Estrogen Deficiency Bone Loss Research Articles

Transplantation of osteoporotic bone marrow stromal cells rejuvenated by the overexpression of SATB2 prevents alveolar bone loss in ovariectomized rats

Estrogen-deficient osteoporosis is an aging-related disease with high morbidity that not only significantly increases a woman's risk of fragility fracture but is also associated with tooth and bone loss in the supporting alveolar bone of the jaw. Emerging evidence suggests that the aging of bone marrow stromal cells (BMSCs) contributes to the development of osteoporosis. In this study, we aimed to investigate the role of the special AT-rich sequence-binding protein 2 (SATB2), a stemness and senescence regulator of craniofacial BMSCs, in rat ovariectomy-induced alveolar osteoporosis. We also sought to determine whether transplantation of SATB2-modified BMSCs could ameliorate estrogen deficient alveolar bone loss. Our data revealed that BMSCs from ovariectomy-induced alveolar bone exhibited typical senescence phenotypes such as diminished stemness and osteogenic capacity, increased expression of senescence or osteoclastic markers and enhanced adipogenic potential. These phenotypic changes are a result of SATB2-mediated senescence dysregulation as evidenced by nuclear γH2AX foci formation. Moreover, overexpression of SATB2 significantly alleviated the senescence of osteoporotic BMSCs in vitro. Importantly, transplantation of SATB2-modified BMSCs significantly attenuated ovariectomy-induced alveolar bone loss in vivo. Together, our results revealed that SATB2 is a critical regulator of alveolar BMSC senescence, and its overexpression decreases these senescent changes both in vitro and in vivo. SATB2-modified BMSC delivery could be a viable and promising therapeutic strategy for alveolar bone loss induced by estrogen-deficient osteoporosis.

Methoxyisoflavones formononetin and isoformononetin inhibit the differentiation of Th17 cells and B-cell lymphopoesis to promote osteogenesis in estrogen-deficient bone loss conditions.

Recent studies have shown that immune system plays a major role in pathophysiology of postmenopausal osteoporosis. Previously we have shown that phytoestrogens like daidzein and medicarpin exhibit immunoprotective effects, by virtue of which they alleviate bone loss. With this background, methoxyisoflavones like formononetin (formo) and isoformononetin (isoformo) that have been studied for preventing bone loss in ovariectomized rats were tested for their immunomodulatory effects in estrogen-deficient bone loss mice model. Adult Balb/c mice (N = 8/group) were given oral dose of formo and isoformo at 10 mg/kg body weight, post ovariectomy (Ovx) daily for 6 weeks. Animals were autopsied and long bones were harvested to study bone microarchitecture. Peripheral blood mononuclear cells were isolated for fluorescence-activated cell sorting and RNA analysis. Serum was collected for enzyme-linked immunosorbent assay. It was observed that formo and isoformo treatment to Ovx mice led to significant restoration of Ovx-induced deterioration of trabecular microarchitecture. Pro-osteoclastogenic subset Th17 and B cells were decreased in formo/isoformo-treated Ovx mice in comparison with vehicle-treated Ovx group. Formo and isoformo treatment to Ovx mice also led to decreased expression of Th17 diffentiation factors and promoted T-regulatory cell differentiation. Formo was more effective in enhancing the FOXP3 expression compared with isoformo. IL-17A-induced osteoclastogenesis and inhibition of osteoblast apoptosis were also suppressed by formo and isoformo treatment, with formo having a more potent effect. Our study demonstrates the immunomodulatory activity of methoxyisoflavones, formo, and isoformo, which translate into improved skeletal parameters, thereby preventing Ovx-induced bone loss.

Dose-response estrogen promotes osteogenic differentiation via GPR40 (FFAR1) in murine BMMSCs.

