RESUMO
Women are more prone to develop rheumatoid arthritis, with peak incidence occurring around menopause. Estrogen has major effects on the immune system and is protective against arthritis. We have previously shown that treatment with estrogen inhibits inflammation and joint destruction in murine models of arthritis, although the mechanisms involved remain unclear. Fibroblastic reticular cells (FRCs) are specialized stromal cells that generate the three-dimensional structure of lymph nodes (LNs). FRCs are vital for coordinating immune responses from within LNs and are characterized by the expression of the chemokine CCL19, which attracts immune cells. The aim of this study was to determine whether the influence of estrogen on innate and adaptive immune cells in arthritis is mediated by estrogen signaling in FRCs. Conditional knockout mice lacking estrogen receptor α (ERα) in CCL19-expressing cells (Ccl19-CreERαfl/fl) were generated and tested. Ccl19-CreERαfl/fl mice and littermate controls were ovariectomized, treated with vehicle or estradiol and subjected to the 28-day-long antigen-induced arthritis model to enable analyses of differentiated T- and B-cell populations and innate cells in LNs by flow cytometry. The results reveal that while the response to estradiol treatment in numbers of FRCs per LN is significantly reduced in mice lacking ERα in FRCs, estrogen does not inhibit joint inflammation or markedly affect immune responses in this arthritis model. Thus, this study validates the Ccl19-CreERαfl/fl strain for studying estrogen signaling in FRCs within inflammatory diseases, although the chosen arthritis model is deemed unsuitable for addressing this question.
Assuntos
Imunidade Adaptativa , Receptor alfa de Estrogênio , Estrogênios , Fibroblastos , Imunidade Inata , Camundongos Knockout , Transdução de Sinais , Animais , Estrogênios/metabolismo , Camundongos , Feminino , Fibroblastos/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/genética , Artrite Experimental/imunologia , Quimiocina CCL19/metabolismo , Linfonodos/metabolismo , Linfonodos/imunologia , Camundongos Endogâmicos C57BL , Antígenos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismoRESUMO
Osteoporosis is a common secondary complication in patients with systemic lupus erythematosus (SLE). Current osteoporosis treatment with bisphosphonates has some negative side effects and there is a lack of data regarding newer treatments options for SLE associated osteoporosis. The tissue-selective estrogen complex (TSEC) containing conjugated estrogens and the selective estrogen receptor modulator bazedoxifene (Bza) is approved for treatment of postmenopausal vasomotor symptoms and prevention of osteoporosis. However, it has not been evaluated for treatment of osteoporosis in postmenopausal SLE patients. Ovariectomized MRL/lpr mice constitute a model for postmenopausal lupus that can be used for osteoporosis studies. We used this model in a set of experiments where the mice were treated with different doses of 17ß-estradiol-3-benzoate (E2), Bza, or TSEC (E2 plus Bza), administered in the early or late phases of disease development. The skeleton was analyzed by dual-energy X-ray absorptiometry, peripheral quantitative computed tomography, and high-resolution microcomputed tomography. The lupus disease was assessed by determination of proteinuria, hematuria, and lupus disease markers in serum. Treatment with medium dose TSEC administered in early disease protected ovariectomized MRL/lpr mice from trabecular bone loss, while there were no differences in lupus disease parameters between treatments. This is the first experimental study to investigate TSEC as a potential new therapy for osteoporosis in postmenopausal SLE.
