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Estradiol inhibits adhesion and promotes apoptosis in murine osteoclasts in vitro
Abstract
Osteoporosis caused by estrogen deficiency is characterized by enhanced bone resorption mediated by osteoclasts. Adhesion to bone matrix and survival of differentiated osteoclasts is necessary to resorb bone. The aim of our study was to investigate the in vitro effects of estradiol on murine osteoclasts. RAW 264.7 cells treated with 30 ng/ml RANK-L were used as a model for osteoclastogenesis. Estradiol (10(-8)M) for 5 days induced an inhibition of osteoclast differentiation and beta3 expression. Estradiol inhibited significantly the adhesion of mature osteoclasts by 30%. Furthermore estradiol-induced apoptosis shown by with nuclear condensation and Bax/Bcl2 ratio. In addition, estradiol enhanced caspase-3, -8 and -9 activities. This effect completely disappeared using specific caspase-8 inhibitor. However, increased caspase-3 activity by estradiol was observed in the presence of caspase-9 inhibitor, indicating the preferential involvement of caspase-8 pathway. Fas and FasL mRNA expression was not regulated by estradiol. However, estradiol enhanced caspase-3 activity in Fas-induced apoptosis on mature osteoclasts, suggesting that this might interact with the Fas-signaling pathway. These data suggest that estradiol decreases bone resorption by several mechanisms including adhesion and apoptosis of osteoclasts.
... Studies including different models have suggested that estrogen is able to promote osteoclast apoptosis but the mechanism is still unclear (reviewed by Saintier et al. 2006;Faloni et al. 2007;Cruzoé -Souza et al. 2009). The treatment of young female rats with estrogen has caused a 50% reduction in the number of alveolar bone osteoclasts due to osteoclast death by apoptosis (Faloni et al. 2007;Cruzoé -Souza et al. 2009). ...
... Moreover, the structural integrity of the osteoclasts and the bone resorptive surface were significantly altered, probably due to estrogen-induced cell death by apoptosis. Osteoclast apoptosis involves caspase-3, which has been shown to cleave vimentin, inducing its disruption and collapse of the cytoskeleton in many cell types (Byun et al. 2001;Lee et al. 2002;Saintier et al. 2006;Alam et al. 2010). A cytoskeleton containing vimentin filaments distributed around the nuclei and in the ruffled border region has been described in osteoclasts (Akisaka et al. 2008). ...
... In EG, most of the TRAP-positive cells (about 68%) were small osteoclasts; large osteoclasts were not found. Moreover, in the EG, about 50% of these TRAP-positive cells were mononucleated, supporting in vitro findings (Saintier et al. 2006). It is not possible to exclude the hypothesis that these mononucleated cells are osteoclast precursors that fail to fuse during osteoclastogenesis, probably due to the estrogen action on the RANK ⁄ RANKL ⁄ OPG [receptor activator of nuclear factor (NF)-jB ⁄ RANK ligand ⁄ osteoprotegerin] system (Kawamoto et al. 2002;Rogers et al. 2002;Bord et al. 2003;Kanzaki et al. 2006). ...
This study investigated structural and functional features of apoptotic alveolar bone osteoclasts in estrogen-treated rats. For this purpose, 15 female rats 22 days old were divided into three groups: Estrogen (EG), Sham (SG) and Control (CG). The rats of EG received daily intramuscular injection of estrogen for 7 days. The SG received only the oil vehicle. Maxillary fragments containing alveolar bone were removed and processed for light and transmission electron microscopy. Area (OcA) and number of nuclei (OcN) and bone resorption surface per TRAP-positive osteoclasts (BS/OC) were obtained. Vimentin, caspase-3 and MMP-9 immunoreactions, TUNEL/TRAP and MMP-9/TUNEL combined reactions were performed. In EG, the OcA, OcN and BS/Oc were reduced. Moreover, osteoclasts showed cytoplasm immunolabelled by caspase-3 and a different pattern of vimentin expression in comparison with CG and SG. MMP-9 expression was not affected by estrogen and the TUNEL-positive osteoclasts were MMP-9-immunolabelled. In EG, ultrastructural images showed that apoptotic osteoclasts did not exhibit ruffled borders or clear zones and were shedding mononucleated portions. TRAP-positive structures containing irregular and dense chromatin were partially surrounded by fibroblast-like cells. In conclusion, the reduction in the BS/Oc may be due to reduction in OcA and OcN; these effects seem to be related to vimentin disarrangement rather than to an interference of estrogen with osteoclast MMP-9 expression. Osteoclast apoptosis involves caspase-3 activity and vimentin degradation; these cells release portions containing one apoptotic nucleus and, subsequently, undergo fragmentation, giving rise to apoptotic bodies.
... Exercise has attracted much clinical attention because of its convenience, affordability, and safety, and it has been recommended by many guidelines for the prevention and treatment of osteoporosis [43,44]. It has been proven that exercise can effectively intervene in the symptoms of PMOP [45][46][47][48]. e effect of exercise on estrogen levels may explain its therapeutic effect. ...
... Moreover, estrogen plays a very important role in the mechanism of female bone metabolism. Studies have shown that exercise can promote a slight increase in estrogen concentration [45]. Estrogen inhibits the secretion of thyroid hormone, which, in turn, reduces bone absorption, promotes the secretion of calcitonin, and reduces bone resorption. ...
Background:
Postmenopausal osteoporosis (PMOP) is a common disease in older women that can severely jeopardize their health. Previous studies have demonstrated the effect of Er xian decoction (EXD) or Baduanjin exercise (BE) on PMOP. However, reports on the effect of EXD combined with BE on PMOP are limited. This study aimed to investigate the impact of EXD combined with BE on bone mineral density (BMD), lower limb balance, and mental health in women with PMOP.
Methods:
A 1 : 1 : 1 simple randomization technique was employed. Fifty participants with postmenopausal osteoporosis were allocated to three groups: the EXD group (EXD = 15); the BE group (BE = 18); and the combined group (EXD + BE = 17). After both 8 weeks and 16 weeks of intervention treatment, participants improved significantly with respect to BMD and the one-leg standing test (OLST), Berg balance scale (BBS), timed up and go (TUG) test, self-anxiety scale (SAS), and self-rating depression scale (SDS). The results were used to compare the effect of the intervention on BMD, lower limb balance function, and mental health in patients with PMOP.
Results:
Compared to the EXD and BE groups, the EXD + BE group showed the strongest effects on BMD, lower limb balance function, and mental health (p < 0.01). A correlation between BMD and lower limb balance and mental health was noted in the EXD + BE group. The change in mental health (SAS score) was correlated with BMD (femoral neck) improvement.
Conclusions:
The present study demonstrates that EXD combined with BE (EXD + BE) may have a therapeutic advantage over both monotherapies for treating BMD, lower limb balance function, and mental health in patients with PMOP. The feasibility of the approach for a large-scale RCT was also confirmed. Er xian decoction combined with Baduanjin exercise (EXD + BE) might offer a viable treatment alternative for participants with postmenopausal osteoporosis given its promising effects in disease control and treatment, with good efficacy and safety profiles.
... The finding that mice lacking FasL or Fas are resistant to OVX-induced bone loss supported a role for Fas/FasL in the anti-osteoporotic actions of estrogens 7,18 . However, several laboratories, including ours, have not been able to confirm a stimulatory effect of E 2 on FasL production in primary cultures of murine osteoclasts [19][20][21][22] . Based on in vitro evidence from an osteoblast-like cell line that estrogen activated ERα signaling directly regulates FasL via an ERE-containing transcriptional enhancer, Krum et al. proposed the alternative idea that FasL stimulates osteoclast apoptosis in a paracrine manner 19 . ...
... We show here that E 2 decreases osteoclastogenesis in osteoclast progenitors lacking FasL, but not in cells lacking Bak/Bax suggesting that Bak/Bax stimulation, independently of FasL, is responsible for the decrease in osteoclast formation. These evidence together with the findings that FasL gld/gld mice lose bone mass following OVX indistinguishable from controls does not support the contention that FasL plays a critical role in the effects of estrogens on bone [19][20][21][22] . The reasons for the different results between our study and that of Nakamura et al. with the same FasL gld/gld mice are unclear. ...
Loss of estrogens at menopause is a major cause of osteoporosis and increased fracture risk. Estrogens protect against bone loss by decreasing osteoclast number through direct actions on cells of the myeloid lineage. Here, we investigated the molecular mechanism of this effect. We report that 17β-estradiol (E2) decreased osteoclast number by promoting the apoptosis of early osteoclast progenitors, but not mature osteoclasts. This effect was abrogated in cells lacking Bak/Bax—two pro-apoptotic members of the Bcl-2 family of proteins required for mitochondrial apoptotic death. FasL has been previously implicated in the pro-apoptotic actions of E2. However, we show herein that FasL-deficient mice lose bone mass following ovariectomy indistinguishably from FasL-intact controls, indicating that FasL is not a major contributor to the anti-osteoclastogenic actions of estrogens. Instead, using microarray analysis we have elucidated that ERα-mediated estrogen signaling in osteoclast progenitors decreases “oxidative phosphorylation” and the expression of mitochondria complex I genes. Additionally, E2 decreased the activity of complex I and oxygen consumption rate. Similar to E2, the complex I inhibitor Rotenone decreased osteoclastogenesis by promoting osteoclast progenitor apoptosis via Bak/Bax. These findings demonstrate that estrogens decrease osteoclast number by attenuating respiration, and thereby, promoting mitochondrial apoptotic death of early osteoclast progenitors.
... Previous studies have reported that estrogen promotes osteoclast apoptosis. Saintier et al. demonstrated that estrogen induced nuclear condensation and increased Bax, CASP3, and CASP9 protein expression without altering BCL2 protein expression and FASL and FAS mRNA expression in osteoclasts, eventually inducing osteoclast apoptosis [33]. However, Nakamura et al. showed that estrogen induced osteoclast apoptosis by upregulating FasL expression [5]. ...
... Inhibition of ITGB3 in vitro decreased the ability of osteoclasts to bind and degrade bone and promoted osteoclast apoptosis [18,39,40]. There is evidence that estrogen reduces ITGB3 expression in differentiating and mature osteoclasts in humans and mice and inhibits osteoclast adhesion [9,33]. To confirm the effect of estrogen on integrin-mediated osteoclast adhesion, we further analyzed the differential expression of proteins in the integrin-mediated cell adhesion pathway. ...
Estrogen plays an important role in inhibiting osteoclast differentiation and protecting against bone loss from osteoporosis, especially in postmenopausal women. However, the precise mechanisms underlying the effect of estrogen on osteoclasts are not well known. In the present study, we performed proteomics analysis and bioinformatics analysis to comprehensively compare the differential expression of proteins in receptor activator of nuclear factor-κB ligand RANKL-induced osteoclasts in the presence and absence of estrogen. We identified 6403 proteins, of which 124 were upregulated and 231 were downregulated by estrogen. Bioinformatics analysis showed that estrogen treatment interfered with 77 intracellular pathways, including both confirmed canonical and unconfirmed pathways of osteoclast formation. Our findings validate the inhibitory effect of estrogen on osteoclasts via the promotion of apoptosis and suppression of differentiation and polarization and suggest that estrogen might inhibit osteoclast formation via other pathways, which requires further investigation and verification.
... inhibiting osteoclasts686970. However, in intact mice with 12-hr exposure to dark, the combination therapy did not augment normal bone formation. ...
... Similarly, estradiol/progesterone HRT increased bone density, increased bone volume density, reduced trabecular space, and decreased the number of osteoclasts on mouse tibia following 1 yr of treatment. These positive parameters on bone formation were expected because estrogen is known to improve bone density by inhibiting osteoclasts686970 and estrogen deficiency, as a result of menopause, increases oneÕs risk of osteoporosis [23]. No changes in Runx2, Bmp2, Bmp6, Bglap, and Per2 mRNA occurred, but these findings could be due to the mice being euthanized during the light hours, the time of day when the levels of these genes are lowest (Figs 1 and 2). ...
... inhibiting osteoclasts686970. However, in intact mice with 12-hr exposure to dark, the combination therapy did not augment normal bone formation. ...
... Similarly, estradiol/progesterone HRT increased bone density, increased bone volume density, reduced trabecular space, and decreased the number of osteoclasts on mouse tibia following 1 yr of treatment. These positive parameters on bone formation were expected because estrogen is known to improve bone density by inhibiting osteoclasts686970 and estrogen deficiency, as a result of menopause, increases oneÕs risk of osteoporosis [23]. No changes in Runx2, Bmp2, Bmp6, Bglap, and Per2 mRNA occurred, but these findings could be due to the mice being euthanized during the light hours, the time of day when the levels of these genes are lowest (Figs 1 and 2). ...
... inhibiting osteoclasts686970. However, in intact mice with 12-hr exposure to dark, the combination therapy did not augment normal bone formation. ...
... Similarly, estradiol/progesterone HRT increased bone density, increased bone volume density, reduced trabecular space, and decreased the number of osteoclasts on mouse tibia following 1 yr of treatment. These positive parameters on bone formation were expected because estrogen is known to improve bone density by inhibiting osteoclasts686970 and estrogen deficiency, as a result of menopause, increases oneÕs risk of osteoporosis [23]. No changes in Runx2, Bmp2, Bmp6, Bglap, and Per2 mRNA occurred, but these findings could be due to the mice being euthanized during the light hours, the time of day when the levels of these genes are lowest (Figs 1 and 2). ...
In this study, the effects of the light/dark cycle, hormone replacement therapy (HRT), and nocturnal melatonin supplementation on osteogenic markers and serum melatonin levels were examined in a blind mouse model (MMTV-Neu transgenic mice). Melatonin levels in this mouse strain (FVB/N) with retinal degeneration (rd-/-) fluctuate in a diurnal manner, suggesting that these mice, although blind, still perceive light. Real-time RT-PCR analyses demonstrated that Runx2, Bmp2, Bmp6, Bglap, and Per2 mRNA levels coincide with melatonin levels. The effect of chronic HRT (0.5 mg 17β-estradiol + 50 mg progesterone in 1800 kcal of diet) alone and in combination with melatonin (15 mg/L drinking water) on bone quality and density was also assessed by histomorphometry and microcomputed tomography, respectively. Bone density was significantly increased (P < 0.05) after 1 yr of treatment with the individual therapies, HRT (22% increase) and nocturnal melatonin (20% increase) compared to control. Hormone replacement therapy alone also increased surface bone, decreased trabecular space, and decreased the number of osteoclasts without affecting osteoblast numbers compared to the control group (P < 0.05). Chronic HRT + melatonin therapy did not significantly increase bone density, even though this combination significantly increased Bglap mRNA levels. These data suggest that the endogenous melatonin rhythm modulates markers important to bone physiology. Hormone replacement therapy with or without nocturnal melatonin in cycling mice produces unique effects on bone markers and bone density. The effects of these therapies alone and combined may improve bone health in women in perimenopause and with low nocturnal melatonin levels from too little sleep, too much light, or age.
... The topoisomerase inhibitor etoposide did stimulate osteoclast apoptosis in cells from the FasL gld/gld mice, eliminating the possibility that cells from these mice are resistant to all inducers of apoptosis (Fig. 7E). Strikingly, in spite of the requirement of FasL for the proapoptotic effects of E 2 , DHT, and EDC, none of these compounds was able to stimulate the expression of FasL mRNA in mature osteoclasts (Fig. 7F), confirming several previous reports that estrogens do not stimulate FasL in osteoclasts (18,(25)(26)(27). Consistent with the lack of an effect at the mRNA level, there was no change in FasL protein levels in mature osteoclasts treated with E 2 between 1 and 24 h (Fig. 7G). ...
... Likewise, E 2 could not in-duce the apoptosis of osteoclasts derived from FasLdeficient mice. However, in contrast to the results of Nakamura et al. (17), but in agreement with the findings of Krum et al. (18) as well as several other studies (25)(26)(27), we were unable to demonstrate a stimulatory effect of E 2 on FasL production in primary cultures of murine osteoclasts. FasL-deficient osteoclasts were also resistant to the proapoptotic effect of the nonaromatizable androgen DHT. ...
