Hakeda Y, Shiokawa M, Mano H, Kameda T, Raisz L G, Kumegawa M
Department of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama, Japan.
Endocrinology. 1997 May;138(5):1821-8. doi: 10.1210/endo.138.5.5107.
PGF2alpha stimulates the proliferation of clonal osteoblastic MC3T3-E1 cells via PGF2alpha receptor linked to phospholipase C activation. To elucidate intracellular events elicited by this receptor, we examined the effects of PGF2alpha on tyrosine phosphorylation and mitogen-activated protein kinase (MAPK) activity in MC3T3-E1 cells. PGF2alpha rapidly raised the level of phosphotyrosine of cellular proteins with Mr values of 62, 68, 72, 76, 82, 125, and 150 kDa. This PGF2alpha-induced tyrosine phosphorylation of proteins (except for pp62) was blocked by down-regulating protein kinase C (PKC) by 12-O-tetradecanoylphorbol 13-acetate pretreatment and by GF 109203X, a potent specific PKC inhibitor. The addition of PGF2alpha also transiently activated MAPK in the same range of concentrations that stimulated tyrosine phosphorylation. In addition, PGF2alpha augmented the MAPK kinase kinase activity of Raf-1, whereas basal activity of MAPK/extracellular signal-regulated protein kinase kinase was less than that of Raf-1 and was little affected by PGF2alpha. Like the tyrosine phosphorylation, these activations of Raf-1 and MAPK activities were reduced by inhibition and down-regulation of PKC. Genistein, a potent inhibitor of tyrosine kinases, did not block the Raf-1 induced by PGF2alpha, indicating a tyrosine kinase-independent pathway for Raf-1 activation. However, the tyrosine kinase inhibitor partially inhibited the MAPK activity, suggesting an involvement of another Raf-1-independent kinase cascade for activation of MAPK by PGF2alpha. Fluprostenol, a specific agonist of PGF2alpha receptor, mimicked the actions of PGF2alpha consistent with a PGF2alpha receptor pathway. Thus, the action of PGF2alpha on osteoblastic MC3T3-E1 cells appears to involve a single receptor that uses diverse interacting signal transduction systems.
前列腺素F2α（PGF2α）通过与磷脂酶C激活相关的PGF2α受体刺激克隆性成骨细胞MC3T3-E1细胞的增殖。为了阐明该受体引发的细胞内事件，我们研究了PGF2α对MC3T3-E1细胞中酪氨酸磷酸化和丝裂原活化蛋白激酶（MAPK）活性的影响。PGF2α迅速提高了分子量为62、68、72、76、82、125和150 kDa的细胞蛋白的磷酸酪氨酸水平。PGF2α诱导的蛋白质酪氨酸磷酸化（pp62除外）可通过12-O-十四酰佛波醇13-乙酸酯预处理下调蛋白激酶C（PKC）以及通过强效特异性PKC抑制剂GF 109203X来阻断。在刺激酪氨酸磷酸化的相同浓度范围内，添加PGF2α也能瞬时激活MAPK。此外，PGF2α增强了Raf-1的MAPK激酶激酶活性，而MAPK/细胞外信号调节蛋白激酶激酶的基础活性低于Raf-1，且受PGF2α的影响较小。与酪氨酸磷酸化一样，PKC的抑制和下调可降低Raf-1和MAPK活性的这些激活。酪氨酸激酶的强效抑制剂染料木黄酮并未阻断PGF2α诱导的Raf-1，表明存在一条不依赖酪氨酸激酶的Raf-1激活途径。然而，酪氨酸激酶抑制剂部分抑制了MAPK活性，提示存在另一条不依赖Raf-1的激酶级联反应参与PGF2α对MAPK的激活。氟前列醇是PGFα受体的特异性激动剂，模拟了PGF2α的作用，这与PGF2α受体途径一致。因此，PGF2α对成骨细胞MC3T3-E1细胞的作用似乎涉及一个单一受体，该受体利用多种相互作用的信号转导系统。
