The PERK-eIF2?-ATF4 Signaling Branch Regulates Osteoblast Differentiation And Proliferation By PTH

Osteoporosis is a very common systemic skeletal disease in the elderly.It has the characteristics of low bone density and quality,destruction of bone microstructure,which leads to increased bone fragility and increased fracture risk.Nowadays,the aging of population is becoming more and more serious,which has become a global problem.The incidence of osteoporosis is also increasing.The therapeutic drugs for osteoporosis can be divided into anti-bone absorption drugs and bone-promoting drugs drugs.Anti-bone resorption drugs can inhibit the activity and differentiation of osteoclasts,and then reducing the rate of bone remodeling and reversing the temporary bone loss caused by bone resorption greater than bone formation,but anti-bone resorption drugs can not improve the integrity of bone structure.Therefore,the combination of anti-bone resorption drugs and bone-promoting drugs has become a hotspot.PTH1-34 are one of FDA-approved bone-promoting drugs for age-related osteoporosis.Treatment with PTH1-34 can effectively stimulates bone formation.But the molecular mechanisms of PTH-mediated osteoblast differentiation and cell proliferation are still not completely understood.In this study,we showed that PTH induced endoplasmic reticulum stress in osteoblasts through the PERK-EIF2?-ATF4 signaling pathway,and then regulates osteoblast differentiation and proliferation.After 4-6 hours treatment with PTH,the mRNA levels of ER-stress markers such as Bip,Chop,Gadd34,Grp94,P4 hb and Pdia3 were doubled.The phosphorylation of PERK and EIF2?,and protein level of ATF4 also increased.To verify the role of this PERK-eIF2?-ATF4 signaling pathway in regulating PTH-induced differentiation and proliferation of osteocytes,We used ATF4-siRNA to knock down ATF4 expression.We found that the proliferation and differentiation of osteoblasts were significantly inhibited.The mRNA levels of osteoblast differentiation and proliferation markers were significantly decreased,alkaline phosphatase activity decreased nearly twice,mineralization ability and osteocalcin secretion level also decreased significantly.To further explore the upstream of ATF4,we used ISRIB to inhibit the phosphorylation of eIF2?.The results showed that the expression of ATF4 was decreased and PTH-mediated osteoblast differentiation and cell proliferation was inhibited.Next,AMG'44 was used to block PERK-eIF2? pathway.The expression of ATF4 and the mRNA levels of osteoblast differentiation and proliferation markers were decreased,alkaline phosphatase activity was reduced nearly two-fold,mineralization ability and osteocalcin secretion were also decreased.PTH promoted the proliferation and differentiation of osteoblasts through PERK-eIF2?-ATF4 signaling pathway.These findings were further verified by knocking down PERK expression with a specific siNRA for PERK in MC3T3-E1 cells.Interestingly,we found that salubrinal enhanced PTH-induced osteoblast differentiation and proliferation by maintenance of phosphorylation of EIF2?.Furthermore,we observed that PTH increased the association between HSP90 and PERK and maintained PERK protein stabilization in the early stages of PTH-induced ER stress.Treatment of MC3T3-E1 cells with geldanamycin,an HSP90 inhibitor,decreased PERK protein expression and inhibited osteoblast differentiation and cell proliferation upon PTH treatment.Taken together,our data demonstrate that PTH regulates osteoblast differentiation and cell proliferation,partly by activating the HSP90-dependent PERK-EIF2?-ATF4 signaling pathway.The activation of PERK by accumulated unfolded proteins in the ER lumen leads to phosphorylation of the EIF2? and inhibits global protein translation.Concurrently,phosphorylated EIF2? can activate ATF4 translation to regulate the expression of bone-formation related genes,and then stimulating osteoblast proliferation and differentiation.