Abstract 608: c-Myb is Critical for Contractile Smooth Muscle Cell Differentiation
Both in vitro and in vivo studies have implicated the c-Myb transcription factor in vascular smooth muscle cells (SMC) proliferation and in hematopoiesis. However, its role in the differentiation and maturation of contractile- as opposed to proliferating-SMC has not been investigated. In this study we demonstrate that c-myb−/− ES cells (ESC) are incapable of producing embryoid bodies (EB) with spontaneously contracting SMC, but exhibit unimpaired development of contracting cardiomyocytes. By quantitative real-time RT-PCR we found that myocardin, a critical determinant of SMC differentiation, became upregulated at day 7 in wild-type (WT), but not in c-myb−/− EB. Interestingly, we found three consensus binding motifs for c-Myb within the putative myocardin promoter. SMC-specific genes SMa-actin, SM22a and SM-myosin heavy chain (SM-MHC) reached peak mRNA expression levels by day 15 of differentiation, and were 2 to 3-fold higher in WT as compared to c-myb−/− derived EB. Likewise, FACS analysis confirmed significant differences in percentage of SMa-actin positive cells in WT (26.8±0.7%) versus c-myb−/− EB (12.3±0.4%). Temporal induction of these SMC-specific markers preceded and paralleled contractile SMC appearance, and predicted the relative (in)ability of c-myb−/− and WT ESC lines to generate EB with contracting SMC. Using chimaeric EB composed of 50% WT and 50% c-myb−/− ESC or 90% WT and 10% c-myb−/− ESC, we could demonstrate that SM-like contractions rarely occurred in the former, but were at WT levels in the latter. Importantly, data from EB faithfully predicted limited c-myb−/− cell contribution to SMC lineage in vivo, in chimaeric E11.5 embryo and adult aortas. Moreover, visceral SMC population in chimaeric embryos was nearly devoid of c-myb−/− cells. Our data are the first to implicate c-Myb in SMC differentiation from precursor stem cell-derived populations, reinforcing its role in phenotypic modulation of SMC and vascular disease.