Early stage-specific inhibitions of cardiomyocyte differentiation and expression of Csx/Nkx-2.5 and GATA-4 by phosphatidylinositol 3-kinase inhibitor LY294002.
Inhibition of phosphatidylinositol 3-kinase (PI3-kinase) has been reported to block cardiomyocyte differentiation. However, at which stage PI3-kinase plays this important role and what its molecular targets are remain unknown. To answer these questions, we induced cardiomyocyte differentiation of P19CL6 mouse embryonal carcinoma cells and investigated the activation of PI3-kinase by analyzing phospho-Akt. We also treated P19CL6 cells with the PI3-kinase-specific inhibitor LY294002 either continuously or at various time points and monitored the expression of cardiac contractile proteins and transcription factors. Most cells differentiated into sarcomeric myosin heavy chain (MHC)-positive cardiomyocytes on day 16 after induction. An increase in phospho-Akt was observed after induction and was maintained throughout the differentiation. LY294002 treatment restricted to the phase from days 0 to 4 was sufficient to inhibit cardiomyocyte differentiation in a dose-dependent manner. In contrast, LY294002 treatment either from days 4 to 8 or from days 8 to 12 did not cause significant changes in sarcomeric MHC expression. LY294002 treatment from days 0 to 4 also suppressed Csx/Nkx-2.5 and GATA-4 expression. These results demonstrate that PI3-kinase becomes activated and plays a pivotal role at a very early stage of cardiomyocyte differentiation, possibly by modulating the expression of the cardiac transcription factors.
Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Atsuhiko T
AT
Naito
Aki
A
Tominaga
Masahito
M
Oyamada
Yumiko
Y
Oyamada
Isao
I
Shiraishi
Koshiro
K
Monzen
Issei
I
Komuro
Tetsuro
T
Takamatsu
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004334:Drug Administration Schedule; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050991:GATA4 Transcription Factor; D006321:Heart / Q000196:embryology*; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D014157:Transcription Factors / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*
D002867:Chromones; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D050991:GATA4 Transcription Factor; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D009025:Morpholines; D018995:Myosin Heavy Chains; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D014157:Transcription Factors; C095552:NKX2-5 protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one