Assuntos
Lúpus Eritematoso Discoide , Lúpus Eritematoso Sistêmico , Osteoporose , Animais , Estrogênios/química , Estrogênios Conjugados (USP)/química , Humanos , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Camundongos , Camundongos Endogâmicos MRL lpr , Osteoporose/induzido quimicamente , Osteoporose/tratamento farmacológico , Microtomografia por Raio-XRESUMO
Mutation of arginine 264 in ERα has been shown to abrogate rapid membrane ERα-mediated endothelial effects. Our novel finding that mutation of R264 is dispensable for ERα-mediated skeletal effects supports the concept that R264 determines tissue specificity of ERα. Estrogen protects against bone loss but is not a suitable treatment due to adverse effects in other tissues. Therefore, increased knowledge regarding estrogen signaling in estrogen-responsive tissues is warranted to aid the development of bone-specific estrogen treatments. Estrogen receptor-α (ERα), the main mediator of estrogenic effects in bone, is widely subjected to posttranslational modifications (PTMs). In vitro studies have shown that methylation at site R260 in the human ERα affects receptor localization and intracellular signaling. The corresponding amino acid R264 in murine ERα has been shown to have a functional role in endothelium in vivo, although the methylation of R264 in the murine gene is yet to be empirically demonstrated. The aim of this study was to investigate whether R264 in ERα is involved in the regulation of the skeleton in vivo. Dual-energy X-ray absorptiometry (DEXA) analysis at 3, 6, 9, and 12 mo of age showed no differences in total body areal bone mineral density (BMD) between R264A and wild type (WT) in either female or male mice. Furthermore, analyses using computed tomography (CT) demonstrated that trabecular bone mass in tibia and vertebra and cortical thickness in tibia were similar between R264A and WT mice. In addition, R264A females displayed a normal estrogen treatment response in trabecular bone mass as well as in cortical thickness. Furthermore, uterus, thymus, and adipose tissue responded similarly in R264A and WT female mice after estrogen treatment. In conclusion, our novel finding that mutation of R264 in ERα does not affect the regulation of the skeleton, together with the known role of R264 for ERα-mediated endothelial effects, supports the concept that R264 determines tissue specificity of ERα.NEW & NOTEWORTHY Mutation of arginine 264 in ERα has been shown to abrogate rapid membrane ERα-mediated endothelial effects. Our novel finding that mutation of R264 is dispensable for ERα-mediated skeletal effects supports the concept that R264 determines tissue specificity of ERα.
Assuntos
Arginina/genética , Arginina/fisiologia , Osso e Ossos/fisiologia , Receptor alfa de Estrogênio/genética , Absorciometria de Fóton , Envelhecimento/fisiologia , Animais , Densidade Óssea , Osso e Ossos/diagnóstico por imagem , Endotélio/metabolismo , Estrogênios/farmacologia , Feminino , Metilação , Camundongos , Tamanho do Órgão/genética , Ovariectomia , Coluna Vertebral/química , Coluna Vertebral/metabolismo , Tíbia/química , Tíbia/metabolismo , Tomografia Computadorizada por Raios XRESUMO
Immunoglobulin G (IgG) is important in clearance and recognition of previously presented antigens and after activation, IgGs can interact with the Fc gamma receptors (FcγRs) on haematopoietic cells, including bone-resorbing osteoclasts. The pathogenicity of IgG, that is the ability to elicit stimulatory effects via FcγRs, can be modulated by attachment of sugar moieties, including sialic acids. Human IgGs and autoantibodies are associated with bone loss in autoimmune disease. However, the impact of polyclonal murine IgG via FcγRs on bone loss is poorly understood. Here, we investigate if heat-aggregated activated murine polyclonal IgG complexes have any direct effects on murine osteoclasts and if they modulate arthritis-mediated bone loss. Using cell cultures of murine osteoclasts, we show that IgG complexes without sialic acids (de-IgG complexes) enhance receptor activator of nuclear factor kappa-Β ligand (RANKL)-stimulated osteoclastogenesis, an effect associated with increased FcγRIII expression. Using an in vivo model of arthritis-mediated bone loss, where IgG complexes were injected into arthritic knees, no effect on the severity of arthritis or the degree of arthritis-mediated bone loss was detected. Interestingly, injection of de-IgG complexes into non-arthritic knees increased osteoclast formation and enhanced bone erosions. Our findings show that activated de-IgG complexes have no additive effect on arthritis-mediated bone loss. However, de-IgG complexes potentiate murine osteoclastogenesis and enhance local bone erosion in non-arthritic bones, further confirming the link between the adaptive immune system and bone.
