Buitrago Claudia Graciela, Pardo Veronica González, de Boland Ana R, Boland Ricardo
Departamento de Biología, Bioquímica and Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahia Blanca, Argentina.
J Biol Chem. 2003 Jan 24;278(4):2199-205. doi: 10.1074/jbc.M205732200. Epub 2002 Nov 1.
We have previously shown that stimulation of proliferation of avian embryonic muscle cells (myoblasts) by 1alpha,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 1alpha,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 1alpha,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 C (PKC) inhibitors calphostin C, bisindolylmaleimide I, and Ro 318220 blocked 1alpha,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 1alpha,25(OH)(2)D(3). The increase in MAPK activity and tyrosine phosphorylation caused by 1alpha,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 1alpha,25(OH)(2)D(3) 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.
我们之前已经表明，1α,25(OH)₂-维生素D₃（1α,25(OH)₂D₃）对禽胚胎肌肉细胞（成肌细胞）增殖的刺激作用是由丝裂原活化蛋白激酶（MAPK；ERK1/2）的激活介导的。为了了解1α,25(OH)₂D₃如何上调MAPK级联反应，我们研究了该激素是否通过刺激Raf-1在其上游起作用以及这种作用可能发生的信号传导机制。用1α,25(OH)₂D₃（1 nM）处理鸡成肌细胞导致Raf-1丝氨酸磷酸化快速增加（分别在1分钟和2分钟时比基础水平高1倍和3倍），表明该激素激活了Raf-1。用一种特异性Ras抑制肽对细胞进行预孵育可消除这些效应，该肽在1α,25(OH)₂D₃对Raf-1的刺激中涉及Ras。1α,25(OH)₂D₃迅速诱导Ras-GTP酶激活蛋白的酪氨酸去磷酸化，表明抑制Ras-GTP水解是1α,25(OH)₂D₃在成肌细胞中激活Ras的机制的一部分。蛋白激酶C（PKC）抑制剂钙泊三醇C、双吲哚马来酰胺I和Ro 318220阻断了1α,25(OH)₂D₃诱导的Raf-1丝氨酸磷酸化，表明激素对Raf-1的刺激也涉及PKC。此外，用针对PKCα mRNA的反义寡脱氧核苷酸转染肌肉细胞可抑制1α,25(OH)₂D₃引起的丝氨酸磷酸化。1α,25(OH)₂D₃引起的MAPK活性增加和酪氨酸磷酸化可被Ras抑制肽、化合物PD 98059（其可阻止Raf-1激活MEK）或在1α,25(OH)₂D₃处理前用抗Raf-1抗体孵育细胞裂解物所消除。总之，这些结果首次在1α,25(OH)₂D₃靶细胞中证明，通过Ras和PKCα依赖性丝氨酸磷酸化激活Raf-1在激素刺激导致肌肉细胞增殖的MAPK信号通路中起核心作用。
