初级纤毛通过sAC/cAMP/PKA途径启动低频脉冲电磁场促进骨形成信号级联反应的研究

Although low frequency pulsed electromagnetic fields (PEMFs) are characterized by no damage and easy to use in the treatment of osteoporosis, they have not been widely applied clinically today because of the unclear mechanism of action and unstable therapeutic effects. Basing on our previous report that primary cilia are indispensable for the PEMFs-induced osteogenic differentiation of osteoblasts, and the recent finding that sAC, p-PKA, BMPRII and IGF-1R are localized at primary cilia, here we hypothesized that the whole process of PEMFs-induced osteogenic differentiation was like the following: The TRP channel at the membrane of primary cilium was firstly opened by PEMFs and caused the influx of Ca2+ into cilium. The increased concentration of Ca2+ stimulated sAC to produce more cAMP from ATP and lead to the higher level of phosphorylation of PKA, a kinase specially activated by cAMP. BMPRII and IGF-1 located at primary cilium were then activated by p-PKA and left for cytoplasm accompanied by p-PKA. The p-PKA and these receptors with serine/threonine kinase activity or tyrosine kinase activity would initiate their respective downstream signal pathways like PI3K/AKT, Ras/Raf/MAPK, cAKP/PKA/CREB et al, and finally caused the happening of whole cell osteogenic signal transduction cascades. To verify this hypothesis, we would perform the following experiments using the techniques like the FRET-based Ca2+ biosensor and cAMP biosensor, RNA interference and immunofluorescence staining et al. First, we would find out whether sAC is the main adenylyl cyclase which determines the activity of cAMP/PKA pathway. Second, we would screen for the TRP channel at cilium which was sensitive to PEMFs and gave way for the influx of Ca2+. Third, at least three complete osteogenic signal transduction pathways would be clarified beginning from the primary cilia and ending at the expression of osteogenic genes. What we do would greatly help to understand the mechanism of osteogenic action of PEMFs.

低频脉冲电磁场（PEMFs）防治骨质疏松症具有无损伤和使用方便等优点，但由于作用机制不明、疗效不确切而影响了推广应用。我们已报道PEMFs促进骨形成活性离不开初级纤毛的参与，近期又发现成骨细胞初级纤毛内存在sAC、p-PKA、BMPRII和IGF-1R等，据此提出假说：PEMFs激活初级纤毛膜上的TRP通道而致Ca2+内流，使sAC及cAMP/PKA信号途径被激活，p-PKA又激活BMPRII和IGF-1R及其在胞体内的多条下游信号途径，从而启动了骨形成信号级联反应。为证明该假说，本项目拟采用基于FRET的Ca2+和cAMP生物传感器、RNA干扰和免疫荧光染色等技术开展如下研究：⑴初级纤毛内的cAMP/PKA信号途径是否由sAC主导；⑵筛选确定初级纤毛膜上对PEMFs敏感的TRP通道；⑶确定至少3条由PEMFs激活的骨形成信号途径。本项目将为阐明PEMFs促进骨形成作用机制奠定扎实基础。

低频脉冲电磁场（PEMFs）防治骨质疏松症具有无损伤和使用方便等优点，但由于作用机制不明、疗效不确切而影响了推广应用。我们在前期研究的基础上提出假说：PEMFs激活成骨细胞初级纤毛膜上的TRP通道而致Ca2+内流，使sAC及cAMP/PKA信号途径被激活，p-PKA或CREB又激活BMPRII、IGF-1R及其在胞体内的下游信号途径，从而启动了骨形成信号级联反应。为证明该假说而开展的研究工作有：⑴ sAC是否在cAMP/PKA信号途径中发挥主导作用；⑵筛选确定初级纤毛膜上对PEMFs敏感的TRP通道；⑶确定至少3条由PEMFs激活的骨形成信号途径。本项目研究获得的重要结果及其科学意义有：⑴新生大鼠颅骨成骨细胞（ROBs）共表达9种腺苷酸环化酶，其中AC2、AC4、AC6、AC8和sAC对PEMFs处理敏感，尤以sAC蛋白表达量的增加幅度最大，且只有sAC被定位于初级纤毛。敲低sAC基因表达，发现PEMFs的促骨活性消失；⑵发现PC1、PC2和TRPV4三种定位于初级纤毛的信号蛋白或Ca2+通道，其中PC1、PC2对PEMFs处理非常敏感，如敲低或抑制其功能，则PEMFs的促骨活性消失；⑶发现NO/sGC/PKG信号途径的所有成员都分布于初级纤毛有，其上游的 PI3K/AKT/eNOS也位于初级纤毛内；⑷发现IGF-1R平时不在初级纤毛中，但被PEMFs激活后发生磷酸化并进入纤毛根部，其下游是PI3K/AKT信号途径；⑸发现Wnt10b平时位于初级纤毛根部，但被PEMFs激活后全部离开初级纤毛，并激活了Wnt10b/β-catenin信号途径；⑹所有能够促进骨形成PEMFs都可显著延长初级纤毛长度。如设法抑制PEMFs的促骨活性，则其延长纤毛作用消失。以上结果的科学意义在于：⑴PEMFs促进成骨细胞分化成熟的过程可能是：PEMFs首先激活初级纤毛表面的PC1/PC2复合体，导致纤毛内Ca2+浓度升高，激活了sAC/PKA/CREB信号途径，p-PKA即可进一步激活下游信号途径，启动成骨相关信号级联反应，也可直接发生核转位，激活成骨相关基因的表达；⑵初级纤毛犹如细胞的“引信”，PEMFs须先激活初级纤毛内的sAC/PKA/CREB、等信号途径，才能激活胞体内的成骨相关信号级联反应，等于是给细胞加了一个保险。同时，初级纤毛对PEMFs等胞外信号更加敏感，只需极低能量即可被