Endogenously activated mGlu5 metabotropic glutamate receptors sustain the increase in c-Myc expression induced by leukaemia inhibitory factor in cultured mouse embryonic stem cells.
We have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read-out the immediate early gene, c-Myc. The selective mGlu5 receptor antagonist, 2-methyl-6-(phenylenthynyl)pyridine (MPEP; 1 mum), reduced the increase in c-Myc protein levels induced by LIF by enhancing c-Myc ubiquitination. A reduction in c-Myc levels was also observed following small interfering RNA-mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase-3beta phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol-3-kinase (PI-3-K) substrate, AKT. In our hands, activated PI-3-K reduced the stability of c-Myc, because (i) the PI-3-K inhibitor, LY294002, prevented the reduction in c-Myc levels induced by MPEP; and (ii) over-expression of AKT promoted c-Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol-13-acetate. We conclude that endogenous activation of mGlu5 receptors sustains the increase in c-Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase-3beta and PI-3-K, both effects resulting from the activation of PKC.
Spinsanti
Paola
P
Department of Human Physiology and Pharmacology, University of Rome, Rome, Italy.
De Vita
Teresa
T
Di Castro
Sara
S
Storto
Marianna
M
Formisano
Pietro
P
Nicoletti
Ferdinando
F
Melchiorri
Daniela
D
D000818:Animals; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental; D016259:Genes, myc; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D015850:Interleukin-6 / Q000502:physiology*; D053605:Leukemia Inhibitory Factor; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*
D018691:Excitatory Amino Acid Antagonists; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D011725:Pyridines; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; C121465:6-methyl-2-(phenylethynyl)pyridine; D019869:Phosphatidylinositol 3-Kinases; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3