Cellular signal transduction system in prostaglandin-stimulated osteoblast-like MC3T3-E1 cells : Phospholipid turnover and protein tyrosine phosphorylation

联合应用β-甘油磷酸和抗坏血酸可增加成骨细胞克隆细胞系MC3T3-E1中碱性磷酸酶(ALP)的活性，碱性磷酸酶是成骨细胞分化的标志物。在加入这些药剂后24小时检测到细胞增殖，在培养5天时几乎达到最高水平。虽然细胞内环磷酸腺苷水平的升高可以诱导成骨细胞分化，但这些处理并没有引起环磷酸腺苷的显著增加。β-甘油磷酸和抗坏血酸的处理分别抑制了前列腺素F_&lt；2pha&gt；(PGF_&lt；2pha&gt；)对磷脂酶C(PI-PLC)和磷脂酶D(PLD)的激活20-30%和15%。用亚型特异性PI-PLC抗体进行Western印迹分析表明，在这些分化诱导剂处理5d的细胞中，PI-PLCbeta1和PI-PLCbeta3的表达水平分别下降到未分化对照细胞的37%和67%。然而，PLC_1和蛋白激酶C同工酶(α、β1、β2、Delta、epsilon、Rheta、Zeta)的水平没有变化。PGF还可刺激蛋白酪氨酸的磷酸化和丝裂原活化蛋白(MAP)的激活。这些反应不受分化诱导剂的影响。这些结果提示PI-PLCbeta1和PI-PLCbeta3的抑制表达与成骨细胞的分化密切相关。

The combination of beta-glycerophosphate and ascorbic acid increased alkaline phosphate (ALP) activity, a maraker for osteoblastic differentiation, in clonal osteoblast like cell line MC3T3-E1. The increase was detected as 24 hours after the addition of these agents to growth medium and reached almost maximal level at 5 days of culture. Although increase in intracellular cyclic AMP level is known to induce osteoblastic acell differentiation, these treatment did not cause significant increase in cyclic AMP.Treatment of beta-glycerophsphate and ascorbic acid suppressed activaaton of phosphoinositide-specific phospholipase C (PI-PLC) and phospholipase D (PLD) in response to prostaglandin F_<2alpha> (PGF_<2alpha>) by 20-30% and 15%, respectively. Western blot analyzes with subtype-specific PI-PLC antibodies revealed that in cells treated for 5 days with these differentiation inducers, the levels of PI-PLCbeta1 and PI-PLCbeta3 decreased to 37% and 67% compared to those of undifferentiated control cells, respectively. However, the level of PLC_<gamma>1 and those of protein kinase C isozymes (alpha, beta1, beta2, delta, epsilon, rheta, zeta) remained unaltered. PGF_<2alpha> also stimulated protein tyrosine phosphorilations and activation of mitogen-activated protein (MAP) kinases. These responses were not affected by differentiation induceres. These results suggest that the suppressed expression of PI-PLCbeta1 and PI-PLCbeta3 are closely related to osteoblast cell differentiation.