Abstract:Objective To determine the role of Orai2 in regulating osteoblast proliferation,apoptosis and differentiation in osteoblasts.Methods Western blot and real-time PCR were used to detect the protein expression and mRNA transcription levels of Orai2 after osteoblastic induction.The expression of Orai2 was silenced in MC3T3-E1 osteoblasts by transfecting Orai2 shRNA,and unrelated interference shRNA was transfected as negative control.MC3T3-E1 osteoblasts were divided into control group and Orai2 shRNA group.In the control group and Orai2 shRNA group,cell proliferation and apoptosis,the transcription of Runx2,Osterix and ALP,and Ras-ERK1/2 signaling pathway activity were tested to dentify whether Orai2 is involved in the regulation of osteogenesis.Results After osteogenic induction,Orai2 protein expression level and mRNA transcription level were gradually increased (P<0.05).Compared with the control group,Orai2 was knockdown successfully after Orai2 shRNA transfected in MC3T3-E1 cells.Furthermore,compared with the control group,the proliferation were decreased and apoptosis were increased after Orai2 knockdown.Moreover,compared with the control group,the transcription of Runx2,Osterix and ALP,were decreased after Orai2 knockdown.We also found that Ras-ERK1/2 signaling pathway activity were inhibited compared with the control group.Conclusion The inhibition of Orai2 expression can significantly reduce the activity of Ras-Erk1/2 signaling pathway,reduce the proliferation of osteoblasts,increase the apoptosis of osteoblasts,and inhibit the differentiation of osteoblasts,resulting in the decrease in the number of osteoblasts and the involvement in the occurrence and development of osteoporosis.
[1]Donohue D,Decker S,Ford J,et al.Opportunistic CT screening for osteoporosis in patients with pelvic and acetabular trauma:technique and potential clinical impact[J].J Orthop Trauma,2018,32(8):408-413.
[2]Jaleel A,Saag KG,Danila MI.Improving drug adherence in osteoporosis:an update on more recent studies[J].Ther Adv Musculoskelet Dis,2018,10(7):141-149.
[3]Li Y,Xuan M,Wang B,et al.Comparison of parathyroid hormone (1~34)and elcatonin in postmenopausal women with osteoporosis:an 18-month randomized,multicenter controlled trial in China[J].Chin Med J,2013,126(3):457-463.
[4]Pagani S,Fini M,Giavaresi G,et al.The active role of osteoporosis in the interaction between osteoblasts and bone metastases[J].Bone,2015,79(10):176-182.
[5]Scimeca M,Salustri A,Bonanno E,et al.Impairment of PTX3 expression in osteoblasts:a key element for osteoporosis[J].Cell Death Dis,2017,8(10):e3125.
[6]Prakriya M,Feske S,Gwack Y,et al.Orai1 is an essential pore subunit of the CRAC channel[J].Nature,2006,443(7108):230-233.
[7]Nguyen NT,Han W,Cao WM,et al.Storeoperated calcium entry mediated by ORAI and STIM[J].Compr Physiol,2018,8(3):981-1002.
[8]Choi H,Srikanth S,Atti E,et al.Deletion of Orai1 leads to bone loss aggravated with aging and impairs function of osteoblast lineage cells[J].Bone Rep,2018,8(4):147-155.
[9]Robinson LJ1,Mancarella S,Songsawad D,et al.Gene disruption of the calcium channel orai1 results in inhibition of osteoclast and osteoblast differentiation and impairs skeletal development[J].Lab Invest,2012,92(7):1071-1083.
[10]Sawai CM,Sisirak V,Ghosh HS,et al.Transcription factor Runx2 controls the development and migration of plasmacytoid dendritic cells[J].J Exp Med,2013,210(11):2151-1259.
[11]Han X,Zhou J,Peng W.Orexins facilitates osteogenic differentiation of MC3T3-E1 cells[J].IUBMB Life,2018,70(7):633-641.
[12]Choi YH,Han Y,Jin SW,et al.Pseudoshikonin I enhances osteoblast differentiation by stimulating Runx2 and Osterix[J].J Cell Biochem,2018,119(1):748-757.
[13]Kanno T,Takahashi T,Tsujisawa T,et al.Mechanical stress-mediated Runx2 activation is dependent on Ras/ERK1/2 MAPK signaling in osteoblasts[J].J Cell Biochem,2007,101(5):1266-1277.
[14]Peng S,Zhou G,Luk KD,et al.Strontium promotes osteogenic differentiation of mesenchymal stem cells through the Ras/MAPK signaling pathway[J].Cell Physiol Biochem,2009,23(1-3):165-174.
[15]Keiper M,Stope MB,Szatkowski D,et al.Epac-and Ca2+ controlled activation of ras and extracellular signal-regulated kinases by Gs-coupled receptors[J].J Biol Chem,2004,279(45):46497-46508.
[16]Clapham DE.Calcium Signaling[J].Cell,2007,131(6):1047-1058.
[17]Zhou P,He F,Han Y,et al.Nanosecond pulsed electric field induces calcium mobilization in osteoblasts[J].Bioelectrochemistry,2018,124(12):7-12.
[18]Mitran V,Ion R,Miculescu F,et al.Osteoblast cell response to naturally derived calcium phosphate-based materialsv[J].Materials (Basel),2018,11(7):E1097.
[19]Barradas AM,Fernandes HA,Groen N,et al.A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells[J].Biomaterials,2012,33(11):3205-3215.
[20]Staehlke S,Rebl H,Finke B,et al.Enhanced calcium ion mobilization in osteoblasts on amino group containing plasma polymer nanolayer[J].Cell Biosci,2018,8(3):22-33.
[21]An S,Gao Y,Ling J,et al.Calcium ions promote osteogenic differentiation and mineralization of human dental pulp cells:implications for pulp capping materials[J].J Mater Sci:Mater Med,2012,23(3):789-795.
[22]Yamaguchi T,Kifor O,Chattopadhyay N,et al.Expression of extracellular calcium (Ca2+)sensing receptor in the clonal osteoblast-like cell lines,UMR-106 and SAOS-2[J].Biochem Biophys Res Commun,1998,243(3):753-757.
[23]Mobasheri A,Shakibaei M.Osteogenic effects of resveratrol in vitro:potential for the prevention and treatment of osteoporosis[J].Ann N Y Acad Sci,2013(1290):59-66.
[24]Clines GA.Prospects for osteoprogenitor stem cells in fracture repair and osteoporosis[J].Curr Opin Organ Transplant,2010,15(1):73-78.