低频脉冲电磁场通过 PC2/sAC/PKA/CREB 信号途径促进大鼠颅骨成骨细胞的矿化和成熟

目的 研究低频脉冲电磁场（PEMFs）促进成骨细胞矿化成熟作用是否与初级纤毛及定位于初级纤毛的多囊蛋白2（PC2）和sAC/PKA/CREB信号通路有关。方法 检测新生大鼠颅骨成骨细胞（ROBs）经50 Hz 0.6 mT PEMFs分别处理0、5、15、30、60、90、120 min后PC2、sAC、PKA、CREB及其磷酸化蛋白的表达水平；用盐酸阿米洛利阻断PC2功能，检测sAC/PKA/CREB信号通路活性变化及ROBs矿化成熟是否受到影响；用RNA干扰法敲低PC2表达水平后做同上检查；用免疫荧光染色法观察PC2、sAC、PKA、CREB及其磷酸化蛋白与初级纤毛的共定位情况；用RNA干扰法抑制初级纤毛发生后检测PC2和sAC/PKA/CREB信号途径相关蛋白的表达情况。结果 ROBs经PEMFs处理0、5、15、30、60、90、120 min后，PC2、sAC、p-PKA和 p-CREB表达水平均增加（P<0.01）。阻断PC2功能或敲低PC2表达水平后，sAC/PKA/CREB信号通路未被激活，成骨细胞的矿化成熟受阻（P<0.01）。PC2、sAC、p-PKA和 p-CREB可被定位于整根初级纤毛或其根部，但PKA和CREB在初级纤毛内没有分布。干扰初级纤毛后，PEMFs处理未提高PC2表达量（P<0.01），未激活sAC/PKA/CREB信号通路，也不再能促进成骨细胞矿化成熟。结论 存在于ROBs初级纤毛表面的PC2可感知并传递PEMFs发出的物理信号，通过激活PC2/sAC/PKA/CREB信号途径促进成骨细胞的矿化成熟。

Low-frequency pulsed electromagnetic fields promote osteoblast mineralization and maturation of rats through the PC2/sAC/PKA/CREB signaling pathway

Objective To explore whether the effect of low-frequency pulsed electromagnetic fields (PEMFs) in promoting osteoblast mineralization and maturation is related to the primary cilia, polycystin2 (PC2) and sAC/PKA/CREB signaling pathway. Methods We detected the expression levels of PC2, sAC, PKA, CREB and their phosphorylated proteins in primary rat calvarial osteoblasts exposed to 50 Hz 0.6 mT PEMFs for 0, 5, 15, 30, 60, 90, and 120 min. We blocked PC2 function with amiloride hydrochloride and detected the changes in the activity of sAC/PKA/CREB signal pathway and the mineralization and maturation of the osteoblasts. These examinations were repeated in the osteoblasts after specific knockdown of PC2 via RNA interference and were the co-localization of PC2, sAC, PKA, CREB and their phosphorylated proteins with the primary cilia were using immunofluorescence staining. The expressions of PC2 and the signaling proteins of sAC/PKA/CREB pathway were detected after inhibition of primary ciliation by RNA interference. Results The expression levels of PC2, sAC, p-PKA and p-CREB were significantly increased in the osteoblasts after exposure to PEMFs for different time lengths (P<0.01). Blocking PC2 function or PC2 knockdown in the osteoblasts resulted in failure of sAC/PKA/CREB signaling pathway activation and arrest of osteoblast mineralization and maturation. PC2, sAC, p-PKA and p-CREB were localized to the entire primary cilia or its roots, but PKA and CREB were not detected in the primary cilia. After interference of the primary cilia, PEMFs exposure no longer caused increase of PC2 expression and failed to activate the sAC/PKA/CREB signaling pathway or promote osteoblast mineralization and maturation. Conclusion PC2, located on the surface of the primary cilia of osteoblasts, can perceive and transmit the physical signals from PEMFs and promote the mineralization and maturation of osteoblasts by activating the PC2/sAC/PKA/CREB signaling pathway