Estrogen plays an essential role in bone formation, and estrogen modulation dysfunction is tightly associated with postmenopausal osteoporosis (PMOP). The underlying mechanisms of estrogen-mediated osteogenic differentiation have not been well defined. In this study, murine bone marrow mesenchymal stem cells were induced to undergo osteogenic differentiation, and gene expression analysis or GPR40 expression manipulation was performed. Bilateral ovariectomized or sham-operated C57BL/6 mice were administered GPR40 agonist (GW9508) for bone mineral density analysis. We identified GPR40, a long chain unsaturated fatty acid receptor, to be regulated by estrogen and involved in osteogenic differentiation both invivo and invitro. Mechanistically, the Wnt/β-catenin signaling pathway is essential for GPR40 to promote osteogenic differentiation. Furthermore, invivo GW9508 administration rescued estrogen-deficient bone loss, indicating the essential role of the GPR40 receptor. To our knowledge, this is the first study that provides evidence for GPR40 as a positive regulator of osteogenesis and Wnt/β-catenin signaling. These results indicate that GPR40 may function as an endogenous promoter of estrogen-induced osteogenic differentiation through Wnt/β-catenin signaling activation. Therefore, as the global population of ages and the prevalence of metabolic-related disorders, especially PMOP, increases, our findings suggest that GPR40 is a key in understanding the link between bone and fat. It may also be a useful target for the treatment of bone complications in the future.

Comparing the disease modifying effects of alendronate to risedronate in the rat anterior cruciate ligament transection model of osteoarthritis

Comparing the disease modifying effects of alendronate to risedronate in the rat anterior cruciate ligament transection model of osteoarthritis

Bone Loss in Surgically Ovariectomized Premenopausal Women Is Associated With T Lymphocyte Activation and Thymic Hypertrophy

Postmenopausal osteoporosis is associated with estrogen deficiency and rapid bone loss. The mechanism by which estrogen deficiency results in bone loss has not been fully explained. Studies in mice rendered...

The Polyphenol Fisetin Protects Bone by Repressing NF-κB and MKP-1-Dependent Signaling Pathways in Osteoclasts

Osteoporosis is a bone pathology leading to increase fractures risk and challenging quality of life. Since current treatments could exhibit deleterious side effects, the use of food compounds derived from plants represents a promising innovative alternative due to their potential therapeutic and preventive activities against human diseases. In this study, we investigated the ability of the polyphenol fisetin to counter osteoporosis and analyzed the cellular and molecular mechanisms involved. In vivo, fisetin consumption significantly prevented bone loss in estrogen deficiency and inflammation mice osteoporosis models. Indeed, bone mineral density, micro-architecture parameters and bone markers were positively modulated by fisetin. Consistent with in vivo results, we showed that fisetin represses RANKL-induced osteoclast differentiation and activity as demonstrated by an inhibition of multinucleated cells formation, TRAP activity and differentiation genes expression. The signaling pathways NF-κB, p38 MAPK, JNK and the key transcription factors c-Fos and NFATc1 expressions induced by RANKL, were negatively regulated by fisetin. We further showed that fisetin inhibits the constitutive proteasomal degradation of MKP-1, the phosphatase that deactivates p38 and JNK. Consistently, using shRNA stable cell lines, we demonstrated that impairment of MKP-1 decreases fisetin potency. Taken together, these results strongly support that fisetin should be further considered as a bone protective agent.

Alpha‐1 antitrypsin reduces ovariectomy‐induced bone loss in mice

Proinflammatory cytokines are primary mediators of bone loss in estrogen deficiency. This study determined whether alpha-1 antitrypsin (AAT), a multifunctional protein with proteinase inhibitor and anti-inflammatory activities, mitigates bone loss induced by estrogen deficiency. Mice were either sham-operated or ovariectomized and injected with either AAT or phosphate buffered saline (PBS). Ovariectomy resulted in decreased wet uterus weight, significant bone loss, increased serum leptin concentrations, and higher body weight compared to sham. AAT injection increased tibial trabecular bone volume/total volume and trabecular thickness compared to PBS injection in ovariectomized mice. Ovariectomized mice with AAT treatment had higher uterus weight, lower serum osteocalcin levels, fewer bone marrow tartrate-resistant acid phosphatase-positive osteoclasts, and less expression of calcitonin receptor in bone than that in PBS-injected mice. These data demonstrate that AAT mitigates ovariectomy-induced bone loss in mice possibly through inhibiting osteoclast activity and bone resorption.