Estrogens attenuate osteoclastogenesis and stimulate osteoclast apoptosis, but the molecular mechanism and contribution of these effects to the overall antiosteoporotic efficacy of estrogens remain controversial. We selectively deleted the estrogen receptor (ER)alpha from the monocyte/macrophage cell lineage in mice (ERalpha(LysM)(-/-)) and found a 2-fold increase in osteoclast progenitors in the marrow and the number of osteoclasts in cancellous bone, along with a decrease in cancellous bone mass. After loss of estrogens these mice failed to exhibit the expected increase in osteoclast progenitors, the number of osteoclasts in bone, and further loss of cancellous bone. However, they lost cortical bone indistinguishably from their littermate controls. Mature osteoclasts from ERalpha(LysM)(-/-) were resistant to the proapoptotic effect of 17beta-estradiol. Nonetheless, the effects of estrogens on osteoclasts were unhindered in mice bearing an ERalpha knock-in mutation that prevented binding to DNA. Moreover, a polymeric form of estrogen that is not capable of stimulating the nuclear-initiated actions of ERalpha was as effective as 17beta-estradiol in inducing osteoclast apoptosis in cells with the wild-type ERalpha. We conclude that estrogens attenuate osteoclast generation and life span via cell autonomous effects mediated by DNA-binding-independent actions of ERalpha. Elimination of these effects is sufficient for loss of bone in the cancellous compartment in which complete perforation of trabeculae by osteoclastic resorption precludes subsequent refilling of the cavities by the bone-forming osteoblasts. However, additional effects of estrogens on osteoblasts, osteocytes, and perhaps other cell types are required for their protective effects on the cortical compartment, which constitutes 80% of the skeleton.
... According to other reports, estrogen is able to induce apoptosis in undifferentiated U937 monocytes, but macrophages differentiated from these cells are refractory to such effects of estrogen (21). Further examples of cell types in which estrogen is reported to induce cell death include bone macrophages like murine oste-oclasts, preosteoclastic FLG 29.1 cell line, and mouse peritoneal macrophages (22)(23)(24)(25). ...
... No significant cell death could be recorded with any of the doses as compared with vehicle treated (cyclodextrin dissolved in water) controls. Percentage of survival was at the following: vehicle treated, 94 Ϯ 1; 1 nM E2 treatment, 95 Ϯ 2; 10 nM E2 treatment, 96 Ϯ 1; 100 nM E2 treatment, 96 Ϯ 1; and 1 M E2 treatment, 96 Ϯ 2. Contrary to our findings, some studies show that estrogen causes death in cells of monocytic lineage (19,(21)(22)(23)(24)(25). Arguably, if estrogen is able to induce death in cells of similar lineage, the failure of the hormone to do so in THP-1 macrophages could mean differential regulation of the proapoptotic and antiapoptotic proteins leading to maintenance of viability. ...
Estrogen is a known immunomodulator with pleiotropic effects on macrophage function that partly accounts for the gender bias observed in numerous autoimmune, cardiovascular, and neurodegenerative disorders. The effect of estrogen on the survival of human macrophages is largely unknown, and in this study we demonstrate that 17beta-estradiol (E2) provokes a death response in human THP-1 macrophages by initiating Bax translocation from cytosol to the mitochondria; however, a concomitant up-regulation of Bcl-2 creates a Bax to Bcl-2 ratio favorable for Bcl-2, thus ensuring cell survival. Both Bcl-2 up-regulation and Bax translocation are estrogen receptor-dependent events; however, Bcl-2 augmentation but not Bax translocation is dependent on Ca(2+) increase, activation of protein kinase C, and ERK phosphorylation. This estrogen-induced Bcl-2 increase is crucial for the survival of THP-1 macrophages as well as that of human peripheral blood monocyte-derived macrophages, which is evident from E2-induced cell death under small interfering RNA-mediated Bcl-2 knockdown conditions. Hence, this study demonstrates that E2-induced Bcl-2 up-regulation is a homeostatic survival mechanism necessary for the manifestation of immunomodulatory effect of estrogen on human macrophages.
... Estrogen has been implicated in the regulation of RANKL signaling and inhibits bone resorption in premenopausal women; however, the underlying mechanism has not yet been fully elucidated (Anagnostis et al., 2021). In addition, it has also been proposed that estrogen may regulate bone resorption through its direct effect on osteoclasts, which may be mediated, at least in part, by fast-acting nongenomic mechanisms (Kousteni et al., 2002;Saintier et al., 2006). Robinson LJ et al. found that estrogen inhibited RANKL-stimulated osteoclastic differentiation of human monocytes through estrogen and RANKL-regulated interaction of ER-α with BCAR1 and TRAF6 (Robinson et al., 2009). ...
Osteoporosis is the most common metabolic bone disease in postmenopausal women. As precursors of osteoclasts, peripheral blood mononuclear cells are accessible and considered suitable models for studying osteoporosis pathology. Ubiquitination is a crucial protein degradation system in bone metabolism. The aim of this study was to identify potential ubiquitination-related genes in PBMCs that are related to osteoporosis pathogenesis. Therefore, we performed an integrated analysis of osteoporosis-related microarray datasets. With the obtained ubiquitination-related gene set, weighted gene coexpression network analysis was performed. The results showed that genes in the turquoise module were correlated with menopause, and 48 genes were identified as hub genes. A differential expression analysis revealed 43 differentially expressed genes between pre- and postmenopausal samples. After integrating the information on differentially expressed menopause-related genes, we found that several members of the ubiquitin-specific protease ( USP ) family ( USP1 , USP7 , USP9X , USP16 , and USP25 ) were highly expressed in samples from postmenopausal female and that, USP25 expression was significantly higher in low-BMD samples than in high-BMD samples among samples from premenopausal subjects ( p = 0.0013) and among all samples ( p = 0.013). Finally, we verified the protein expression of USP25 in PBMCs by performing Western blot analysis, which yielded results consistent with the aforementioned results. Moreover, by assessing GTEx datasets, we found that USP25 expression was highly correlated with TRAF6 expression in whole blood ( p < 0.001). We also tested the protein expression levels of TRAF6 in PBMCs and found that it was positively correlated with USP25 expression ( p = 0.036). Our results reveal that the ubiquitin-specific protease family may play important roles in menopause and that USP25 is related to osteoporosis pathogenesis.
... Studies have proven that estrogen suppresses the formation of osteoclasts and activity in cultures of both rat and human osteoclasts 64,65 . Estrogen inhibits bone resorption by directly inducing the apoptosis of murine and rabbit osteoclasts in vitro 66,67 . It has been noticed that estrogens reduce osteoclast's lifespan by inducing their apoptosis, through enhancing Fas-ligand (FasL) gene transcription. ...
... In contrast, estrogen stimulates the expression of OPG in mouse stromal cells (Saika et al. 2001). Moreover, estrogen induces apoptosis of pre-osteoclasts and osteoclasts (Krum et al. 2008;Saintier et al. 2006). In the present study, serum estradiol concentration in quails after E2 administration substantially increased at day 1, then gradually decreased; but concentrations remained higher than in non-treated quails (Supplementary Fig. 4). ...
Osteoclasts are differentiated from hematopoietic mononuclear cells by regulation of the receptor activator of nuclear factor kappa-B (RANK)/receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) system. Medullary bone (MB) that forms in the bone marrow of female birds is remodeled under the control of circulating estrogen (E2) during the laying period. Although the osteoclasts of MB are differentiated from mononuclear cells, the mechanism of osteoclastogenesis is not known. We investigated whether MB osteoclastogenesis is regulated by the RANK/RANKL/OPG system using MB from male quails induced with E2. Bone marrow cells (BMCs) differentiate into osteoclasts that have the ability of bone resorption via stimulation of RANKL/M-CSF, but this ability is suppressed by OPG and differentiation is inhibited by calcinurin inhibitors. We found that BMCs at 3 days after E2 administration had high bone osteoclastogenesis ability and colony forming unit-granulocyte/macrophage (CFU-GM)/colony forming unit-macrophage (CFU-M) formation abilities. We conclude that MB osteoclasts are differentiated from BMCs by the RANK/RANKL/OPG system, and that precursor cells of osteoclasts are increased during MB formation.
... Studies have shown that estrogen plays an important role in bone remodeling by inhibiting osteoclasts formation and differentiation [62]. Estrogen also increases the inhibitory activity of BPs on osteoclasts [63], thereby inducing osteoclasts apoptosis. Based on these findings, La Ferla, et al postulated that estrogen may be associated with development of ONJ [52]. ...
Osteonecrosis of the jaw (ONJ) is a rare but serious drug induced adverse event, mainly associated with the use of antiresorptive medications, such as intravenous (IV) bisphosphonates (BPs) in cancer patients. In this review, we evaluated all the pharmacogenomic association studies for ONJ published up to December 2018. To date, two SNPs (CYP2C8 rs1934951 and RBMS3 rs17024608) were identified to be associated with ONJ by two genome-wide association studies (GWAS). However, all six subsequent candidate gene studies failed to replicate these results. In addition, six discovery candidate gene studies tried to identify the genetic markers in several genes associated with bone remodeling, bone mineral density, or osteoporosis. After evaluating the results of these 6 studies, none of the SNPs was significantly associated with ONJ. Recently, two whole-exome sequencing (WES) analysis (including one from our group) were performed to identify variants associated with ONJ. So far, only our study successfully replicated discovery result indicating SIRT1 SNP rs7896005 to be associated with ONJ. However, this SNP also did not reach genome-wide significance. The major limitations of these studies include lack of replication phases and limited sample sizes. Even though some studies had larger sample sizes, they recruited healthy individuals as controls, not subjects treated with BPs. We conclude that a GWAS with a larger sample size followed by replication phase will be needed to fully investigate the pharmacogenomic markers of ONJ.
... To determine the osteoblasts number (N.Ob) in OS a magnification of 400x was used (Axioimager Z2 microscope, Zeiss, Germany). Osteoclasts were stained with naphthol AS-TR (3-hydroxy-2-naphthoic acid 4-chloro-2-methylanilide) phosphate for tartrate-resistant acid phosphatase (TRAP) detection and counterstained with Toluidine blue (0.5%), [20]. Osteoclasts were counted and expressed relatively to bone surface as Oc.S/B.Ar (mm 2 ). ...
Osteoporosis is associated with chronic iron overload secondary to hereditary hemochromatosis (HH), but the causative mechanisms are incompletely understood. The main objective of this study was to investigate the role of dietary iron on osteoporosis, using as biological model the Hfe-KO mice, which have a systemic iron overload. We showed that these mice show an increased susceptibility for developing a bone loss phenotype compared to WT mice, which can be exacerbated by an iron rich diet. The dietary iron overload caused an increase in inflammation and iron incorporation within the trabecular bone in both WT and Hfe-KO mice. However, the osteoporotic phenotype was only evident in Hfe-KO mice fed the iron-enriched diet. This appeared to result from an imbalance between bone formation and bone resorption driven by iron toxicity associated to Hfe-KO and confirmed by a decrease in bone microarchitecture parameters (identified by micro-CT) and osteoblast number. These findings were supported by the observed downregulation of bone metabolism markers and upregulation of ferritin heavy polypeptide 1 (Fth1) and transferrin receptor-1 (Tfrc), which are associated with iron toxicity and bone loss phenotype. In WT mice the iron rich diet was not enough to promote a bone loss phenotype, essentially due to the concomitant depression of bone resorption observed in those animals. In conclusion the dietary challenge influences the development of osteoporosis in the HH mice model thus suggesting that the iron content in the diet may influence the osteoporotic phenotype in systemic iron overload conditions.
... To determine the osteoblasts number (N.Ob) in OS a magnification of 400x was used (Axioimager Z2 microscope, Zeiss, Germany). Osteoclasts were stained with naphthol AS-TR (3-hydroxy-2-naphthoic acid 4-chloro-2-methylanilide) phosphate for tartrate-resistant acid phosphatase (TRAP) detection and counterstained with Toluidine blue (0.5%), [20]. Osteoclasts were counted and expressed relatively to bone surface as Oc.S/B.Ar (mm 2 ). ...
Iron is an essential element in life, with roles in oxygen transport, cellular respiration and protein structure, however in excess can lead to cellular oxidative stress. The iron metabolism is characterized by a fine-tuning system with a highly regulated homeostasis due to absence of an active excretion mechanism. Bone loss phenotype has been reported as a common complication associated with Hereditary Hemochromatosis (HH), a genetic disorder associated with iron overload establishment. In this study we aimed to characterize the impact of an enriched iron diet on bone metabolism using Hfe-KO mouse as model for HH. The results showed that enriched iron diet led to a decrease in bone microarchitecture parameters (micro-Ct) in Hfe-KO mice which promoted the acceleration of bone loss. In addition, a significant decrease in osteoblast number and downregulation of bone metabolism markers was observed, as well as iron accumulation on bone surface and incorporation within mineralized matrix. The iron metabolism was altered in bone tissues with upregulation of transferrin-receptor-1 and ferritin-H subunit and an increase in expression of Tumor Necrosis Factor alpha and Catalase. These results suggest an increase in osteoblast death which may be a consequence of ferroptosis.
... Detection of BMSCs apoptosis was performed by Hoechst 33258 staining as described previously [17]. The BMSCs were incubated with different concentration of HGE (1, 10, 100 ng/mL) for 48 h, following with fixation with 4% formaldehyde for 15min at room temperature, then stained with Hoescht 33258 for 15min. ...
Elevated adipogenesis of bone marrow stromal cells (BMSCs) is closely associated with non-traumatic osteonecrosis of femoral head (ONFH). Our previous studies have shown that Huogu (HG) formula was effective both in clinic experience and experimental ONFH. How HG impacts the differentiation of BMSCs and what is the underlying molecular mechanism remain largely unknown. Our results showed that ethyl acetate extract of HG (HGE) significantly decreased the adipocyte differentiation as determined by oil red staining, while slightly increased the ALP activity. Investigation of the molecular mechanism revealed that HGE could inhibit the mRNA and protein expression of peroxisome proliferators-activated receptor (PPAR)γ, lipoprotein lipase (LPL) and adipocyteprotein2 (AP2). Interestingly, the inhibition of adipogenic differentiation in BMSCs by HGE could be restored by DKK-1, an inhibitor of Wnts. However, Noggin (an inhibitor of BMPs) displayed an additive role with HGE in suppressing the expression of PPARγ, LPL, and AP2. Furthermore, the bone marrow fat formation, as well as the expression of Wnt3a and PPARγ, was effectively regulated by HGE in the steroid-induced ONFH rats. Our results demonstrated that HGE treatment significantly inhibited adipogenesis and slightly promoted osteogenesis of BMSCs through regulating the BMP and Wnt pathways. The findings shed lights on the molecular mechanism of HGE in the inhibition of adipogenesis and provide scientific rationale for its clinical application of HGE in the treatment of ONFH.
... Consistent with this contention, estrogens stimulate FasL expression in osteoclasts derived from human monocytes (543). However, this conclusion remains controversial because the stimulatory effect of estrogens on FasL expression in osteoclasts has not been observed in some other studies (275,339,438). Furthermore, as we discussed in the previous section, contrary to a report by others (275,339,365), we have found that FasL plays no role in the effects of estrogen deficiency on the murine skeleton or the OVX-induced increase in osteoclast numbers. ...
