Abstract：
We and others have seen that osteocytes sense high-impact osteogenic mechanical loading via transient plasma membrane disruptions (PMDs) which initiate downstream mechanotransduction. However, a PMD must be repaired for the cell to survive this wounding event. Previous work suggested that the protein Prkd1 (also known as PKCμ) may be a critical component of this PMD repair process, but the specific role of Prkd1 in osteocyte mechanobiology had not yet been tested. We treated MLO-Y4 osteocytes with Prkd1 inhibitors (Go6976, kbNB 142-70, staurosporine) and generated an osteocyte-targeted (Dmp1-Cre) Prkd1 conditional knockout (CKO) mouse. PMD repair rate was measured via laser wounding and FM1-43 dye uptake, PMD formation and post-wounding survival were assessed via fluid flow shear stress (50 dyn/cm2), and in vitro osteocyte mechanotransduction was assessed via measurement of calcium signaling. To test the role of osteocyte Prkd1 in vivo, Prkd1 CKO and their wildtype (WT) littermates were subjected to 2 weeks of unilateral axial tibial loading and loading-induced changes in cortical bone mineral density, geometry, and formation were measured. Prkd1 inhibition or genetic deletion slowed osteocyte PMD repair rate and impaired post-wounding cell survival. These effects could largely be rescued by treating osteocytes with the FDA-approved synthetic copolymer Poloxamer 188 (P188), which was previously shown to facilitate membrane resealing and improve efficiency in the repair rate of PMD in skeletal muscle myocytes. In vivo, while both WT and Prkd1 CKO mice demonstrated anabolic responses to tibial loading, the magnitude of loading-induced increases in tibial BMD, cortical thickness, and periosteal mineralizing surface were blunted in Prkd1 CKO as compared to WT mice. Prkd1 CKO mice also tended to show a smaller relative difference in the number of osteocyte PMD in loaded limbs and showed greater lacunar vacancy, suggestive of impaired post-wounding osteocyte survival. While P188 treatment rescued loading-induced increases in BMD in the Prkd1 CKO mice, it surprisingly further suppressed loading-induced increases in cortical bone thickness and cortical bone formation. Taken together, these data suggest that Prkd1 may play a pivotal role in the regulation and repair of the PMD response in osteocytes and support the idea that PMD repair processes can be pharmacologically targeted to modulate downstream responses, but suggest limited utility of PMD repair-promoting P188 in improving bone anabolic responses to loading.
摘要：
我们和其他人已经看到，骨细胞通过瞬时质膜破坏（PMD）感知高影响的成骨机械负荷，从而引发下游机械传导。然而,必须修复PMD以使细胞在该损伤事件中存活。以前的工作表明，蛋白质Prkd1（也称为PKCμ）可能是这种PMD修复过程的关键组成部分，但Prkd1在骨细胞力学生物学中的具体作用尚未得到证实.我们用Prkd1抑制剂（Go6976，kbNB142-70，星形孢菌素）处理MLO-Y4骨细胞，并产生了针对骨细胞的（Dmp1-Cre）Prkd1条件敲除（CKO）小鼠。通过激光损伤和FM1-43染料摄取测量PMD修复率，通过流体流剪切应力(50dyn/cm2)评估PMD形成和创伤后存活率。通过测量钙信号来评估体外骨细胞的机械转导。为了测试骨细胞Prkd1在体内的作用，Prkd1CKO及其野生型（WT）同窝进行了2周的单侧胫骨轴向负荷和负荷诱导的皮质骨矿物质密度变化，几何图形,并对地层进行了测量。Prkd1抑制或遗传缺失减慢了骨细胞PMD修复率并损害了创伤后细胞的存活。这些影响在很大程度上可以通过用FDA批准的合成共聚物泊洛沙姆188（P188）处理骨细胞来挽救，先前已证明可促进膜的重新密封并提高骨骼肌肌细胞中PMD的修复率。在体内,而WT和Prkd1CKO小鼠均表现出对胫骨负荷的合成代谢反应，载荷引起的胫骨BMD增加的幅度，皮质厚度,与WT小鼠相比，Prkd1CKO中的骨膜矿化表面变钝。Prkd1CKO小鼠在加载肢体中骨细胞PMD的数量也倾向于显示较小的相对差异，并显示较大的腔隙空位，提示损伤后骨细胞存活受损。虽然P188治疗挽救了负载诱导的Prkd1CKO小鼠BMD增加，它令人惊讶地进一步抑制了负荷诱导的皮质骨厚度和皮质骨形成的增加。一起来看,这些数据表明，Prkd1可能在调节和修复骨细胞中的PMD反应中起关键作用，并支持PMD修复过程可以在药理学上靶向调节下游反应的观点。但表明PMD修复促进P188在改善骨合成代谢对负荷的反应方面的作用有限。
