The bone-sparing effects of estrogen and WNT16 are independent of each other.

Wingless-type MMTV integration site family (WNT)16 is a key regulator of bone mass with high expression in cortical bone, and Wnt16(-/-) mice have reduced cortical bone mass. As Wnt16 expression is enhanced by estradiol treatment, we hypothesized that the bone-sparing effect of estrogen in females is WNT16-dependent. This hypothesis was tested in mechanistic studies using two genetically modified mouse models with either constantly high osteoblastic Wnt16 expression or no Wnt16 expression. We developed a mouse model with osteoblast-specific Wnt16 overexpression (Obl-Wnt16). These mice had several-fold elevated Wnt16 expression in both trabecular and cortical bone compared with wild type (WT) mice. Obl-Wnt16 mice displayed increased total body bone mineral density (BMD), surprisingly caused mainly by a substantial increase in trabecular bone mass, resulting in improved bone strength of vertebrae L3. Ovariectomy (ovx) reduced the total body BMD and the trabecular bone mass to the same degree in Obl-Wnt16 mice and WT mice, suggesting that the bone-sparing effect of estrogen is WNT16-independent. However, these bone parameters were similar in ovx Obl-Wnt16 mice and sham operated WT mice. The role of WNT16 for the bone-sparing effect of estrogen was also evaluated in Wnt16(-/-) mice. Treatment with estradiol increased the trabecular and cortical bone mass to a similar extent in both Wnt16(-/-) and WT mice. In conclusion, the bone-sparing effects of estrogen and WNT16 are independent of each other. Furthermore, loss of endogenous WNT16 results specifically in cortical bone loss, whereas overexpression of WNT16 surprisingly increases mainly trabecular bone mass. WNT16-targeted therapies might be useful for treatment of postmenopausal trabecular bone loss.

The estrogen receptor antagonist ICI 182,780 can act both as an agonist and an inverse agonist when estrogen receptor α AF-2 is modified.

The bone-sparing effect of estrogen is primarily mediated via estrogen receptor (ER) α, which stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal and AF-2 in the ligand-binding domain. It was recently demonstrated that the ER antagonist ICI 182,780 (ICI) acts as an ER agonist in uterus of mice with mutations in the ERα AF-2. To evaluate the estrogen-like effects of ICI in different tissues, ovariectomized wild-type mice and mice with mutations in the ERα AF-2 (ERαAF-2(0)) were treated with ICI, estradiol, or vehicle for 3 wk. Estradiol increased the trabecular and cortical bone mass as well as the uterine weight, whereas it reduced fat mass, thymus weight, and the growth plate height in wild-type but not in ERαAF-2(0) mice. Although ICI had no effect in wild-type mice, it exerted tissue-specific effects in ERαAF-2(0) mice. It acted as an ERα agonist on trabecular bone mass and uterine weight, whereas no effect was seen on cortical bone mass, fat mass, or thymus weight. Surprisingly, a pronounced inverse agonistic activity was seen on the growth plate height, resulting in enhanced longitudinal bone growth. In conclusion, ICI uses ERα AF-1 in a tissue-dependent manner in mice lacking ERαAF-2, resulting in no effect, agonistic activity, or inverse agonistic activity. We propose that ERα lacking AF-2 is constitutively active in the absence of ligand in the growth plate, enabling ICI to act as an inverse agonist.

SERMs have substance-specific effects on bone, and these effects are mediated via ERαAF-1 in female mice.