Estrogens and androgens influence the growth and maintenance of the mammalian skeleton and are responsible for its sexual dimorphism. Estrogen deficiency at menopause or loss of both estrogens and androgens in elderly men contribute to the development of osteoporosis, one of the most common and impactful metabolic diseases of old age. In the last 20 years, basic and clinical research advances, genetic insights from humans and rodents, and newer imaging technologies have changed considerably the landscape of our understanding of bone biology as well as the relationship between sex steroids and the physiology and pathophysiology of bone metabolism. Together with the appreciation of the side effects of estrogen-related therapies on breast cancer and cardiovascular diseases, these advances have also drastically altered the treatment of osteoporosis. In this article, we provide a comprehensive review of the molecular and cellular mechanisms of action of estrogens and androgens on bone, their influences on skeletal homeostasis during growth and adulthood, the pathogenetic mechanisms of the adverse effects of their deficiency on the female and male skeleton, as well as the role of natural and synthetic estrogenic or androgenic compounds in the pharmacotherapy of osteoporosis. We highlight latest advances on the crosstalk between hormonal and mechanical signals, the relevance of the antioxidant properties of estrogens and androgens, the difference of their cellular targets in different bone envelopes, the role of estrogen deficiency in male osteoporosis, and the contribution of estrogen or androgen deficiency to the monomorphic effects of aging on skeletal involution.
... Although potentiation of ER␣ signaling by the interaction of Cav-1 with ER␣ was reported (23), other investigators found inhibition of estrogen signaling by the interaction (24). As ER␣ is an important regulator of differentiation, activation, and survival of both osteoclasts and osteoblasts (25)(26)(27)(28)(29)(30)(31)(32), critical involvement of Cav-1 in bone cell regulation may be projected. We found that Cav-1 was greatly increased by RANKL during osteoclastogenesis. ...
Lipid raft microdomains have important roles in various cellular responses. Caveolae are a specialized type of lipid rafts that are stabilized by oligomers of caveolin proteins. Here we show that caveolin-1 (Cav-1) plays a crucial role in the regulation of osteoclastogenesis. We found that caveolin-1 was dramatically up-regulated by receptor activator of nuclear factor κB ligand (RANKL), the osteoclast differentiation factor. Knockdown of Cav-1 reduced osteoclastogenesis and induction of NFATc1, the master transcription factor for osteoclastogenesis, by RANKL. Consistent with in vitro results, injection of caveolin-1 siRNA onto mice calvariae showed reduction in RANKL-induced bone resorption and osteoclast formation. Moreover, Cav-1-/- female mice had higher bone volume and lower osteoclast number compared to wild type mice. However, Cav-1-/- male mice had both osteoclast and osteoblast numbers higher than wild type mice with no difference in bone volume. The sex dependency in the effect of Cav-1 deficiency was partly attributed to decreased RANK and increased cFms expression in osteoclast precursors of female and male mice, respectively. Taken together, these data demonstrate that Cav-1 has a complicated, but critical role, for osteoclastogenesis.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... A restoration of the melatonin peak at night by limiting LAN or by supplementing nighttime levels with exogenous melatonin may prove to be of benefit [82]. This idea is supported by rodent studies where melatonin administration enhances bone cell proliferation [83], increases bone mass [33,84], and induces new bone growth [27] and, in fact, shows equal efficacy as an estrogen-progesterone HT at increasing bone density given for 1 yr [52]; HTs are known for their bone-protective functions through estrogen's inhibitory actions on osteoclasts [85][86][87] and progesterone's stimulatory effects on osteoblasts [88]. In humans, specifically perimenopausal women, nighttime supplementation with melatonin (3 mg) for 6 months restored the equilibrium between boneresorbing osteoclasts and bone-forming osteoblasts [26]. ...
An important role for melatonin in bone formation and restructuring has emerged and studies demonstrate the multiple mechanisms for these beneficial actions. Statistical analysis shows that even with existing osteoporotic therapies, bone-related disease and mortality are on the rise creating a huge financial burden for societies worldwide. These findings suggest that novel alternatives need to be developed to either prevent or reverse bone loss to combat osteoporosis-related fractures. The focus of this review describes melatonin's role in bone physiology and discusses how disruption of melatonin rhythms by light exposure at night, shift work and disease can adversely impact on bone. The signal transduction mechanisms underlying osteoblast and osteoclast differentiation and coupling with one another are discussed with a focus on how melatonin, through the regulation of RANKL and osteoprotegerin synthesis and release from osteoblasts, can induce osteoblastogenesis while inhibiting osteoclastogenesis. Also, melatonin's free radical scavenging and antioxidant properties of this indoleamine are discussed as yet an additional mechanism by which melatonin can maintain one's bone health, especially oral health. The clinical use for melatonin in bone grafting procedures, in reversing bone loss due to osteopenia and osteoporosis, and in managing periodontal disease are discussed. This article is protected by copyright. All rights reserved.
... With regard to osteoclasts, many studies have demonstrated that estrogen decreases osteoclast numbers and induces osteoclast apoptosis, (46,47) both of which lead to suppression of bone resorption. (47)(48)(49)(50) A recent study has shown that estrogen treatment of rats leads to increased expression of ERβ in osteoclasts which are undergoing apoptosis (51) suggesting that ERβ, at least in part, mediates this process. ...
TGFβ Inducible Early Gene-1 (TIEG1) knockout (KO) mice display a gender specific osteopenic phenotype characterized by low bone mineral density, bone mineral content and overall loss of bone strength in female mice. We therefore speculated that loss of TIEG1 expression would impair the actions of estrogen on bone in female mice. In order to test this hypothesis, we employed an ovariectomy (OVX) and estrogen replacement model system to comprehensively analyze the role of TIEG1 in mediating estrogen signaling in bone at the tissue, cell and biochemical level. DXA, pQCT and micro-CT analyses revealed that loss of TIEG1 expression diminished the effects of estrogen throughout the skeleton and within multiple bone compartments. Estrogen exposure also led to reductions in bone formation rates and mineralizing perimeter in wild-type mice with little to no effects on these parameters in TIEG1 KO mice. Osteoclast perimeter per bone perimeter, and resorptive activity as determined by serum levels of CTX-1, were differentially regulated following estrogen treatment in TIEG1 KO mice compared to wild-type littermates. No significant differences were detected in serum levels of P1NP between wild-type and TIEG1 KO mice. Taken together, these data implicate an important role for TIEG1 in mediating estrogen signaling throughout the mouse skeleton and suggest that defects in this pathway are likely to contribute to the gender specific osteopenic phenotype observed in female TIEG1 KO mice. © 2013 American Society for Bone and Mineral Research.
... Estrogen exerts its indirect effect through products secreted by osteoblasts that include receptor activator of nuclear factor kB ligand (RANKL), colony-stimulating factor-1 (CSF1), and osteoprotegerin (OPG), which are important in differentiation and maturation of osteoclasts. In addition, estrogen treatment has been shown to inhibit osteoclast formation from mononuclear hematopoietic stem cells (Jilka et al. 2001) and also affects adhesion and apoptosis of osteoclasts (Saintier et al. 2006, Nakamura et al. 2007. Decrease in reactive oxygen species (ROS) generation (Lean et al. 2003, Maggio et al. 2003) and secretion of pro-inflammatory cytokines interleukin 1 (IL1), IL6, and tumor necrosis factor a (TNFa) by marrow monocytes (Riggs et al. 2002) add to the beneficial effects of estrogen. ...
Ormeloxifene (Orm), a triphenylethylene compound, has been established as a selective estrogen receptor modulator (SERM) that suppresses the ovariectomy-induced bone resorption in rats. However, the precise mechanism underlying the bone-preserving action of Orm remains unclear. In this study, we evaluated the effect of Orm on osteoclast formation induced by receptor activator of nuclear factor κB ligand (RANKL) in the murine macrophage cell line RAW264.7. We also explored the mechanism of action of Orm by studying the RANKL-induced signaling pathways required for osteoclast differentiation. We found that Orm inhibited osteoclast formation from murine macrophage RAW264.7 cells induced by RANKL in a dose-dependent manner. Orm was able to abolish RANKL-induced reactive oxygen species (ROS) elevation and inhibited the transcriptional activation of two key RANKL-induced transcription factors namely activator protein-1 (AP-1) and NF-κB through mechanisms involving MAPKs. Activation of two MAPKs, i.e. ERK (MAPK1) and JNK (MAPK8), was alleviated by Orm effectively, which subsequently affected the activation of c-Jun and c-Fos, which are the essential components of the AP-1 transcription complex. Taken together, our results demonstrate that Orm potentially inhibits osteoclastogenesis by inhibiting ROS generation and thereby suppressing the activation of ERK1/2 (MAPK3/MAPK1) and JNK (MAPK8) and transcription factors (NF-κB and AP-1), which subsequently affect the regulation of osteoclastogenesis. These results provide a possible mechanism of action of Orm in regulating osteoclastogenesis, thereby supporting the beneficial bone-protective effects of this compound.
... While evidence for the direct regulation of macrophage proliferation by estrogens is lacking, these hormones were shown to modulate bone marrow M-CSF production and reduced aortic expression of M-CSF was found in Apoe-deficient mice treated with E 2 (Sarma et al. 1998, Lea et al. 1999, Martin-McNulty et al. 2003. E 2 was also demonstrated to promote apoptosis of monocytes, monocyte-derived macrophages, and macrophage-derived osteoclasts, and these effects were largely attributed to the upregulation of Fas and Fas ligand (FasL (FASLG)) as well as the activity of caspases 8 and 3 (Carruba et al. 2003, Mor et al. 2003, Thongngarm et al. 2003, Saintier et al. 2006, Nakamura et al. 2007, Montagna et al. 2009). In this context, it is worth noting that Fas/FasL effectively regulate apoptosis of cholesterol-loaded macrophages and that Fas and FasL-mediated macrophage apoptosis coincides with decreased lesion cellularity and regression of atherosclerosis in mice (Esaki et al. 2000, Yao & Tabas 2000. ...
Estrogens not only play a pivotal role in sexual development but are also involved in several physiological processes in various tissues including vasculature. While several epidemiological studies documented an inverse relationship between plasma estrogen levels and the incidence of cardiovascular disease and related it to the inhibition of atherosclerosis, an interventional trial showed an increase in cardiovascular events among postmenopausal women on estrogen treatment. The development of atherosclerotic lesions involves complex interplay between various pro- or anti-atherogenic processes that can be effectively studied only in vivo in appropriate animal models. With the advent of genetic engineering, transgenic mouse models of atherosclerosis have supplemented classical dietary cholesterol-induced disease models such as the cholesterol-fed rabbit. In the last two decades, these models were widely applied along with in vitro cell systems to specifically investigate the influence of estrogens on the development of early and advanced atherosclerotic lesions. The present review summarizes the results of these studies and assesses their contribution toward better understanding of molecular mechanisms underlying anti- and/or pro-atherogenic effects of estrogens in humans.
... In present study, MTT assay (Fig. 3D) showed that treatment of osteoclast with anthraquinone compounds did not decrease the osteoclast viability, and flow cytometry analysis (Fig. 4A) indicated that these compounds also did not cause the significant increase of the number of necrotic osteoclast (see the left upper quadrant of Fig. 4A), but induced osteoclast apoptosis (see the right upper and right lower quadrant of Fig. 4A and B). The investigation on the effects of estradiol and resveratrol on osteoclastic apoptosis have yield similar results to those reported here [22][23]. The inconsistent of results of MTT assay and flow cytometry analysis may be because that the genes involved in apoptosis regulation are more sensitive to the stimulation of anthraquinone compounds than that of enzyme related with cell viability, such as succinate dehydrogenase in osteoclast, and treatment of osteoclast with anthraquinone compounds at concentration of 0.1-10 lmol/L for 48 h is not enough to cause significant change of osteoclast viability. ...
The root of Morinda officinalis has been claimed to have a protective effect against bone loss in sciatic neurectomized and ovariectomized osteoporotic rats, and this protective effect is supposed to be attributed to anthraquinone compounds in the plant. In the present study, we investigated the effects of three anthraquinones isolated from M. officinalis, including 1, 3, 8-trihydroxy-2-methoxy-anthraquinone (1), 2-hydroxy-1-methoxy-anthraquinone (2) and rubiadin (3) on bone resorption activity in vitro and the mechanism on osteoclasts derived from rat bone marrow cells. Compound 1, 2 and 3 decreased the formation of bone resorption pits, the number of multinucleated osteoclasts, and the activity of tartrate resistant acid phosphates (TRAP) and cathepsin K in the coculture system of osteoblasts and bone marrow cells in the presence of 1, 25-dihydroxyvitamine D(3) and dexamethasone. They also enhanced the apoptosis of osteoclasts induced from bone marrow cells with M-CSF and RANKL. In addition, Compound 1, 2 and 3 improved the ratio of mRNA and protein expression of OPG and RANKL in osteoblasts, interfered with the JNK and NF-κB signal pathway, and reduced the expression of calcitonin receptor (CTR) and carbonic anhydrase/II (CA II) in osteoclasts induced from bone marrow cells with M-CSF and RANKL. These findings indicate that the anthraquinone compounds from M. officinalis are potential inhibitors of bone resorption, and may also serve as evidence to explain the mechanism of the inhibitory effects of some other reported anthraquinones on bone loss.
... The ovarian steroid oestrogen is able to exert pleiotropic effect on macrophages, including modulation of the death pathway, for example, it exerts paradoxical effects on human U937 macrophages where cell death is induced by oestrogen [1], but the same hormone accords protection to these cells from TNF-␣ induced apoptosis [2]. Similar effect is exerted on murine osteoclasts, where oestrogen exposure leads to caspase-dependent apoptosis [3,4]. A previous study from this laboratory demonstrated 17-oestradiol (E2) induced apoptosis in macrophages derived from human peripheral blood monocytes and THP-1 acute monocytic leukaemia cell line, when Bcl-2 was down-regulated [5]. ...
Human macrophages express oestrogen receptors and are therefore competent to respond to the hormone present in their microenvironment, which is implicated in sexual dimorphism observed in several immune and autoimmune phenomena. An earlier study from this laboratory demonstrated 17beta-oestradiol (E2) induced apoptosis in macrophages derived from human peripheral blood monocytes and THP-1 acute monocytic leukaemia cell line when Bcl-2 was down-regulated; however, the involvement of E2 receptor subtypes in the modulation of death pathways in these cells remain unknown. Using macrophages derived from THP-1 human acute monocytic leukaemia cells as a model, we demonstrate that plasma membrane associated oestrogen receptor (ER) -alpha participate in E2 induced Bcl-2 increase, through activation of the mitogen activated protein kinase (MAPK) pathway whereas cytosolic ER-beta transmits signals for the pro-apoptotic event of Bax translocation. The mechanistic basis of Bax translocation comprised of ER-beta mediated increase in intracellular pH, facilitated by activation of the Na(+)-H(+) exchanger. Intracellular alkalinization accompanied by concomitant Bcl-2 increase and Bax migration does not cause cellular apoptosis; however, siRNA mediated down-regulation of ER-alpha during E2 exposure leads to inhibition of Bcl-2 increase and consequently apoptosis due to the unopposed action of mitochondrial Bax. In summary, this study underscores the importance of integrative signalling modality from multiple oestrogen receptor pools in modulating oestrogen effects on human monocyte-derived macrophage apoptotic signalling pathway, which opens new vistas to explore the use of selective oestrogen receptor modulators in apoptosis-based therapies.
... Another difference from the results of Nakamura et al. (178) is that we have been unable to elicit a stimulatory effect of E 2 on FasL production in primary cultures of murine osteoclasts. Lack of an effect of estrogens on FasL production has also been reported by others in the osteoclast-like cell line RAW 264.7, although estrogens do enhance caspase-3 activity in Fas-induced apoptosis of mature osteoclasts (181). Furthermore, it has been shown that endogenous FasL does not have a role in the apoptosis of mature osteoclasts and has only a minimal effect on the apoptosis of osteoclast progenitors from C57BL/6 mice (182,183). ...