Selective deletion of the membrane-bound colony stimulating factor 1 isoform leads to high bone mass but does not protect against estrogen-deficiency bone loss

To better define the biologic function of membrane-bound CSF1 (mCSF1) in vivo, we have generated mCSF1 knockout (k/o) mice. Spinal bone density (BMD) was 15.9% higher in k/o mice compared to wild-type (wt) controls (P < 0.01) and total BMD was increased by 6.8% (P < 0.05). A higher mean femur BMD was also observed but did not reach statistical significance (6.9% P = NS). The osteoclastogenic potential of bone marrow isolated from mCSF1 k/o mice was reduced compared to wt marrow. There were no defects in osteoblast number or function suggesting that the basis for the high bone mass phenotype was reduced resorption. In addition to a skeletal phenotype, k/o mice had significantly elevated serum triglyceride levels (123 ± 7 vs. 88 ± 3.2 mg/dl; k/o vs. wt, P < 0.001), while serum cholesterol levels were similar (122 ± 6 vs. 116 ± 6 mg/dl; k/o vs. wt, P = NS). One month after surgery, 5-month-old k/o and wt female mice experienced the same degree of bone loss following ovariectomy (OVX). OVX induced a significant fourfold increase in the expression of the soluble CSF1 isoform (sCSF1) in the bones of wt mice while expression of mCSF1 was unchanged. These findings indicate that mCSF1 is essential for normal bone remodeling since, in its absence, BMD is increased. Membrane-bound CSF1 does not appear to be required for estrogen-deficiency bone loss while in contrast; our data suggest that sCSF1 could play a key role in this pathologic process. The reasons why mCSF1 k/o mice have hypertriglyceridemia are currently under study.

Teriparatide [rhPTH (1-34)], But Not Strontium Ranelate, Demonstrated Bone Anabolic Efficacy in Mature, Osteopenic, Ovariectomized Rats

We compared teriparatide (TPTD) and strontium ranelate (SR) efficacy on bone formation activity in a mature rat model of estrogen-deficiency bone loss. Rats were ovariectomized (OVX) at age 6 months and permitted to lose bone for 2 months to establish osteopenia before initiation of treatment with TPTD (5 or 15 μg/kg · d sc) or SR (150 or 450 mg/kg · d oral gavage). After 3 wk, RT-PCR analyses of bone formation genes in the distal femur metaphysis showed significant elevation of collagen 1α2, osteocalcin, bone sialoprotein, alkaline phosphatase, and Runx2 gene expression at both TPTD doses, relative to OVX controls. SR had no significant effect on expression of these genes. TPTD treatment for 12 wk dose dependently increased lumbar vertebral (LV) and femoral midshaft bone mineral content (BMC) and bone mineral density over pretreatment and age-matched OVX controls. SR 150 increased BMC, and SR 450 increased BMC and bone mineral density of femoral midshaft and LV over OVX controls. There were significant dose-dependent TPTD increases of LV and femoral neck strength, and TPTD 15 also increased midshaft strength compared with pretreatment and age-matched OVX controls. SR did not enhance bone strength relative to pretreatment or age-matched OVX controls. Histomorphometry of the proximal tibial metaphysis showed dose-dependent effects of TPTD on trabecular area, number, width, and osteoblast surface, bone mineralizing surface, and bone formation rate relative to pretreatment and age-matched OVX controls, whereas SR had no effect on these parameters. These findings confirmed the bone anabolic efficacy of teriparatide, but not SR in mature, osteopenic, OVX rats.

Immune System and Postmenopausal Bone Loss

The relation between immune system, estrogen deficiency and postmenopausal bone loss is an intriguing and yet unexplained challenge of the past two decades. Here we summarize the evidences that link estrogen deficiency, T and B cells proliferation and activation, cytokines production, and bone loss with particular regard to humans.