Assuntos
Artrite Experimental/patologia , Reabsorção Óssea/patologia , Imunoglobulina G/imunologia , Osteogênese/fisiologia , Receptores de IgG/imunologia , Animais , Artrite Experimental/imunologia , Reabsorção Óssea/imunologia , Feminino , Imunoglobulina G/química , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Receptores de IgG/química , Ácidos Siálicos/metabolismoRESUMO
Estrogen treatment increases bone mass and reduces fat mass but is associated with adverse effects in postmenopausal women. Knowledge regarding tissue-specific estrogen signaling is important to aid the development of new tissue-specific treatments. We hypothesized that the posttranslational modification phosphorylation in estrogen receptor alpha (ERα) may modulate ERα activity in a tissue-dependent manner. Phosphorylation of site S122 in ERα has been shown in vitro to affect ERα activity, but the tissue-specific role in vivo is unknown. We herein developed and phenotyped a novel mouse model with a point mutation at the phosphorylation site 122 in ERα (S122A). Female S122A mice had increased fat mass and serum insulin levels but unchanged serum sex steroid levels, uterus weight, bone mass, thymus weight, and lymphocyte maturation compared to WT mice. In conclusion, phosphorylation site S122 in ERα has a tissue-dependent role with an impact specifically on fat mass in female mice. This study is the first to demonstrate in vivo that a phosphorylation site in a transactivation domain in a nuclear steroid receptor modulates the receptor activity in a tissue-dependent manner.
Assuntos
Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Fosforilação/genética , Animais , Densidade Óssea/genética , Osso e Ossos/metabolismo , Estrogênios/genética , Estrogênios/metabolismo , Feminino , Insulina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/genética , Mutação Puntual/genética , Transdução de Sinais/genéticaRESUMO
Sexually dimorphic bone structure emerges largely during puberty. Sex steroids are critical for peak bone mass acquisition in both genders. In particular, the biphasic effects of estrogens mediate the skeletal sexual dimorphism. However, so far the stimulatory vs inhibitory actions of estrogens on bone mass are not fully explained by direct effects on bone cells. Recently, it has become evident that there is possible neuroendocrine action of estrogen receptor alpha (ERα) on the skeleton. Based on these considerations, we hypothesized that neuronal ERα-signaling may contribute to the skeletal growth during puberty. Here, we generated mice with tamoxifen-inducible Thy1-Cre mediated ERα inactivation during late puberty specifically in extrahypothalamic neurons (N-ERαKO). Inactivation of neuronal ERα did not alter the body weight in males, whereas N-ERαKO females exhibited a higher body weight and increased body and bone length compared to their control littermates at 16 weeks of age. Ex vivo microCT analysis showed increased radial bone expansion of the midshaft femur in female N-ERαKO along with higher serum levels of insulin-like growth factor (IGF)-1 as well as IGF-binding protein (IGFBP)-3. Furthermore, the 3-point bending test revealed increased bone strength in female N-ERαKO. In contrast, inactivation of neuronal ERα had no major effect on bone growth in males. In conclusion, we demonstrate that central ERα-signaling limits longitudinal bone growth and radial bone expansion specifically in females potentially by interacting with the GH/IGF-1 axis.
