Mechanism Of Endoplasmic Reticulum Stress Signal Molecule PERK Regulating Osteoclast Differentiation And Function

Objective:Osteoclasts are multinucleated giant cells with the ability to degrade bone tissue,and are closely related to abnormal bone metabolic diseases.Their differentiation is regulated by two key cytokines: M-CSF and RANKL.Endoplasmic reticulum is an organelle responsible for protein modification,quality control,and transportation.The accumulation of unfolded or misfolded proteins in endoplasmic reticulum cavity induces endoplasmic reticulum stress.PERK is an endoplasmic reticulum stress-sensing protein that is ubiquitous in eukaryotic cells.Systemic knockout of PERK mice shows severe bone loss,and the expression of the osteoclast marker genes TRAP and CSTK is significantly reduced,suggesting that PERK is of great significance for maintaining the normal growth and development of bone tissue.Endoplasmic reticulum stress is associated with many diseases,but its role in osteoclast differentiation is still unclear.The purpose of this project is to study the role of endoplasmic reticulum stress and its key signal molecule PERK on osteoclast differentiation and function.Methods:1.During the RANKL induced osteoclastogenesis of BMMs,TRAP staining and western blot were used to detect the effects of Thapsigargin and GSK2606414 on osteoclast formation and the expression of osteoclast differentiation-related proteins.2.After silencing and inhibiting PERK and induced osteoclastogenesis of BMMs by RANKL,TRAP staining was used to detect the number of osteoclasts.RT-q PCR was used to detect the expression level of osteoclast differentiation marker genes.The expression of NF-?B and MAPK signal pathway was detected by Westrern blot,and the function of osteoclast was detected by bone resorption experiment and F-actin ring staining.3.The female mice were ovariectomized and intragastrically administered GSK2606414 for 6 weeks.The femurs of one side were collected for mcro-CT to detect bone mass,and the femurs of the other side were decalcified for TRAP staining to detect the number of osteoclasts.4.Western blot was used to detect the effect of Thapsigargin and GSK2606414 on the expression of autophagy-related proteins during RANKL-induced osteoclast differentiation.LC3 marker fluorescence staining and transmission electron microscopy were used to detect the effects of GSK2606414 on the autophagy flux and the formation of autophagosome.5.After inducing osteoclastogenesis of BMMs by RANKL,Westrern blot was used to detect the expression levels of osteoclast-related proteins and autophagy-related proteins after using NAC and CCT020312.Results:1.Thapsigargin activates endoplasmic reticulum stress in the concentration range of 0.05-0.1n M to promote osteoclast differentiation;PERK is significantly activated during RANKL-induced osteoclastogenesis;the use of PERK inhibitor GSK2606414 can inhibit the facilitating effects of Thapsigargin on osteoclast differentiation and expression of osteoclast-related proteins.2.Knockdown of PERK by si RNA and inhibition of PERK by GSK2606414,respectively,have significant negative regulatory effects on the formation and bone resorption of mature osteoclasts.3.GSK2606414 inhibits the formation of osteoclast F-actin ring,down-regulates the m RNA and protein expression levels of osteoclast differentiation marker genes,and inhibits RANKL-induced activation of NF-?B and MAPK signaling pathways.4.GSK2606414 at 50 mg/kg was administered orally every 2 day to inhibit bone loss and osteoclast formation in femurs of ovariectomized mice.5.During RANKL-induced osteoclast differentiation,the expression of autophagy related proteins increases.Thapsigargin activates endoplasmic reticulum stress to enhance autophagy,while GSK2606414 has a significant inhibitory effect on autophagy flux and autophagosome formation.6.Antioxidant NAC can inhibit the expression of PERK phosphorylation and osteoclast-related proteins and autophagy-related proteins.The use of PERK activator CCT020312 can reverse inhibition effect of NAC on osteoclast-related proteins and autophagy-related proteins expression.Conclusion:Endoplasmic reticulum stress is important to osteoclast differentiation,and its signaling molecule PERK plays a key regulatory role.Inhibition of PERK cuts off the effect of activating endoplasmic reticulum stress to promote osteoclast differentiation.GSK2606414 significantly inhibits RANKL-induced osteoclast differentiation and activation of NF-?B,MAPK,autophagy,and ROS.In terms of mechanism,activating PERK can partially reverse the anti-osteoclastic effect and autophagy inhibition of NAC.This study reveals the regulatory mechanism of ER stress signal molecule PERK during osteoclast differentiation,which will provide new ideas for treating diseases related to abnormal activation of osteoclasts.