Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu, Japan.
Department of Dermatology, Central Japan International Medical Center, Minokamo, Japan.
Connect Tissue Res. 2023 Mar;64(2):139-147. doi: 10.1080/03008207.2022.2109468. Epub 2022 Aug 19.
Oncostatin M produced by osteal macrophages, a cytokine that belongs to the interleukin-6 family, is implicated in bone fracture healing. Macrophage colony-stimulating factor (M-CSF) secreted from osteoblasts plays an important role in osteoclastogenesis. We have previously reported that tumor necrosis factor-α (TNF-α), a potent bone resorptive agent, stimulates the activation of p44/p42 mitogen-activated protein (MAP) kinase, Akt, and p70 S6 kinase in osteoblast-like MC3T3-E1 cells, and induces the synthesis of M-CSF at least in part via Akt.
In the present study, we investigated whether oncostatin M affects the TNF-α-induced M-CSF synthesis in MC3T3-E1 cells and the underlying mechanisms.
Clonal osteoblast-like MC3T3-E1 cells were treated with oncostatin M or rapamycin and then stimulated with TNF-α. M-CSF release was assessed by ELISA. M-CSF mRNA expression level was assessed by real-time RT-PCR. Phosphorylation of Akt, p44/p42 MAP kinase, and p70 S6 kinase was detected by Western blot analysis.
Oncostatin M dose-dependently reduced the TNF-α-stimulated M-CSF release. The expression of M-CSF mRNA induced by TNF-α was significantly suppressed by oncostatin M. Rapamycin, an inhibitor of mTOR/p70 S6 kinase, had little effect on the M-CSF release by TNF-α. Oncostatin M significantly reduced the TNF-α-induced phosphorylation of Akt and p44/p42 MAP kinase. However, the p70 S6 kinase phosphorylation by TNF-α was not affected by oncostatin M.
These results strongly suggest that oncostatin M attenuates TNF-α-stimulated synthesis of M-CSF in osteoblasts, and the inhibitory effect is exerted at a point upstream of Akt and p44/p42 MAP kinase but not p70 S6 kinase.
破骨细胞产生的细胞因子白细胞介素-6 家族成员之一的骨细胞生成素 M，与骨骨折愈合有关。成骨细胞分泌的巨噬细胞集落刺激因子 (M-CSF) 在破骨细胞生成中发挥重要作用。我们之前曾报道过，肿瘤坏死因子-α（TNF-α）是一种有效的骨吸收剂，可刺激成骨样 MC3T3-E1 细胞中 p44/p42 丝裂原活化蛋白 (MAP) 激酶、Akt 和 p70 S6 激酶的激活，并通过 Akt 诱导至少部分 M-CSF 的合成。
在本研究中，我们研究了骨细胞生成素 M 是否影响 MC3T3-E1 细胞中 TNF-α诱导的 M-CSF 合成及其潜在机制。
克隆成骨样 MC3T3-E1 细胞用骨细胞生成素 M 或雷帕霉素处理，然后用 TNF-α刺激。通过 ELISA 评估 M-CSF 释放。通过实时 RT-PCR 评估 M-CSF mRNA 表达水平。通过 Western blot 分析检测 Akt、p44/p42 MAP 激酶和 p70 S6 激酶的磷酸化。
骨细胞生成素 M 呈剂量依赖性降低 TNF-α刺激的 M-CSF 释放。TNF-α诱导的 M-CSF mRNA 表达明显受骨细胞生成素 M 抑制。mTOR/p70 S6 激酶抑制剂雷帕霉素对 TNF-α诱导的 M-CSF 释放影响不大。骨细胞生成素 M 显著降低了 TNF-α诱导的 Akt 和 p44/p42 MAP 激酶的磷酸化。然而，TNF-α 对 p70 S6 激酶的磷酸化没有影响。
这些结果强烈表明，骨细胞生成素 M 减弱了 TNF-α刺激的成骨细胞中 M-CSF 的合成，抑制作用发生在 Akt 和 p44/p42 MAP 激酶上游，但不发生在 p70 S6 激酶上。
