title: Regulated expression and role of c-Myb in the cardiovascular-directed differentiation of mouse embryonic stem cells.
abstract: BACKGROUND
  c-myb null (knockout) embryonic stem cells (ESC) can differentiate into cardiomyocytes but not contractile smooth muscle cells (SMC) in embryoid bodies (EB).
  
  
  OBJECTIVE
  To define the role of c-Myb in SMC differentiation from ESC.
  
  
  RESULTS
  In wild-type (WT) EB, high c-Myb levels on days 0-2 of differentiation undergo ubiquitin-mediated proteosomal degradation on days 2.5-3, resurging on days 4-6, without changing c-myb mRNA levels. Activin-A and bone morphogenetic protein 4-induced cardiovascular progenitors were isolated by FACS for expression of vascular endothelial growth factor receptor (VEGFR)2 and platelet-derived growth factor receptor (PDGFR)α. By day 3.75, hematopoesis-capable VEGFR2+ cells were fewer, whereas cardiomyocyte-directed VEGFR2+/PDGFRα+ cells did not differ in abundance in knockout versus WT EB. Importantly, highest and lowest levels of c-Myb were observed in VEGFR2+ and VEGFR2+/PDGFRα+ cells, respectively. Proteosome inhibitor MG132 and lentiviruses enabling inducible expression or knockdown of c-myb were used to regulate c-Myb in WT and knockout EB. These experiments showed that c-Myb promotes expression of VEGFR2 over PDGFRα, with chromatin immunopreciptation and promoter-reporter assays defining specific c-Myb-responsive binding sites in the VEGFR2 promoter. Next, FACS-sorted VEGFR2+ cells expressed highest and lowest levels of SMC- and fibroblast-specific markers, respectively, at days 7-14 after retinoic acid (RA) as compared with VEGFR2+/PDGFRα+ cells. By contrast, VEGFR2+/PDGFRα+ cells cultured without RA beat spontaneously, like cardiomyocytes between days 7 and 14, and expressed cardiac troponin. Notably, RA was required to more fully differentiate SMC from VEGFR2+ cells and completely blocked differentiation of cardiomyocytes from VEGFR2+/PDGFRα+ cells.
  
  
  CONCLUSIONS
  c-Myb is tightly regulated by proteosomal degradation during cardiovascular-directed differentiation of ESC, expanding early-stage VEGFR2+ progenitors capable of RA-responsive SMC formation.
authors: Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, Canada, M5G 1L7.
authors: Masayoshi
authors: M
authors: Ishida
authors: Omar
authors: O
authors: El-Mounayri
authors: Steven
authors: S
authors: Kattman
authors: Peter
authors: P
authors: Zandstra
authors: Hiroshi
authors: H
authors: Sakamoto
authors: Minetaro
authors: M
authors: Ogawa
authors: Gordon
authors: G
authors: Keller
authors: Mansoor
authors: M
authors: Husain
mesh_terms: D028341:Activins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015415:Biomarkers / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods; D047369:Chromatin Immunoprecipitation; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology; D014336:Troponin / Q000378:metabolism; D054875:Ubiquitination; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism
chemical_list: D015415:Biomarkers; D055415:Bone Morphogenetic Protein 4; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; D020598:Proto-Oncogene Proteins c-myb; D012333:RNA, Messenger; D014336:Troponin; C422405:activin A; D016222:Fibroblast Growth Factor 2; D028341:Activins; D014212:Tretinoin; D020796:Receptor, Platelet-Derived Growth Factor alpha; D040301:Vascular Endothelial Growth Factor Receptor-2; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde