A Neutralizing Antibody Targeting Oxidized Phospholipids Promotes Bone Anabolism in Chow-Fed Young Adult Mice.

Oxidized phospholipids containing phosphocholine (OxPL) are pro-inflammatory lipid peroxidation products that bind to scavenger receptors (SRs), such as Scarb1, and toll-like receptors (TLRs). Excessive OxPL, as found in oxidized low-density lipoprotein (OxLDL), overwhelm these defense mechanisms and become pathogenic in atherosclerosis, nonalcoholic steatohepatitis (NASH), and osteoporosis. We previously reported that the innate IgM natural antibody E06 binds to OxPL and neutralizes their deleterious effects; expression of the single-chain (scFv) form of the antigen-binding domain of E06 (E06-scFv) as a transgene increases trabecular bone in male mice. We show herein that E06-scFv increases trabecular and cortical bone in female and male mice by increasing bone formation and decreasing osteoblast apoptosis in vivo. Homozygous E06-scFv mice have higher bone mass than hemizygous, showing a dose effect of the transgene. To investigate how OxPL restrain bone formation under physiologic conditions, we measured the levels of SRs and TLRs that bind OxPL. We found that osteoblastic cells primarily express Scarb1. Moreover, OxLDL-induced apoptosis and reduced differentiation were prevented in bone marrow-derived or calvaria-derived osteoblasts from Scarb1 knockout mice. Because Scarb1-deficient mice are reported to have high bone mass, our results suggest that E06 may promote bone anabolism in healthy young mice, at least in part, by neutralizing OxPL, which in turn promote Scarb1-mediated apoptosis of osteoblasts or osteoblast precursors. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

Increased marrow adipogenesis does not contribute to age-dependent appendicular bone loss in female mice.

Marrow adipocytes and osteoblasts differentiate from common mesenchymal progenitors in a mutually exclusive manner, and diversion of these progenitors toward adipocytes in old age has been proposed to account for the decline in osteoblasts and the development of involutional osteoporosis. This idea has been supported by evidence that thiazolidinedione (TZD)-induced activation of PPARγ, the transcription factor required for adipocyte differentiation, increases marrow fat and causes bone loss. We functionally tested this hypothesis using C57BL/6J mice with conditional deletion of PPARγ from early mesenchymal progenitors targeted by the Prx1-Cre transgene. Using a longitudinal littermate-controlled study design, we observed that PPARγ is indispensable for TZD-induced increase in marrow adipocytes in 6-month-old male mice, and age-associated increase in marrow adipocytes in 22-month-old female mice. In contrast, PPARγ is dispensable for the loss of cortical and trabecular bone caused by TZD or old age. Instead, PPARγ restrains age-dependent development of cortical porosity. These findings do not support the long-standing hypothesis that increased marrow adipocyte differentiation contributes to bone loss in old age but reveal a novel role of mesenchymal cell PPARγ in the maintenance of cortical integrity.

Osteocyte RANKL is required for cortical bone loss with age and is induced by senescence.

In aging mice, osteoclast number increases in cortical bone but declines in trabecular bone, suggesting that different mechanisms underlie age-associated bone loss in these 2 compartments. Osteocytes produce the osteoclastogenic cytokine RANKL, encoded by Tnfsf11. Tnfsf11 mRNA increases in cortical bone of aged mice, suggesting a mechanism underlying the bone loss. To address this possibility, we aged mice lacking RANKL in osteocytes. Whereas control mice lost cortical bone between 8 and 24 months of age, mice lacking RANKL in osteocytes gained cortical bone during this period. Mice of both genotypes lost trabecular bone with age. Osteoclasts increased with age in cortical bone of control mice but not in RANKL conditional knockout mice. Induction of cellular senescence increased RANKL production in murine and human cell culture models, suggesting an explanation for elevated RANKL levels with age. Overexpression of the senescence-associated transcription factor Gata4 stimulated Tnfsf11 expression in cultured murine osteoblastic cells. Finally, elimination of senescent cells from aged mice using senolytic compounds reduced Tnfsf11 mRNA in cortical bone. Our results demonstrate the requirement of osteocyte-derived RANKL for age-associated cortical bone loss and suggest that increased Tnfsf11 expression with age results from accumulation of senescent cells in cortical bone.

Estrogens decrease osteoclast number by attenuating mitochondria oxidative phosphorylation and ATP production in early osteoclast precursors.

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 17ß-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.

Elevation of the unfolded protein response increases RANKL expression.

Increased production of the osteoclastogenic cytokine RANKL is a common feature of pathologic bone loss, but the underlying cause of this increase is poorly understood. The unfolded protein response (UPR) is activated in response to accumulation of misfolded proteins in the endoplasmic reticulum (ER). Failure to resolve misfolding results in excess UPR signaling that stimulates cytokine production and cell death. We therefore investigated whether RANKL is one of the cytokines stimulated in response to elevated UPR in bone cells. Pharmacologic induction of UPR with tunicamycin (Tm)-stimulated RANKL expression in cultures of primary osteoblastic cells and in osteoblast and osteocyte cell lines. Pharmacologic inhibition of the UPR blunted Tm-induced RANKL production. Silencing Edem1 or Sel1l, proteins that aid in degradation of misfolded proteins, also induced UPR and increased RANKL mRNA. Moreover, Tm or hypoxia increased RANKL and bone resorption in cultures of neonatal murine calvaria. Administration of Tm to adult mice caused dilation of ER in osteoblasts and osteocytes, elevated the UPR, and increased RANKL expression and osteoclast number. These findings support the hypothesis that excessive UPR signaling stimulates the expression of RANKL by osteoblasts and osteocytes, and thereby facilitates excessive bone resorption and bone loss in pathologic conditions.

Oxidation-specific epitopes restrain bone formation.

Atherosclerosis and osteoporosis are epidemiologically linked and oxidation specific epitopes (OSEs), such as phosphocholine (PC) of oxidized phospholipids (PC-OxPL) and malondialdehyde (MDA), are pathogenic in both. The proatherogenic effects of OSEs are opposed by innate immune antibodies. Here we show that high-fat diet (HFD)-induced bone loss is attenuated in mice expressing a single chain variable region fragment of the IgM E06 (E06-scFv) that neutralizes PC-OxPL, by increasing osteoblast number and stimulating bone formation. Similarly, HFD-induced bone loss is attenuated in mice expressing IK17-scFv, which neutralizes MDA. Notably, E06-scFv also increases bone mass in mice fed a normal diet. Moreover, the levels of anti-PC IgM decrease in aged mice. We conclude that OSEs, whether produced chronically or increased by HFD, restrain bone formation, and that diminished defense against OSEs may contribute to age-related bone loss. Anti-OSEs, therefore, may represent a novel therapeutic approach against osteoporosis and atherosclerosis simultaneously.

Old age causes de novo intracortical bone remodeling and porosity in mice.

Decreased cortical thickness and increased cortical porosity are the key anatomic changes responsible for osteoporotic fractures in elderly women and men. The cellular basis of these changes is unbalanced endosteal and intracortical osteonal remodeling by the osteoclasts and osteoblasts that comprise the basic multicellular units (BMUs). Like humans, mice lose cortical bone with age, but unlike humans, this loss occurs in the face of sex steroid sufficiency. Mice are therefore an ideal model to dissect age-specific osteoporotic mechanisms. Nevertheless, lack of evidence for endosteal or intracortical remodeling in mice has raised questions about their translational relevance. We show herein that administration of the antiosteoclastogenic cytokine osteoprotegerin to Swiss Webster mice ablated not only osteoclasts, but also endosteal bone formation, demonstrating the occurrence of BMU-based endosteal remodeling. Femoral cortical thickness decreased in aged male and female C57BL/6J mice, as well as F1 hybrids of C57BL/6J and BALB/cBy mice. This decrease was greater in C57BL/6J mice, indicating a genetic influence. Moreover, endosteal remodeling became unbalanced because of increased osteoclast and decreased osteoblast numbers. The porosity of the femoral cortex increased with age but was much higher in females of both strains. Notably, the increased cortical porosity resulted from de novo intracortical remodeling by osteon-like structures. Age-dependent cortical bone loss was associated with increased osteocyte DNA damage, cellular senescence, the senescence-associated secretory phenotype, and increased levels of RANKL. The demonstration of unbalanced endosteal and intracortical remodeling in old mice validates the relevance of this animal model to involutional osteoporosis in humans.

DNA damage and senescence in osteoprogenitors expressing Osx1 may cause their decrease with age.

