Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan.
Life Sci. 2012 Jan 2;90(1-2):71-6. doi: 10.1016/j.lfs.2011.10.023. Epub 2011 Nov 9.
We have previously reported that platelet-derived growth factor (PDGF)-BB stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that the activation of p44/p42 mitogen-activated protein (MAP) kinase, p38MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is implicated in the IL-6 synthesis. In the present study,we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in the PDGF-BB-stimulated IL-6 synthesis in MC3T3-E1 cells.
The levels of IL-6 were measured by ELISA. The phosphorylation of each protein kinases was analyzed by Western blotting. The mRNA levels of IL-6 were determined by real-time RT-PCR.
PDGF-BB time-dependently induced the phosphorylation of AMPK. Compound C, an inhibitor of AMPK, which reduced PDGF-BB-induced acetyl-CoA carboxylase phosphorylation, dose-dependently suppressed the PDGF-BB-stimulated IL-6 release. In addition, the PDGF-BB-stimulated IL-6 release in human osteoblasts was also inhibited by compound C. The mRNA expression of IL-6 induced by PDGF-BB was markedly reduced by compound C. The PDGF-BB-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK was inhibited by compound C.
These results strongly suggest that AMPK positively regulates PDGF-BB-stimulated IL-6 synthesis via the MAP kinases in osteoblasts.
我们之前曾报道过血小板衍生生长因子（PDGF）-BB 可刺激成骨样 MC3T3-E1 细胞中白细胞介素 6（IL-6）的合成，IL-6 是一种强有力的破骨细胞因子，而丝裂原激活蛋白激酶（MAPK）p44/p42、p38MAPK 和应激激活蛋白激酶/c-Jun N-末端激酶（SAPK/JNK）的激活与 IL-6 的合成有关。在本研究中，我们研究了能量代谢调节剂 AMP 激活蛋白激酶（AMPK）是否参与 PDGF-BB 刺激的 MC3T3-E1 细胞中 IL-6 的合成。
通过 ELISA 法测定 IL-6 水平。通过 Western 印迹法分析各蛋白激酶的磷酸化情况。通过实时 RT-PCR 法测定 IL-6 的 mRNA 水平。
PDGF-BB 可时间依赖性诱导 AMPK 磷酸化。AMPK 的抑制剂 Compound C 可降低 PDGF-BB 诱导的乙酰辅酶 A 羧化酶磷酸化，且呈剂量依赖性抑制 PDGF-BB 刺激的 IL-6 释放。此外，Compound C 也可抑制人成骨细胞中 PDGF-BB 刺激的 IL-6 释放。Compound C 可明显降低 PDGF-BB 诱导的 IL-6 mRNA 表达。PDGF-BB 诱导的 p44/p42 MAPK、p38 MAPK 和 SAPK/JNK 磷酸化被 Compound C 抑制。
这些结果强烈表明，在成骨细胞中，AMPK 通过 MAPK 正向调控 PDGF-BB 刺激的 IL-6 合成。
