Nitric oxide as a mediator of estrogen effects in osteocytes.

Postmenopausal osteoporosis due to estrogen deficiency is a major health problem, and available therapies rely largely on the inhibition of bone resorption, because estrogen replacement is associated with risks. Estrogen promotes bone health in large part by increasing osteocyte survival, but the molecular mechanisms involved are only partly understood. We showed that estradiol stimulates nitric oxide (NO) production in osteocytes, leading to increased cGMP synthesis and activation of cGMP-dependent protein kinases (PKGs). Moreover, we found that 17ß-estradiol protects osteocytes against apoptosis via the NO/cGMP signaling pathway: type II PKG mediates estradiol-induced activation of the prosurvival kinases Erk and Akt, whereas type I PKG contributes to prosurvival signaling by directly phosphorylating and inactivating the cell death protein BAD. Preclinical data support an important role of NO in bone biology, and clinical trials suggest that NO donors may prevent bone loss in postmenopausal women. Our data provide novel insights into estrogen signaling through the NO/cGMP/PKG pathway and a rationale for using NO donors and other cGMP-elevating agents for treating postmenopausal osteoporosis.

Acetylation of p53 stimulates miRNA processing and determines cell survival following genotoxic stress.

It is widely accepted that different forms of stress activate a common target, p53, yet different outcomes are triggered in a stress-specific manner. For example, activation of p53 by genotoxic agents, such as camptothecin (CPT), triggers apoptosis, while non-genotoxic activation of p53 by Nutlin-3 (Nut3) leads to cell-cycle arrest without significant apoptosis. Such stimulus-specific responses are attributed to differential transcriptional activation of various promoters by p53. In this study, we demonstrate that CPT, but not Nut3, induces miR-203, which downregulates anti-apoptotic bcl-w and promotes cell death in a p53-dependent manner. We find that acetylation of K120 in the DNA-binding domain of p53 augments its association with the Drosha microprocessor and promotes nuclear primary miRNA processing. Knockdown of human orthologue of Males absent On the First (hMOF), the acetyltransferase that targets K120 in p53, abolishes induction of miR-203 and cell death mediated by CPT. Thus, this study reveals that p53 acetylation at K120 plays a critical role in the regulation of the Drosha microprocessor and that post-transcriptional regulation of gene expression by p53 via miRNAs plays a role in determining stress-specific cellular outcomes.

Pro-survival effects of 17ß-estradiol on osteocytes are mediated by nitric oxide/cGMP via differential actions of cGMP-dependent protein kinases I and II.

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 17ß-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 17ß-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 17ß-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 17ß-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, 17ß-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.

Cyclic GMP and protein kinase G control a Src-containing mechanosome in osteoblasts.

Mechanical stimulation is crucial for bone growth and remodeling, and fluid shear stress promotes anabolic responses in osteoblasts through multiple second messengers, including nitric oxide (NO). NO triggers production of cyclic guanosine 3',5'-monophosphate (cGMP), which in turn activates protein kinase G (PKG). We found that the NO-cGMP-PKG signaling pathway activates Src in mechanically stimulated osteoblasts to initiate a proliferative response. PKGII was necessary for Src activation, a process that also required the interaction of Src with ß3 integrins and dephosphorylation of Src by a complex containing the phosphatases SHP-1 (Src homology 2 domain-containing tyrosine phosphatase 1) and SHP-2. PKGII directly phosphorylated and stimulated SHP-1 activity, and fluid shear stress triggered the recruitment of PKGII, Src, SHP-1, and SHP-2 to a mechanosome containing ß3 integrins. PKGII-null mice showed defective Src and ERK (extracellular signal-regulated kinase) signaling in osteoblasts and decreased ERK-dependent gene expression in bone. Our findings reveal a convergence of NO-cGMP-PKG and integrin signaling and establish a previously unknown mechanism of Src activation. These results support the use of PKG-activating drugs to mimic the anabolic effects of mechanical stimulation of bone in the treatment of osteoporosis.

Type II cGMP-dependent protein kinase mediates osteoblast mechanotransduction.

Continuous bone remodeling in response to mechanical loading is critical for skeletal integrity, and interstitial fluid flow is an important stimulus for osteoblast/osteocyte growth and differentiation. However, the biochemical signals mediating osteoblast anabolic responses to mechanical stimulation are incompletely understood. In primary human osteoblasts and murine MC3T3-E1 cells, we found that fluid shear stress induced rapid expression of c-fos, fra-1, fra-2, and fosB/DeltafosB mRNAs; these genes encode transcriptional regulators that maintain skeletal integrity. Fluid shear stress increased osteoblast nitric oxide (NO) synthesis, leading to activation of cGMP-dependent protein kinase (PKG). Pharmacological inhibition of the NO/cGMP/PKG signaling pathway blocked shear-induced expression of all four fos family genes. Induction of these genes required signaling through MEK/Erk, and Erk activation was NO/cGMP/PKG-dependent. Treating cells with a membrane-permeable cGMP analog partly mimicked the effects of fluid shear stress on Erk activity and fos family gene expression. In cells transfected with small interfering RNAs (siRNA) specific for membrane-bound PKG II, shear- and cGMP-induced Erk activation and fos family gene expression was nearly abolished and could be restored by transducing cells with a virus encoding an siRNA-resistant form of PKG II; in contrast, siRNA-mediated repression of the more abundant cytosolic PKG I isoform was without effect. Thus, we report a novel function for PKG II in osteoblast mechanotransduction, and we propose a model whereby NO/cGMP/PKG II-mediated Erk activation and induction of c-fos, fra-1, fra-2, and fosB/DeltafosB play a key role in the osteoblast anabolic response to mechanical stimulation.