Activation of RAF-1 through Ras and protein kinase Cα mediates 1α,25(OH)2-vitamin D3 regulation of the mitogen-activated protein kinase pathway in muscle cells

We have previously shown that stimulation of proliferation of avian embryonic muscle cells (myoblasts) by lalpha,25(OH)(2)-vitamin D-3 (1alpha,,25(OH)(2)D-3) is mediated by activation of the mitogen-activated protein kinase (MAPK; ERK1/2). To understand how lalpha,25(OH)(2)D-3 up-regulates the MAPK cascade, we have investigated whether the hormone acts upstream through stimulation of Raf-1 and the signaling mechanism by which this effect might take place. Treatment of chick myoblasts with lalpha,25(OH)(2)D-3 (1 nM) caused a fast increase of Raf-1 serine phosphorylation (1- and 3-fold over basal at 1 and 2 min, respectively), indicating activation of Raf-1 by the hormone. These effects were abolished by preincubation of cells with a specific Ras inhibitor peptide that involves Ras in 1alpha,25(OH)(2)D-3 stimulation of Raf-1. 1alpha,25(OH)(2)D-3 rapidly induced tyrosine de-phosphorylation of Ras-GTPase-activating protein, suggesting that inhibition of Ras-GTP hydrolysis is part of the mechanism by which 1alpha,25(OH)(2)D-3 activates Ras in myoblasts. The protein kinase CPKC inhibitors calphostin C, bisindolylmaleimide I, and Ro 318220 blocked lalpha,25(OH)(2)D-3-induced Raf-1 serine phosphorylation, revealing that hormone stimulation of Raf-1 also involves PKC. In addition, transfection of muscle cells with an antisense oligodeoxynucleotide against PKCalpha mRNA suppressed serine phosphorylation by lalpha,25(OH)(2)D-3. The increase in MAPK activity and tyrosine phosphorylation caused by lalpha,25(OH)(2)D-3 could be abolished by Ras inhibitor peptide, compound PD 98059, which prevents the activation of MEK by Raf-1, or incubation of cell lysates before 1alpha,25(OH)(2)D-3 exposure with an anti-Raf-1 antibody. In conclusion, these results demonstrate for the first time in a la,25(011)2% target cell that activation of Raf-1 via Ras and PKCalpha-dependent serine phosphorylation plays a central role in hormone stimulation of the MAPK-signaling pathway leading to muscle cell proliferation.