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Institute of Pharmacology, University of Messina (S.C., L.D., T.G., R.D.P., A.P.C.), Department of Biomorphology, University of Messina School of Medicine (E.M., D.P.), and Department of Obstetrical and Gynecological Sciences, University of Messina (Z.R.), 98100 Messina, Italy; Center of Excellence on Neurodegenerative Diseases and Center for Pharmacology and Biotechnology, University of Milan (E.V., A.M.), 20129 Milan, Italy; and Department of Rheumatology, University Hospital Nijmegen (F.A.J.V.d.L.), 6500 HB, Nijmegen, The Netherlands
Address all correspondence and requests for reprints to: Salvatore Cuzzocrea, Ph.D., Institute of Pharmacology, School of Medicine, University of Messina, Torre Biologica-Policlinico Universitario Via C. Valeria-Gazzi, 98100 Messina, Italy. E-mail: salvator{at}unime.it; or to: Prof. Adriana Maggi, Center MPL, Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Balzaretti 9, 20129 Milan, Italy. E-mail: adriana.maggi{at}unimi.it.
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Abstract |
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plasma levels showed that ovariectomy fails to elicit the increase observed in WT animals and suggests that iNOS plays a primary role in the protective effects of estrogens. To further support this hypothesis, we show that estradiol-dependent activation of estrogen receptor- blocks phorbol 12-acetate 13-myristate-induced transcription of iNOS promoter in transfected cells, thus demonstrating that the promoter of iNOS is under estrogen negative control. Our findings point to a key role of iNOS in mediating the negative effects of estrogen depletion on bones and provide a novel mechanistic explanation for the effects of menopause in osteoporosis and possibly also in other diseases in which the inflammatory component is elevated.
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Introduction |
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/TNF receptor families, named RANK [receptor activator of nuclear factor-
B (NF-B)] and its ligand (RANKL), play a fundamental role. RANKL triggers osteoclast differentiation and is secreted by stromal bone cells (4, 5). RANKL is also expressed in abundance by activated T lymphocytes; these cells can, in fact, trigger osteoclastogenesis, and this mechanism is probably pivotal in the joint destruction typical of rheumatoid arthritis (6). This observation is consistent with clinical data showing abundant osteoclast proliferation in inflammatory lesions of bone. In addition, systemic administration of bacterial lipopolysaccharide prompts rapid production of osteoclasts through the p55 TNF receptor (7). Whether TNF in the absence of RANKL directly targets macrophages to induce their differentiation into osteoclasts is still controversial. It is clear, however, that TNF stimulates osteoblasts to express RANKL and macrophage colony-stimulating factor (2). ILs, interferon-
, and prostaglandin E stimulate macrophage colony-stimulating factor expression by marrow stroma cells, thus participating in the proliferation of osteoclast precursors (8, 9). Physiologically, bone remodeling is under endocrine control; PTH favors bone resorption, and 17ß-estradiol (E2) is necessary for maintaining the bone mass, as indicated by the rapid appearance of osteoporosis with surgical or natural ablation of ovarian functions and blockade of such an effect with E2 administration. The accelerated osteoclastogenesis induced by PTH is due to a direct effect of the hormone on its receptors located on the membrane of osteoblasts and on certain stromal cells that produce RANKL (10). Less understood at the moment is the role of estrogen. Apparently, this hormone does not directly modulate the synthesis of RANKL or of its decoy osteoprotegerin, nor does it affect the activity of RANK. The studies of estrogen’s role in bone metabolism focused on its action on proinflammatory cytokines such as IL-1, IL-6, TNF, granulocyte colony-stimulating factor, and prostaglandin E2. All of these factors, which increase bone resorption by increasing the pool of preosteoclasts in bone marrow, are down-regulated by estrogen (3, 11, 12). It has also been proposed that estrogen inhibits bone resorption by inducing small, but cumulative, changes in multiple estrogen-dependent regulatory factors (13). Even if this were the case, however, it remains to be clarified how these effects are effectively coordinated to achieve the final protective effects of estrogen in the progression of osteoporosis.
