The Research Of Orai1Regulated Osteoblast Differentiation And Apoptosis In The Development Of Postmenopausal Osteoporosis

Background:The main pathogenesis of postmenopausal osteoporosis is the imbalance in boneremodeling and resorption when estrogen levels decrease, which lead to destruction of themicrostructure of bone tissue, bone fragility and susceptibility to fracture. Currently,clinical treatment for osteoporosis is mainly focused on the inhibition of the osteoclastsactivity and bone resorption, and treatment methods for improving the ability of osteoblastbone formation is still very rare. It is mainly due to the regulation mechanisms ofosteogenesis cell bone-forming ability is very complex, the drug targets for improvingosteoblast bone formation have also poorly understood. Therefore, study the molecularmechanisms of osteoblast bone formation and find the drug targets, will provide newstrategies and approaches to the treatment of osteoporosis. Orai1is a recently identifiedintracellular calcium channel which mediates store-operated Ca2+entry and is a fourtransmembrane protein. A study found that the Orai1gene knockout mice appeared osteogenesis imperfecta symptoms, such as size decrease and reduction of cancellous bone,indicating that calcium channel Orai1abnormalities may be involved in bone formationprocess. However, the molecular mechanisms of orai1mediated bone formation have notbeen reported. Our research studied Orai1regulated osteoblast differentiation andapoptosis, and identified the molecular mechanism of Orai1promoted bone formation.The study may provide new strategies and drug targets for clinical treatment ofosteoporosis.Aims:Identify the mechanism of Orai1regulated osteoblasts differentiation and apoptosis inpost-menopausal osteoporosis.Methods:1. Construct osteoporosis mouse model by ovariectomy, and then the Orai1expression levels and calcium ions flow were detected. By constructing Orai1stableknockdown cell model, test the transcription of bone formation key indicators: alkalinephosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), Runx2, Osterix, and test thealkaline phosphatase activity and calcium nodule formation ability. Then detectcalcium-dependent Ras-ERK1/2and CaMKII-NF-ATc signaling pathway activity todentify whether Orai1is involved in the regulation of osteogenesis.2. By17β-E2stimulated murine MC3T3-E1cells, detect Orai1protein expression andmRNA transcript levels and detect AKT-mTOR signaling pathway activity. Applicationwith the specific inhibitors of AKT and mTOR to inhibit their activities, detect the role of17β-E2to Orai1protein and mRNA levels, thus clarifying the mechanism of estrogenregulated Orai1change.17β-E2induced Orai1knockdown cells bone formation, test thetranscription of bone formation key indicators: alkaline ALP, OCN, OPN, Runx2, Osterix,and test the alkaline phosphatase activity and calcium nodule formation ability. Thendetect calcium-dependent Ras-ERK1/2and CaMKII-NF-ATc signaling pathway activity todentify whether estrogen may promote bone formation through Orai1. 3. After induction of endoplasmic reticulum stress by Tg treatment, we tested the roleof17β-E2on the activity of Caspase-12and Caspase-3, and the expression of Grp78andCHOP. We further detection the role of17β-E2on Grp78key transcription factor ATF6,YY1, IRE1TFII-I and Ras-ERK1/2signaling pathway in endoplasmic reticulum stresscondition, to clarify the molecular mechanism of17β-E2against endoplasmic reticulumstress-induced osteoblast apoptosis.4. Test the transcription of bone formation key indicators: ALP, OCN, OPN, Runx2,Osterix, and test the alkaline phosphatase activity and calcium nodule formation ability inBMP2stimulated Orai1stable knockdown cells. Then detect calcium-dependentRas-ERK1/2and CaMKII-NF-ATc signaling pathway activity to identify whether BMP2may promote bone formation through Orai1. By testing the activity of key signalingmolecules regulating Orai1expression or activity, clarify the molecular mechanisms ofOrai1regulation by BMP2.Results:1. We construct osteoporosis mouse model by ovariectomy, and found that the Orai1expression levels and calcium ions flow were decreased. Then we used shRNAinterference in human MG63cells and mouse MC3T3-E1cells to successfully constructOrai1stable knockdown cell models. We found that the transcription of bone formationkey indicators: ALP, OCN, OPN, Runx2, Osterix were decreased and the alkalinephosphatase activity and calcium nodule formation ability were inhibited after Orai1knockdown. We also found that Ras-ERK1/2and CaMKII-NF-ATc signaling pathwayactivity were inhibited. The results identified that by inducing Ca2+entry, Orai1mayactivate Ras-ERK1/2and CaMKII-NF-ATc signaling pathway, thus promoting osteoblastdifferentiation.2. We used17β-E2stimulates murine MC3T3-E1cells, and found that Orai1proteinand mRNA levels increased in a time-dependent manner.17β-E2could also increaseactivity of AKT and mTOR. We applied with the specific inhibitors of AKT and mTOR to inhibit their activities, and found that17β-E2could not increase Orai1protein and mRNAlevels. These results imply that by activating AKT-mTOR signaling pathway,17β-E2mayincrease Orai1expression. Further study indicated that the Orai1inhibition significantlyblocked17β-E2-increased transcription of bone formation key indicators: ALP, OCN, OPN,Runx2, Osterix, and17β-E2-promoted the alkaline phosphatase activity, calcium noduleformation ability and Ras-CaMKII signaling pathway activation, suggesting that bypromoting Orai1expression,17β-E2promote osteoblast bone formation.3. We found that in endoplasmic reticulum stress conditions, by promoting Grp78expression,17β-E2reduced Caspase-12and Caspase-3activity, thus inhibiting theendoplasmic reticulum stress-induced osteoblast apoptosis. We further found that bypromoting Orai1expression,17β-E2activated Ras-ERK1/2signaling pathway andenhanced TFII-I tyrosine phosphorylation,17β-E2also induced p-TFII-I nuclearlocalization and binding to the Grp78promoter, thus increasing transcription of Grp78.4. We found the in BMP2stimulated conditions, knockdown Orai1inhibitedosteoblast-specific transcription factor expressions of Runx2and Osterix, and furtherinhibited the expressions of bone formation key indicators: ALP, OPN and OCN, therebyinhibiting alkaline phosphatase activity and calcium nodule formation ability. Also foundthat inhibit the expression of Orai1blocked BMP2-promoted store-operated Ca2+entry,and BMP2-promoted CaMKII, Ras signaling pathway activity, thereby inhibitingosteoblast-specific transcription factor Runx2and Osterix transcription, as well as thetranscription of bone formation key indicators: ALP, OCN and OPN transcription. Furtherstudies showed that by activating Integrinβ1-FAK-Src signaling pathway, BMP2activatesOrai1, and increased store-operated Ca2+entry and activation of CaMKII, Ras signalingpathway and promote the osteoblasts differentiation and extracellular mineral calcification.We also confirmed the direct interaction of Integrinβ1with BMPR-I and BMPR-II on thecell membrane. These results revealed that by regulation Orai1activity, BMP2promotedosteoblast differentiation. Conclusion:In summary, we found that due to the decrease of estrogen levels after menopause,AKT-mTOR signaling pathway activity gave rise to reduced Orai1expression. Thereduced Orai1expression reduced the role of BMP2on ossification and on the other handincreased the apoptosis of osteoblasts, leading to osteoporosis. This study clarifies the roleand mechanism of Orai1on bone formation, and may provides new strategies and drugtargets for clinical treatment of osteoporosis.