Involvement of tyrosine kinase activity in 1α,25(OH)2-vitamin D3 signal transduction in skeletal muscle cells

Abstract

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 Ca2+ release from internal stores and extracellular Ca2+ influx induced by 1α,25(OH)2-vitamin D3 (1α,25(OH)2D3). Daidzein, an inactive analog of genistein, was without effects. Tyrosine phosphatase inhibition by orthovanadate increased cytosolic Ca2+. Anti-phosphotyrosine immunoblot analysis revealed that 1α,25(OH)2D3 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 Cγ (127 kDa), which mediates intracellular store Ca2+ mobilization and external Ca2+ 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 1α,25(OH)2D3. 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 1α,25(OH)2D3 regulation of Ca2+ homeostasis in hormone target cells. In addition, the data involve tyrosine kinase cascades in the mitogenic effects of 1α,25(OH)2D3 on skeletal muscle cells.