle–associated IP3R1 transcription in VSMCs via specific highly conserved Myb-binding sites in the IP3R1 promoter.

The IP3R1 promoter contains several putative binding sites for the c-Myb transcription factor, two of which are shown by gel shift and ChIP to bind c-Myb. Point mutations in these abolished promoter activity in immortalized and primary VSMCs. These data provide a mechanism for cell cycle– and c-Myb–responsive IP3R1 expression.

Key Words: inositol 1,4,5-trisphosphate receptor • c-myb • cell cycle • VSMC

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