Abstract 1163: IP3-Regulated Ca2+ Release and c-Myb Dependent IP3R1 Expression During Cell Cycle Progression of Vascular Smooth Muscle Cells
Background: Inositol 1,4,5 trisphosphate receptor subtype-1 (IP3R1) is the principal mediator of IP3-induced Ca2+ release (IICR)and capacitative Ca2+ entry, signals critical to the proliferation of vascular smooth muscle cells (VSMC). The cell cycle-associated transcription factor c-Myb is known to mediate increased resting [Ca2+]i and stored Ca2+ release at the G1/S interface of VSMC. Here we show the mechanism and relevance of c-Myb-dependent IP3R1 expression in cell cycle.
Methods & Results: Ratiometric calcium imaging in cell cycle synchronized mouse VSMC revealed a 2-fold rise in basal [Ca2+]i (G0: 67±9 vs. G1/S: 133±9 nM, p < 0.001) and 2.2- and 1.2-fold increases in UTP-stimulated IICR at G0 (152 ± 14 nM, p < 0.001) and G1/S (163 ± 11, p < 0.001) respectively, suggesting depletion of IP3R-regulated Ca2+ stores at G1/S. Ribonuclease protection assays (RPA) confirmed transcriptional start and qRT-PCR revealed a 6-fold increase in IP3R1 mRNA as cell cycle-synchronized mouse VSMC progress from G0 to G1/S. Electroporation of a construct encoding a c-Myb neutralizing antibody led to a 3-fold decrease in IP3R1 mRNA level compared to electroporation with vector DNA (p = 0.003; n > 5). Sequence analysis of a 3.1 Kb mouse IP3R1 promoter fragment revealed 17 putative c-Myb binding sites, 12 upstream and 5 downstream of transcriptional start. Reporter assays employing this fragment demonstrated a 2-fold increase in promoter activity in G1/S- vs. G0- synchronized VSMC, which was abolished by c-Myb knockdown. Deletion analysis showed that promoter sequences upstream, as well as downstream, of transcriptional start severely curtailed G1/S-associated reporter activity, while point mutations introduced into Myb sites-13 and -15 specifically abolished IP3R1 promoter activity. Gel shift assays as well as chromatin immunoprecipitation experiments confirmed that Myb sites-13 and -15 bind the c-Myb present in nuclear extracts of G1/S stage VSMC.
Conclusion: c-Myb regulates cell cycle-associated IP3R1 expression in VSMC via specific highly conserved Myb-binding sites in the IP3R1 promoter.