We have recently shown that estrogen receptors (ER and ERß) are expressed in monocyte-derived cells such as macrophages and microglia (14) and that their estradiol-dependent activation blocks inflammatory reactions induced by lipopolysaccharide (15). In vivo studies with a well known model of inflammation (carrageenan-induced pleurisy) (16, 17) further demonstrated the antiinflammatory potential of estradiol. In all of these model systems, estradiol blocked the synthesis of inducible nitric oxide (NO) synthase (iNOS). In inflammation and septic shock, iNOS synthesis is stimulated by proinflammatory cytokines and the bacterial wall in endothelial cells, smooth muscle cells, macrophages, and other cell types (18, 19). iNOS causes a massif production of NO. NO can have both direct effects on cell signaling as well as indirect actions mediated by the reaction products formed when NO interacts with other molecules, such as oxygen or superoxide, thereby playing a central role in the pathophysiology of inflammation and oxidant stress (19, 20). Thus, it is conceivable that the negative control by E2 of iNOS production might be a key element in E2-dependent prevention of osteoporosis and a series of other pathologies associated with menopause, all characterized by a high inflammatory component (such as arteriosclerosis, cardiovascular risk, uveitis, and dementia-like Alzheimer’s disease).
Although the effects of E2 have been previously studied in endothelial cells (21) and bone (22), little is known about the effects of E2 on iNOS and the role of this enzyme isoform in bone turnover. In the present study we investigated the role of iNOS in osteoclasteogenesis as well as in bone formation. To this aim, we compared the onset of osteoporosis induced by estrogen ablation in wild-type mice (iNOSWT) and in mice in which the inos gene had been knocked out (iNOSKO). The study shows that iNOS plays a role in estrogen bone protective action, thus opening novel therapeutic perspectives.
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Materials and Methods |
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Animals
The homozygous iNOS-/- and iNOS+/+ [wild-type (WT) C57BL/6 x 129/Sv] male mice (20–25 g) were supplied by Fons A. J. Van de Loo (University Hospital Nijmegen, Nijmegen, The Netherlands) (21). All animals were allowed access to food and water ad libitum. Animal care and treatments were in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes as well as with the European Economic Community regulations.
Ovariectomy
All surgical procedures were performed under halothane (2%) anesthesia, followed by nitrous oxygen/O2 anesthesia for about 18 min. Ovariectomy was performed as previously described (12). Controls (sham) underwent anesthesia and surgery without removal of the ovaries.
Nitrite/nitrate
Nitrite/nitrate (NOx) production, an indicator of NO synthesis, was measured in the plasma as previously described (23). Briefly, the nitrate in the exudate was first reduced to nitrite by incubation with nitrate reductase (670 mU/ml) and NADPH (160 µM) at room temperature for 3 h. The nitrite concentration in the samples was then measured by the Griess reaction, by adding 100 µl Griess reagent (0.1% naphthylethylenediamide dihydrochloride in H2O and 1% sulfanilamide in 5% concentrated H2PO4; 1:1, vol/vol) to 100-µl samples. The OD at 550 nm (OD550) was measured using an ELISA microplate reader (SLT-Labinstruments, Salzburg, Austria). Nitrate concentrations were calculated by comparison with the OD550 of standard solutions of DMEM.
Cytokines
The plasma contents of TNF, IL-6, and IL-1ß were measured 32 d after ovariectomy by immunoenzymatic assays (Calbiochem, La Jolla, CA).
Tissue processing and analysis
After killing the animals by CO2 narcosis, calvariae and hind limbs were removed, and soft tissues were gently dissected. Bones were fixed in 10% phosphate-buffered formalin (pH 7.4) for 24 h and decalcified in EDTA for 7–8 d. After being dehydrated in different graded alcohol concentrations, tibiae and femurs were paraffin embedded. Longitudinal sections of femur and tibial metaphyses (5 µm thick) were prepared on silanized slides and stained with Masson’s Trichrome as previously reported (12). To facilitate histomorphometric measurements, longitudinal sections through the epiphyses of the tibias and femurs were also obtained for analysis.
Structural morphometry
Blind histomorphometric analysis was carried out by the Measure System (Lucia, Nikon, Florence, Italy). Structural analyses were performed using digital two-dimensional images captured from Masson’s Trichrome-stained sections of the proximal tibia. Bone histomorphometry was performed under a microscope using UV light (Optiphot, Nikon, Tokyo, Japan) to obtain static and dynamic parameters. The histomorphometry parameters used were derived from Parfitt and colleagues and were approved by an ASBMR committee (24, 25). To assess bone structure, trabecular bone volume and osteoid volume were measured relative to total volume (BV/TV and OV/TV), we also measured trabecular thickness and trabecular number. To assess bone resorption we quantified the eroded surface, osteoclast surface, and osteoclast number as relative to bone surface. To evaluate bone formation, osteoblast surface was quantified relative to bone surface. Cells with one or more nuclei that formed resorption lacunae at the surface of trabeculae were identified as osteoclasts.
