title: Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER-ERK-mTOR pathway.
abstract: Estradiol could protect osteoblast against apoptosis, and apoptosis and autophagy were extensively and intimately connected. The aim of the present study was to test the hypothesis that autophagy was present in osteoblasts under serum deprivation and estrogen protected against osteoblast apoptosis via promotion of autophagy. MC3T3-E1 osteoblastic cells were cultured in a serum-free and phenol red-free minimal essential medium (α-MEM). Ultrastructural analysis, lysosomal activity assessment and monodansycadaverine (MDC) staining were employed to determine the presence of autophagy, and real time PCR was used to evaluate the expression of autophagic markers. Meanwhile, the osteoblasts were transferred in a serum-free and phenol red-free α-MEM containing either vehicle or estradiol. Apoptosis and autophagy was assessed by using the techniques of real-time PCR, Western blot, immunofluorescence assay, and flow cytometry. The possible pathway through which estrogen promoted autophagy in the serum-deprived osteoblasts was also investigated. Real-time PCR demonstrated the expression of LC3, beclin1 and ULK1 genes in osteoblasts under serum deprivation, and immunofluorescence assay verified high expression of proteins of these three autophagic bio-markers. Lysosomes and autolysosomes accumulated in the cytoplasm of osteoblasts were also detected under transmission electron microscopy, MDC staining and lysosomal activity assessment. Meanwhile, estradiol significantly decreased the expression of proteins of the bio-markers of apoptosis, and at the same time increased the expression of proteins of the bio-markers of autophagy in the serum-deprived osteoblasts. Furthermore, the estradiol-promoted autophagy in serum-deprived osteoblasts could be blocked by estrogen receptor (ER) antagonist (ICI 182780), and estradiol failed to rescue the cells pretreated with an inhibitor of vacuolar ATPase (bafilomycin A) from apoptosis. Serum deprivation resulted in apoptosis through activation of Caspase-3 and induced autophagy through inhibition of phospho-mammalian target of rapamycin (p-mTOR). Both 3-methyladenine (3MA) and U0126 led to increase of apoptosis in osteoblasts with serum deprivation. Estradiol failed to over-ride the inhibitory effect of 3MA on phosphorylation of AKT but directly led to dephosphorylation of mTOR and upregulation of LC3 protein expression. However, the estradiol-enhanced LC3 protein expression was significantly suppressed by U0126 through inhibition of phosphorylation of extracellular signal-regulated kinase (ERK). Estradiol rescued osteoblast apoptosis via promotion of autophagy through the ER-ERK-mTOR pathway.
authors.affiliation: Department of Orthopaedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, No. 1665, Kongjiang Road, Shanghai, 200092, China.
authors.forename: Yue-Hua
authors.initials: YH
authors.lastname: Yang
authors.affiliation: Department of Orthopaedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, No. 1665, Kongjiang Road, Shanghai, 200092, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Chen
authors.affiliation: Department of Orthopaedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, No. 1665, Kongjiang Road, Shanghai, 200092, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Orthopaedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, No. 1665, Kongjiang Road, Shanghai, 200092, China.
authors.forename: Jiang-Wei
authors.initials: JW
authors.lastname: Chen
authors.affiliation: Department of Orthopaedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, No. 1665, Kongjiang Road, Shanghai, 200092, China.
authors.forename: Xin-Feng
authors.initials: XF
authors.lastname: Zheng
authors.affiliation: Department of Orthopaedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, No. 1665, Kongjiang Road, Shanghai, 200092, China.
authors.forename: Yu-Ren
authors.initials: YR
authors.lastname: Wang
authors.affiliation: Department of Orthopaedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, No. 1665, Kongjiang Road, Shanghai, 200092, China. jiangsd@126.com.
authors.forename: Sheng-Dan
authors.initials: SD
authors.lastname: Jiang
authors.affiliation: Department of Orthopaedic Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, No. 1665, Kongjiang Road, Shanghai, 200092, China. jiangleisheng@126.com.
authors.forename: Lei-Sheng
authors.initials: LS
authors.lastname: Jiang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071189:Autophagy-Related Protein-1 Homolog; D000071186:Beclin-1; D002070:Butadienes / Q000494:pharmacology; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003470:Culture Media; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology*; D004965:Estrogen Antagonists / Q000494:pharmacology; D000077267:Fulvestrant; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D051379:Mice; D009570:Nitriles / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011960:Receptors, Estrogen / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; C115010:Becn1 protein, mouse; D002070:Butadienes; D003470:Culture Media; D004965:Estrogen Antagonists; D018942:Macrolides; D009570:Nitriles; D011960:Receptors, Estrogen; C113580:U 0126; C057620:bafilomycin A; D000077267:Fulvestrant; D004958:Estradiol; D005700:Galactosyltransferases; C053372:glucosaminylgalactosylglucosylceramide beta-galactosyltransferase; C546843:mTOR protein, mouse; D000071189:Autophagy-Related Protein-1 Homolog; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases; C112444:Ulk1 protein, mouse; D053148:Caspase 3