Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
Department of Rehabilitation Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
Connect Tissue Res. 2022 Jul;63(4):359-369. doi: 10.1080/03008207.2021.1939323. Epub 2021 Jun 8.
We have demonstrated that epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells is mediated through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/ c- N-terminal kinase (SAPK/JNK), and Akt.The molecular chaperone heat shock protein 90 (HSP90) is abundantly expressed in osteoblasts. However, the role of HSP90 in osteoblast migration remains obscure.
In this study, we investigated the effect of HSP90 inhibitors on the EGF-induced migration of MC3T3-E1 cells and the mechanism.
Clonal osteoblast-like MC3T3-E1 cells were treated with the HSP90 inhibitors geldanamycin or onalespib and then stimulated with EGF. Cell migration was evaluated using the transwell cell migration assay and wound-healing assay. The viability of MC3T3-E1 cells was analyzed using the Cell Counting Kit-8. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, Akt, and protein kinase-like endoplasmic reticulum kinase (PERK) was evaluated by western blot analysis.
EGF-induced migration was significantly suppressed by geldanamycin and onalespib, evaluated by both transwell cell migration assay and wound-healing assay. Geldanamycin and onalespib did not significantly alter cell viability. Geldanamycin and onalespib markedly reduced the EGF-induced phosphorylation of p44/p42 MAP kinase, but not p38 MAP kinase or Akt. By contrast, geldanamycin and onalespib increased the EGF-induced phosphorylation of SAPK/JNK. PERK phosphorylation was not significantly affected by geldanamycin or onalespib.
Our results strongly suggest that HSP90 inhibitors reduce the EGF-induced osteoblast migration through the p44/p42 MAP kinase.
我们已经证明表皮生长因子（EGF）诱导的成骨样 MC3T3-E1 细胞迁移是通过 p44/p42 丝裂原活化蛋白（MAP）激酶、p38 MAP 激酶、应激激活蛋白激酶/ c- N 端激酶（SAPK/JNK）和 Akt 介导的。分子伴侣热休克蛋白 90（HSP90）在成骨细胞中大量表达。然而，HSP90 在成骨细胞迁移中的作用尚不清楚。
在这项研究中，我们研究了 HSP90 抑制剂对 EGF 诱导的 MC3T3-E1 细胞迁移的影响及其机制。
用 HSP90 抑制剂格尔德霉素或 onalespib 处理克隆成骨样 MC3T3-E1 细胞，然后用 EGF 刺激。用 Transwell 细胞迁移实验和划痕愈合实验评估细胞迁移。用细胞计数试剂盒-8 分析 MC3T3-E1 细胞的活力。通过 Western blot 分析评估 p44/p42 MAP 激酶、p38 MAP 激酶、SAPK/JNK、Akt 和蛋白激酶样内质网激酶（PERK）的磷酸化。
用 Transwell 细胞迁移实验和划痕愈合实验评估，EGF 诱导的迁移均被格尔德霉素和 onalespib 显著抑制。格尔德霉素和 onalespib 对细胞活力没有显著影响。格尔德霉素和 onalespib 显著降低了 EGF 诱导的 p44/p42 MAP 激酶磷酸化，但不影响 p38 MAP 激酶或 Akt。相比之下，格尔德霉素和 onalespib 增加了 EGF 诱导的 SAPK/JNK 磷酸化。PERK 磷酸化不受格尔德霉素或 onalespib 的显著影响。
我们的研究结果强烈表明，HSP90 抑制剂通过 p44/p42 MAP 激酶减少 EGF 诱导的成骨细胞迁移。