Calcein labeling
To define bone growth, a modified histomorphometric analysis was performed using labeling with calcein as marker of mineralization. Oxytetracycline (30 mg/kg body weight) was administered on d 22 after ovariectomy and calcein (20 mg/kg; Sigma-Aldrich) was injected ip on d 28 after surgery. Mice were killed 4 d after the calcein injection. Calcein labeling was measured in the tibiae and femurs using samples formalin-fixed and embedded in methyl methacrylate.
The double labeling was visualized with a Nikon fluorescence microscope equipped with both visible and UV light sources (LSM 510, Zeiss, Milan, Italy). Histomorphometric measurements of cancellous bone were made in the secondary spongiosa of the proximal tibia metaphysis. To study bone formation, the following parameters were measured and calculated: double-label surface; single-label surface; mineralizing surface, equal to double-label surface/bone surface plus one half of single label surface/bone surface; mineral apposition rate (MAR), calculated as the distance between double labels divided by interval labeling time and then multiplied by 
/4; and formation rate per surface reference, equal to mineralizing surface/bone surface times MAR.
Immunohistochemical localization of iNOS, nitrotyrosine, and poly(ADP-ribose) synthetase (PARS)
Sections from decalcified femur and tibia were used for immunohistochemical evaluation as previously described (26). After deparaffinization and rehydration, endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min; sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Nonspecific Ab absorption was reduced by incubating the section in 2% preimmune goat serum in PBS for 20 min. Endogenous biotin- or avidin-binding sites were blocked by sequential incubation for 15 min with avidin and biotin. Sections were incubated overnight with 1) anti-iNOS polyclonal antibody (1:500 in PBS, vol/vol); or 2) with antinitrotyrosine antibody (1:500 in PBS, vol/vol; DBA, Milan, Italy), or 3) with anti-PARS goat polyclonal antibody (1:500 in PBS, vol/vol). After washing with PBS, the incubation with the secondary antibody was carried out for 2 h at room temperature. Specific labeling was detected with an avidin-biotin peroxidase complex.
Control sections were also incubated with the primary antibody (antinitrotyrosine) in the presence of excess nitrotyrosine (10 mM). To verify the specificity of PARS and iNOS immunoreactivity, either the primary or secondary antibodies were omitted from the sequelae of reactions.
iNOS promoter activity in transiently transfected cells
Twenty-four hours before transfection, 8 x 104 cells were plated in 24-well plates containing 1 ml phenol red-free RPMI 1640 medium supplemented with 10% dextran-coated charcoal/fetal bovine serum (FBS). Six hours before addition of the calcium phosphate-DNA mix, medium was replaced with DMEM with 10% dextran-coated charcoal/FBS. Plasmids containing 3.5 kb of the upstream sequence of the iNOS promoter linked to luciferase (LUC) were used to cotransfect COS-1 cells grown in 24-well plates. One thousand micrograms of DNA (pBKCMV) mixes were added to each well using the carrier DNA to reach the same final DNA concentration in each transfection mix. Sixteen hours after transfection, medium was replaced with phenol red-free RPMI 1640 containing 1% charcoal-stripped FBS in the presence or absence of hormone and phorbol 12-myristate 13-acetate (PMA) as specified in each figure. Twenty-four hours later, the medium was removed, cells were washed three times with PBS, and cells extracts were prepared with cell culture lysis reagent 5x (Promega Corp., Madison, WI), as specified by the manufacturer. Luciferase activity was measured by integrating the luminescence signal for 20 sec; experimental values are expressed as arbitrary luminescence units. Protein content was measured according to the method of Bradford. The reported LUC activity is calculated by normalizing the LUC levels with the protein content. Each experiment was performed on triplicate samples and repeated at last three times.
Statistical analysis
All values in the figures and text are expressed as mean ± SEM of n observations. For the in vivo studies, n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way ANOVA, followed by a Bonferroni post hoc test for multiple comparisons. P < 0.05 was considered significant.
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Results |
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shows that inhibition of iNOS activity protected by the significant decrease in bone volume and turnover induced by ovariectomy. It is of interest to underline that genetic and pharmacological ablation of iNOS activity by itself did not modify any of the histomorphometric parameters.