Assuntos
Desenvolvimento Ósseo/fisiologia , Receptor alfa de Estrogênio/metabolismo , Neurônios/metabolismo , Maturidade Sexual/fisiologia , Animais , Fenômenos Biomecânicos , Densidade Óssea/genética , Densidade Óssea/fisiologia , Desenvolvimento Ósseo/genética , Osso e Ossos/anatomia & histologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/fisiologia , Receptor alfa de Estrogênio/deficiência , Receptor alfa de Estrogênio/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Caracteres Sexuais , Maturidade Sexual/genética , Transdução de Sinais , Microtomografia por Raio-XRESUMO
Mouse models with lifelong inactivation of estrogen receptor-α (ERα) show that ERα is the main mediator of estrogenic effects in bone, thymus, uterus, and fat. However, ERα inactivation early in life may cause developmental effects that confound the adult phenotypes. To address the specific role of adult ERα expression for estrogenic effects in bone and other nonskeletal tissues, we established a tamoxifen-inducible ERα-inactivated model by crossing CAGG-Cre-ER and ERαflox/flox mice. Tamoxifen-induced ERα inactivation after sexual maturation substantially reduced ERα mRNA levels in cortical bone, trabecular bone, thymus, uterus, gonadal fat, and hypothalamus, in CAGG-Cre-ERαflox/flox (inducible ERαKO) compared with ERαflox/flox (control) mice. 17ß-estradiol (E2) treatment increased trabecular bone volume fraction (BV/TV), cortical bone area, and uterine weight, while it reduced thymus weight and fat mass in ovariectomized control mice. The estrogenic responses were substantially reduced in inducible ERαKO mice compared with control mice on BV/TV (-67%), uterine weight (-94%), thymus weight (-70%), and gonadal fat mass (-94%). In contrast, the estrogenic response on cortical bone area was unaffected in inducible ERαKO compared with control mice. In conclusion, using an inducible ERαKO model, not confounded by lack of ERα during development, we demonstrate that ERα expression in sexually mature female mice is required for normal E2 responses in most, but not all, tissues. The finding that cortical, but not trabecular bone, responds normally to E2 treatment in inducible ERαKO mice strengthens the idea of cortical and trabecular bone being regulated by estrogen via different mechanisms.
Assuntos
Densidade Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Útero/efeitos dos fármacos , Animais , Osso e Ossos/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Camundongos , Camundongos Transgênicos , Tamanho do Órgão/efeitos dos fármacos , Ovariectomia , Timo/efeitos dos fármacos , Timo/metabolismo , Útero/metabolismoRESUMO
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.
Assuntos
Densidade Óssea/fisiologia , Osteoblastos/metabolismo , Coluna Vertebral/metabolismo , Proteínas Wnt/biossíntese , Animais , Estrogênios , Feminino , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Proteínas Wnt/genéticaRESUMO
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.
Assuntos
Estradiol/análogos & derivados , Receptor alfa de Estrogênio/química , Receptores de Estrogênio/antagonistas & inibidores , Tecido Adiposo/metabolismo , Animais , Células da Medula Óssea/citologia , Osso e Ossos/metabolismo , Estradiol/química , Antagonistas de Estrogênios/química , Feminino , Fulvestranto , Fator de Crescimento Insulin-Like I/metabolismo , Ligantes , Camundongos , Mutação , Tamanho do Órgão , Estrutura Terciária de Proteína , Pirrolidinas/química , Cloridrato de Raloxifeno/química , Tetra-Hidronaftalenos/química , Timo/efeitos dos fármacos , Distribuição Tecidual , Tomografia Computadorizada por Raios X , Útero/efeitos dos fármacos , Microtomografia por Raio-XRESUMO
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.
Assuntos
Densidade Óssea/fisiologia , Moduladores de Receptor Estrogênico/administração & dosagem , Receptor alfa de Estrogênio/metabolismo , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/fisiologia , Animais , Densidade Óssea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , Tamanho do Órgão/fisiologia , Ovariectomia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Desenvolvimento Ósseo/fisiologia , Receptor alfa de Estrogênio/fisiologia , Osteócitos/fisiologia , Animais , Desenvolvimento Ósseo/genética , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/genética , Remodelação Óssea/fisiologia , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Contagem de Células , Estradiol/farmacologia , Receptor alfa de Estrogênio/deficiência , Receptor alfa de Estrogênio/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Osteoclastos/citologia , Osteoclastos/fisiologia , Osteócitos/citologia , Osteogênese/genética , Osteogênese/fisiologia , Ovariectomia , Ovário/fisiologia , Caracteres Sexuais , Estresse MecânicoRESUMO
Interleukin-17 (IL-17) drives inflammation and destruction of joints in rheumatoid arthritis (RA). The female sex hormone 17ß-estradiol (E2) inhibits experimental arthritis. γδT cells are significant producers of IL-17, thus the aim of this study was to investigate if E2 influenced IL-17(+) γδT cells during arthritis development using a variety of experimental RA models: collagen-induced arthritis (CIA); antigen-induced arthritis (AIA); and collagen antibody-induced arthritis (CAIA). We demonstrate that E2 treatment decreases IL-17(+) γδT cell number in joints, but increases IL-17(+) γδT cells in draining lymph nodes, suggesting an E2-mediated prevention of IL-17(+) γδT cell migration from lymph nodes to joints, in concert with our recently reported effects of E2 on Th17 cells (Andersson et al., 2015). E2 did neither influence the general γδT cell population nor IFNγ(+) γδT cells, implying a selective regulation of IL-17-producing cells. In conclusion, this study contributes to the understanding of estrogen's role in autoimmune disease.
