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


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.


CONCLUSIONS
c-Myb regulates cell cycle-associated IP3R1 transcription in VSMCs via specific highly conserved Myb-binding sites in the IP3R1 promoter.
Afroze
Talat
T
Division of Cell and Molecular Biology, Toronto General Hospital Research Institute, Toronto, Ontario, Canada.
Sadi
Al Muktafi
AM
Momen
M Abdul
MA
Gu
Steven
S
Heximer
Scott
S
Husain
Mansoor
M
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002339:Carotid Arteries / Q000601:surgery; D002340:Carotid Artery Diseases / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004195:Disease Models, Animal; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008156:Luciferases; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020598:Proto-Oncogene Proteins c-myb / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; C025389:Itpr1 protein, mouse; D008562:Membrane Glycoproteins; D020598:Proto-Oncogene Proteins c-myb; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D004247:DNA; D008156:Luciferases; D002118:Calcium