Regulation of osteoblast differentiation by Pasteurella multocida toxin (PMT):: A role for Rho GTPase in bone formation
Regulation of osteoblast differentiation by Pasteurella multocida toxin (PMT):: A role for Rho GTPase in bone formation
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DOI：
10.1359/jbmr.040105
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发表时间：
2004-04-01
影响因子：
6.2
通讯作者：
Grigoriadis, AE
中科院分区：
文献类型：
--
作者：
Harmey, D;Stenbeck, G;Grigoriadis, AE
使用原代小鼠颅骨细胞研究Rho-Rho激酶信号通路对成骨细胞分化的作用。细菌毒素PMT抑制，而Rho-ROK抑制剂刺激，成骨细胞分化和骨结节形成。这些作用与改变的BMP-2和-4表达相关。这些数据表明Rho-ROK信号在成骨细胞分化和骨形成中的重要性。引言：控制成骨细胞分化的信号转导途径还不清楚。在这项研究中，我们使用多杀性巴氏杆菌毒素（PMT），一种独特的细菌毒素，激活小的GT-Rho，和特定的Rho抑制剂，以探讨Rho在成骨细胞分化和骨形成的作用，在vitro.Materials和Methods：原代小鼠颅骨成骨细胞培养，以探讨重组PMT和Rho-Rho激酶（ROK）抑制剂对成骨细胞分化和骨结节形成的影响。用北方印迹和RT-PCR分析成骨细胞基因表达，鬼笔环肽染色和共聚焦microscope.Results后，肌动蛋白重排可视化：PMT刺激原代小鼠颅骨细胞的增殖，并显着抑制成骨细胞前体细胞向骨结节的分化，同时抑制成骨细胞标志物基因的表达。增殖的刺激和分化的抑制之间没有明显的因果关系。PMT引起细胞骨架重排，因为激活的Rho，和抑制骨结节完全逆转的Rho抑制剂C3转移酶和部分逆转的Rho效应，ROK的抑制剂。有趣的是，Rho和ROK抑制剂单独有效地刺激成骨细胞分化、基因表达和骨结节形成。最后，PMT抑制而ROK抑制剂刺激骨形态发生蛋白（BMP）-2和-4 mRNA的表达，为它们影响骨结节形成提供了可能的机制。结论：这些结果表明PMT通过涉及Rho-ROK途径的机制抑制成骨细胞分化，该途径是成骨细胞分化的重要负调节因子。相反，ROK抑制剂刺激成骨细胞分化，可能是潜在的有用的骨合成代谢剂。
The role of the Rho-Rho kinase signaling pathway on osteoblast differentiation was investigated using primary mouse calvarial cells. The bacterial toxin PMT inhibited, whereas Rho-ROK inhibitors stimulated, osteoblast differentiation and bone nodule formation. These effects correlated with altered BMP-2 and -4 expression. These data show the importance of Rho-ROK signaling in osteoblast differentiation and bone formation.Introduction: The signal transduction pathways controlling osteoblast differentiation are not well understood. In this study, we used Pasteurella multocida toxin (PMT), a unique bacterial toxin that activates the small GTPase Rho, and specific Rho inhibitors to investigate the role of Rho in osteoblast differentiation and bone formation in vitro.Materials and Methods: Primary mouse calvarial osteoblast cultures were used to investigate the effects of recombinant PMT and Rho-Rho kinase (ROK) inhibitors on osteoblast differentiation and bone nodule formation. Osteoblast gene expression was analyzed using Northern blot and RT-PCR, and actin rearrangements were visualized after phalloidin staining and confocal microscopy.Results: PMT stimulated the proliferation of primary mouse calvarial cells and markedly inhibited the differentiation of osteoblast precursors to bone nodules with a concomitant inhibition of osteoblastic marker gene expression. There was no apparent causal relationship between the stimulation of proliferation and inhibition of differentiation. PMT caused cytoskeletal rearrangements because of activation of Rho, and the inhibition of bone nodules was completely reversed by the Rho inhibitor C3 transferase and partly reversed by inhibitors of the Rho effector, ROK. Interestingly, Rho and ROK inhibitors alone potently stimulated osteoblast differentiation, gene expression, and bone nodule formation. Finally, PMT inhibited, whereas ROK inhibitors stimulated, bone morphogenetic protein (BMP)-2 and -4 mRNA expression, providing a possible mechanism for their effects on bone nodule formation.Conclusions: These results show that PMT inhibits osteoblast differentiation through a mechanism involving the Rho-ROK pathway and that this pathway is an important negative regulator of osteoblast differentiation. Conversely, ROK inhibitors stimulate osteoblast differentiation and may be potentially useful as anabolic agents for bone.