Estrogen deficiency has been considered the seminal mechanism of osteoporosis in both women and men, but epidemiological evidence in humans and recent mechanistic studies in rodents indicate that aging and the associated increase in reactive oxygen species (ROS) are the proximal culprits. ROS greatly influence the generation and survival of osteoclasts, osteoblasts, and osteocytes. Moreover, oxidative defense by the FoxO transcription factors is indispensable for skeletal homeostasis at any age. Loss of estrogens or androgens decreases defense against oxidative stress in bone, and this accounts for the increased bone resorption associated with the acute loss of these hormones. ROS-activated FoxOs in early mesenchymal progenitors also divert ss-catenin away from Wnt signaling, leading to decreased osteoblastogenesis. This latter mechanism may be implicated in the pathogenesis of type 1 and 2 diabetes and ROS-mediated adverse effects of diabetes on bone formation. Attenuation of Wnt signaling by the activation of peroxisome proliferator-activated receptor gamma by ligands generated from lipid oxidation also contributes to the age-dependent decrease in bone formation, suggesting a mechanistic explanation for the link between atherosclerosis and osteoporosis. Additionally, increased glucocorticoid production and sensitivity with advancing age decrease skeletal hydration and thereby increase skeletal fragility by attenuating the volume of the bone vasculature and interstitial fluid. This emerging evidence provides a paradigm shift from the "estrogen-centric" account of the pathogenesis of involutional osteoporosis to one in which age-related mechanisms intrinsic to bone and oxidative stress are protagonists and age-related changes in other organs and tissues, such as ovaries, accentuate them.
... Remarkably, there is another dimension to estrogen action which contrasts with its ability to stimulate growth and inhibit apoptosis. Physiologic E2 is also capable of inducing apoptosis in breast cancer cells that have been long-term estrogen-deprived (LTED) or have been treated exhaustively with anti-estrogens [15][16][17][18][19][20][21][22][23], prostate cancer cells [24], neuronal cells [25], bone-derived cells [26], thymocytes [27], and ER-transfected cells [28,29]. These data are particularly interesting because high-dose estrogen therapy was used as a treatment for post-menopausal patients with metastatic breast cancer from the 1940s until the introduction of the safer anti-estrogen tamoxifen in the 1970s [30]. ...
The link between estrogen and the development and proliferation of breast cancer is well documented. Estrogen stimulates growth and inhibits apoptosis through estrogen receptor-mediated mechanisms in many cell types. Interestingly, there is strong evidence that estrogen induces apoptosis in breast cancer and other cell types. Forty years ago, before the development of tamoxifen, high-dose estrogen was used to induce tumor regression of hormone-dependent breast cancer in post-menopausal women. While the mechanisms by which estrogen induces apoptosis were not completely known, recent evidence from our laboratory and others demonstrates the involvement of the extrinsic (Fas/FasL) and the intrinsic (mitochondria) pathways in this process. We discuss the different apoptotic signaling pathways involved in E2 (17beta-estradiol)-induced apoptosis, including the intrinsic and extrinsic apoptosis pathways, the NF-kappaB (nuclear factor-kappa-B)-mediated survival pathway as well as the PI3K (phosphoinositide 3-kinase)/Akt signaling pathway. Breast cancer cells can also be sensitized to estrogen-induced apoptosis through suppression of glutathione by BSO (L-buthionine sulfoximine). This finding has implications for the control of breast cancer with low-dose estrogen and other targeted therapeutic drugs.
... The ovarian steroid oestrogen is able to exert pleiotropic effect on macrophages, including modulation of the death pathway, for example, it exerts paradoxical effects on human U937 macrophages where cell death is induced by oestrogen [1], but the same hormone accords protection to these cells from TNF-␣ induced apoptosis [2]. Similar effect is exerted on murine osteoclasts, where oestrogen exposure leads to caspase-dependent apoptosis [3,4]. A previous study from this laboratory demonstrated 17-oestradiol (E2) induced apoptosis in macrophages derived from human peripheral blood monocytes and THP-1 acute monocytic leukaemia cell line, when Bcl-2 was down-regulated [5]. ...
Abstract Human macrophages express estrogen receptors and are therefore competent to respond to the hormone present in their microenvironment which is implicated in sexual dimorphism observed in several immune and autoimmune phenomenon. An earlier study from this laboratory demonstrated 17beta-estradiol (E2) induced apoptosis in macrophages derived from human peripheral blood monocytes and THP-1 acute monocytic leukemia cell line when Bcl-2 was downregulated, however, the involvement of E2 receptor subtypes in the modulation of death pathways in these cells remain unknown. Using macrophages derived from THP-1 human acute monocytic leukemia cells as a model, we demonstrate that plasma membrane associated estrogen receptor (ER)-alpha participate in E2 induced Bcl-2 increase through activation of the MAP-kinase pathway while cytosolic ER-beta transmits signals for the pro-apoptotic event of Bax translocation. The mechanistic basis of Bax translocation comprised of ER-beta mediated increase in intracellular pH facilitated by activation of the Na(+)-H(+) exchanger. Intracellular alkalinization accompanied by concomitant Bcl-2 increase and Bax migration does not cause cellular apoptosis, however, siRNA mediated downregulation of ER-alpha during E2 exposure leads to inhibition of Bcl-2 increase and consequently apoptosis due to the unopposed action of mitochondrial Bax. In summary, this study underscores the importance of integrative signaling modality from multiple estrogen receptor pools in modulating estrogen effects on human monocyte derived macrophage apoptotic signaling pathway which opens new vistas to explore the use of selective estrogen receptor modulators in apoptosis-based therapies.
... Both estrogen and PTHrP regulate the expression of OPG and/or RANKL by BM stromal cells and osteoblasts (37)(38)(39). Furthermore, estrogen can trigger osteoclast apoptosis directly (51)(52)(53). In addition, we observed a transient increase in circulating calcium levels and a simultaneous decrease in PTH levels upon weaning. ...
A significant portion of milk calcium comes from the mother's skeleton, and lactation is characterized by rapid bone loss. The most remarkable aspect of this bone loss is its complete reversibility, and the time after weaning is the most rapid period of skeletal anabolism in adults. Despite this, little is known of the mechanisms by which the skeleton repairs itself after lactation. We examined changes in bone and calcium metabolism defining the transition from bone loss to bone recovery at weaning in mice. Bone mass decreases during lactation and recovers rapidly after weaning. Lactation causes changes in bone microarchitecture, including thinning and perforation of trabecular plates that are quickly repaired after weaning. Weaning causes a rapid decline in urinary C-telopeptide levels and stimulates an increase in circulating levels of osteocalcin. Bone histomorphometry documented a significant reduction in the numbers of osteoclasts on d 3 after weaning caused by a coordinated wave of osteoclast apoptosis beginning 48 h after pup removal. In contrast, osteoblast numbers and bone formation rates, which are elevated during lactation, remain so 3 d after weaning. The cessation of lactation stimulates an increase in circulating calcium levels and a reciprocal decrease in PTH levels. Finally, weaning is associated with a decrease in levels of receptor activator of nuclear factor-kappaB ligand mRNA in bone. In conclusion, during lactation, bone turnover is elevated, and bone loss is rapid. Weaning causes selective apoptosis of osteoclasts halting bone resorption. The sudden shift in bone turnover favoring bone formation subsequently contributes to the rapid recovery of bone mass.
Introduction
Estrogens inhibit bone resorption and preserve bone mass, at least in part, via direct effects on osteoclasts. The binding of RANKL, the critical cytokine for osteoclast differentiation, to its receptor in osteoclast precursor cells of the monocyte lineage recruits the adaptor protein TRAF6 and activates multiple signaling pathways. Early effects of RANKL include stimulation of mitochondria. 17β-estradiol (E 2 ) prevents the effects of RANKL on mitochondria and promotes mitochondria mediated apoptotic cell death. However, the molecular mechanisms responsible for the actions of RANKL and estrogens on mitochondria remain unknown. Evolutionarily Conserved Signaling Intermediate in Toll Pathway (ECSIT) is a complex I-associated protein that regulates immune responses in macrophages following the engagement of Toll-like receptors, which also recruit TRAF6. Here, we examined whether ECSIT could be implicated in the rapid effects of RANKL and E 2 on osteoclast progenitors.
Methods
Bone marrow-derived macrophages (BMMs) from C57BL/6 mice were cultured with RANKL (30 ng/ml) with or without E 2 (10 ⁻⁸ M). ECSIT-TRAF6 interaction was evaluated by co-immunoprecipitation and ECSIT levels in mitochondria and cytosolic fractions by Western blot. ShRNA lentivirus particles were used to knockdown ECSIT. Osteoclasts were enumerated after tartrate-resistant acid phosphatase staining. Oxygen consumption and extracellular acidification rates were measured with Seahorse XFe96 Analyzer. ATP, lactate, and NAD/NADH were measured with commercial assay kits. NADH oxidation to NAD was used to evaluate Complex I activity. Total and mitochondrial ROS, and mitochondrial membrane potential were measured with H2DCFDA, MitoSOX, and TMRM probes, respectively. Degradation of DEVD-AFC was used to measure Caspase-3 activity.
Results
We found that RANKL promoted ECSIT-TRAF6 interaction and increased the levels of ECSIT in mitochondria. E 2 abrogated these effects of RANKL. Silencing of ECSIT decreased osteoclast differentiation and abrogated the inhibitory effects of E 2 on osteoclastogenesis. Loss of ECSIT decreased complex I activity, oxygen consumption, NAD ⁺ /NADH redox ratio, and ATP production and increased mitochondrial ROS. In the absence of ECSIT, the stimulatory actions of RANKL on complex I activity and all other markers of oxidative phosphorylation, as well as their inhibition by E 2 , were prevented. Instead, RANKL stimulated apoptosis of osteoclast progenitors.
Discussion
These findings suggest that dysregulated mitochondria cause a switch in RANKL signaling from pro-survival to pro-apoptotic. In addition, our results indicate that ECSIT represents a central node for the early effects of RANKL on mitochondria and that inhibition of ECSIT-mediated mitochondria stimulation might contribute to the bone protective actions of estrogens.
Background:
Equol, a metabolite of daidzein, binds to the estrogen receptor with greater affinity than daidzein and exhibits various biological properties. It exists as an enantiomer, either (S)-equol or (R)-equol.
Objective:
We have previously shown that the inhibitory effect of (S)-equol on bone fragility is stronger than that of racemic equol in ovariectomized (OVX) mice; however, the effect of (R)-equol has not been elucidated. The aim of this study was to compare the activities of equol enantiomers on bone metabolism in vitro and in vivo.
Methods:
Bone marrow cells (BMCs) and RAW 264.7 cells were treated with equol enantiomers. The number of osteoclasts and caspase-3/7 activity were measured. We examined the effect of equol enantiomers on osteoblast differentiation in MC3T3-E1 cells. In vivo, 8-week-old female ddY mice were assigned to 4 groups: sham-operated (sham), OVX, OVX + 0.5 mg/day of (S)-equol (S-eq), and OVX + 0.5 mg/day of (R)-equol (R-eq). Four weeks after the intervention, femoral bone mineral density (BMD) and osteoclastic gene expression were analyzed, along with concentrations of equol enantiomers in the serum and tissues.
Results:
(S)-equol and (R)-equol inhibited osteoclast differentiation in BMCs (97% and 60%, P < 0.05) and RAW 264.7 cells (83% and 68%, P < 0.05). (S)-equol promoted apoptosis of mature osteoclasts by inducing caspase-3/7 activity (29%, P < 0.05) and enhanced osteoblast differentiation (29%, P < 0.05). In OVX mice, BMD was ameliorated in (S)-equol-treated mice (11%, P < 0.05), but not in (R)-equol-treated mice. The concentrations of (S)-equol were greater than those of (R)-equol in the serum, tibia, liver, and kidney (148%, 80%, 22%, and 139%).
Conclusions:
These results suggest that (S)-equol is more effective than (R)-equol in inhibiting osteoclast formation and enhancing osteoclast apoptosis in vitro, supporting the beneficial effect of (S)-equol to reduce estrogen deficiency-induced bone loss in OVX mice.
Osteoporosis, a systemic skeletal disease prevalent in elderly women, is associated with post-menopausal estrogen deficiency. Although systemic administration of exogenous estradiol (E2) reduced fragility fractures, the treatment has adverse effects. Localized delivery technologies of E2 could be utilized to circumvent systemic adverse effects of systemic administration. In this study, a localized E2 delivery system is developed. Mesoporous bioactive glass nanoparticles (MBGNPs) with inherent osteogenic properties are modified with β-cyclodextrin (CD-MBGNPs) to enhance its affinity for E2. To ensure mechanical stability and integrity, E2 loaded CD-MBGNPs are further electrospun with silk fibroin (SF) to produce nanofibrous mesh (E2@CD-MBGNPs/SF). The incorporation of MBGNPs in SF enhances in vitro apatite formation and sustains constant release of E2. Moreover, osteoblast proliferation and differentiation markers such as alkaline phosphatase activity, collagen 1 and osteocalcin expression of MC3T3-E1 are augmented in CD-MBGNPs/SF and E2@CD-MBGNPs/SF as compared to SF nanofibers. On the other hand, osteoclast DNA, tartrate resistant acid phosphatase activity and multinucleated cell formation are reduced in E2@CD-MBGNPs/SF as compared to CD-MBGNPs/SF and SF. Hence the presence of CD-MBGNPs in SF stimulates osteoblast function whereas E2 incorporation in CD-MBGNPs/SF reduces osteoclast activity. This is the first report to develop CD-MBGNPs/SF as a localized delivery system for hydrophobic molecule such as estradiol to treat osteoporosis.
It has long been appreciated that most autoimmune disorders are characterized by increased prevalence in females, suggesting a potential role for sex hormones in the etiology of autoimmunity. To study how estrogen receptor α (ERα) contributes to autoimmune diseases, we generated mice in which ERα was deleted specifically in T lymphocytes. We found that ERα deletion in T cells reduced their pathogenic potential in a mouse model of colitis and correlated with transcriptomic changes that affected T cell activation. ERα deletion in T cells contributed to multiple aspects of T cell function, including reducing T cell activation and proliferation and increasing the expression of Foxp3, which encodes a critical transcription factor for the differentiation and function of regulatory T cells. Thus, these data demonstrate that ERα in T cells plays an important role in inflammation and suggest that ERα-targeted immunotherapies could be used to treat autoimmune disorders.
Estrogen, a steroid hormone, plays an important role in modulating osteoclast proliferation and development. Estrogen deficiency boosts osteoclast activity, leading to osteoporosis in elderly women. In this study, 17-ß estradiol (E2) loaded poly(ε-caprolactone) (PCL)/silk fibroin (SF) electrospun microfibers was developed as a proposed localized E2 delivery system to treat osteoporotic fractures. PCL is a synthetic polymer known for its biocompatibility and excellent mechanical properties. The bioactivity of PCL was enhanced by mixing it with natural silk fibroin (SF) polymer that has low immunogenicity and inherent bioactivity. Different ratios of PCL/SF blends were electrospun and characterized by scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR), and water contact angle measurement. PCL and SF at ratio of 50:50 (PCL50/SF50) augmented cell proliferation of murine preosteoblast MC3T3-E1 cells and murine preosteoclast RAW 264.7 cells. Hence, PCL50/SF50 was selected and mixed with three concentrations of E2 to produce electrospun fiber mesh (0.1%E2@PCL/SF, 1%E2@PCL/SF and 5%E2@PCL/SF). Sustained release of E2 was obtained for about 3 weeks at higher E2 concentration 5%E2@PCL/SF. E2 loaded PCL50/SF50 elecrospun microfiber (1%E2@PCL/SF and 5%E2@PCL/SF) reduced tartrate-resistant acid phosphate activity, total DNA and multinucleated cell formation of osteoclasts. On the other hand, the alkaline phosphatase activity and collagen I expression of osteoblasts were retained on all E2 loaded electrospun microfibers. The E2@PCL/SF system shows potential to be used for localized E2 delivery for the treatment of osteoporotic fractures.