Increased circulating heat shock protein 60 induced by menopause, stimulates apoptosis of osteoblast-lineage cells via up-regulation of toll-like receptors

Postmenopausal osteoporosis is a heterogeneous disorder characterized by accelerated bone loss after natural or surgical menopause and an increased risk of fractures. The bone loss in estrogen deficiency results from the increased bone resorption and impaired ability of osteoblastic bone formation. Previous studies have reported that the HSP60 stimulates osteoclast formation and bone resorption. Here we found that plasma HSP60 levels were significantly higher in postmenopausal (median 1152.4 ng/ml; range 724.7–2123.4 ng/ml) than in premenopausal (median 316.3 ng/ml; range 164.6–638.4 ng/ml) women. In primary human bone marrow stromal cells (hBMSC) and the HS-5 hBMSC cell line, HSP60 significantly reduced cell viability and increased caspase-dependent apoptosis. Consistent with these observations, HSP60 activated caspase-3 and -9, but not caspase-8 in HS-5 cells, and increased the release of mitochondrial cytochrome c into the cytosol. In addition, HSP60 activated p38 and NFκB, but not ERK or JNK; importantly, inhibitors of p38 (SB203580) and NFκB (PDTC) abolished HSP60-induced apoptosis. Furthermore, Western blotting showed that HSP60 up-regulated TLR-2 and TLR-4 expression, and pretreatment with blocking antibodies for TLR-2 and TLR-4 almost completely eliminated the effects of HSP60 on apoptosis, caspase-3 and -9 activation, and activation of NFκB and p38 MAPK. Most notably, ovariectomy-induced bone loss was attenuated in TLR-2 KO mice. In conclusion, up-regulation of TLR-2 by HSP60 may play a critical role in promoting bone loss in the estrogen-deficient state.

Targeted overexpression of the two colony-stimulating factor-1 isoforms in osteoblasts differentially affects bone loss in ovariectomized mice

Colony-stimulating factor-1 (CSF1) is one of two cytokines required for normal osteoclastogenesis. There are two major isoforms of CSF1, the cell-surface or membrane-bound isoform (mCSF1) and soluble CSF1 (sCSF1). Whether these isoforms serve nonredundant functions in bone is unclear. To explore this question, we generated transgenic mice expressing human sCSF1, human mCSF1, or both (s/mCSF1) in osteoblasts using the 2.3-kb rat alphaI-collagen promoter. Bone density determined by peripheral quantitative computed tomography was significantly reduced in mCSF1, sCSF1, and s/mCSF1 transgenic mice compared with wild-type animals. When analyzed by sex, sCSF1, and s/mCSF1, female animals but not mCSF1 female mice were found to have greater bone loss than their male littermates (-20 vs. -9.2%; P<0.05 for sCSF1 and -21.6 vs. -11.2% for s/mCSF1; P<0.01). By breeding CSF1 isoform-selective transgenic mice to an op/op background, mice were generated in which a single CSF1 isoform was the only source of the cytokine (sCSF1op/op and mCSF1op/op). Unlike osteoblast-targeted overexpression of mCSF1, selective transgenic expression of sCSF1 did not completely correct the op/op phenotype in 5-mo-old animals. Interestingly, compared with sham-ovariectomized mice of the same genotype, ovariectomy in sCSF1op/op mice led to a greater loss of spinal bone mineral density (22.1%) than was seen in either mCSF1op/op mice (12.9%) or in wild-type animals (10.9%). Our findings support the conclusion that sCSF1 and mCSF1 serve nonredundant functions in bone and that sCSF1 may play a role in mediating estrogen-deficiency bone loss.

Effects of cod bone gelatin on bone metabolism and bone microarchitecture in ovariectomized rats

Several animal studies have showed that gelatin may be effective for minimizing bone loss in OVX rats with established osteopenia. To gain insight into how cod bone gelatin administration affects bone loss after ovariectomy, studies were carried out focusing on bone quality and the molecular mechanisms. Eighty-four female rats were ovariectomized, 12 sham-operated, divided into six groups of 12 each and treated one week after ovariectomy either with vehicle or cod bone gelatin (0.375, 0.75, 1.5, 3, 6 mg/kg body weight) for 90 days. Bone densitometry, μCT analysis, real-time PCR analysis and biochemical analysis were used at the end of the study. After 90 days, BMD of proximal tibia and femoral neck decreased in OVX rats, whereas the loss of BMD in those regions was prevented at 3 g/kg (P<0.05). However, the BMD of midshaft femurs showed no significant differences. BV/TV, Tb.N. and Tb.Th. in the 3 g/kg group were, respectively, 30.4% (P<0.05), 145.5% (P<0.05) and 81.5% (P<0.05) higher than in the OVX group. A significant decrease was detected in urine CTX, NTX and DPD, suggesting decreased bone resorption. Treatment with 3 g/kg and 6 g/kg cod bone gelatin attenuated the increase in serum IL-1β, IL-6 and TNF-α observed in the OVX group. Real-time PCR showed significantly decreased levels of mRNA expression for RANKL at the dosage of 6 g/kg and the RANKL/OPG mRNA ratio in the 3 g/kg and 6 g/kg group significantly decreased compared to the OVX group (P<0.05). In conclusion, our data confirmed that the cod bone gelatin treatment at 3 g/kg is effective in the prevention of estrogen deficient bone loss by modulating the expression of RANKL and OPG and suppressing the release of proinflammatory cytokines.