Age-related bone loss in mice results from a decrease in bone formation and an increase in cortical bone resorption. The former is accounted by a decrease in the number of postmitotic osteoblasts which synthesize the bone matrix and is thought to be the consequence of age-dependent changes in mesenchymal osteoblast progenitors. However, there are no specific markers for these progenitors, and conclusions rely on results from in vitro cultures of mixed cell populations. Moreover, the culprits of such changes remain unknown. Here, we have used Osx1-Cre;TdRFP mice in which osteoprogenitors express the TdRFP fluorescent protein. We report that the number of TdRFP-Osx1 cells, freshly isolated from the bone marrow, declines by more than 50% between 6 and 24 months of age in both female and male mice. Moreover, TdRFP-Osx1 cells from old mice exhibited markers of DNA damage and senescence, such as γH2AX foci, G1 cell cycle arrest, phosphorylation of p53, increased p21CIP1 levels, as well as increased levels of GATA4 and activation of NF-κB - two major stimulators of the senescence-associated secretory phenotype (SASP). Bone marrow stromal cells from old mice also exhibited elevated expression of SASP genes, including several pro-osteoclastogenic cytokines, and increased capacity to support osteoclast formation. These changes were greatly attenuated by the senolytic drug ABT263. Together, these findings suggest that the decline in bone mass with age is the result of intrinsic defects in osteoprogenitor cells, leading to decreased osteoblast numbers and increased support of osteoclast formation.

The Effects of Aging and Sex Steroid Deficiency on the Murine Skeleton Are Independent and Mechanistically Distinct.

Old age and sex steroid deficiency are the two most critical factors for the development of osteoporosis. It remains unknown, however, whether the molecular culprits of the two conditions are similar or distinct. We show herein that at 19.5 months of age-a time by which the age-dependent decline of cortical and cancellous bone mass and cortical porosity were fully manifested in C57BL/6J mice-these animals remained functionally estrogen sufficient. Transgenic mice with conditional expression of mitochondria-targeted catalase-a potent H2 O2 inactivating enzyme-in cells of the myeloid lineage (mitoCAT;LysM-Cre mice) were protected from the loss of cortical, but not cancellous, bone caused by gonadectomy in either sex. Consistent with these findings, in vitro studies with ERα-deficient Prx1+ cells and gonadectomized young adult mice showed that in both sexes decreased ERα signaling in Prx1+ cells leads to an increase in SDF1, a.k.a. CXCL12, an osteoclastogenic cytokine whose effects were abrogated in macrophages from mitoCAT;LysM-Cre mice. In contrast to sex steroid deficiency, the adverse effects of aging on either cortical or cancellous bone were unaffected in mitoCAT;LysM-Cre mice. On the other hand, attenuation of H2 O2 generation in cells of the mesenchymal lineage targeted by Prx1-Cre partially prevented the loss of cortical bone caused by old age. Our results suggest the effects of sex steroid deficiency and aging on the murine skeleton are independent and result from distinct mechanisms. In the former, the prevailing mechanism of the cortical bone loss in both sexes is increased osteoclastogenesis caused by estrogen deficiency; this is likely driven, at least in part, by mesenchymal/stromal cell-derived SDF1. Decreased osteoblastogenesis, owing in part to increased H2 O2, combined with increased osteoclastogenesis caused by aging mechanisms independent of estrogen deficiency, are the prevailing mechanisms of the loss of cortical bone with old age. © 2016 American Society for Bone and Mineral Research.

The Road to Reproducibility in Animal Research.

Reproducibility of research findings is the hallmark of scientific advance. However, the recently noted lack of reproducibility and transparency of published research using animal models of human biology and disease has alarmed funders, scientists, and the public. Improved reporting of methodology and better use of statistical tools are needed to enhance the quality and utility of published research. Reporting guidelines like Animal Research: Reporting In Vivo Experiments (ARRIVE) have been devised to achieve these goals, but most biomedical research journals, including the JBMR, have not been able to obtain high compliance. Cooperative efforts among authors, reviewers and editors-empowered by increased awareness of their responsibilities, and enabled by user-friendly guidelines-are needed to solve this problem. © 2016 American Society for Bone and Mineral Research.

Skeletal inflammation and attenuation of Wnt signaling, Wnt ligand expression, and bone formation in atherosclerotic ApoE-null mice.