The demands of modern society are posing serious effects on our bone health. People are working longer hours, working through the night, getting less sleep, and eating at irregular hours. This is causing more stress and less time to spend outdoors. All of these factors are contributing to circadian disruption “in general” but more importantly to circadian disruption of bone rhythms. Bone metabolism displays circadian variation that is coincident with clock rhythms in bone, with the light/dark cycle and with circulating melatonin levels. Light exposure at night, shift work, and poor quality sleep can lead to weakened bones attributed, in part, to altered clock rhythms in bone and to changes in circulating melatonin and cortisol rhythms in the body. The intent of this review is not to describe bone metabolism “in general” and then to discuss the effect of melatonin in these processes. There are many reviews on this subject matter described throughout the chapter. Rather, the focus of this chapter is to describe clock gene expression and function in bone and how their rhythms impact on osteoblast and osteoclast activity and differentiation and on bone metabolism; and then discuss variables that lead to circadian disruption of bone rhythms and describe ways to maintain healthy bone in a society that continually promotes circadian disruption.
Estrogen withdrawal following menopause results in an increase of osteoclasts formation and bone resorption, which is one of the most important mechanisms of postmenopausal osteoporosis. Recently, growing evidence has suggested that receptor-interacting protein 140 was implicated in estrogen-regulated metabolic disease, including fat metabolism and lipid metabolism. However, little is known regarding the role of receptor-interacting protein 140 in the regulation of bone metabolic by estrogen. In the present study, Western blotting disclosed that estrogen brings a significant increasing expression of receptor-interacting protein 140 in osteoclasts, but not in osteoblasts and bone marrow mesenchymal stem cells. Furthermore, analysis of TRAP staining and bone resorption assay showed that depletion of receptor-interacting protein 140 could significantly alleviate the inhibitory effects of estrogen on osteoclasts formation and bone resorption activity. Moreover, estrogen could induce osteoclasts apoptosis by increasing receptor-interacting protein 140 expression through the Fas/FasL pathway. Taken together, receptor-interacting protein 140 might be a critical player in estrogen-mediated osteoclastogenesis and bone resorption.
The aim of this study was to evaluate the effect of raloxifene (RLX) during progression of periapical lesions in ovariectomized (OVX) rats.
Female Wistar rats were OVX or subjected to sham surgery and received vehicle or RLX by gavage for 90 days. The treatment groups were as follows: sham surgery and treated with vehicle (SHAM-veh), OVX and treated with vehicle (OVX-veh), and OVX and treated with RLX (OVX-RLX). During treatment, the pulp of lower first molar was exposed to the oral environment for induction of periapical lesion that was analyzed 7 or 30 days after procedure. Blood samples were taken from jugular vein for measurement of estradiol, and the mandibles were removed and prepared for radiographic, histopathologic, histometric, and immunohistochemical analysis.
Estradiol plasma concentration showed hypoestrogenism in OVX rats. The histopathologic analysis of the OVX/RLX group was similar to that of the SHAM-veh group, whereas OVX-veh group showed larger periapical lesions with more intense inflammatory response and more cells positive for tartrate-resistant acid phosphatase. Radiographically, the groups were similar, but lesions on day 7 were smaller than lesions on day 30.
The results suggest that hypoestrogenism potentiates the progression of periapical lesions, and such condition was reversed by treatment with RLX.
Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.
Estradiol is a major drug used clinically to alleviate osteoporosis, partly through inhibition of the activity of osteoclasts, which play a crucial role in bone resorption. So far, little is known about the effects of estradiol on osteoclast metabolism. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC/MS)-based metabolomics strategy was used to investigate the metabolite response to 17β-estradiol in mouse osteoclast RAW264.7, a commonly used cell model for studying osteoporosis. Our results showed that the application of estradiol altered the levels of 27 intracellular metabolites, including lysophosphatidylcholines (LysoPCs), other lipids and amino acid derivants. The changes of all the 27 metabolites were observed in the study of estradiol induced osteoclast proliferation inhibition (1 μM estradiol applied), while the changes of only 18 metabolites were observed in the study of differentiation inhibition (0.1 μM estradiol applied). Further pathway impact analysis determined glycerophospholipid metabolism as the main potential target pathway of estradiol, which was further confirmed by LCAT (phosphatidylcholine-sterol acyltransferase) activity changes and lipid peroxidative product (MDA, methane dicarboxylic aldehyde) changes caused by estradiol. Additionally, we found that estradiol significantly decreased intracellular oxidative stress during cell proliferation but not during cell differentiation. Our study suggested that estradiol generated a highly condition-dependent influence on osteoclast metabolism.
Estrogen can promote or inhibit cellular proliferation depending on tissue cell types and physiological condition and acts through the signal transduction pathways mediated primarily by estrogen receptors. This study examined the effects of fulvestrant (Ful), a well-known antagonist for the estrogen receptor, on the action of -estradiol (E2) with respect to the proliferation and apoptosis of Chinese hamster ovarian (CHO) cells. We used different concentrations of E2, Ful, and E2 plus Ful during different treatment durations. Treatment with 15-40 E2 significantly inhibited proliferation in a time-dependent manner, although it had no influence in concentrations up to 1 . Interestingly, Ful at 10-40 also inhibited cellular proliferation in both a concentration- and time-dependent manner. In addition, Ful enhanced rather than decreased the inhibitory effect on cellular proliferation by E2 in combined treatment for 10 days. Thus, Ful does not appear to have an antagonistic effect on estrogen's anti-proliferative action in CHO cells. In TUNEL assays to confirm DNA fragmentation by E2 and/or Ful, CHO cells treated with 20 E2 showed a TUNEL-positive reaction in most DAPI-stained nuclei, and cells treated with either 40 Ful or 40 Ful plus 20 E2 also exhibited a TUNEL-positive reaction but at a lower rate compared to the E2-treated cells. These results indicate that Ful does not have an antagonistic effect on estrogen's anti-proliferative action in CHO cells, suggesting that the anti-proliferative and apoptosis-related mechanism(s) through DNA fragmentation by E2 and Ful may be mediated by different signal transduction pathways.
Osteoclasts play a crucial role in bone resorption. Since osteoclast differentiation/activation is involved in orthodontic tooth movement at the compression sites, the investigation on osteoclasts is very important to the field of orthodontics. It is well known that estrogen has the protective effect on bone. However, the mechanisms by which estrogen prevents bone loss remain to be elucidated. Although estrogen was recently reported to induce apoptosis of osteoclasts, the precise mechanisms of estrogen-induced osteoclast apoptosis remained controversial with regard to whether estrogen affects osteoclasts directly or not. Here we investigated whether estrogen directly induces differentiation and apoptosis of osteoclasts in vitro using mouse monocytic RAW264 cells differentiated into osteoclasts by RANKL. It was observed that estrogen inhibited RANKL-induced osteoclast differentiation of RAW264 cells in a dose-dependent manner. Estrogen suppressed p38 phosphorylation while it enhanced ERK phosphorylation induced by RANKL, suggesting that modulation of MAPK signaling may be involved in inhibition of osteoclast differentiation by estrogen. Next, it was shown that estrogen dose-dependently augmented caspase-3 activation in osteoclasts differentiated from RAW264 cells by RANKL, demonstrating that estrogen directly enhanced apoptosis of osteoclasts. Estrogen-induced caspase-3 activation was attenuated by ICI 182,780, suggesting that the effects of estrogen on osteoclast apoptosis is mediated through estrogen receptors. Thus, these results suggest that estrogen may directly inhibit differentiation and induce apoptosis of osteoclasts.
The effects of estrogen on osteoclast survival and differentiation were studied using CD14-selected mononuclear osteoclast precursors from peripheral blood. Estradiol at ∼1 nM reduced RANKL-dependent osteoclast differentiation by 40–50%. Osteoclast differentiation was suppressed 14 days after addition of RANKL even when estradiol was withdrawn after 18 h. In CD14+ cells apoptosis was rare and was not augmented by RANKL or by 17-β-estradiol. Estrogen receptor-α (ERα) expression was strongly down-regulated by RANKL, whether or not estradiol was present. Mature human osteoclasts thus cannot respond to estrogen via ERα. However, ERα was present in CD14+ osteoclast progenitors, and a scaffolding protein, BCAR1, which binds ERα in the presence of estrogen, was abundant. Immunoprecipitation showed rapid (∼5 min) estrogen-dependent formation of ERα–BCAR1 complexes, which were increased by RANKL co-treatment. The RANKL-signaling intermediate Traf6, which regulates NF-κB activity, precipitated with this complex. Reduction of NF-κB nuclear localization occurred within 30 min of RANKL stimulation, and estradiol inhibited the phosphorylation of IκB in response to RANKL. Inhibition by estradiol was abolished by siRNA knockdown of BCAR1. We conclude that estrogen directly, but only partially, curtails human osteoclast formation. This effect requires BCAR1 and involves a non-genomic interaction with ERα.
The purpose of the CoE is to discover the mechanism(s) of estrogen-induced apoptosis in advanced antihormone-resistant breast cancers using our unique models, and to establish the clinical value of short-term low-dose estrogen to reverse antihormone resistance in patients exhaustively treated with antihormone therapy. The clinical trial has begun enrolling patients, but enrollment has been low. We have addressed this by amending patient eligibility and expanding trial site locations. We have completed gene expression microarray hybridizations covering extended E2-treatment time courses of wild-type MCF-7:WS8, and of estrogen deprivation-resistant MCF-7:5C and MCF-7:2A cells, which undergo E2-induced apoptosis. Multiple custom methods have been developed for analyses of time-course microarray data. We have also conducted proteomic analyses, and identified proteins which differentially co-immunoprecipitate with the co-activator AIB1 or phospho-tyrosine complexes in an E2-dependent manner. Using the gene expression microarray and proteomic data, networks were built that highlight differential growth versus apoptosis pathways regulated by E2. Further, based on the microarray analyses, we investigated the G protein coupled-receptor GPR30, and the endoplasmic reticulum stress-associated factors caspase-4 and XBP1 in E2-induced growth or apoptosis. Finally, we surprisingly found that long-term treatment of MCF-7:5C cells with the c-Src inhibitor PP2 reversed E2-induced apoptosis.
Bone is a highly metabolically active tissue and its formation and resorption is at the base of bone remodelling. The critical importance of a balanced bone remodelling is demonstrated by human diseases, i.e. osteoporosis, in which a net increase in bone resorption is responsible of skeleton weakening and fracture risk. Estrogens display anti-resorptive properties on bone metabolism. Indeed, the so-called post-menopausal osteoporosis occurs after interruption of gonad function and benefits from hormonal replacement treatment. Recently, an important role for the endocannabinoid system in the regulation of skeletal remodelling in human has also been shown. In particular, we showed that CB2 stimulation is able to reduce the number of human OCs in vitro. Here, we provide unprecedented evidence that 17-β-estradiol administration inhibits activity and formation of human OCs in vitro, demonstrating that estrogens are able to induce an increase of CB2 expression probably through the recruitment of a putative estrogens responsive element in the CB2 encoding for gene.
The aim of this study was to determine whether receptor activator of nuclear factor NF-κB ligand (RANKL), osteoprotegerin (OPG) and a calcium:phosphorus (Ca:P) ratio of 2:1 could affect survival and activation of Muscovy duck osteoclasts (OCs). Bone marrow cells were obtained from 5-day-old Muscovy ducks and cultured with (Group A) No added factors, (B) 30 ng/mL soluble RANKL (sRANKL), (C) 30 ng/mL sRANKL and 10 ng/mL OPG, (D) 10 ng/mL OPG, (E) 50 ng/mL OPG, (F) 100 ng/mL OPG and (G) 30 ng/mL sRANKL, 6 mmol/L Ca and 3 mmol/L P.sRANKL promoted the survival of OCs on day 2, whereas the number of OCs decreased with addition of OPG in a dose-dependent manner. OPG and Ca:P (2:1) both inhibited OC survival induced by RANKL. RANKL stimulated bone resorption by OCs, whereas OPG, but not Ca:P (2:1), inhibited the activity of OCs induced by RANKL. RANKL promotes the survival and activation of OCs from Muscovy ducks, whereas OPG and, to a lesser extent, Ca:P (2:1) reduce the life span and inhibited the activation of OCs induced by RANKL.
Introduction: Bone resorption and formation are closely linked during bone remodeling. The cessation of bone resorption precedes bone formation, and is characterized by osteoclast apoptosis. Although apoptosis has been studied in rodent models of osteoclast differentiation, little is known about osteoclast apoptosis in humans.
Osteoclast apoptosis: The two most common apoptotic pathways are the activation of one of the membrane death receptors, and that of the mitochondrion-activated pathway involving members of the Bcl-2 family. The induction of apoptosis by extracellular signals involves ligands related to TNF. Some death receptors have been described in detail, including CD95 (Fas) that binds the CD95 ligand (FasL), and the TRAIL receptors that bind TRAIL (TNF-Related Apoptosis-Inducing Ligand). Among others, caspase-8, -9, and -3 are situated at pivotal junctions in apoptotic pathways. Caspase-8 activation is initiated during receptor-induced cell death. Caspase-9 is activated in response to agents or insults that impair the internal Bcl-2 family members balance. Both caspase-8 and caspase-9 eventually lead to the activation of executor caspases, such as caspase-3 or caspase-7. When apoptosis occurs in osteoclasts, caspases are likely to be activated. Osteoclast apoptosis and its down-regulation by the survival factor M-CSF, may involve members of the Bcl-2 family, particularly Bcl-xL. The Fas-FasL and TRAIL-TRAILR systems may also be involved in osteoclast apoptosis; however TRAIL-/- or Fas-/- deficient mouse models suggest that these factors cannot be essential for bone biology under normal conditions. However, the two main apoptotic pathways, involving TRAIL receptors and Fas, could be implicated in human osteoclast apoptosis. The regulation of this process may be important in controlling bone homeostasis, and could be altered in-vivo under conditions characterized by a high level of osteoclast formation.
Conclusion: A better knowledge of the role of apoptosis as a major point of control of bone resorption, would fairly increase our understanding of osteoclast biology. This field is of considerable clinical importance, and can be expected to advance our therapeutic approach to diseases characterized by a high level of bone resorption.
The resorption and formation of bone are closely linked during bone remodeling. The cessation of bone resorption precedes bone formation, and is characterized by osteoclast apoptosis. The two most common apoptotic pathways consist of the activation of one of the membrane death receptors, including Fas that binds FasL, and the TRAIL receptors that bind TRAIL (TNF-Related Apoptosis-Inducing Ligand), and the mitochondrion-activated pathway involving members of the Bcl-2 family. Apoptosis occurs in osteoclasts, and is an important point of control of bone resorption. M-CSF and RANKL, the two critical factors involved in osteoclast formation and activation, are osteoclast survival factors and down-regulate osteoclast apoptosis. The regulation of this process may be important in controlling bone homeostasis, and could be altered in vivo under conditions characterized by a high level of osteoclast formation. Bone diseases, such as osteoporosis, malignant osteolysis, and Paget's disease of bone, are characteristically associated with bone hyper-resorption, as rheumatoid arthritis where peri-articular or subchondral bone resorption may occur. There is now increasing evidence suggesting that changes in the regulation of osteoclast death may contribute to these clinically important bone diseases, and that the induction of osteoclast apoptosis is a potential therapeutic tool for treating them.
Bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) is an unpredictable, debilitating adverse effect. Recently, genetic polymorphisms have arisen as promising tools to identify patients with a higher risk of drug-related adverse events.
We aimed to examine the association between the aromatase polymorphism g.132810C>T, and the estrogen receptor polymorphisms g.156705T>C and g.156751A>G, and the risk of BP-related ONJ.
Eighty-three subjects were included in the study. A clinical and radiological examination was conducted on oncologic subjects treated with zoledronic acid. Subjects with histologically confirmed ONJ were included in the test group (n = 30) whereas subjects with good oral health were included in control group (n = 53). Aromatase and estrogen receptor polymorphisms from blood samples were analyzed.
The aromatase g.132810C>T polymorphism displayed an over-representation of the TT genotype in the test group (36.67 vs 16.98%; p < 0.05). There was no significant difference in either estrogen receptor polymorphism genotype frequency between the test and control groups.
Our data suggest a role for the g.132810C>T polymorphism in predicting ONJ risk. These results can pave the way to the personalization of BP therapy, based on individual genotype.