Lipopolysaccharide‐Mediated Enhancement of Bone Metabolism in Estrogen‐Deficient Mice

Osteoporosis may be a risk factor in periodontal disease. However, biologic mechanisms explaining the apparent interaction between these two diseases have not been defined. It is well known that lipopolysaccharide (LPS) increases the resorption of alveolar bone. We hypothesized that LPS and estrogen deficiency have synergistic effects on bone metabolism and may lead to enhanced bone resorption. Eighty 8-week-old female ddY mice were divided into two groups and underwent a sham operation or bilateral ovariectomy. They were maintained for 4 weeks to assess estrogen-deficient bone loss. Osteoblasts and bone marrow cells (BMCs) were collected from ovariectomized (OVX) and sham-operated mice. Osteoclast differentiation in a coculture of osteoblasts and BMCs was investigated by tartrate-resistant acid phosphatase staining. Receptor activator of nuclear factor-kappa B ligand (RANKL) mRNA expression in LPS-treated osteoblasts was investigated using real-time polymerase chain reaction. Interferon-gamma (IFN-gamma) and interleukin (IL)-6 and -10 levels in the culture supernatants were evaluated by enzyme-linked immunosorbent assay. Group means were compared by analysis of variance followed by Tukey's honestly significant difference. There was a significant increase in the number of osteoclasts in LPS-treated cocultures from OVX mice compared to sham controls (P <0.05). RANKL mRNA expression in LPS-treated osteoblasts from OVX mice was greater than in sham mice (P <0.05). In contrast, the production of IFN-gamma in LPS-treated coculture from OVX mice was significantly lower than in sham mice (P <0.05). LPS-bearing Gram-negative organisms are abundant in periodontal disease, and the results supported the hypothesis that bone resorption is increased in estrogen-deficient patients.

Runx2 determines bone maturity and turnover rate in postnatal bone development and is involved in bone loss in estrogen deficiency

Runx2 is an essential transcription factor for osteoblast differentiation. However, the functions of Runx2 in postnatal bone development remain to be clarified. Introduction of dominant-negative (dn)-Runx2 did not inhibit Col1a1 and osteocalcin expression in mature osteoblastic cells. In transgenic mice that expressed dn-Runx2 in osteoblasts, the trabecular bone had increased mineralization, increased volume, and features of compact bone, and the expression of major bone matrix protein genes was relatively maintained. After ovariectomy, neither osteolysis nor bone formation was enhanced and bone was relatively conserved. In wild-type mice, Runx2 was strongly expressed in immature osteoblasts but downregulated during osteoblast maturation. These findings indicate that the maturity and turnover rate of bone are determined by the level of functional Runx2 and Runx2 is responsible for bone loss in estrogen deficiency, but that Runx2 is not essential for maintenance of the expression of major bone matrix protein genes in postnatal bone development and maintenance.

Effect of ethanol extract of Lepidium meyenii Walp. on osteoporosis in ovariectomized rat

Maca ( Lepidium meyenii Walp.) is a cruciferous plant from the Andes of Peru. The root of Maca is traditionally employed for its supposed properties in aphrodisiacs and improving fertility, it also has been widely used to help alleviate the symptoms of menopause. The purpose of this study was to evaluate the effect of ethanol extract of Maca on postmenopausal osteoporosis in ovariectomized rats. Female Sprague-Dawley rats were divided into four groups: Sham-operated and ovariectomized groups were fed with equivolume of distilled water, and the remaining ovariectomized groups were orally administrated with ethanol extract of Maca at 0.096 and 0.24 g/kg for 28 weeks. The findings derived from the basis of bone mineral density, biomechanical, biochemical and histopathological parameters indicated that higher dose of ethanol extract of Maca was effective in the prevention of estrogen deficient bone loss.