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shows that ovariectomy in iNOSWT mice severely affected bone structure by decreasing BV/TV and the number of trabecular cells (respectively, -40% and -28% vs. sham-operated). In ovariectomized (OVX) iNOSKO and L-NIL-treated mice, neither BV/TV nor trabecular number was statistically different from those in sham-operated mice. In addition, a significant reduction of the OV/TV was observed in the OVX iNOSWT in comparison with sham animals. A significant inhibition of the loss of OV/TV was observed in OVX iNOSKO and L-NIL-treated mice. However, the osteoid volume was still reduced in OVX iNOSKO and L-NIL-treated mice in comparison with sham mice. Analysis of bone resorption parameters indicates that ovariectomy severely affected bone eroded surface (+71%), the active resorption surface (+366%), and the number of osteoclasts (+228%). In OVX iNOSKO and L-NIL-treated mice the eroded surface was not statistically different from that in sham-operated mice, and the genetic ablation of iNOS or treatment with L-NIL protected by about 80% against the increased bone resorption and osteoclast number. Bone formation was also impaired after ovariectomy, with about a 50% decrease in MAR. Ovariectomy did not significantly alter this parameter in iNOS or L-NIL-treated mice. Consistent with the above parameters, the rate of bone formation was severely reduced by ovariectomy only in mice with viable iNOS activity.
Histological examination of the femur and tibia epiphyses further demonstrated that iNOSKO mice are protected by ovariectomy-induced bone damage. After ovariectomy, the femur (Fig. 1A) and tibia (Fig. 1C) epiphyses of wild-type mice have necrosis and sloughing of the articular space, bone erosion (see arrow). Therefore, alteration of the trabecular architecture (Fig. 2A) was observed in iNOSWT mice after ovariectomy. These damages are not observed in femur (Fig. 1B) and tibia (Fig. 1D) ephiphysis, as well as in the trabecular architecture (Fig. 2B) of iNOSKO mice whose morphology was indistinguishable from non-OVX iNOSWT mice.
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) 32 d after ovariectomy, strong staining was observed in chondrocytes (see arrows, Fig. 3A1), indicating a response of the enzyme to ovary ablation; conversely, no staining was found in sham-operated mice (not shown) or in OVX iNOSKO mice (Fig. 3, B and B1). No positive staining was observed in the negative control section (Fig. 3C). In addition, after ovariectomy, plasma NOx levels were significantly increased (+170% vs. sham-operated; Fig. 4), further supporting the concept of induction of iNOS as a consequence of lowered levels of circulating estradiol. The ovariectomy-dependent increase in NOx, however, was not observed in animals lacking a functional iNOS gene or chronically treated with the specific iNOS inhibitor L-NIL (1 mg/kg daily, ip, for 32 d). This observation suggests that the changes induced by ovariectomy require a functional iNOS gene to occur and are not triggered by NOS isoenzymes. Interestingly, genetic or pharmacological ablation of iNOS did not result in altered physiological levels of NOx in plasma. This is in accordance with previous reports (27) and is predicted on the basis of the well demonstrated presence of constitutive eNOS in lumina endothelium determining the levels of NO in plasma.
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) in the femur metaphysis portion of WT OVX mice (Fig. 5B), but not in OVX iNOSKO (Fig. 5, C and C1) or sham-operated WT mice (not shown). In agreement with these results we found that PARS staining was significantly augmented in chondrocytes (see arrows in Fig. 5E1) in the femur metaphysis portion of OVX WT mice (Fig. 5E), but was not revealed in sections from OVX iNOSKO (Fig. 5, F and F1) or sham-operated WT (not shown) mice. No positive staining was observed in the negative control section for nitrotyrosine (Fig. 5A) and PARS (Fig. 5D) reaction.
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, which have been previously shown to be modulated by estrogens. After ovariectomy a severalfold increase in IL-1ß, IL-6, and TNF plasma levels was found in WT mice (Fig 6). Interestingly, in mice not expressing iNOS or treated as before with the iNOS inhibitor L-NIL, the effect of ovariectomy on plasma IL-1ß, IL-6, and TNF was severely reduced, suggesting that the lack of estrogen stimulated iNOS production, which, in turn, up-regulated the synthesis of proinflammatory cytokines.