Estrogen and androgen are both critical for the maintenance of bone, but the target cells, mechanisms, and responses could be sex-specific. To compare sex-specific actions of estrogen and androgen on osteoclasts, human peripheral blood mononuclear precursor cells from adult Caucasian males (n = 3) and females (n = 3) were differentiated into osteoclasts and then treated for 24 h with 17β-estradiol (10 nM) or testosterone (10 nM). Gene expression was studied with a custom designed qPCR-based array containing 94 target genes related to bone and hormone action. In untreated osteoclasts, 4 genes showed significant gender differences. 17β-estradiol significantly affected 12 genes in osteoclasts from females and 6 genes in osteoclasts from males. Fifteen of the 18 17β-estradiol-responsive genes were different in the cells from the two sexes; 2 genes affected by 17β-estradiol in both sexes were regulated oppositely in the two sexes. Testosterone significantly affected 6 genes in osteoclasts from females and 2 genes in osteoclasts from males; all except one were different in the two sexes. 17β-estradiol and testosterone largely affected different genes, suggesting that conversion of testosterone to 17β-estradiol had a limited role in the responses. The findings indicate that although osteoclasts from both sexes respond to 17β-estradiol and testosterone, the effects of both 17β-estradiol and testosterone differ in the two sexes, highlighting the importance of considering gender in the design of therapy.
The heterogeneous nuclear ribonucleoprotein (hnRNP)-like estrogen response element-binding protein (ERE-BP) competes with estrogen receptor α (ERα) for occupancy of estrogen response elements (EREs). Here we report that ERE-BP potently stimulates osteoclastogenesis. ERE-BP mRNA and protein were found to be expressed ubiquitously in bone. Overexpression of ERE-BP in cultured osteoblasts stimulated expression of the receptor activator of NF-κB ligand (RANKL) and decreased osteoprotegerin (OPG). The effect of ERE-BP on RANKL was shown to be transcriptional in transient transfection assay and competed with via the ER. Constitutive expression of ERE-BP increased the sensitivity of cells toward 1,25-dihydroxyvitamin D(3) stimulation of RANKL expression. In contrast, knockdown of ERE-BP in stromal ST-2 cells decreased basal RANKL promoter activity. Cocultures of ERE-BP lentivirus-transduced ST-2 cells with spleen monocytes induced formation of multinucleated osteoclasts (OCs) characterized by tartrate-resistant acid phosphatase, calcitonin receptors, and functional calcium resorption from bone slices. Although ERα competed with ERE-BP for an ERE in a dose-dependent manner, ERE-BP was an independent and potent regulator of RANKL and osteoclastogenesis. In preosteoclastic RAW cells, overexpression of ERE-BP increased RANK, upregulated NF-κB signaling, and enhanced differentiation toward a mature OC phenotype independent of RANKL. These results identify ERE-BP as a potent modulator of osteoclastogenesis. We hypothesize that ERE-BP may play a critical role in the regulation of bone homeostasis as a modulator of estrogen sensitivity as well as by direct action on the transcription of critical osteoclastogenic genes.
Introduction:
Osteocyte apoptosis has been linked to bone resorption resulting from estrogen depletion and other resorptive stimuli; however, precise spatial and temporal relationships between the two events have not been clearly established. The purpose of this study was to characterize the patterns of osteocyte apoptosis in relation to bone resorption following ovariectomy to test whether osteocyte apoptosis occurs preferentially in areas known to activate resorption. Moreover, we report that osteocyte apoptosis is necessary to initiate endocortical remodeling in response to estrogen withdrawal.
Materials and methods:
Adult female C57BL/6J mice (17 weeks old) underwent either bilateral ovariectomy (OVX), or sham surgery (SHAM) and were euthanized on days 3, 7, 14, or 21 days after OVX. Diaphyseal cross-sections were stained by immunohistochemistry for activated caspase-3 as a marker of apoptosis. The percentages of caspase-positive stained osteocytes (Casp+Ot.) were measured along major and minor anatomical axes around the femoral diaphysis to evaluate the distribution of osteocyte apoptosis after estrogen loss; resorption surface was measured at the adjacent endocortical regions. In a second study to test whether osteocyte apoptosis plays a regulatory role in the initiation of bone resorption, a group of OVX mice received the pan-caspase inhibitor, QVDOPh, to inhibit osteocyte apoptosis. Remaining experimental and sham groups received either QVD or Vehicle.
Results:
OVX increased osteocyte apoptosis in a non-uniform distribution throughout the femoral diaphyses. Increases in Casp+osteocytes were predominantly located in the posterior diaphyseal cortex. Here, the number of apoptotic osteocytes 4- to 7-fold higher than sham controls (p<0.005) by day 3 post-OVX and remained elevated. Increases in resorption post-OVX also occurred along the posterior endocortical surface overlying the region of osteocyte apoptosis, but these increases occurred only at 14 and 21 days post-OVX (p<0.002) well after the increases in osteocyte apoptosis. Treatment with QVD in OVX animals suppressed osteocyte apoptosis, with levels in QVD-treated samples equivalent to baseline. Moreover, the increases in osteoclastic resorption normally observed after estrogen loss did not occur in OVX mice treated with QVD.
Conclusions:
The results of this study demonstrate that osteocyte apoptosis following estrogen loss occur regionally, rather than uniformly throughout the cortex. We also showed that estrogen loss increased osteocyte apoptosis. Apoptotic osteocytes were overwhelmingly localized within the posterior cortical region, the location where endocortical resorption was subsequently activated in ovariectomized mice. Finally, the increases in osteoclastic resorption normally observed after estrogen withdrawal did not occur in the absence of osteocyte apoptosis indicating that this apoptosis is necessary to activate endocortical remodeling following estrogen loss.
Antiresorptive agents have proven to be effective therapies for the treatment of bone diseases associated with excessive osteoclast activity. Decreased osteoclast formation, inhibition of osteoclast actions, and reduced osteoclast survival represent mechanisms by which antiresorptive agents could act. The goals of this article are to present the evidence that antiresorptive agents can decrease osteoclast survival through apoptosis, to review the mechanisms by which they are thought to activate the apoptotic process, and to consider whether the actions on apoptosis fully account for the antiresorptive effects. As background, the apoptotic process will be briefly summarized together with the evidence that factors that promote osteoclast survival affect steps in the process. Following this, therapeutic agents that are both antiresorptive and can stimulate osteoclast apoptosis will be discussed. Other bone therapeutic agents that are either antiresorptive or apoptotic, but not both, will be described. Finally, newer antiresorptive compounds that elicit apoptosis and could represent potential therapeutic agents will be noted.
Osteoclasts express the αvβ3 integrin, an adhesion receptor that has been implicated in bone resorption and that is therefore a potential therapeutic target. To assess the role of this heterodimer in skeletal development in vivo, we engineered mice in which the gene for the β3 integrin subunit was deleted. Bone marrow macrophages derived from these mutants differentiate in vitro into numerous osteoclasts, thus establishing that αvβ3 is not necessary for osteoclast recruitment. Furthermore, the closely related integrin, αvβ5, does not substitute for αvβ3 during cytokine stimulation or authentic osteoclastogenesis. β3 knockout mice, but not their heterozygous littermates, develop histologically and radiographically evident osteosclerosis with age. Despite their increased bone mass, β3-null mice contain 3.5-fold more osteoclasts than do heterozygotes. These mutant osteoclasts are, however, dysfunctional, as evidenced by their reduced ability to resorb whale dentin in vitro and the significant hypocalcemia seen in the knockout mice. The resorptive defect in β3-deficient osteoclasts may reflect absence of matrix-derived intracellular signals, since their cytoskeleton is distinctly abnormal and they fail to spread in vitro, to form actin rings ex vivo, or to form normal ruffled membranes in vivo. Thus, although it is not required for osteoclastogenesis, the integrin αvβ3 is essential for normal osteoclast function.
Osteoclastic bone degradation requires intimacy between the matrix and the resorptive cell. While the precise role the integrin alpha(v)beta3 plays in the process is not yet understood, occupancy of the heterodimer by soluble ligand or by blocking antibody effectively inhibits bone resorption in vitro and in vivo, suggesting that alpha(v)beta3 blockade may prevent postmenopausal osteoporosis. Thus, we identified a synthetic chemical peptide mimetic, beta-[2-[[5-[(aminoiminomethyl)amino]-1-oxopentyl]amino]-1-+ ++oxoethyl]amino-3-pyridinepropanoic acid, bistrifluoroacetate (SC56631) based upon the alpha(v)beta3 ligand, Arg-Gly-Asp (RGD), which recognizes the isolated integrin, and its relative, alpha(v)beta5, as effectively as does the natural peptide. The mimetic dampens osteoclastic bone resorption in vitro and in vivo. Most importantly, intravenous administration of the mimetic prevents the 55% loss of trabecular bone sustained by rats within 6 wk of oophorectomy. Histological examination of bones taken from SC56631-treated, oophorectomized animals also demonstrates the compound's bone sparing properties and its capacity to decrease osteoclast number. Thus, an RGD mimetic prevents the rapid bone loss that accompanies estrogen withdrawal.
Estrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy. Using a recently developed technique for isolation of highly purified mammalian osteoclasts, we showed that 17 beta-estradiol (E2) was able to directly inhibit osteoclastic bone resorption. At concentrations effective for inhibiting bone resorption, E2 also directly induced osteoclast apoptosis in a dose- and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E2 on both inhibition of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptor- mediated mechanism.
Central to the bone-sparing effect of estrogen (E(2)) is its ability to block the monocytic production of the osteoclastogenic cytokine TNF-alpha (TNF). However, the mechanism by which E(2) downregulates TNF production is presently unknown. Transient transfection studies in HeLa cells, an E(2) receptor-negative line, suggest that E(2) inhibits TNF gene expression through an effect mediated by estrogen receptor beta (ERbeta). We also report that in RAW 264.7 cells, an E(2) receptor-positive murine monocytic line, E(2) downregulates cytokine-induced TNF gene expression by decreasing the activity of the Jun NH(2)-terminal kinase (JNK). The resulting diminished phosphorylation of c-Jun and JunD at their NH(2)-termini decreases the ability of these nuclear proteins to autostimulate the expression of the c-Jun and JunD genes, thus leading to lower production of c-Jun and JunD. The consequent decrease in the nuclear levels of c-Jun and JunD leads to diminished binding of c-Jun/c-Fos and JunD/c-Fos heterodimers to the AP-1 consensus sequence in the TNF promoter and, thus, to decreased transactivation of the TNF gene.
The ability of estrogen to prevent glucocorticoid-induced apoptosis in osteoblasts was studied both in vitro and in vivo. Glucocorticoid treatment for 72 h produced a dose-dependent increase in the number of apoptotic cells, determined by acridine orange/ethidium bromide staining, with a maximal response of 31+/-2% and 26+/-3% with 100 nM corticosterone in primary rat and mouse osteoblasts, respectively. Simultaneous administration of varying concentrations of 17beta-estradiol and 100 nM corticosterone decreased apoptotic osteoblasts in a dose-dependent manner, with a maximal decrease of 70% with 0.01 nM 17beta-estradiol. Terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling also demonstrated glucocorticoid-induced DNA fragmentation that was inhibited by estrogen. Estrogen was shown to inhibit apoptosis induced by lipopolysaccharide treatment. As early as 6 h, Western blots demonstrated a dose-dependent decrease in the Bcl-2/Bax ratio, which reached a minimum of 0.18 in osteoblasts treated with 1000 nM corticosterone for 72 h. This reduction in Bcl-2/Bax was abolished by treating osteoblasts simultaneously with 17beta-estradiol, but not with 17alpha-estradiol. In 7-day-old mice, administration of varying concentrations of dexamethasone for 72 h resulted in a dose-dependent increase in the number of apoptotic osteoblasts as demonstrated by in situ terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling staining of calvaria. A maximum of 22+/-1% apoptotic osteoblasts on the bone surface was found with 1 mg/kg BW dexamethasone compared with 2+/-1% in vehicle-treated mice. Injection of varying concentrations of 17beta-estradiol (0.5-5 mg/kg BW), but not 17alpha-estradiol, with 1 mg/kg dexamethasone produced a dose-dependent decrease in the number of apoptotic osteoblasts to 5+/-1% with 5 mg/kg 17beta-estradiol. Thus, glucocorticoid-induced apoptosis of osteoblasts may be prevented at least in part by 17beta-estradiol.
Loss of ovarian function following menopause results in a substantial increase in bone turnover and a critical imbalance between bone formation and resorption. This imbalance leads to a progressive loss of trabecular bone mass and eventually osteoporosis, in part the result of increased osteoclastogenesis. Enhanced formation of functional osteoclasts appears to be the result of increased elaboration by support cells of osteoclastogenic cytokines such as IL-1, tumor necrosis factor, and IL-6, all of which are negatively regulated by estrogens. We show here that estrogen can suppress receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)-induced differentiation of myelomonocytic precursors into multinucleated tartrate-resistant acid phosphatase-positive osteoclasts through an estrogen receptor-dependent mechanism that does not require mediation by stromal cells. This suppression is dose-dependent, isomer-specific, and reversed by ICI 182780. Furthermore, the bone-sparing analogues tamoxifen and raloxifene mimic estrogen's effects. Estrogen blocks RANKL/M-CSF-induced activator protein-1-dependent transcription, likely through direct regulation of c-Jun activity. This effect is the result of a classical nuclear activity by estrogen receptor to regulate both c-Jun expression and its phosphorylation by c-Jun N-terminal kinase. Our results suggest that estrogen modulates osteoclast formation both by down-regulating the expression of osteoclastogenic cytokines from supportive cells and by directly suppressing RANKL-induced osteoclast differentiation.
The differentiation of cells of the monocytic lineage into mature osteoclasts (OC) is specifically induced by the tumor necrosis
factor-related factor, RANKL (receptor activator of NF-κB ligand; also known as OPGL, ODF, or TRANCE). Because inhibition of osteoclastogenesis is one of the main mechanisms by
which estrogen (E2) prevents bone loss, it is likely that E2 may regulate either the production of, or the target cell responsiveness
to RANKL. We found that E2 decreases the differentiation into OC of both murine bone marrow monocytes and RAW 264.7 cells,
a monocytic line, by down-regulating the activation of Jun N-terminal kinase 1 (JNK1). Diminished JNK1 activity results in
decreased nuclear levels of the key osteoclastogenic transcription factors, c-Fos and c-Jun, and lower binding of these transcriptional
inducers to DNA. Thus, one novel mechanism by which E2 down-regulates osteoclastogenesis is by decreasing the responsiveness
of OC precursors to RANKL.
Osteoclastic bone resorption requires cell-matrix contact, an event mediated by the alpha v beta 3 integrin. The structural components of the integrin that mediate osteoclast function are, however, not in hand. To address this issue, we generated mice lacking the beta 3 integrin gene, which have dysfunctional osteoclasts. Here, we show the full rescue of beta 3(-/-) osteoclast function following expression of a full-length beta 3 integrin. In contrast, truncated beta 3, lacking a cytoplasmic domain (h beta 3c), is completely ineffective in restoring function to beta 3(-/-) osteoclasts. To identify the components of the beta 3 cytoplasmic domain regulating osteoclast function, we generated six point mutants known, in other circumstances, to mediate beta integrin signaling. Of the six, only the S(752)P substitution, which also characterizes a form of the human bleeding disorder Glanzmann's thrombasthenia, fails to rescue beta 3(-/-) osteoclasts or restore ligand-activated signaling in the form of c-src activation. Interestingly, the double mutation Y(747)F/Y(759)F, which disrupts platelet function, does not affect the osteoclast. Thus similarities and distinctions exist in the mechanisms by which the beta 3 integrin regulates platelets and osteoclasts.
