Different roles of H-ras for regulation of myosin heavy chain promoters in satellite cell-derived muscle cell culture during proliferation and differentiation.
The effect of constitutively activated proto-oncogene H-ras (H-rasQ61L) on the regulation of myosin heavy chain (MHC) promoter activities was investigated in rabbit satellite cell-derived muscle cell culture during the proliferation stage and early and later stages of differentiation, respectively. During proliferation, overexpression of H-rasQ61L did not affect basal level of activity of the slow MHCI/beta or the fast MHCIId/x promoter luciferase reporter gene construct in transient transfection assays. By contrast, H-rasQ61L affected both MHC promoter activities during differentiation, and this effect changes from inactivation after 2 days to activation after 4 days of differentiation. The activating effect of H-rasQ61L on both MHC promoters after 4 days of differentiation was significantly reduced by LY-294002, a specific inhibitor of the phosphoinositol-3-kinase (PI3K), a downstream target of Ras. Furthermore, the protein kinase Akt (protein kinase B), a downstream target of PI3k, was activated 4 days after initiation of differentiation in myotubes overexpressing H-rasQ61L. By contrast, inhibition of another Ras downstream pathway, mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated protein kinase 1/2 (MKK1/2-ERK1/2-MAPK), increased activities of both MHC promoters, indicating a suppressive role of this pathway. Moreover, the Ras-PI3K-Akt signaling pathway is involved in the activation of MHCI/beta and IId/x promoters in a later stage of differentiation of muscle cells, presumably by a known inhibiting effect of activated Akt on the MKK1/2-ERK1/2-MAPK pathway. The experiments demonstrate that during differentiation of muscle cells activated H-ras is an important regulator of MHC isoform promoter function with opposite effects during early and later stages.
Scholz
Michael E
ME
Department of Physiology, Hannover Medical School, Hannover, Germany.
Meissner
Joachim D
JD
Scheibe
Renate J
RJ
Umeda
Patrick K
PK
Chang
Kin-Chow
KC
Gros
Gerolf
G
Kubis
Hans-Peter
HP
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D011905:Genes, ras; D020935:MAP Kinase Signaling System / Q000502:physiology; D009025:Morpholines / Q000494:pharmacology; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011401:Promoter Regions, Genetic; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011817:Rabbits; D032496:Satellite Cells, Skeletal Muscle / Q000166:cytology / Q000502:physiology*; D015398:Signal Transduction
D002867:Chromones; D009025:Morpholines; D018995:Myosin Heavy Chains; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D016283:Proto-Oncogene Proteins p21(ras); C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one