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or ERß in the presence of the inos promoter driving the luciferase reporter gene. Figure 7 shows that estradiol-activated ERs do not up- or down-regulate the activity of the promoter; however, they significantly opposed luciferase accumulation induced by a well-known positive regulator of the promoter PMA (Fig. 7). In the absence of cotransfected receptor, estradiol does not alter iNOS promoter activity. Thus, in agreement with the results of the present study, the iNOS promoter may be a target for negative regulation by the estradiol-receptor complex.
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Discussion |
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and ERß blocks the transcription of iNOS promoter induced by phorbol esters. This is, to the best of our knowledge, the first evidence of a direct effect of estrogen on iNOS gene activity. Bone metabolism is thought to be locally regulated by a network of cytokines produced by osteoblasts, bone marrow stoma cells, and peripheral blood monocytes. Our results, in accordance with previous reports, show that in mice with intact ovarian function, iNOS depletion does not affect bone turnover. Indeed, sham-operated iNOSKO mice have a normal number of osteoclasts and normally active bone resorption, as measured by the extent of eroded bone surface containing osteoclasts. Accordingly, iNOS is apparently not required for normal trabecular bone development, as neither gross structural abnormalities nor significant differences in trabecular bone volume were observed in the bones of iNOSKO mice.
The absence of iNOS becomes crucial when bone loss is induced by estrogen depletion, suggesting a predominant role of this enzyme in the osteoporosis consequent to deficient ovarian function. Our findings, suggesting a relevant role for iNOS in osteoporosis, are in agreement with recent studies carried out on two different models of osteoporosis. Armour and colleagues (30) demonstrated that iNOS is indispensable for the development of osteoporosis consequent to systemic inflammation and proposed that the reduced bone formation mediated by iNOS is due to the dramatic increase in osteoblast apoptosis. Watanuki and colleagues (31) showed that the decreased bone volume induced by tail suspension is not observed in iNOSKO mice and suggested that the iNOS synthesized in osteoblasts plays a critical role in increasing osteogenic activity in response to the acute increase in mechanical loading after tail suspension. Our morphometric data point to a strong role for iNOS in osteoclast activation after ovariectomy. The complexity of our in vivo study, however, does not allow full definition of the mechanisms involved in this effect.
By investigating other cell systems relevant for iNOS activity, such as smooth muscle cells, we previously showed that prolonged exposure to a medium deprived of estrogens by itself is a stimulus sufficient to induce iNOS synthesis (32). In line with this observation, it may be proposed that with ovariectomy the prolonged absence of estrogens causes a depression of iNOS synthesis in osteoblasts. Osteoblasts express ERs (33, 34), and it is conceivable that the circulating levels of estradiol maintain the receptors in a state of activation sufficient to repress transcription of the iNOS promoter. Indeed, we here show that in transiently transfected cells estradiol opposes the synthesis of iNOS induced by a phorbol ester, PMA, via a receptor-mediated mechanism. Sequence analysis of the mouse iNOS promoter does not reveal any canonical estrogen-responsive element; however, this promoter contains NF-B and activating protein I-1-responsive elements. As it is well known that estradiol may modulate the activities of promoters controlled by these two transcription factors, it is possible that in the presence of estradiol the ER impairs their positive action on inos gene transcription. The absence of the hormone would release the ER inhibitory influence, with consequent pathological augmentation of iNOS production.
In osteoblasts, an increased iNOS content may lead to local accumulation of nitrites and other products of the inflammatory cascade, which trigger osteoclastogenesis, or alternatively, the NO produced by osteoblasts may also activate p21ras activity in the monocytes and the NF-B signal transduction pathway necessary for osteoclast differentiation.
The effect of estradiol may not be restricted to osteoblasts. In previous studies we showed that monocytes and macrophages express ERs (14). It could be hypothesized that estradiol represses osteoclast function, in line with recent reports demonstrating that this hormone blocks microglia activation induced by inflammatory compounds such as lipopolysaccharides (15).
We here report that ovariectomy results in increased immunostaining of PARS and nitrotyrosine. This effect is mostly observed in chondrocytes, and this could impair their viability; however, in disagreement with Armour et al. we observed no evidence of an increase in chondrocytes and/or osteocyte apoptosis. Despite the increased levels of iNOS here reported in chondrocytes of iNOSWT mice after ovariectomy, we cannot conclude that estrogens modulate iNOS synthesis directly in these cells.