The bone-sparing effect of estrogens is mediated primarily via estrogen receptor (ER)α, which stimulates gene transcription through activation function (AF)-1 and AF-2. The role of ERαAF-1 for the estradiol (E2) effects is tissue specific. The selective ER modulators (SERMs) raloxifene (Ral), lasofoxifene (Las), and bazedoxifene (Bza) can be used to treat postmenopausal osteoporosis. They all reduce the risk for vertebral fractures, whereas Las and partly Bza, but not Ral, reduce the risk for nonvertebral fractures. Here, we have compared the tissue specificity of Ral, Las, and Bza and evaluated the role of ERαAF-1 for the effects of these SERMs, with an emphasis on bone parameters. We treated ovariectomized (OVX) wild-type (WT) mice and OVX mice lacking ERαAF-1 (ERαAF-1(0)) with E2, Ral, Las, or Bza. All three SERMs increased trabecular bone mass in the axial skeleton. In the appendicular skeleton, only Las increased the trabecular bone volume/tissue volume and trabecular number, whereas both Ral and Las increased the cortical bone thickness and strength. However, Ral also increased cortical porosity. The three SERMs had only a minor effect on uterine weight. Notably, all evaluated effects of these SERMs were absent in ovx ERαAF-1(0) mice. In conclusion, all SERMs had similar effects on axial bone mass. However, the SERMs had slightly different effects on the appendicular skeleton since only Las increased the trabecular bone mass and only Ral increased the cortical porosity. Importantly, all SERM effects require a functional ERαAF-1 in female mice. These results could lead to development of more specific treatments for osteoporosis.

Estrogen receptor-α in osteocytes is important for trabecular bone formation in male mice.

The bone-sparing effect of estrogen in both males and females is primarily mediated via estrogen receptor-α (ERα), encoded by the Esr1 gene. ERα in osteoclasts is crucial for the trabecular bone-sparing effect of estrogen in females, but it is dispensable for trabecular bone in male mice and for cortical bone in both genders. We hypothesized that ERα in osteocytes is important for trabecular bone in male mice and for cortical bone in both males and females. Dmp1-Cre mice were crossed with ERα(flox/flox) mice to generate mice lacking ERα protein expression specifically in osteocytes (Dmp1-ERα(-/-)). Male Dmp1-ERα(-/-) mice displayed a substantial reduction in trabecular bone volume (-20%, P < 0.01) compared with controls. Dynamic histomorphometry revealed reduced bone formation rate (-45%, P < 0.01) but the number of osteoclasts per bone surface was unaffected in the male Dmp1-ERα(-/-) mice. The male Dmp1-ERα(-/-) mice had reduced expression of several osteoblast/osteocyte markers in bone, including Runx2, Sp7, and Dmp1 (P < 0.05). Gonadal intact Dmp1-ERα(-/-) female mice had no significant reduction in trabecular bone volume but ovariectomized Dmp1-ERα(-/-) female mice displayed an attenuated trabecular bone response to supraphysiological E2 treatment. Dmp1-ERα(-/-) mice of both genders had unaffected cortical bone. In conclusion, ERα in osteocytes regulates trabecular bone formation and thereby trabecular bone volume in male mice but it is dispensable for the trabecular bone in female mice and the cortical bone in both genders. We propose that the physiological trabecular bone-sparing effect of estrogen is mediated via ERα in osteocytes in males, but via ERα in osteoclasts in females.

Estrogen receptor-α expression in neuronal cells affects bone mass.

It has generally been assumed that bone mass is controlled by endocrine mechanisms and the local bone environment. Recent findings demonstrate that central pathways are involved in the regulation of bone mass. Estrogen is involved in the regulation of bone homeostasis and the CNS is also a target for estrogen actions. The aim of this study was to investigate in vivo the role of central estrogen receptor-α (ERα) expression for bone mass. Nestin-Cre mice were crossed with ERα(flox) mice to generate mice lacking ERα expression specifically in nervous tissue (nestin-ERα(-/-)). Bone mineral density was increased in both the trabecular and cortical bone compartments in nestin-ERα(-/-) mice compared with controls. Femoral bone strength was increased in nestin-ERα(-/-) mice, as demonstrated by increased stiffness and maximal load of failure. The high bone mass phenotype in nestin-ERα(-/-) mice was mainly caused by increased bone formation. Serum leptin levels were elevated as a result of increased leptin expression in white adipose tissue (WAT) and slightly increased amount of WAT in nestin-ERα(-/-) mice. Leptin receptor mRNA levels were reduced in the hypothalamus but not in bone. In conclusion, inactivation of central ERα signaling results in increased bone mass, demonstrating that the balance between peripheral stimulatory and central inhibitory ERα actions is important for the regulation of bone mass. We propose that the increased bone mass in nestin-ERα(-/-) mice is mediated via decreased central leptin sensitivity and thereby increased secretion of leptin from WAT, which, in turn, results in increased peripheral leptin-induced bone formation.

