title: Pro-survival effects of 17β-estradiol on osteocytes are mediated by nitric oxide/cGMP via differential actions of cGMP-dependent protein kinases I and II.
abstract: Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.
authors: Department of Medicine and Cancer Center and the Biomedical Sciences Graduate Progam, University of California, San Diego, California 92093, USA.
authors: Nisha
authors: N
authors: Marathe
authors: Hema
authors: H
authors: Rangaswami
authors: Shunhui
authors: S
authors: Zhuang
authors: Gerry R
authors: GR
authors: Boss
authors: Renate B
authors: RB
authors: Pilz
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004958:Estradiol / Q000494:pharmacology*; D004967:Estrogens / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D010011:Osteocytes / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000378:metabolism
chemical_list: C494820:Bad protein, mouse; D004967:Estrogens; D019253:Proto-Oncogene Proteins c-bcl-2; D051025:bcl-Associated Death Protein; D009569:Nitric Oxide; D004958:Estradiol; D051057:Proto-Oncogene Proteins c-akt; D017869:Cyclic GMP-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D006152:Cyclic GMP