The present study also shows that after estrogen depletion the plasma levels of NO are significantly increased; as it is well known that eNOS is positively regulated by estrogens, this increase could be ascribed to a deficit of the estrogen-negative control on the expression of the smooth muscle cell iNOS. Interestingly, using either the iNOS inhibitor L-NIL or iNOS knockout mice, we demonstrated that iNOS contributes to the increased expression of the cytokines IL-6, IL-1ß, and TNF consequent to estrogen depletion. IL-1, IL-6, and TNF are known to be potent stimulators of bone resorption. The finding that the basal levels of IL-1-ß, IL-6, and TNF were not decreased in knockout mice or after prolonged administration of L-NIL indicates that in the absence of an appropriate stimulus (e.g. estrogen depletion) iNOS does not have any influence of the systemic synthesis of these proinflammatory compounds. These cytokines had been proposed to play an important role in the induction of osteoclastogenesis and bone loss after estrogen depletion. It was shown that secretion of both IL-1 and TNF by peripheral blood monocytes is increased in postmenopausal women and in both men and women with osteoporosis (35). Interestingly, the release of these cytokines can be strongly reduced by iNOS inhibition in shock (36, 37, 38). The current data support the idea that NO can increase cytokine expression (39, 40). An association between the up-regulation of these proinflammatory events by NO and bone erosion is shown by our experiments demonstrating reduced bone alteration in the joint with iNOS suppression. These data provide compelling evidence not only that iNOS is in part responsible for bone erosion after estrogen depletion, but also that the induction of NO synthesis is a key event in the subsequent activation of inflammatory cascades after estrogen depletion.
NO can have both direct effects on cell signaling as well as indirect actions mediated by the reaction products formed when NO interacts with other molecules such as oxygen or superoxide (9, 20). ROS and peroxynitrite produce cellular injury and necrosis via several mechanisms, including peroxidation of membrane lipids, protein denaturation, and DNA damage. ROS produce strand breaks in DNA that trigger energy-consuming DNA repair mechanisms and activate the nuclear enzyme PARS, resulting in the depletion of its substrate NAD in vitro and a reduction in the rate of glycolysis. As NAD functions as a cofactor in glycolysis and the tricarboxylic acid cycle, NAD depletion leads to a rapid fall in intracellular ATP. This process has been termed the PARS suicide hypothesis (41). There is recent evidence that the activation of PARS may also play an important role in inflammation (42, 43, 44). We demonstrate here that absence of the inos gene attenuates the increase in PARS activity caused by estrogen depletion in the joint. Thus, we propose that iNOS induction is also important, at least in part, for the activation of PARS. Therefore, we suggest that PARS play a role in the bone erosion after estrogen depletion.
In conclusion, this study identifies iNOS as a key signaling molecule (Fig. 8) in determining the imbalance between bone resorption and bone formation caused by estrogen depletion in mice and indicates that it is a potential target for therapy of postmenopausal osteoporosis in women. We believe that the interest of the observations here presented is not restricted to osteoporosis. Our findings may indicate a novel, unifying hypothesis to explain the pleiotropic effects of estrogens in diseases etiologically very unlike, but all characterized by the presence of an inflammatory components that drives their progression or maintenance; by blocking iNOS activity, estrogens would oppose the inflammatory process and delay the onset of Alzheimer’s, improve the symptomatology of multiple sclerosis, limit the brain damage induced by ischemia, and prevent cardiovascular disease.
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Footnotes |
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B, nuclear factor-B; NO, nitric oxide; NOS, NO synthase; NOx, nitrite/nitrate; OVX, ovariectomized; PARS, poly(ADP-ribose) synthetase; PMA, phorbol 12-myristate 13-acetate; RANK, receptor activator of NF-B; RANKL, ligand of RANK; WT, wild-type. Received June 7, 2002.
Accepted for publication November 18, 2002.
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, and prostaglandin E impact the osteoclastic cell-forming potential of murine bone marrow macrophages. Endocrinology 136:2367–2376[Abstract]
-induced apoptosis. FASEB J 13:793–803 (ERR) is expressed throughout osteoblast differentiation and regulates bone formation in vitro. J Cell Biol 153:971–984This article has been cited by other articles:
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V. Garcia Palacios, L. J. Robinson, C. W. Borysenko, T. Lehmann, S. E. Kalla, and H. C. Blair Negative Regulation of RANKL-induced Osteoclastic Differentiation in RAW264.7 Cells by Estrogen and Phytoestrogens J. Biol. Chem., April 8, 2005; 280(14): 13720 - 13727. [Abstract] [Full Text] [PDF]
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0.001 vs. PMA-treated cells.