Enzalutamide Reduces the Bone Mass in the Axial But Not the Appendicular Skeleton in Male Mice.

Testosterone is a crucial regulator of the skeleton, but the role of the androgen receptor (AR) for the maintenance of the adult male skeleton is unclear. In the present study, the role of the AR for bone metabolism and skeletal growth after sexual maturation was evaluated by means of the drug enzalutamide, which is a new AR antagonist used in the treatment of prostate cancer patients. Nine-week-old male mice were treated with 10, 30, or 100 mg/kg·d of enzalutamide for 21 days or were surgically castrated and were compared with vehicle-treated gonadal intact mice. Although orchidectomy reduced the cortical bone thickness and trabecular bone volume fraction in the appendicular skeleton, these parameters were unaffected by enzalutamide. In contrast, both enzalutamide and orchidectomy reduced the bone mass in the axial skeleton as demonstrated by a reduced lumbar spine areal bone mineral density (P < .001) and trabecular bone volume fraction in L5 vertebrae (P < .001) compared with vehicle-treated gonadal intact mice. A compression test of the L5 vertebrae revealed that the mechanical strength in the axial skeleton was significantly reduced by enzalutamide (maximal load at failure -15.3% ± 3.5%; P < .01). The effects of enzalutamide in the axial skeleton were associated with a high bone turnover. In conclusion, enzalutamide reduces the bone mass in the axial but not the appendicular skeleton in male mice after sexual maturation. We propose that the effect of testosterone on the axial skeleton in male mice is mainly mediated via the AR.

Androgens regulate bone marrow B lymphopoiesis in male mice by targeting osteoblast-lineage cells.

Testosterone has profound immune-modulatory actions, which may be important for the sexual dimorphism in immune-related disorders, such as autoimmune diseases. A well-known effect of androgens is inhibition of bone marrow B lymphopoiesis; however, a plausible target cell for this effect has not yet been presented. The aim of this study was to determine the target cell for androgen-mediated regulation of bone marrow B lymphopoiesis in males. We confirm higher number of bone marrow B cells in male mice with global inactivation of the androgen receptor (AR) and these global AR knockout (G-ARKO) mice had increased number of B cell precursors from the pro-B stage. Because osteoblast-lineage cells are known to support B lymphopoiesis at the pro-B stage, we investigated the effect on B lymphopoiesis in osteoblast-lineage cell-specific ARKO (O-ARKO) mice; O-ARKO mice had increased number of B cells in the bone marrow, and the number of B cell precursors was increased from the pro-B stage, demonstrating that O-ARKO mimics the bone marrow B lymphopoiesis pattern of G-ARKO mice. By contrast, O-ARKO mice displayed only minor changes in B cell numbers in the splenic compartment compared with G-ARKO. Further, O-ARKO mice had moderately reduced number of bone trabeculae in the vertebrae, whereas cortical bone was unaffected. In conclusion, androgens exert inhibitory effects on bone marrow B lymphopoiesis in males by targeting the AR in osteoblast-lineage cells. The identification of the likely target cell for androgen-mediated regulation of bone marrow B lymphopoiesis will contribute to elucidation of the mechanisms by which androgens modulate immune-related disorders.

Measurement of a Comprehensive Sex Steroid Profile in Rodent Serum by High-Sensitive Gas Chromatography-Tandem Mass Spectrometry.

