Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan.
Bone. 2010 Apr;46(4):1068-74. doi: 10.1016/j.bone.2010.01.378. Epub 2010 Jan 28.
We previously reported that basic fibroblast growth factor (FGF-2) stimulates the release of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates the VEGF release in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is involved in FGF-2-stimulated VEGF release in MC3T3-E1 cells. FGF-2 induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632, a specific inhibitor of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly enhanced the FGF-2-stimulated VEGF release. Fasudil, another Rho-kinase inhibitor, also amplified the VEGF release. FGF-2 significantly stimulated VEGF accumulation and fasudil enhanced FGF-2-stimulated VEGF accumulation also in whole cell lysates. Neither Y27632 nor fasudil affected the phosphorylation levels of p44/p42 MAP kinase or p38 MAP kinase. Y27632 and fasudil markedly strengthened the FGF-2-induced phosphorylation of SAPK/JNK. Y27632 as well as fasudil enhanced FGF-2-stimulated VEGF release and Y27632 enhanced the FGF-2-induced phosphorylation levels of SAPK/JNK also in human osteoblasts. These results strongly suggest that Rho-kinase negatively regulates FGF-2-stimulated VEGF release in osteoblasts.
我们之前曾报道过，碱性成纤维细胞生长因子（FGF-2）通过 p44/p42 有丝分裂原激活蛋白（MAP）激酶和应激激活蛋白激酶/ c-Jun N 末端激酶（SAPK/JNK）刺激骨细胞样 MC3T3-E1 细胞中血管内皮生长因子（VEGF）的释放，并且 FGF-2 激活的 p38 MAP 激酶负调节骨细胞样 MC3T3-E1 细胞中 VEGF 的释放。在本研究中，我们研究了 Rho-激酶是否参与 FGF-2 刺激的 MC3T3-E1 细胞中 VEGF 的释放。FGF-2 诱导肌球蛋白磷酸酶靶向亚单位（MYPT-1）的磷酸化，MYPT-1 是 Rho-激酶的底物。Rho-激酶的特异性抑制剂 Y27632 减弱了 MYPT-1 的磷酸化，显著增强了 FGF-2 刺激的 VEGF 释放。另一种 Rho-激酶抑制剂法舒地尔也放大了 VEGF 的释放。FGF-2 显著刺激 VEGF 积累，法舒地尔增强了 FGF-2 刺激的 VEGF 积累，也在全细胞裂解物中。Y27632 或法舒地尔均不影响 p44/p42 MAP 激酶或 p38 MAP 激酶的磷酸化水平。Y27632 和法舒地尔明显增强了 SAPK/JNK 的磷酸化。Y27632 和法舒地尔增强了 FGF-2 刺激的 VEGF 释放，Y27632 还增强了 FGF-2 诱导的 SAPK/JNK 磷酸化水平，在人成骨细胞中也是如此。这些结果强烈表明，Rho-激酶负调节成骨细胞中 FGF-2 刺激的 VEGF 释放。