Assuntos
Artrite Experimental/imunologia , Estradiol/farmacologia , Interleucina-17/imunologia , Linfócitos T/efeitos dos fármacos , Animais , Artrite Experimental/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , ELISPOT , Estrogênios/farmacologia , Feminino , Interleucina-17/metabolismo , Articulações/efeitos dos fármacos , Articulações/imunologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Contagem de Linfócitos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores CCR6/imunologia , Receptores CCR6/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/metabolismoRESUMO
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.
Assuntos
Osso e Ossos/metabolismo , Osso e Ossos/patologia , Receptor alfa de Estrogênio/metabolismo , Neurônios/metabolismo , Animais , Densidade Óssea , Remodelação Óssea , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/cirurgia , Receptor alfa de Estrogênio/deficiência , Feminino , Hormônio Foliculoestimulante/metabolismo , Deleção de Genes , Proteínas de Filamentos Intermediários/metabolismo , Leptina/sangue , Hormônio Luteinizante/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Nestina , Tamanho do Órgão , Ovariectomia , Radiografia , Serotonina/metabolismo , Transdução de Sinais , Esteroides/metabolismo , Linfócitos T/metabolismoRESUMO
The effects of estrogen on bone are mediated mainly via estrogen receptor (ER)α. ERα in osteoclasts (hematopoietic origin) is involved in the trabecular bone-sparing effects of estrogen, but conflicting data are reported on the role of ERα in osteoblast lineage cells (nonhematopoietic origin) for bone metabolism. Because Cre-mediated cell-specific gene inactivation used in previous studies might be confounded by nonspecific and/or incomplete cell-specific ERα deletion, we herein used an alternative approach to determine the relative importance of ERα in hematopoietic (HC) and nonhematopoietic cells (NHC) for bone mass. Chimeric mice with selective inactivation of ERα in HC or NHC were created by bone marrow transplantations of wild-type (WT) and ERα-knockout (ERα(-/-)) mice. Estradiol treatment increased both trabecular and cortical bone mass in ovariectomized WT/WT (defined as recipient/donor) and WT/ERα(-/-) mice but not in ERα(-/-)/WT or ERα(-/-)/ERα(-/-) mice. However, estradiol effects on both bone compartments were reduced (â¼50%) in WT/ERα(-/-) mice compared with WT/WT mice. The effects of estradiol on fat mass and B lymphopoiesis required ERα specifically in NHC and HC, respectively. In conclusion, ERα in NHC is required for the effects of estrogen on both trabecular and cortical bone, but these effects are enhanced by ERα in HC.
Assuntos
Osso e Ossos/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/fisiologia , Estrogênios/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Animais , Densidade Óssea/efeitos dos fármacos , Transplante de Medula Óssea , Quimera , Feminino , Camundongos , Camundongos Knockout , OvariectomiaRESUMO
OBJECTIVE: Bone loss in arthritis is a complex process characterized by bone erosions and periarticular and generalized bone loss. The antigen-induced arthritis (AIA) model is mainly used to study synovitis and joint destruction, including bone erosions; however, periarticular bone loss has been less extensively investigated. The objectives of this study were to characterize and establish AIA as a model for periarticular bone loss, and to determine the importance of NADPH oxidase 2 (NOX-2)-derived reactive oxygen species (ROS) in periarticular bone loss. METHODS: Arthritis was induced in mice by local injection of antigen in one knee; the other knee was used as a nonarthritis control. At study termination, the knees were collected for histologic assessment. Periarticular bone mineral density (BMD) was investigated by peripheral quantitative computed tomography. Flow cytometric analyses were performed using synovial and bone marrow cells. RESULTS: AIA resulted in decreased periarticular trabecular BMD and increased frequencies of preosteoclasts, neutrophils, and monocytes in the arthritic synovial tissue. Arthritis induction resulted in an increased capability to produce ROS. However, induction of arthritis in Ncf1 / mice, which lack NOX-2-derived ROS, and control mice resulted in similar reductions in periarticular trabecular BMD. CONCLUSION: The initiation of AIA resulted in periarticular bone loss associated with local effects on inflammatory cells and osteoclasts. Furthermore, based on our observations using this model, we conclude that NOX-2-derived ROS production is not essential for inflammation-mediated periarticular bone loss. Thus, AIA can be used as a model to investigate the pathogenesis of local inflammation-mediated bone loss.