Accurate measurement of sex steroid concentrations in rodent serum is essential to evaluate mouse and rat models for sex steroid-related disorders. The aim of the present study was to develop a sensitive and specific gas chromatography-tandem mass spectrometry (GC-MS/MS) method to assess a comprehensive sex steroid profile in rodent serum. A major effort was invested in reaching an exceptionally high sensitivity for measuring serum estradiol concentrations. We established a GC-MS/MS assay with a lower limit of detection for estradiol, estrone, T, DHT, progesterone, androstenedione, and dehydroepiandrosterone of 0.3, 0.5, 4.0, 1.6, 8, 4.0, and 50 pg/mL, respectively, whereas the corresponding values for the lower limit of quantification were 0.5, 0.5, 8, 2.5, 74, 12, and 400 pg/mL, respectively. Calibration curves were linear, intra- and interassay coefficients of variation were low, and accuracy was excellent for all analytes. The established assay was used to accurately measure a comprehensive sex steroid profile in female rats and mice according to estrous cycle phase. In addition, we characterized the impact of age, sex, gonadectomy, and estradiol treatment on serum concentrations of these sex hormones in mice. In conclusion, we have established a highly sensitive and specific GC-MS/MS method to assess a comprehensive sex steroid profile in rodent serum in a single run. This GC-MS/MS assay has, to the best of our knowledge, the best detectability reported for estradiol. Our method therefore represents an ideal tool to characterize sex steroid metabolism in a variety of sex steroid-related rodent models and in human samples with low estradiol levels.

The role of total and cartilage-specific estrogen receptor alpha expression for the ameliorating effect of estrogen treatment on arthritis.

INTRODUCTION: Estrogen (E2) delays onset and decreases severity of experimental arthritis. The aim of this study was to investigate the importance of total estrogen receptor alpha (ERα) expression and cartilage-specific ERα expression in genetically modified mice for the ameliorating effect of estrogen treatment in experimental arthritis. METHODS: Mice with total (total ERα-/-) or cartilage-specific (Col2α1-ERα-/-) inactivation of ERα and wild-type (WT) littermates were ovariectomized, treated with E2 or placebo, and induced with antigen-induced arthritis (AIA). At termination, knees were collected for histology, synovial and splenic cells were investigated by using flow cytometry, and splenic cells were subjected to a T-cell proliferation assay. RESULTS: E2 decreased synovitis and joint destruction in WT mice. Amelioration of arthritis was associated with decreased frequencies of inflammatory cells in synovial tissue and decreased splenic T-cell proliferation. E2 did not affect synovitis or joint destruction in total ERα-/- mice. In Col2α1-ERα-/- mice, E2 protected against joint destruction to a similar extent as in WT mice. In contrast, E2 did not significantly ameliorate synovitis in Col2α1-ERα-/- mice. CONCLUSIONS: Treatment with E2 ameliorates both synovitis and joint destruction in ovariectomized mice with AIA via ERα. This decreased severity in arthritis is associated with decreased synovial inflammatory cell frequencies and reduced splenic T-cell proliferation. ERα expression in cartilage is not required for estrogenic amelioration of joint destruction. However, our data indicate that ERα expression in cartilage is involved in estrogenic effects on synovitis, suggesting different mechanisms for the amelioration of joint destruction and synovitis by E2.

Sex steroid actions in male bone.