Interleukin-11 Receptor Signaling Is Required for Normal Bone Remodeling

IL-6 and -11 regulate bone turnover and have been implicated in estrogen deficiency-related bone loss. In this study, deletion of IL-11 signaling, but not that of IL-6, suppressed osteoclast differentiation, resulting in high trabecular bone volume and reduced bone formation. Furthermore, IL-11 signaling was not required for the effects of estradiol or estrogen deficiency on the mouse skeleton. Interleukin (IL)-6 and -11 stimulate osteoclastogenesis and bone formation in vitro and have been implicated in bone loss in estrogen deficiency. Because of their common use of the gp130 co-receptor signaling subunit, the roles of these two cytokines are linked, and each may compensate for the absence of the other to maintain trabecular bone volume and bone cell differentiation. To determine the interactions in bone between IL-11 and IL-6 in vivo and whether IL-11 is required for normal bone turnover, we examined the bone phenotype of mature male and female IL-11 receptor knockout mice (IL-11Ralpha1-/-) and compared with the bone phenotype of IL-6-/- mice and mice lacking both IL-6 and IL-11Ralpha. To determine whether IL-11 is required for the effects of estrogen on trabecular bone, mature IL-11Ralpha1-/- mice were ovariectomized and treated with estradiol. In both male and female IL-11Ralpha1-/- mice, trabecular bone volume was significantly higher than that of wildtype controls. This was associated with low bone resorption and low bone formation, and the low osteoclast number generated by IL-11Ralpha1-/- precursors was reproduced in ex vivo cultures, whereas elevated osteoblast generation was not. Neither trabecular bone volume nor bone turnover was altered in IL-6-/- mice, and compound IL-6-/- :IL-11Ralpha1-/- mice showed an identical bone phenotype to IL-11Ralpha1-/- mice. The responses of IL-11Ralpha1-/- mice to ovariectomy and estradiol treatment were the same as those observed in wildtype mice. IL-11 signaling is clearly required for normal bone turnover and normal trabecular bone mass, yet not for the effects of estradiol or estrogen deficiency on the skeleton. In the absence of IL-11Ralpha, increased trabecular bone mass seems to result from a cell lineage-autonomous reduction in osteoclast differentiation, suggesting a direct effect of IL-11 on osteoclast precursors. The effects of IL-11Ralpha deletion on the skeleton are not mediated or compensated for by changes in IL-6 signaling.

An Uncoupling Agent Containing Strontium Prevents Bone Loss by Depressing Bone Resorption and Maintaining Bone Formation in Estrogen‐Deficient Rats

AbstractTrabecular bone loss in estrogen deficiency is associated with enhanced bone resorption with a smaller increase in bone formation. We previously reported that low doses of strontium can increase trabecular bone volume in rodents by affecting bone resorption and formation. In this study we determined the effect of a new divalent strontium salt (S12911) on bone loss induced by E2 deficiency. Sprague‐Dawley female rats (230 g, n = 15–25 per group) were sham operated or ovariectomized (OVX) and treated with 17β‐estradiol (E2, 10 μg/kg/day, sc) or S12911 by gavage at the dose of 77, 154, or 308 mg/kg/day or the vehicle. Treatment for 60 days with S12911 resulted in a dose‐dependent increase in plasma, urine, and bone strontium concentrations without any deleterious effect on total or skeletal growth. OVX rats were osteopenic compared to sham rats as shown by decreased femoral dry bone weight and mineral content measured on bone ash and by DXA. Treatment of OVX rats with S12911 prevented bone loss as bone ash and bone mineral content were restored to the values in sham rats. Trabecular bone volume measured by histomorphometry on the tibial metaphysis was decreased by 46% in OVX rats and was corrected by E2. Treatment of OVX rats with S12911 increased the trabecular bone volume by 30–36%. Histomorphometric indices of bone resorption (osteoclast surface and number) were increased in OVX rats and were reduced by S12911 to the levels in sham rats. In contrast to this inhibitory effect on bone resorption, the osteoid surface, osteoblast surface, mineral apposition rate, and bone formation rate were as high in OVX rats treated with S12911 as in untreated OVX rats. In addition, plasma osteocalcin (OC) and alkaline phosphatase (ALP) levels remained elevated or were further increased in OVX rats treated with S12911. In contrast, treatment with E2 reduced both bone resorption and formation and plasma ALP and OC to the levels in sham rats. The data indicate that the divalent strontium salt S12911 is acting as an uncoupling agent that can prevent the femoral osteopenia and partially prevent the trabecular bone loss in E2‐deficient rats by inhibiting bone resorption without reducing bone formation.

