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Departments of Molecular Genetics and Biochemistry (M.A.M., S.J.S., T.P.D., G.J.N., T.E.S.) and Medicine (G.J.N.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Self-renewal and differentiation of embryonic stem (ES) cells are regulated by cytokines and growth factors through tyrosine kinase-dependent signaling pathways. In murine ES cells, signals for self-renewal are generated by the leukemia inhibitory factor (LIF). LIF and other growth factors are linked to the activation of the Src family of cytoplasmic protein-tyrosine kinases (SFKs), which consists of eight members having shared structural architecture. In this article, we show that murine ES cells express seven SFKs, three of which (Hck, Src, and Fyn) exhibit constitutive activity in self-renewing ES cells. Differentiation of ES cells to embryoid bodies was associated with rapid transcriptional silencing of Hck and Lck and with the loss of the corresponding kinase proteins. The expression of other family members remained relatively constant, although some loss of Fgr and Lyn proteins was observed during differentiation. Like ES cells, embryoid bodies maintained constitutive Src and Fyn kinase activity. Partial inhibition of endogenous SFK activity with the ATP-competitive inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or Src kinase inhibitor-1 induced differentiation of ES cells in the presence of LIF. In contrast, suppression of all SFK activity with higher concentrations of these inhibitors, or with the more potent compound A-419259 (Bioorg Med Chem Lett 12:1683-1686, 2002) blocked differentiation in response to LIF withdrawal. It is surprising that these inhibitor-treated cells remained pluripotent despite the absence of LIF. Our results implicate individual members of the Src kinase family in distinct ES cell renewal and differentiation pathways and show that small-molecule SFK inhibitors can control ES cell fate.
Address correspondence to: Dr. Thomas E. Smithgall, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, E1240 Biomedical Science Tower, Pittsburgh, PA 15261. E-mail: tsmithga{at}pitt.edu
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