Sex steroids are chief regulators of gender differences in the skeleton, and male gender is one of the strongest protective factors against osteoporotic fractures. This advantage in bone strength relies mainly on greater cortical bone expansion during pubertal peak bone mass acquisition and superior skeletal maintenance during aging. During both these phases, estrogens acting via estrogen receptor-α in osteoblast lineage cells are crucial for male cortical and trabecular bone, as evident from conditional genetic mouse models, epidemiological studies, rare genetic conditions, genome-wide meta-analyses, and recent interventional trials. Genetic mouse models have also demonstrated a direct role for androgens independent of aromatization on trabecular bone via the androgen receptor in osteoblasts and osteocytes, although the target cell for their key effects on periosteal bone formation remains elusive. Low serum estradiol predicts incident fractures, but the highest risk occurs in men with additionally low T and high SHBG. Still, the possible clinical utility of serum sex steroids for fracture prediction is unknown. It is likely that sex steroid actions on male bone metabolism rely also on extraskeletal mechanisms and cross talk with other signaling pathways. We propose that estrogens influence fracture risk in aging men via direct effects on bone, whereas androgens exert an additional antifracture effect mainly via extraskeletal parameters such as muscle mass and propensity to fall. Given the demographic trends of increased longevity and consequent rise of osteoporosis, an increased understanding of how sex steroids influence male bone health remains a high research priority.

Osteoblast-derived WNT16 represses osteoclastogenesis and prevents cortical bone fragility fractures.

The WNT16 locus is a major determinant of cortical bone thickness and nonvertebral fracture risk in humans. The disability, mortality and costs caused by osteoporosis-induced nonvertebral fractures are enormous. We demonstrate here that Wnt16-deficient mice develop spontaneous fractures as a result of low cortical thickness and high cortical porosity. In contrast, trabecular bone volume is not altered in these mice. Mechanistic studies revealed that WNT16 is osteoblast derived and inhibits human and mouse osteoclastogenesis both directly by acting on osteoclast progenitors and indirectly by increasing expression of osteoprotegerin (Opg) in osteoblasts. The signaling pathway activated by WNT16 in osteoclast progenitors is noncanonical, whereas the pathway activated in osteoblasts is both canonical and noncanonical. Conditional Wnt16 inactivation revealed that osteoblast-lineage cells are the principal source of WNT16, and its targeted deletion in osteoblasts increases fracture susceptibility. Thus, osteoblast-derived WNT16 is a previously unreported key regulator of osteoclastogenesis and fracture susceptibility. These findings open new avenues for the specific prevention or treatment of nonvertebral fractures, a substantial unmet medical need.

The role of activation functions 1 and 2 of estrogen receptor-α for the effects of estradiol and selective estrogen receptor modulators in male mice.

Estradiol (E2) is important for male skeletal health and the effect of E2 is mediated via estrogen receptor (ER)-α. This was demonstrated by the findings that men with an inactivating mutation in aromatase or a nonfunctional ERα had osteopenia and continued longitudinal growth after sexual maturation. The aim of the present study was to evaluate the role of different domains of ERα for the effects of E2 and selective estrogen receptor modulators (SERMs) on bone mass in males. Three mouse models lacking either ERαAF-1 (ERαAF-1(0)), ERαAF-2 (ERαAF-2(0)), or the total ERα (ERα(-/-)) were orchidectomized (orx) and treated with E2 or placebo. E2 treatment increased the trabecular and cortical bone mass and bone strength, whereas it reduced the thymus weight and bone marrow cellularity in orx wild type (WT) mice. These parameters did not respond to E2 treatment in orx ERα(-/-) or ERαAF-2(0). However, the effects of E2 in orx ERαAF-1(0) [corrected] were tissue-dependent, with a clear response in cortical bone parameters and bone marrow cellularity, but no response in trabecular bone. To determine the role of ERαAF-1 for the effects of SERMs, we treated orx WT and ERαAF-1(0) mice with raloxifene (Ral), lasofoxifene (Las), bazedoxifene (Bza), or vehicle. These SERMs increased total body areal bone mineral density (BMD) and trabecular volumetric BMD to a similar extent in orx WT mice. Furthermore, only Las increased cortical thickness significantly and only Bza increased bone strength significantly. However, all SERMs showed a tendency toward increased cortical bone parameters. Importantly, all SERM effects were absent in the orx ERαAF-1(0) mice. In conclusion, ERαAF-2 is required for the estrogenic effects on all evaluated parameters, whereas the role of ERαAF-1 is tissue-specific. All evaluated effects of Ral, Las and Bza are dependent on a functional ERαAF-1. Our findings might contribute to the development of bone-specific SERMs in males.