ApoE-null (ApoE-KO) mice fed a high-fat diet (HFD) develop atherosclerosis, due in part to activation of vascular inflammation by oxidized low-density lipoprotein. Since bone loss also occurs in these mice, we used them to investigate the impact of oxidized lipids on bone homeostasis and to search for underlying pathogenic pathways. Four-month-old female ApoE-KO mice fed a HFD for three months exhibited increased levels of oxidized lipids in bone, as well as decreased femoral and vertebral trabecular and cortical bone mass, compared with ApoE-KO mice on normal diet. Despite HFD-induced increase in expression of Alox15, a lipoxygenase that oxidizes LDL and promotes atherogenesis, global deletion of this gene failed to ameliorate the skeletal impact of HFD. Osteoblast number and function were dramatically reduced in trabecular and cortical bone of HFD-fed mice, whereas osteoclast number was modestly reduced only in trabecular bone, indicating that an imbalance in favor of osteoclasts was responsible for HFD-induced bone loss. These changes were associated with decreased osteoblast progenitors and increased monocyte/macrophages in the bone marrow as well as increased expression of IL-1ß, IL-6, and TNF. HFD also attenuated Wnt signaling as evidenced by reduced expression of Wnt target genes, and it decreased expression of pro-osteoblastogenic Wnt ligands. These results suggest that oxidized lipids decrease bone mass by increasing anti-osteoblastogenic inflammatory cytokines and decreasing pro-osteoblastogenic Wnt ligands.

The Role of Osteocytes in Age-Related Bone Loss.

The decrease in bone mass and strength during aging has multiple causes. Osteocytes are long-lived cells within the bone matrix that perform a variety of functions, including the control of bone remodeling. Because of their longevity, osteocytes are more likely than osteoclasts or osteoblasts to accumulate molecular damage over time. Osteocytes utilize quality-control pathways like autophagy to remove damaged organelles and macromolecules, and thereby maintain function. When the damage is excessive, cell death pathways such as apoptosis minimize the impact of potential osteocyte dysfunction on the skeleton. The goal of this review is to discuss how dysregulation of these pathways in osteocytes may contribute to the decline in bone mass and strength with age.

The Effects of Androgens on Murine Cortical Bone Do Not Require AR or ERα Signaling in Osteoblasts and Osteoclasts.

In men, androgens are critical for the acquisition and maintenance of bone mass in both the cortical and cancellous bone compartment. Male mice with targeted deletion of the androgen receptor (AR) in mature osteoblasts or osteocytes have lower cancellous bone mass, but no cortical bone phenotype. We have investigated the possibility that the effects of androgens on the cortical compartment result from AR signaling in osteoprogenitors or cells of the osteoclast lineage; or via estrogen receptor alpha (ERα) signaling in either or both of these two cell types upon conversion of testosterone to estradiol. To this end, we generated mice with targeted deletion of an AR or an ERα allele in the mesenchymal (AR(f/y);Prx1-Cre or ERα(f/f);Osx1-Cre) or myeloid cell lineage (AR(f/y);LysM-Cre or ERα(f/f);LysM-Cre) and their descendants. Male AR(f/y);Prx1-Cre mice exhibited decreased bone volume and trabecular number, and increased osteoclast number in the cancellous compartment. Moreover, they did not undergo the loss of cancellous bone volume and trabecular number caused by orchidectomy (ORX) in their littermate controls. In contrast, AR(f/y);LysM-Cre, ERα(f/f);Osx1-Cre, or ERα(f/f);LysM-Cre mice had no cancellous bone phenotype at baseline and lost the same amount of cancellous bone as their controls following ORX. Most unexpectedly, adult males of all four models had no discernible cortical bone phenotype at baseline, and lost the same amount of cortical bone as their littermate controls after ORX. Recapitulation of the effects of ORX by AR deletion only in the AR(f/y);Prx1-Cre mice indicates that the effects of androgens on cancellous bone result from AR signaling in osteoblasts-not on osteoclasts or via aromatization. The effects of androgens on cortical bone mass, on the other hand, do not require AR or ERα signaling in any cell type across the osteoblast or osteoclast differentiation lineage. Therefore, androgens must exert their effects indirectly by actions on some other cell type(s) or tissue(s).