Decreased E2 levels after menopause cause bone loss through increased penetrative resorption. The reversal effect of E2 substitution therapy is well documented in vivo, although the detailed mechanism of action is not fully understood. To study the effects of E2 on bone resorption, we developed a novel in vitro bone resorption assay in which degradation of inorganic and organic matrix could be measured separately. E2 treatment significantly decreased the depth of resorption pits, although the area resorbed was not changed. Electron microscopy further revealed that the resorption pits were filled with nondegraded collagen, suggesting that E2 disturbed the organic matrix degradation. Two major groups of proteinases, matrix metalloproteinases (MMPs) and cysteine proteinases, have been suggested to participate in organic matrix degradation by osteoclasts. We show here that MMP-9 released a cross-linked carboxyl-terminal telopeptide of type I collagen from bone collagen, and cathepsin K released another C-terminal fragment, the C-terminal cross-linked peptide of type I collagen. E2 significantly inhibited the release of the C-terminal cross-linked peptide of type I collagen into the culture medium without affecting the release of cross-linked carboxyl-terminal telopeptide of type I collagen in osteoclast cultures. These results suggest that organic matrix degradation is initiated by MMPs and continued by cysteine proteases; the latter event is regulated by E2.
The beta3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various beta3 integrins with cytoplasmic tail mutations in beta3-deficient OC precursors. We find that S752 in the beta3 cytoplasmic tail is required for growth factor-induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional beta3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on beta3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional beta3. Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin. Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.
The release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members and is considered to take place through voltage-dependent anion channels (VDACs) on the outer membranes of mitochondria, results in activation of effector caspases, such as caspase-3, which induce apoptosis. We studied the involvement of the mitochondrial apoptosis pathway in uterine epithelial apoptosis. Estradiol-17beta pellets were implanted into ovariectomized mice and removed 4 days later (Day 0). The apoptotic index (percentage of apoptotic cells) of the luminal epithelium increased markedly, peaking on Day 2, whereas that of the glandular epithelium increased much less. Expression of VDAC1, 2, and 3 mRNAs increased in the luminal epithelium in correlation with the apoptotic index of the luminal epithelium. No increases in VDAC1, 2, and 3 mRNA levels were observed in the stroma or muscle, where no apoptosis occurs. VDAC1 protein levels in the uterus also correlated well with the apoptotic index of the luminal epithelium. In addition, the apoptotic index showed good correlation with the release of cytochrome c from mitochondria, activation of caspase-3, which was immunohistochemically detected only in the epithelium, and the mRNA and protein ratios of Bax:Bcl-2 and Bax:Bcl-X in the uterus. The present results suggest that the release of cytochrome c from mitochondria, which is regulated by Bcl-2 family members, plays a role in uterine epithelial apoptosis after estrogen deprivation. The increase in VDAC expression may facilitate the release of cytochrome c during apoptosis.
Cell/matrix detachment is a general inducer of programmed cell death, an event mediated by loss of integrin/ligand association. Because alpha(v)beta3 is the major integrin expressed by the osteoclast, we asked whether its occupancy promotes survival of the resorptive cell. Thus, we generated wild-type preosteoclasts and placed them on selective matrix proteins. Consistent with the posture that alpha(v)beta3 occupancy promotes survival, preosteoclasts plated on native collagen, a matrix not recognized by the integrin, undergo apoptosis 4-fold faster than those on the alpha(v)beta3 ligand, vitronectin. To further explore the role of alpha(v)beta3 in osteoclast apoptosis, wild-type and beta3-/- preosteoclasts were suspended and apoptosis determined, with time. Beta3-/- preosteoclasts, in suspension, undergo a rate of apoptosis only 40-60% of that of their wild-type counterparts, indicating that unoccupied alpha(v)beta3 transmits a positive death signal that we find regulated by caspase-8. Attesting to specificity of the unoccupied integrin-transmitted death signal, apoptosis in the absence of alpha(v)beta3 is mediated by capsase-9. We have shown that the resorptive defect of beta3-/- osteoclasts is rescued by wild-type beta3 cDNA but not by one bearing a S752P mutation. To determine whether the same holds true regarding osteoclast apoptosis, we constructed lentivirus vectors encoding green fluorescent protein, wild-type beta3, or beta3S752P. Once again, native beta3-/- preosteoclasts were protected against apoptosis. Similar to its effect on bone resorption, transduced wild-type beta3 normalizes the apoptotic rate of beta3-/- preosteoclasts. Unexpectedly, however, beta3S752P transductants also die at a rate indistinguishable from wild type. Thus, unoccupied alpha(v)beta3 integrin regulates osteoclast apoptosis via a component of the integrin that is different than that regulating resorption.
We studied estrogen effects on osteoclastic differentiation using RAW264.7, a murine monocytic cell line. Differentiation, in response to RANKL and colony-stimulating factor 1, was evaluated while varying estrogen receptor (ER) stimulation by estradiol or nonsteroidal ER agonists was performed. The RAW264.7 cells were found to express ERalpha but not ERbeta. In contrast to RANKL, which decreased ERalpha expression and induced osteoclast differentiation, 10 nm estradiol, 3 microm genistein, or 3 microm daidzein all increased ERalpha expression, stimulated cell proliferation, and decreased multinucleation, with the effects of estrogen > or = daidzein > genistein. However, no estrogen agonist reduced RANKL stimulation of osteoclast differentiation markers or its down-regulation of ERalpha expression by more than approximately 50%. Genistein is also an Src kinase antagonist in vitro, but it did not decrease Src phosphorylation in RAW264.7 cells relative to other estrogen agonists. However, both phytoestrogens and estrogen inhibited RANKL-induced IkappaB degradation and NF-kappaB nuclear localization with the same relative potency as seen in proliferation and differentiation assays. This study demonstrates, for the first time, the direct effects of estrogen on osteoclast precursor differentiation and shows that, in addition to effecting osteoblasts, estrogen may protect bone by reducing osteoclast production. Genistein, which activates ERs selectively, inhibited osteoclastogenesis less effectively than the nonselective phytoestrogen daidzein, which effectively reproduced effects of estrogen.
Both the number and the activity of osteoclasts are critical for maintaining normal bone turnover. The number is determined by rates of cell differentiation and death. Fas-mediated apoptosis is a dominant mechanism for apoptosis. Here, we show the presence of the Fas receptor on mouse, human, avian, and cultured RAW264.7 (murine) derived osteoclasts and the up-regulation of its expression during mouse osteoclast differentiation. Additionally, Fas is a fully functional death receptor in osteoclasts, and its signaling pathway is consistent with classical Fas signaling in other cell systems, involving mitochondrial release of cytochrome c and activation of caspases 3 and 9. This demonstration of Fas-mediated apoptosis in mature osteoclasts provides a new and potent mechanism for the regulation of osteoclast life span. The in vivo significance of Fas-mediated apoptosis in bone (osteoclasts) was demonstrated in aged Lpr and Gld mice, which have a dysfunctional immune system. Lpr mice, which have a defect in the Fas gene, have decreased bone mineral density, bone volume, trabecular thickness, and increased osteoclast number. Gld mice, which have a Fas ligand mutation, have a slight yet insignificant decrease in bone mineral density, but a highly significant increase in osteoclast number. Taken together, these data demonstrate that the Fas/Fas ligand system is important in the regulation of bone turnover and may represent a critical link between the immune system and bone remodeling in development and in various diseases.
Identification of receptor activator of nuclear factor-κB (RANK) and RANK-ligand (RANKL) has provided new insights into the osteoclast differentiation pathway. Osteoclast precursor cells were isolated using monoclonal antibodies against c-Fms and RANK, and the effect of adherence on the in vitro differentiation and proliferation of these cells was examined in 2 different types of stromal-cell–free culture systems: a semisolid culture medium (a nonadherent system) and a liquid culture medium (an adherent system). Osteoclast precursor cells were not able to differentiate into mature osteoclasts efficiently in the semisolid culture system. Trimerized RANKL enhanced osteoclast differentiation in semisolid cultures, but not to the extent seen when cells were allowed to adhere to plastic. Initial precursor cells were capable of differentiating into macrophages or osteoclasts. Once these cells were transferred to adherent conditions, striking differentiation was induced. Multinuclear cells were observed even after they had displayed phagocytic activity, which suggests that cell adhesion plays an important role in the differentiation of osteoclast precursor cells. Integrins, especially the arginine-glycine-aspartic acid (RGD)–recognizing integrins αv and β3, were needed for osteoclast-committed precursor cells to proliferate in order to form multinuclear osteoclasts, and the increase in cell density affected the formation of multinuclear cells. A model of osteoclast differentiation with 2 stages of precursor development is proposed: (1) a first stage, in which precursor cells are bipotential and capable of anchorage-independent growth, and (2) a second stage, in which the further proliferation and differentiation of osteoclast-committed precursor cells is anchorage-dependent.
The process of apoptosis is responsible for normal cellular turnover in numerous tissues throughout the body. The endometrial layer of the uterus shows steroid-dependent cyclic changes in structure and function. After a proliferative and secretory phase, steroid support is withdrawn and the uterine epithelium is shed. We hypothesize that the apoptosis observed in endometrial cells following hormonal withdrawal is mediated by the Fas/Fas ligand (FasL) system. Normal endometrial cells and endometrial cancer cells were cultured in the presence of estrogen and progesterone. In order to mimic physiological hormonal changes, estrogen and progesterone were removed from the media. Apoptosis was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) assay and propidium iodide staining, while Fas and FasL expression were evaluated by Western blot analysis. The endometrial cells expressed Fas and low levels of FasL. Withdrawal of estrogen and/or progesterone from the culture induced apoptosis causing an ~50% decrease in cell viability. This coincided with increased Fas and FasL expression. Treatment of the cells with anti-FasL antibody prevented cell death following hormonal withdrawal. Estrogen and progesterone therefore represent survival factors which hamper cell death by impeding the expression of apoptotic factors. Our results indicate that Fas-mediated apoptosis is important for endometrial cycling and suggest that dysregulation of the Fas/FasL interactions may have an important role in the development of endometrial cancer.
Background: Resistance to tamoxifen, a selective estrogen receptor modulator (SERM), involves changes that prevent apoptosis and enhance cell proliferation and survival. Paradoxically, estrogen treatment inhibits the growth of long-term tamoxifen-treated breast tumors. Because of the increasing use of raloxifene, another SERM, to prevent osteoporosis and potentially reduce breast cancer risk, some women will develop raloxifene-resistant breast cancer. We developed a raloxifene-resistant MCF-7 cell model (MCF-7/Ral) and investigated the nature of raloxifene-resistant breast cancer and its response to estradiol. Methods: Raloxifene resistance and hormone responsiveness were assessed by proliferation assays and cell cycle analysis in parental MCF-7 and MCF-7/Ral cells. Nuclear factor κB (NF-κB) activity was investigated with a transient transfection assay. Apoptosis was investigated by annexin V staining, mRNA was measured by real-time polymerase chain reaction, and protein was measured by western blotting. Tumorigenesis was studied by injecting MCF-7 or MCF-7/Ral cells into ovariectomized athymic mice (10 per group) and monitoring tumor size weekly. All statistical tests were two-sided. Results: Basal NF-κB activity was higher in MCF-7/Ral cells (1.6 U, 95% confidence interval [CI] = 1.2 to 2.0 U) than in MCF-7 cells (0.8 U, 95% CI = 0.4 to 1.1 U; P = .004). When cultured with 1 μM raloxifene, MCF-7/Ral cells grew statistically significantly (P<.001) faster than MCF-7 cells. Estradiol treatment of MCF-7/Ral cells arrested cells in G2/M phase of the cell cycle, decreased NF-κB activity (0.2 U, 95% CI = 0.2 to 0.3 U; P<.001), increased expression of Fas protein and mRNA (4.5-fold, 95% CI = 2.8- to 6.3-fold versus 0.5-fold, 95% CI = 0.3- to 0.8-fold for control treatment; P<.001), and induced apoptosis. Treatment with either raloxifene or tamoxifen stimulated MCF-7/Ral tumor growth, suggesting that such tumors were resistant to both drugs. When a 9-week raloxifene or tamoxifen treatment was followed by a 5-week estradiol treatment, estradiol statistically significantly reduced the size of tumors stimulated by raloxifene or tamoxifen (at week 14, P = .004 for raloxifene and P<.001 for tamoxifen). Conclusions: Growth of raloxifene-resistant MCF-7/Ral cells in vitro and in vivo is repressed by estradiol treatment by a mechanism involving G2/M-phase arrest, decreased NF-κB activity, and increased Fas expression to induce apoptosis.
The relationship of the classical receptors and their transcriptional activity to nongenotropic effects of steroid hormones is unknown. We demonstrate herein a novel paradigm of sex steroid action on osteoblasts, osteocytes, embryonic fibroblasts, and HeLa cells involving activation of a Src/Shc/ERK signaling pathway and attenuating apoptosis. This action is mediated by the ligand binding domain and eliminated by nuclear targeting of the receptor protein; ERα, ERβ, or AR can transmit it with similar efficiency irrespective of whether the ligand is an estrogen or an androgen. This antiapoptotic action can be dissociated from the transcriptional activity of the receptor with synthetic ligands, providing proof of principle for the development of function-specific—as opposed to tissue-selective—and gender-neutral pharmacotherapeutics.
Several observational studies have shown that estrogen replacement therapy decreases cardiovascular mortality and morbidity in postmenopausal women. However, The Women's Health Initiative (WHI) study has found that women receiving estrogen plus progestin had a significantly higher risk of breast cancer, coronary heart disease, stroke, and pulmonary embolus. In the present study, we examined whether estrogen prevents mechanisms that relate to plaque formation by inhibiting monocyte adhesion to endothelial cells. ECV304 cells, an endothelial cell line that normally expresses minimal estrogen receptor (ER)α, were transfected with an ERα expression plasmid. Treatment with tumor necrosis factor (TNF)-α increased expression of vascular cell adhesion molecule (VCAM)-1 mRNA, activation of nuclear factor-κB (NF-κB), and U937 cell adhesion in ECV304 cells. These effects of TNF-α were not significantly inhibited by pretreatment of native ECV304 cells with 17β-estradiol (E2). In ECV304 cells overexpressing ERα, E2 significantly inhibited the effects of TNF-α on NF-κB activation, VCAM-1 expression, and U937 cell adhesion. These findings suggest E2 suppresses inflammatory cell adhesion to vascular endothelial cells that possess functional estrogen receptors. The mechanism of suppression may involve inhibition of NF-κB-mediated up-regulation of VCAM-1 expression induced by atherogenic stimuli. E2 may prevent plaque formation, as first stage of atheroscrelosis through inhibiting adhesion monocytes to endothelial cell. Actions of estrogen replacement therapy can be assessed in terms of densities of functional ERα.
Postmenopausal osteoporosis, the most common bone disease in the developed world, is associated with estrogen deficiency. This deficiency induces increased generation and activity of osteoclasts, which perforate bone trabeculae, thus reducing their strength and increasing fracture risk. Estrogen replacement prevents these effects, indicating that estrogen negatively regulates osteoclast formation and function, but how it does this is unclear. Because functional osteoclast life span and thus the amount of bone that osteoclasts resorb could also be enhanced following estrogen deficiency, and since sex steroids regulate apoptosis in other target tissues, we investigated whether estrogen may affect osteoclast function by promoting apoptosis. 17 beta-Estradiol promoted apoptosis of murine osteoclasts in vitro and in vivo by two- to threefold. Tamoxifen, which has estrogenic effects on bone resorption, and transforming growth factor-beta 1 (TGF-beta), whose production by osteoblasts is increased by estrogen, had similar effects in vitro. Anti-TGF-beta antibody inhibited TGF-beta-, estrogen- and tamoxifen-induced osteoclast apoptosis, indicating that TGF-beta might mediate this effect. These findings suggest that estrogen may prevent excessive bone loss before and after the menopause by limiting osteoclast life span through promotion of apoptosis. The development of analogues to promote this mechanism specifically could be a useful and novel therapeutic approach to prevent postmenopausal osteoporosis.