Estrogen receptor-α is required for the osteogenic response to mechanical loading in a ligand-independent manner involving its activation function 1 but not 2.

Estrogen receptor-α (ERα) is crucial for the adaptive response of bone to loading but the role of endogenous estradiol (E2) for this response is unclear. To determine in vivo the ligand dependency and relative roles of different ERα domains for the osteogenic response to mechanical loading, gene-targeted mouse models with (1) a complete ERα inactivation (ERα(-/-) ), (2) specific inactivation of activation function 1 (AF-1) in ERα (ERαAF-1(0) ), or (3) specific inactivation of ERαAF-2 (ERαAF-2(0) ) were subjected to axial loading of tibia, in the presence or absence (ovariectomy [ovx]) of endogenous E2. Loading increased the cortical bone area in the tibia mainly as a result of an increased periosteal bone formation rate (BFR) and this osteogenic response was similar in gonadal intact and ovx mice, demonstrating that E2 (ligand) is not required for this response. Female ERα(-/-) mice displayed a severely reduced osteogenic response to loading with changes in cortical area (-78% ± 15%, p < 0.01) and periosteal BFR (-81% ± 9%, p < 0.01) being significantly lower than in wild-type (WT) mice. ERαAF-1(0) mice also displayed a reduced response to mechanical loading compared with WT mice (cortical area -40% ± 11%, p < 0.05 and periosteal BFR -41% ± 8%, p < 0.01), whereas the periosteal osteogenic response to loading was unaffected in ERαAF-2(0) mice. Mechanical loading of transgenic estrogen response element (ERE)-luciferase reporter mice did not increase luciferase expression in cortical bone, suggesting that the loading response does not involve classical genomic ERE-mediated pathways. In conclusion, ERα is required for the osteogenic response to mechanical loading in a ligand-independent manner involving AF-1 but not AF-2.

Sex steroids and bone health in men.

The influence of sex steroids on bone in both men and women has long been recognized. In men, however, the relative contribution of androgens versus estrogens in the regulation of bone metabolism remains uncertain. Animal studies demonstrate that both estradiol (E2), via activation of estrogen receptor-α, and testosterone (T), via activation of the androgen receptor, regulate bone mass in male rodents. The main focus of this review is to summarize and discuss recent findings from the osteoporotic fractures in men (MrOS) cohorts regarding the impact of serum sex steroids on bone health in elderly men. Collectively, these data demonstrate that serum E2 is directly associated with bone mineral density (BMD) and that low serum E2 associates with higher rates of bone loss and fracture. In addition, they substantiate the concept of a threshold E2 level that determines fracture risk in elderly men. We propose that the effect of E2 on fracture risk is at least partly mediated by its effect on BMD, whereas the more modest effect of T on fracture risk mainly is mediated by effects on muscle strength and risk of falls. Findings from the MrOS cohorts also demonstrate that racial and genetic variations in aromatase activity influence serum E2 levels in men. In conclusion, there is compelling evidence that not only androgens, but also estrogens, are important regulators of bone health in men. Consequently, E2 should not exclusively be regarded as the 'female hormone' but as a sex steroid that is necessary for maintenance of bone health in men.

Reduced bone mass and muscle strength in male 5α-reductase type 1 inactivated mice.

