Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells.
Knockout of transforming growth factor (TGF)-beta1 or components of its signaling pathway leads to embryonic death in mice due to impaired yolk sac vascular development before significant smooth muscle cell (SMC) maturation occurs. Thus the role of TGF-beta1 in SMC development remains unclear. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) recapitulate many of the events of early embryonic development and represent a more physiological context in which to study SMC development than most other in vitro systems. The present studies showed induction of the SMC-selective genes smooth muscle alpha-actin (SMalphaA), SM22alpha, myocardin, smoothelin-B, and smooth muscle myosin heavy chain (SMMHC) within a mouse ESC-EB model system. Significantly, SM2, the SMMHC isoform associated with fully differentiated SMCs, was expressed. Importantly, the results showed that aggregates of SMMHC-expressing cells exhibited visible contractile activity, suggesting that all regulatory pathways essential for development of contractile SMCs were functional in this in vitro model system. Inhibition of endogenous TGF-beta with an adenovirus expressing a soluble truncated TGF-beta type II receptor attenuated the increase in SMC-selective gene expression in the ESC-EBs, as did an antibody specific for TGF-beta1. Of interest, the results of small interfering (si)RNA experiments provided evidence for differential TGF-beta-Smad signaling for an early vs. late SMC marker gene in that SMalphaA promoter activity was dependent on both Smad2 and Smad3 whereas SMMHC activity was Smad2 dependent. These results are the first to provide direct evidence that TGF-beta1 signaling through Smad2 and Smad3 plays an important role in the development of SMCs from totipotential ESCs.
Dept. of Molecular Physiology and Biological Physics, Univ. of Virginia, 415 Lane Rd., MR5, Rm. 1220, PO Box 801394, Charlottesville, VA 22908, USA.
Sanjay
S
Sinha
Mark H
MH
Hoofnagle
Paul A
PA
Kingston
Mary E
ME
McCanna
Gary K
GK
Owens
D000199:Actins / Q000235:genetics; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015415:Biomarkers; D002454:Cell Differentiation / Q000502:physiology; D019070:Cell Lineage / Q000502:physiology; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology; D004727:Endothelium / Q000166:cytology; D015870:Gene Expression / Q000502:physiology; D051379:Mice; D016296:Mutagenesis; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering / Q000494:pharmacology; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051899:Smad2 Protein; D051900:Smad3 Protein; D039901:Totipotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000276:immunology / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D015854:Up-Regulation / Q000502:physiology
D000199:Actins; D000906:Antibodies; D015415:Biomarkers; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; C508961:Tgfb1 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II