Osteoblastic cells have been shown to be involved in osteoclast formation through cell to cell contacts. This study was designed to examine the possible function of vascular cell adhesion molecule 1 (VCAM-1) during osteoclastogenesis. As a source for stromal cells we used the recently established mouse bone marrow stromal cell line mBMS-B1 which has the ability to support osteoclastogenesis when used in co-culture with a crude spleen cell suspension. mBMS-B1 cells express a single approximately 3.9 kb VCAM-1 mRNA species. Expression was low under basal culture conditions and a 5-10-fold increase was observed in the presence of 1,25(OH)2D3. Cell surface expression of VCAM-1 examined by FACS analysis was increased about 2-fold after 1,25(OH)2D3 treatment. Immunoprecipitation of cell surface expressed VCAM-1 or total VCAM-1 protein using the anti-VCAM-1 monoclonal antibody MK2.7 resulted in a single approximately 110 kDa protein on SDS-PAGE. Induction by 1,25(OH)2D3 was about 2-5-fold on day 3. The stromal cell-osteoclast precursor cell interaction was investigated in a co-culture of the mBMS-B1 and mouse spleen cells in the presence of 1,25(OH)2D3. The monoclonal antibody MK2.7 which is known to block hemopoietic-stromal cell recognition inhibited the formation of osteoclasts when added to the co-culture at day 2 but not day 4. These data suggest that VCAM-1 is involved in the interaction between stromal cells and osteoclastic precursor cells during osteoclastogenesis presumably most important during early stages of the formation of osteoclasts.
Osteoclast activation is initiated by adhesion to the bone surface, followed by cytoskeletal rearrangement, the formation of the sealing zone, and a polarized ruffled membrane. This study shows that PYK2/CAKbeta/RAFTK, a cytoplasmic kinase related to the focal adhesion kinase, is highly expressed in rat osteoclasts in vivo. Using murine osteoclast-like cells (OCLs) or their mononuclear precursors (pOCs), generated in a coculture of bone marrow and osteoblastic MB1.8 cells, we show: (a) tyrosine phosphorylation of PYK2 upon ligation of beta3 integrins or adhesion of pOCs to serum, vitronectin, osteopontin, or fibronectin but not to laminin or collagen; (b) coimmunoprecipitation of PYK2 and c-Src from OCLs; (c) PYK2 binding to the SH2 domains of Src; (d) marked reduction in tyrosine phosphorylation and kinase activity of PYK2 in OCLs derived from Src (-/-) mice, which do not form actin rings and do not resorb bone; (e) PYK2 phosphorylation by exogeneous c-Src; (f) translocation of PYK2 to the Triton X-100 insoluble cytoskeletal fraction upon adhesion; (g) localization of PYK2 in podosomes and the ring-like structures in OCLs plated on glass and in the sealing zone in OCLs plated on bone; and (h) activation of PYK2, in the presence of MB1.8 cells, parallels the formation of sealing zones and pit resorption in vitro and is reduced by echistatin or calcitonin and cytochalasin D. Taken together, these findings suggest that Src-dependent tyrosine phosphorylation of PYK2 is involved in the adhesion-induced formation of the sealing zone, required for osteoclastic bone resorption.
Although compelling data have demonstrated the effectiveness of estrogen replacement therapy for the treatment of accelerated bone loss in postmenopausal osteoporosis and ovariectomized animals, the mechanisms by which estrogens reduce bone resorption remain to be elucidated. To address this issue, in the present study we investigated whether estrogens were able to induce programmed cell death or apoptosis in osteoclast precursors. To this purpose, a preosteoclastic cell line (FLG 29.1) was cultured in the absence or presence of nanomolar concentrations of 17beta-estradiol (17betaE2). Using time-lapse videomicroscopy, it was shown that 17betaE2 induced FLG 29.1 cell apoptosis in a dose- and time-dependent manner. Furthermore, a significant increase in the activity of caspase 3 enzyme and in the number of nuclei undergoing DNA fragmentation was observed in FLG 29.1 cells treated with 17betaE2 compared to untreated cells. Finally, transmission electron microscopy of the treated cells showed typical apoptotic morphology. These data indicate that 17betaE2 is able to promote in vitro apoptosis in preosteoclastic cells and suggest that estrogenic molecules may exert in vivo a direct role in negatively modulating the pool of undifferentiated bone marrow cells capable ultimately of maturing into osteoclasts.
The present study demonstrates that loss of cell adhesion potently promotes apoptosis in osteoclasts, a process termed "anoikis." When purified mature rabbit osteoclasts were cultured on plastic for 18 h, about 25% of them were spontaneously committed to apoptosis. The death rate increased more than twofold, after osteoclasts were subjected to suspension culture in inverted Terasaki plates. The osteoclast anoikis was significantly prevented by bongkrekic acid, an inhibitor of mitochondrial permeability transition (PT), and z-VAD-FMK, a caspase inhibitor, suggesting involvement of mitochondrial PT and caspase activation in the death process. Colony-stimulating factor-1 (CSF-1), receptor activator of NF-kappaB ligand (RANKL), and calcitonin protected adherent osteoclasts, but not floating osteoclasts from anoikis. These data show that adhesion is a primary requirement for osteoclast survival.
Identification of receptor activator of nuclear factor-kappaB (RANK) and RANK-ligand (RANKL) has provided new insights into the osteoclast differentiation pathway. Osteoclast precursor cells were isolated using monoclonal antibodies against c-Fms and RANK, and the effect of adherence on the in vitro differentiation and proliferation of these cells was examined in 2 different types of stromal-cell-free culture systems: a semisolid culture medium (a nonadherent system) and a liquid culture medium (an adherent system). Osteoclast precursor cells were not able to differentiate into mature osteoclasts efficiently in the semisolid culture system. Trimerized RANKL enhanced osteoclast differentiation in semisolid cultures, but not to the extent seen when cells were allowed to adhere to plastic. Initial precursor cells were capable of differentiating into macrophages or osteoclasts. Once these cells were transferred to adherent conditions, striking differentiation was induced. Multinuclear cells were observed even after they had displayed phagocytic activity, which suggests that cell adhesion plays an important role in the differentiation of osteoclast precursor cells. Integrins, especially the arginine-glycine-aspartic acid (RGD)-recognizing integrins alpha(v) and beta(3), were needed for osteoclast-committed precursor cells to proliferate in order to form multinuclear osteoclasts, and the increase in cell density affected the formation of multinuclear cells. A model of osteoclast differentiation with 2 stages of precursor development is proposed: (1) a first stage, in which precursor cells are bipotential and capable of anchorage-independent growth, and (2) a second stage, in which the further proliferation and differentiation of osteoclast-committed precursor cells is anchorage-dependent. (Blood. 2000;96:4335-4343)
The relationship of the classical receptors and their transcriptional activity to nongenotropic effects of steroid hormones is unknown. We demonstrate herein a novel paradigm of sex steroid action on osteoblasts, osteocytes, embryonic fibroblasts, and HeLa cells involving activation of a Src/Shc/ERK signaling pathway and attenuating apoptosis. This action is mediated by the ligand binding domain and eliminated by nuclear targeting of the receptor protein; ERalpha, ERbeta, or AR can transmit it with similar efficiency irrespective of whether the ligand is an estrogen or an androgen. This antiapoptotic action can be dissociated from the transcriptional activity of the receptor with synthetic ligands, providing proof of principle for the development of function-specific-as opposed to tissue-selective-and gender-neutral pharmacotherapeutics.
Estrogen deficiency caused by the menopause or ovariectomy leads to stimulation of osteoclastogenesis. The adhesion molecules, leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1), are necessary for osteoclast formation. In this study, the expression of LFA-1 and ICAM-1 on osteoclast precursors during osteoclast differentiation, and the involvement of ovariectomy in the expression, were investigated.
Spleen cells isolated from normal or ovariectomized (OVX) mice were co-cultured with TMS14, stromal cells derived from mouse bone marrow, in the absence or presence of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) for 7 days. On days 3, 5 and 7 of culture, the expression of LFA-1 and ICAM-1 on osteoclast precursors was quantitated using indirect immunofluorescence and confocal laser cytometry, and, on day 7, the number of formed osteoclasts was measured by tartrate-resistant acid phosphatase (TRAP) stain.
The level of ICAM-1 expression on osteoclast precursors gradually increased with osteoclast differentiation, whereas that of LFA-1 did not change. A high level of ICAM-1 was observed on the integrin beta 3-positive mononuclear cells. On the osteoclast precursors isolated from OVX mice, both the level of ICAM-1 expression per cell and the number of cells showing a high expression of ICAM-1 significantly increased, with an increase in the number of osteoclast-like cells. However, the level of LFA-1 did not change.
These results indicate that the expression level of ICAM-1, but not that of LFA-1, is involved in osteoclast differentiation. Estrogen deficiency results in an increase in ICAM-1 expression on osteoclast precursors, which may be one of the mechanisms underlying bone loss following the menopause or ovariectomy.
Inflammation, and especially mononuclear cell adhesion to endothelium, is an important physiopathological component of atherosclerosis. Since coronary heart disease in women of reproductive age and/or with estrogen replacement therapy is reduced, our aim was to determine if 17beta-estradiol had a regulatory effect on the adhesion of lymphocytes to the endothelium. We performed U-937 cells adhesion assays in TNF-alpha-stimulated HUVECs, and we also quantitated IL-8 and MCP-1 in culture supernatants, in the presence or not of 17beta-estradiol. The presence of alpha- and beta-estrogen receptors was determined by Western blot and RT-PCR, respectively, whereas the transcription of both chemokines was evaluated by RT-PCR. The results showed a 35% decrease in the adhesion of U-937 monocyte cells to TNF-alpha-stimulated HUVECs, and a 54% and 65% inhibition of TNF-alpha-induced IL-8 and MCP-1 secretion by physiological and physiologically high doses of 17beta-estradiol. The hormone did not affect the transcription of both chemokine genes. Tamoxifen reverted the inhibitory effect induced by 17beta-estradiol. In conclusion, 17beta-estradiol modifies the adhesion of leukocytes to endothelial cells by inhibiting the secretion, but not the gene transcription, of proinflammatory chemokines.
Several observational studies have shown that estrogen replacement therapy decreases cardiovascular mortality and morbidity in postmenopausal women. However, The Women's Health Initiative (WHI) study has found that women receiving estrogen plus progestin had a significantly higher risk of breast cancer, coronary heart disease, stroke, and pulmonary embolus. In the present study, we examined whether estrogen prevents mechanisms that relate to plaque formation by inhibiting monocyte adhesion to endothelial cells. ECV304 cells, an endothelial cell line that normally expresses minimal estrogen receptor (ER)alpha, were transfected with an ERalpha expression plasmid. Treatment with tumor necrosis factor (TNF)-alpha increased expression of vascular cell adhesion molecule (VCAM)-1 mRNA, activation of nuclear factor-kappaB (NF-kappaB), and U937 cell adhesion in ECV304 cells. These effects of TNF-alpha were not significantly inhibited by pretreatment of native ECV304 cells with 17beta-estradiol (E(2)). In ECV304 cells overexpressing ERalpha, E(2) significantly inhibited the effects of TNF-alpha on NF-kappaB activation, VCAM-1 expression, and U937 cell adhesion. These findings suggest E(2) suppresses inflammatory cell adhesion to vascular endothelial cells that possess functional estrogen receptors. The mechanism of suppression may involve inhibition of NF-kappaB-mediated up-regulation of VCAM-1 expression induced by atherogenic stimuli. E(2) may prevent plaque formation, as first stage of atheroscrelosis through inhibiting adhesion monocytes to endothelial cell. Actions of estrogen replacement therapy can be assessed in terms of densities of functional ERalpha.
Osteoblast survival is one of the determinants of postmenopausal osteoporosis development. Recent data from animal experiments suggest that cytokines, in particular Fas ligand (FasL), contribute to postmenopausal osteoporosis. We now address the effect of Fas activation in postmenopausal osteoblast survival and the potential modulatory effect of estrogen and raloxifene analog (LY117018). The expression of Fas mRNA, Fas protein, and the sensitivity to Fas-induced apoptosis were studied in primary cultures of human osteoblasts from postmenopausal women and in osteoblastic MG-63 cells. Human postmenopausal osteoblasts constitutively expressed Fas receptors in the cell surface. TNFalpha increased the expression of Fas mRNA and cell surface Fas expression. Neither estradiol nor raloxifene analog prevented this increase in Fas expression. In addition, activation of Fas receptor resulted in apoptosis of postmenopausal osteoblasts. While TNFalpha did not induce human osteoblast apoptosis, it did increase the lethal effect of Fas activation. Therapeutic concentrations of estradiol or raloxifene analog did not modulate lethal cytokine-induced apoptosis. Both postmenopausal osteoblasts and MG-63 cells express FasL. FasL expression was not modulated by TNFalpha. In conclusion, estrogen and raloxifene analog do not appear to affect the sensitivity of postmenopausal osteoblasts to Fas-mediated apoptosis.
Osteoclast apoptosis is an influential determinant of osteoclast bone-resorbing activity. RANKL, a critical factor for osteoclastogenesis, is also important in osteoclast survival. However, the mechanisms by which RANKL prevents osteoclast apoptosis remain largely unknown.
Fas, a death receptor, mediates apoptosis in multiple types of cells including osteoclasts. Here we report that RANKL acts as a survival factor in osteoclasts by downregulating Fas-mediated apoptosis and Fas expression in mature osteoclasts.
RAW264.7 and mouse bone marrow macrophage/monocyte progenitors and progenitor-derived osteoclasts, in the presence of various concentrations of RANKL, were used in this study. Western blotting, semiquantitative RT-PCR, flow cytometry, nuclear staining, and a fluorescent caspase-3 activity assay were used to assess the effect of RANKL on Fas expression and Fas-mediated apoptosis. The involvement of NF-kappaB in the regulation of Fas by RANKL was analyzed by luciferase assay and EMSA.
Mature osteoclasts generated in the presence of a high concentration of RANKL (3.33 nM) failed to respond to Fas-induced apoptosis. The lack of responsiveness in mature osteoclasts is caused by the low level of Fas expression, as detected by both semiquantitative PCR and Western blotting. Fas protein and mRNA expression are inhibited by RANKL in concentration-dependent manners. The downregulation of Fas expression by RANKL is not because of modulation of the stability of Fas protein or mRNA. The regulation of Fas expression by RANKL is biphasic. During the early stage of osteoclastogenesis (1 day) when Fas is expressed at a very low level, RANKL upregulates Fas promoter activity by 2.4 +/- 0.1-fold in a concentration-dependent manner and increases Fas mRNA and protein. This event correlates with regulation of the binding activity of NF-kappaB to the Fas promoter by RANKL, as detected by EMSA. In osteoclast precursors, the induction of Fas promoter activity by RANKL was dramatically reduced when NF-kappaB binding sites on the Fas promoter were mutated.
RANKL upregulates Fas expression in osteoclast progenitors through NF-kappaB, making osteoclasts targets of Fas-stimulated apoptosis. In differentiated mature osteoclasts, RANKL reduces the levels of Fas expression and Fas-mediated apoptosis, acting as a survival factor.
The increased bone resorption observed after estrogen withdrawal is responsible for bone loss and may lead to osteoporosis. The mechanism by which estradiol inhibits bone resorption is known to involve decreased osteoclastogenesis, however, the effect on osteoclast adhesion remains unclear. We examined the in vitro effect of estradiol and raloxifene on human osteoclast differentiation and function. Human peripheral blood mononuclear cells were cultured with M-CSF/RANK-L for 18 days, and we evaluated bone resorption, the expression of the protein and mRNA of the integrins, c-jun and c-fos in the presence or absence of estradiol. In this human model, beta3-integrin expression increased at the mRNA and protein levels during osteoclast differentiation, whereas that of beta5-integrin did not. We found that estradiol and raloxifene directly inhibited bone resorption on bone slices by 50%, and decreased the expression of beta3-integrin mRNA (60%) and protein (20%) in a time-dependent manner. Moreover, the mRNAs of c-fos and c-jun were both diminished by estradiol and raloxifene, particularly in early osteoclasts, but also to a lesser extent in mature cells. These findings suggest that the direct inhibitory action of estradiol on bone resorption may affect human osteoclast differentiation through downregulation of c-fos and c-jun and adhesion through modulation of beta3-integrin.