Androgens are important regulators of bone mass but the relative importance of testosterone (T) versus dihydrotestosterone (DHT) for the activation of the androgen receptor (AR) in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2), encoded by separate genes (Srd5a1 and Srd5a2). 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1⁻/⁻ mice. Four-month-old male Srd5a1⁻/⁻ mice had reduced trabecular bone mineral density (-36%, p<0.05) and cortical bone mineral content (-15%, p<0.05) but unchanged serum androgen levels compared with wild type (WT) mice. The cortical bone dimensions were reduced in the male Srd5a1⁻/⁻ mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05) in orchidectomized WT mice but not in orchidectomized Srd5a1⁻/⁻ mice. Male Srd5a1⁻/⁻ mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05). Female Srd5a1⁻/⁻ mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1⁻/⁻ mice, is an indirect effect mediated by elevated circulating androgen levels.

Amelioration of collagen-induced arthritis and immune-associated bone loss through signaling via estrogen receptor alpha, and not estrogen receptor beta or G protein-coupled receptor 30.

OBJECTIVE: The effects of estrogen may be exerted via the nuclear estrogen receptors (ERs) ERalpha or ERbeta or via the recently proposed transmembrane estrogen receptor G protein-coupled receptor 30 (GPR-30). The purpose of this study was to elucidate the ER specificity for the ameliorating effects of estrogen on arthritis and bone loss in a model of postmenopausal rheumatoid arthritis (RA). METHODS: Female DBA/1 mice underwent ovariectomy or sham operation, and type II collagen-induced arthritis was induced. Mice were treated subcutaneously 5 days/week with the specific agonists propylpyrazoletriol (PPT; for ERalpha), diarylpropionitrile (DPN; for ERbeta), G1 (for GPR-30), or with a physiologic dose of estradiol. Clinical arthritis scores were determined continuously. At termination of the study, bone mineral density (BMD) was analyzed, paws were collected for histologic assessment, serum was analyzed for cytokines and markers of bone and cartilage turnover, and bone marrow was subjected to fluorescence-activated cell sorting. RESULTS: Treatment with PPT as well as estradiol dramatically decreased the frequency and severity of arthritis. Furthermore, estradiol and PPT treatment resulted in preservation of bone and cartilage, as demonstrated by increased BMD and decreased serum levels of bone resorption markers and cartilage degradation markers, whereas no effect was seen after DPN or G1 treatment. CONCLUSION: In a well-established model of postmenopausal RA, ERalpha, but not ERbeta or GPR-30 signaling, was shown to ameliorate the disease and the associated development of osteoporosis. Since long-term treatment with estrogen has been associated with significant side effects, increased knowledge about the mechanisms behind the beneficial effects of estrogen is useful in the search for novel treatments of postmenopausal RA.

The role of estrogen receptor α in growth plate cartilage for longitudinal bone growth.

Estrogens enhance skeletal growth during early sexual maturation, whereas high estradiol levels during late puberty result in growth plate fusion in humans. Although the growth plates do not fuse directly after sexual maturation in rodents, a reduction in growth plate height is seen by treatment with a high dose of estradiol. It is unknown whether the effects of estrogens on skeletal growth are mediated directly via estrogen receptors (ERs) in growth plate cartilage and/or indirectly via other mechanisms such as the growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis. To determine the role of ERα in growth plate cartilage for skeletal growth, we developed a mouse model with cartilage-specific inactivation of ERα. Although mice with total ERα inactivation displayed affected longitudinal bone growth associated with alterations in the GH/IGF-1 axis, the skeletal growth was normal during sexual maturation in mice with cartilage-specific ERα inactivation. High-dose estradiol treatment of adult mice reduced the growth plate height as a consequence of attenuated proliferation of growth plate chondrocytes in control mice but not in cartilage-specific ERα(-/-) mice. Adult cartilage-specific ERα(-/-) mice continued to grow after 4 months of age, whereas growth was limited in control mice, resulting in increased femur length in 1-year-old cartilage-specific ERα(-/-) mice compared with control mice. We conclude that during early sexual maturation, ERα in growth plate cartilage is not important for skeletal growth. In contrast, it is essential for high-dose estradiol to reduce the growth plate height in adult mice and for reduction of longitudinal bone growth in elderly mice.