Involvement of tyrosine kinase activity in 1alpha,25(OH)2-vitamin D3 signal transduction in skeletal muscle cells.
In cultured chick skeletal muscle cells loaded with Fura-2, the tyrosine kinase inhibitors herbimycin A and genistein abolished both the fast inositol 1,4,5-trisphosphatedependent Ca(2+) release from internal stores and extracellular Ca(2+) influx induced by 1alpha, 25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)). Daidzein, an inactive analog of genistein, was without effects. Tyrosine phosphatase inhibition by orthovanadate increased cytosolic Ca(2+). Anti-phosphotyrosine immunoblot analysis revealed that 1alpha, 25(OH)(2)D(3) rapidly (0.5-10 min) stimulates in a concentrationdependent fashion (0.1-10 nm) tyrosine phosphorylation of several myoblast proteins, among which the major targets of the hormone could be immunochemically identified as phospholipase Cgamma (127 kDa), which mediates intracellular store Ca(2+) mobilization and external Ca(2+) influx, and the growth-related proteins mitogen-activated protein (MAP) kinase (42/44 kDa) and c-myc (65 kDa). Genistein suppressed the increase in phosphorylation and concomitant elevation of MAPK activity elicited by the sterol. Both genistein and the MAPK kinase (MEK) inhibitor PD98059 abolished stimulation of DNA synthesis by 1alpha,25(OH)(2)D(3). The sterol-induced increase in tyrosine phosphorylation of c-myc, a finding not reported before for cell growth regulators, was totally suppressed by the specific Src inhibitor PP1. These results demonstrate that tyrosine phosphorylation is a previously unrecognized mechanism involved in 1alpha,25(OH)(2)D(3) regulation of Ca(2+) homeostasis in hormone target cells. In addition, the data involve tyrosine kinase cascades in the mitogenic effects of 1alpha, 25(OH)(2)D(3) on skeletal muscle cells.
Morelli
S
S
Departamento de Biologia, Bioquimica y Farmacia, Universidad Nacional del Sur, (8000) Bahia Blanca, Argentina.
Buitrago
C
C
Vazquez
G
G
De Boland
A R
AR
Boland
R
R
D000818:Animals; D016227:Benzoquinones; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D047029:Lactams, Macrocyclic; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D051966:Phospholipase C gamma; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction / Q000187:drug effects*; D010738:Type C Phospholipases / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D016227:Benzoquinones; D005419:Flavonoids; D007527:Isoenzymes; D047029:Lactams, Macrocyclic; D016271:Proto-Oncogene Proteins c-myc; D011809:Quinones; D017828:Rifabutin; D019000:Phosphotyrosine; D014638:Vanadates; C020754:herbimycin; D004247:DNA; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases; D017027:Protein Tyrosine Phosphatases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002117:Calcitriol; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium