Hurley M M, Marcello K, Abreu C, Kessler M
Department of Medicine, University of Connecticut Health Center, Farmington, USA.
J Bone Miner Res. 1996 Sep;11(9):1256-63. doi: 10.1002/jbmr.5650110910.
Basic fibroblast growth factor (bFGF) is a potent mitogen for bone. In this study, we utilized the clonal rat osteoblastic cell line, Py1a, to examine signal transduction by bFGF and to determine the role of mitogen activated protein kinases (MAPK) and induction of c-fos mRNA in the mitogenic response to bFGF. Stimulation of [3H]thymidine incorporation (TDR) into DNA by bFGF was determined in the presence of phorbol myristate acetate of (PMA) to down-regulate the protein kinase C (PKC) pathway, genistein, an inhibitor of tyrosine kinase and H-7, a PKC inhibitor, bFGF 10(-8) M and PMA 10(-7) M increased TDR by 242 and 245%, respectively. Treatment with bFGF or PMA for 5 or 30 minutes increased tyrosine phosphorylation of multiple proteins, and immunoblotting with MAPK-specific antibody revealed that two of these bands were the 42 and 44 kD isoforms of MAPK. PMA and bFGF induced c-fos mRNA expression at 30 minutes. Genistein at 10 micrograms/ml blocked the mitogenic effect of bFGF and partially inhibited the mitogenic effect of PMA. Genistein at 100 micrograms/ml also blocked both bFGF- and PMA-induced increases in c-fos mRNA. A 24 h pretreatment with PMA at 10(-7) M inhibited the mitogenic response, tyrosine phosphorylation of MAPK, and induction of c-fos mRNA subsequent to the addition of PMA, but not bFGF. H-7 at 50 microM blocked bFGF-induced mitogenesis and c-fos induction, but did not inhibit bFGF-induced tyrosine phosphorylation of MAPK. In this study, we show that the signaling pathway of bFGF and PMA are similar in that they both induce tyrosine phosphorylation of MAP kinases and activate c-fos. However, the signaling pathways ultimately diverge in that once the PKC pathway is down-regulated by PMA pretreatment or blocked by the PKC inhibitor H-7, tyrosine phosphorylation of MAP kinase, c-fos induction, and the mitogenic effect of PMA is blocked. In contrast, down-regulation of the PKC pathway inhibits c-fos and the mitogenic response to bFGF, but not bFGF's effects on tyrosine phosphorylation of MAP kinase.
碱性成纤维细胞生长因子（bFGF）是一种有效的骨促分裂原。在本研究中，我们利用克隆大鼠成骨细胞系Py1a来研究bFGF的信号转导，并确定丝裂原活化蛋白激酶（MAPK）的作用以及c-fos mRNA的诱导在对bFGF的促有丝分裂反应中的作用。在佛波醇肉豆蔻酸酯（PMA）存在的情况下测定bFGF对[3H]胸腺嘧啶核苷掺入（TDR）到DNA中的刺激作用，以下调蛋白激酶C（PKC）途径，还使用了酪氨酸激酶抑制剂染料木黄酮和PKC抑制剂H-7，bFGF 10(-8) M和PMA 10(-7) M分别使TDR增加了242%和245%。用bFGF或PMA处理5或30分钟可增加多种蛋白质的酪氨酸磷酸化，用MAPK特异性抗体进行免疫印迹显示其中两条带是MAPK的42 kD和44 kD同工型。PMA和bFGF在30分钟时诱导c-fos mRNA表达。10微克/毫升的染料木黄酮阻断了bFGF的促有丝分裂作用并部分抑制了PMA的促有丝分裂作用。100微克/毫升的染料木黄酮也阻断了bFGF和PMA诱导的c-fos mRNA增加。用10(-7) M的PMA进行24小时预处理可抑制促有丝分裂反应、MAPK的酪氨酸磷酸化以及在加入PMA后（但不是bFGF后）c-fos mRNA的诱导。50微摩尔的H-7阻断了bFGF诱导的有丝分裂和c-fos诱导，但不抑制bFGF诱导的MAPK酪氨酸磷酸化。在本研究中，我们表明bFGF和PMA的信号通路相似，因为它们都诱导MAP激酶的酪氨酸磷酸化并激活c-fos。然而，信号通路最终有所不同，即一旦PKC途径被PMA预处理下调或被PKC抑制剂H-7阻断，MAP激酶的酪氨酸磷酸化、c-fos诱导以及PMA的促有丝分裂作用就会被阻断。相比之下，PKC途径的下调抑制了c-fos和对bFGF的促有丝分裂反应，但不影响bFGF对MAP激酶酪氨酸磷酸化的作用。
