Abstract 5595: Regulated Expression and Function of c-Myb During Cardiovascular-directed Differentiation of Mouse Embryonic Stem Cells
Background: c-Myb plays a pivotal role in hematopoiesis and in proliferation of vascular smooth muscle cells (VSMC). We showed that c-myb null (KO) mouse embryonic stem cells (ESC) were unable to form contractile VSMC, but formed contracting cardiomyocytes (CM) in embryoid bodies (EB) with seemingly greater efficiency. This suggested lineage-defining effects of c-Myb during CM and VSMC differentiation.
Methods & Results: To investigate underlying mechanisms, we first studied c-Myb levels in wild-type (WT) ESC. In serum-conditioned spontaneously differentiating WT-ESC, Western blot revealed high c-Myb levels on days 0 –2, rapid/complete loss of c-Myb on days 2.5 to 3, and subsequent re-expression during days 4 – 6 of differentiation. The loss of c-Myb between days 2.5–3.0 did not occur with any change in mRNA, but was completely blocked by proteosome inhibitor MG132. We next studied ESC-derived cardiovascular progenitor populations by flow cytometry using VEGFR2 and PDGFRα makers. By day 3, VEGFR2+/PDGFRα- cells were significantly fewer, while VEGFR2-/PDGFRα + and VEGFR2+/PDGFRα+ cells were more frequent, in KO vs. WT EB. In serum-free cardiac-directed differentiation cultures of WT ESC, Western blot revealed the highest levels of c-Myb expression at day 2, with rapid loss by day 3 in VEGFR2+/PDGFRα+ cells. Of interest, VEGFR2+/PDGFRα- cells showed the highest levels of c-Myb, while VEGFR2+/PDGFRα+ cells expressed the lowest. Subsequently, c-Myb was not detectable in beating CM populations and differentiating CM that emerged under these conditions. Lentiviral constructs enabling inducible expression of a shRNA against c-Myb were used to temporally knock down c-Myb expression and observe corresponding effects on cardiovascular differentiation. Finally, co-IP and mass spectrometry identified TIF1β, a known negative regulator of c-Myb, as a putative c-Myb partner proteins affecting c-Myb-modulated cardiovascular lineage specification.
Summary: These data show that c-Myb is tightly regulated by proteosomal degradation and protein-protein interactions during ESC differentiation, and that it plays a role in the differentiation of VEGFR2+/PDGFRα- cells, which may represent the source of the contractile VSMC observed in EBs.