Loading history changes the morphology and compressive force-induced expression of receptor activator of nuclear factor kappa B ligand/osteoprotegerin in MLO-Y4 osteocytes

Background: In this study, we investigated the effect of the mechanical loading history on the expression of receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) in MLO-Y4 osteocyte-like cells. Methods: Three hours after MLO-Y4 osteocytes were seeded, a continuous compressive force (CCF) of 31 dynes/cm2 with or without additional CCF (32 dynes/cm2) was loaded onto the osteocytes. After 36 h, the additional CCF (loading history) was removed for a recovery period of 10 h. The expression of RANKL, OPG, RANKL/OPG ratio, cell numbers, viability, and morphology were time-dependently examined at 0, 3, 6, and 10 h. Then, the same additional CCF was applied again for 1 h to all osteocytes with or without the gap junction inhibitor to examine the expression of RANKL, OPG, the RANKL/OPG ratio, and other genes that essential to characterize the phenotype of MLO-Y4 cells. Fluorescence recovery after photobleaching (FRAP) technique was also applied to test the differences of gap-junctional intercellular communications (GJIC) among MLO-Y4 cells. Results: The expression of RANKL and OPG by MLO-Y4 osteocytes without a loading history was dramatically decreased and increased, respectively, in response to the 1-h loading of additional weight. However, the expression of RANKL, OPG, and the RANKL/OPG ratio were maintained at the same level as in the control group in the MLO-Y4 osteocytes with a loading history but without gap junction inhibitor treatment. Treatment of loading history significantly changed the capacity of GJIC and protein expression of connexin 43 (Cx43) but not the mRNA expression of Cx43. No significant difference was observed in the cell number or viability between the MLO-Y4 osteocyte-like cells with and without a loading history or among different time checkpoints during the recovery period. The cell morphology showed significant changes and was correlated with the expression of OPG, Gja1, and Dmp1 during the recovery period. Conclusion: Our findings indicated that the compressive force-induced changes in the RANKL/OPG expression could be habituated within at least 11 h by 36-h CCF exposure. GJIC and cell morphology may play roles in response to loading history in MLO-Y4 osteocyte-like cells.

背景：本研究探讨了机械加载史对MLO-Y4类骨细胞中核因子κB受体活化因子配体（Receptor Activator of Nuclear Factor Kappa B Ligand, RANKL）与骨保护素（Osteoprotegerin, OPG）表达的影响。方法：将MLO-Y4类骨细胞接种3小时后，对其施加31达因/平方厘米的持续压缩载荷（continuous compressive force, CCF），并辅以或不辅以额外的32达因/平方厘米持续压缩载荷。加载36小时后，移除额外持续压缩载荷（即加载史），设置10小时的恢复期。分别于0、3、6、10小时时点，时间依赖性地检测RANKL、OPG的表达，RANKL/OPG比值，细胞数量、活性及形态。随后，对所有经或未经间隙连接抑制剂处理的骨细胞，再次施加1小时的相同额外持续压缩载荷，检测RANKL、OPG的表达，RANKL/OPG比值，以及用于表征MLO-Y4细胞表型的其他基因的表达情况。同时采用荧光漂白后恢复（Fluorescence Recovery After Photobleaching, FRAP）技术，检测MLO-Y4细胞间间隙连接细胞间通讯（Gap-Junctional Intercellular Communications, GJIC）的差异。结果：未经历加载史的MLO-Y4类骨细胞，在接受1小时额外载荷刺激后，其RANKL表达显著下调，OPG表达则显著上调。然而，经历过加载史且未接受间隙连接抑制剂处理的MLO-Y4类骨细胞，其RANKL、OPG的表达及RANKL/OPG比值均维持与对照组一致的水平。加载史处理可显著改变GJIC功能与连接蛋白43（Connexin 43, Cx43）的蛋白表达水平，但对Cx43的mRNA表达无显著影响。在恢复期内，无论是否经历加载史的MLO-Y4类骨细胞，其细胞数量与活性均无显著差异；不同检测时点间也未观察到显著差异。恢复期内，细胞形态出现显著变化，且该变化与OPG、Gja1及Dmp1的表达呈相关性。结论：本研究结果表明，经36小时持续压缩载荷暴露后，压缩载荷诱导的RANKL/OPG表达变化可在至少11小时内产生适应。GJIC与细胞形态可能在MLO-Y4类骨细胞响应机械加载史的过程中发挥作用。