Ability of circulating human hematopoietic lineage negative cells to support hematopoiesis.

Hematopoietic stem cell (HSC) self-renewal is regulated by osteoblast and/or endothelial cells within the hematopoietic niche. However, the true identity of the supporting cells and the nature of the secreted factors remain uncertain. We developed a novel mouse model and analyzed whether circulating human peripheral hematopoietic lineage negative/AP+ (lin-/AP+) cells support hematopoiesis in vivo. Thus, immunocompromised (Rag) mice expressing thymidine kinase (Tk) under the control of the 3.6Col1α1 promoter (Tk-Rag) were treated with ganciclovir, resulting in osteoblast progenitor cell ablation and subsequent loss of hematopoiesis (evaluated by measuring mouse Ter119+ erythroid cells). Following hematopoietic cell depletion, human bone marrow-derived marrow stromal cells (MSCs) or lin-/AP+ cells were infused into Tk-Rag mice and compared with saline infusions. Ganciclovir significantly reduced (7.4-fold) Ter119+ cells in the bone marrow of Tk-Rag mice compared to saline injections. Infusion of either MSCs or lin-/AP+ cells into ganciclovir-treated mice resulted in a 3.3-fold and 2.7-fold increase (P < 0.01), respectively, in Ter119+ cells compared to mice receiving saline. Relative to lin-/AP- cells, lin-/AP+ cells expressed high levels of mesenchymal, endothelial, and hematopoiesis supporting genes. Thus, human peripheral blood lin-/AP+ cells represent a novel cell type capable of supporting hematopoiesis in a manner comparable to MSCs.

FoxO proteins restrain osteoclastogenesis and bone resorption by attenuating H2O2 accumulation.

Besides their cell-damaging effects in the setting of oxidative stress, reactive oxygen species (ROS) play an important role in physiological intracellular signalling by triggering proliferation and survival. FoxO transcription factors counteract ROS generation by upregulating antioxidant enzymes. Here we show that intracellular H2O2 accumulation is a critical and purposeful adaptation for the differentiation and survival of osteoclasts, the bone cells responsible for the resorption of mineralized bone matrix. Using mice with conditional loss or gain of FoxO transcription factor function, or mitochondria-targeted catalase in osteoclasts, we demonstrate this is achieved, at least in part, by downregulating the H2O2-inactivating enzyme catalase. Catalase downregulation results from the repression of the transcriptional activity of FoxO1, 3 and 4 by RANKL, the indispensable signal for the generation of osteoclasts, via an Akt-mediated mechanism. Notably, mitochondria-targeted catalase prevented the loss of bone caused by loss of oestrogens, suggesting that decreasing H2O2 production in mitochondria may represent a rational pharmacotherapeutic approach to diseases with increased bone resorption.

Dysapoptosis of osteoblasts and osteocytes increases cancellous bone formation but exaggerates cortical porosity with age.

Skeletal aging is accompanied by decreased cancellous bone mass and increased formation of pores within cortical bone. The latter accounts for a large portion of the increase in nonvertebral fractures after age 65 years in humans. We selectively deleted Bak and Bax, two genes essential for apoptosis, in two types of terminally differentiated bone cells: the short-lived osteoblasts that elaborate the bone matrix, and the long-lived osteocytes that are immured within the mineralized matrix and choreograph the regeneration of bone. Attenuation of apoptosis in osteoblasts increased their working lifespan and thereby cancellous bone mass in the femur. In long-lived osteocytes, however, it caused dysfunction with advancing age and greatly magnified intracortical femoral porosity associated with increased production of receptor activator of nuclear factor-κB ligand and vascular endothelial growth factor. Increasing bone mass by artificial prolongation of the inherent lifespan of short-lived osteoblasts, while exaggerating the adverse effects of aging on long-lived osteocytes, highlights the seminal role of cell age in bone homeostasis. In addition, our findings suggest that distress signals produced by old and/or dysfunctional osteocytes are the culprits of the increased intracortical porosity in old age.

FOXOs attenuate bone formation by suppressing Wnt signaling.