Hydrogen Peroxide Is Essential for Estrogen-Deficiency Bone Loss and Osteoclast Formation

We recently found that estrogen deficiency leads to a lowering of thiol antioxidant defenses in rodent bone. Moreover, administration of agents that increase the concentration in bone of glutathione, the main intracellular antioxidant, prevented estrogen-deficiency bone loss, whereas depletion of glutathione by buthionine sulfoximine administration provoked substantial bone loss. To analyze further the mechanism by which antioxidant defenses modulate bone loss, we have now compared expression of the known antioxidant enzymes in osteoclasts. We found that glutathione peroxidase 1 (Gpx), the enzyme primarily responsible for the intracellular degradation of hydrogen peroxide, is overwhelmingly the predominant antioxidant enzyme expressed by osteoclasts and that its expression was increased in bone marrow macrophages by receptor activator of nuclear factor-kappaB ligand (RANKL) and in osteoclasts by 17beta-estradiol. We therefore tested the effect of overexpression of Gpx in osteoclasts by stable transfection of RAW 264.7 (RAW) cells, which are capable of osteoclastic differentiation in response to RANKL, with a Gpx-expression construct. Osteoclast formation was abolished. The Gpx expression construct also suppressed RANKL-induced nuclear factor-kappaB activation and increased resistance to oxidation of dihydrodichlorofluorescein by exogenous hydrogen peroxide. We therefore tested the role of hydrogen peroxide in the loss of bone caused by estrogen deficiency by administering pegylated catalase to mice. We found that catalase prevented ovariectomy-induced bone loss. These results suggest that hydrogen peroxide is the reactive oxygen species responsible for signaling the bone loss of estrogen deficiency.

Tumor necrosis factor-alpha mediates osteopenia caused by depletion of antioxidants.

We recently found that estrogen deficiency leads to a lowering of thiol antioxidant defenses in rodent bone. Moreover, administration of agents that increase the concentration in bone of glutathione, the main intracellular antioxidant, prevented estrogen-deficiency bone loss, whereas depletion of glutathione by buthionine sulfoximine (BSO) administration provoked substantial bone loss. It has been shown that the estrogen-deficiency bone loss is dependent on TNFalpha signaling. Therefore, a model in which estrogen deficiency causes bone loss by lowering antioxidant defenses predicts that the osteopenia caused by lowering antioxidant defenses should similarly depend on TNFalpha signaling. We found that the loss of bone caused by either BSO administration or ovariectomy was inhibited by administration of soluble TNFalpha receptors and abrogated in mice deleted for TNFalpha gene expression. In both circumstances, lack of TNFalpha signaling prevented the increase in bone resorption and the deficit in bone formation that otherwise occurred. Thus, depletion of thiol antioxidants by BSO, like ovariectomy, causes bone loss through TNFalpha signaling. Furthermore, in ovariectomized mice treated with soluble TNFalpha receptors, thiol antioxidant defenses in bone remained low, despite inhibition of bone loss. This suggests that the low levels of antioxidants in bone seen after ovariectomy are the cause, rather than the effect, of the increased resorption. These experiments are consistent with a model for estrogen-deficiency bone loss in which estrogen deficiency lowers thiol antioxidant defenses in bone cells, thereby increasing reactive oxygen species levels, which in turn induce expression of TNFalpha, which causes loss of bone.