Wnt/ß-catenin/TCF signaling stimulates bone formation and suppresses adipogenesis. The hallmarks of skeletal involution with age, on the other hand, are decreased bone formation and increased bone marrow adiposity. These changes are associated with increased oxidative stress and decreased growth factor production, which activate members of the FOXO family of transcription factors. FOXOs in turn attenuate Wnt/ß-catenin signaling by diverting ß-catenin from TCF- to FOXO-mediated transcription. We show herein that mice lacking Foxo1, -3, and -4 in bipotential progenitors of osteoblast and adipocytes (expressing Osterix1) exhibited increased osteoblast number and high bone mass that was maintained in old age as well as decreased adiposity in the aged bone marrow. The increased bone mass in the Foxo-deficient mice was accounted for by increased proliferation of osteoprogenitor cells and bone formation resulting from upregulation of Wnt/ß-catenin signaling and cyclin D1 expression, but not changes in redox balance. Consistent with this mechanism, ß-catenin deletion in Foxo null cells abrogated both the increased cyclin D1 expression and proliferation. The elucidation of a restraining effect of FOXOs on Wnt signaling in bipotential progenitors suggests that FOXO activation by accumulation of age-associated cellular stressors may be a seminal pathogenetic mechanism in the development of involutional osteoporosis.

Suppression of autophagy in osteocytes mimics skeletal aging.

Bone mass declines with age but the mechanisms responsible remain unclear. Here we demonstrate that deletion of a conditional allele for Atg7, a gene essential for autophagy, from osteocytes caused low bone mass in 6-month-old male and female mice. Cancellous bone volume and cortical thickness were decreased, and cortical porosity increased, in conditional knock-out mice compared with control littermates. These changes were associated with low osteoclast number, osteoblast number, bone formation rate, and wall width in the cancellous bone of conditional knock-out mice. In addition, oxidative stress was higher in the bones of conditional knock-out mice as measured by reactive oxygen species levels in the bone marrow and by p66(shc) phosphorylation in L6 vertebra. Each of these changes has been previously demonstrated in the bones of old versus young adult mice. Thus, these results demonstrate that suppression of autophagy in osteocytes mimics, in many aspects, the impact of aging on the skeleton and suggest that a decline in autophagy with age may contribute to the low bone mass associated with aging.

The relevance of mouse models for investigating age-related bone loss in humans.

Mice are increasingly used for investigation of the pathophysiology of osteoporosis because their genome is easily manipulated, and their skeleton is similar to that of humans. Unlike the human skeleton, however, the murine skeleton continues to grow slowly after puberty and lacks osteonal remodeling of cortical bone. Yet, like humans, mice exhibit loss of cancellous bone, thinning of cortical bone, and increased cortical porosity with advancing age. Histologic evidence in mice and humans alike indicates that inadequate osteoblast-mediated refilling of resorption cavities created during bone remodeling is responsible. Mouse models of progeria also show bone loss and skeletal defects associated with senescence of early osteoblast progenitors. Additionally, mouse models of atherosclerosis, which often occurs in osteoporotic participants, also suffer bone loss, suggesting that common diseases of aging share pathophysiological pathways. Knowledge of the causes of skeletal fragility in mice should therefore be applicable to humans if inherent limitations are recognized.

Non-nuclear-initiated actions of the estrogen receptor protect cortical bone mass.

Extensive evidence has suggested that at least some of the effects of estrogens on bone are mediated via extranuclear estrogen receptor α signaling. However, definitive proof for this contention and the extent to which such effects may contribute to the overall protective effects of estrogens on bone maintenance have remained elusive. Here, we investigated the ability of a 17ß-estradiol (E2) dendrimer conjugate (EDC), incapable of stimulating nuclear-initiated actions of estrogen receptor α, to prevent the effects of ovariectomy (OVX) on the murine skeleton. We report that EDC was as potent as an equimolar dose of E2 in preventing bone loss in the cortical compartment that represents 80% of the entire skeleton, but was ineffective on cancellous bone. In contrast, E2 was effective in both compartments. Consistent with its effect on cortical bone mass, EDC partially prevented the loss of both vertebral and femoral strength. In addition, EDC, as did E2, prevented the OVX-induced increase in osteoclastogenesis, osteoblastogenesis, and oxidative stress. Nonetheless, the OVX-induced decrease in uterine weight was unaltered by EDC but was restored by E2. These results demonstrate that the protection of cortical bone mass by estrogens is mediated, at least in part, via a mechanism that is distinct from the classic mechanism of estrogen action on reproductive organs.