Assuntos
Antígenos/farmacologia , Artrite Experimental/patologia , Osteoartrite do Joelho/patologia , Osteoporose/patologia , Sinovite/patologia , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/metabolismo , Densidade Óssea/imunologia , Modelos Animais de Doenças , Feminino , Fêmur/metabolismo , Fêmur/patologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Monócitos/metabolismo , Monócitos/patologia , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Neutrófilos/metabolismo , Neutrófilos/patologia , Osteoartrite do Joelho/induzido quimicamente , Osteoartrite do Joelho/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteoporose/induzido quimicamente , Osteoporose/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Soroalbumina Bovina/farmacologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Sinovite/induzido quimicamente , Sinovite/metabolismoRESUMO
The link between antibodies and bone mass is debated. Activated IgG, which interacts directly with Fc gamma receptors, stimulates osteoclastogenesis in vitro, and local injection in immune-activated mice leads to bone loss. Multiple myeloma patients with high serum IgG levels have induced osteoclast activation and display bone loss. In addition, bone loss has been linked to serum autoantibodies in autoimmune diseases, including anti-citrullinated protein antibodies (ACPA) in individuals with rheumatoid arthritis (RA). Whether serum IgG or autoantibodies regulate bone mass under healthy conditions is poorly studied. In elderly men, neither serum levels of polyclonal IgG nor autoantibody were associated with areal bone mineral density in the MrOS Sweden study. Repetitive systemic injections of high-dose polyclonal IgG complexes in mice did not exert any discernible impact on bone mineral density. However, repetitive local intra-articular injection of the same IgG complexes led to a localized reduction of trabecular bone density. These results indicate antibodies may only impact bone density when close to the bone, such as within the synovial joint.
Assuntos
Artrite Reumatoide , Masculino , Humanos , Animais , Camundongos , Idoso , Artrite Reumatoide/metabolismo , Autoanticorpos , Anticorpos Antiproteína Citrulinada , Receptores de IgG/metabolismo , Imunoglobulina GRESUMO
Estradiol (E2) affects both reproductive and non-reproductive tissues, and the sensitivity to different doses of E2 varies between tissues. Membrane estrogen receptor α (mERα)-initiated signaling plays a tissue-specific role in mediating E2 effects, however, it is unclear if mERα signaling modulates E2 sensitivity. To determine this, we treated ovariectomized C451A females, lacking mERα signaling, and wildtype (WT) littermates with physiological (0.05 µg/mouse/day (low); 0.6 µg/mouse/day (medium)) or supraphysiological (6 µg/mouse/day (high)) doses of E2 (17ß-estradiol-3-benzoate) for three weeks. Low-dose treatment increased uterus weight in WT, but not C451A mice, while non-reproductive tissues (gonadal fat, thymus, trabecular and cortical bone) were unaffected in both genotypes. Medium-dose treatment increased uterus weight and bone mass and decreased thymus and gonadal fat weights in WT mice. Uterus weight was also increased in C451A mice, but the response was significantly attenuated (- 85%) compared to WT mice, and no effects were triggered in non-reproductive tissues. High-dose treatment effects in thymus and trabecular bone were significantly blunted (- 34% and - 64%, respectively) in C451A compared to WT mice, and responses in cortical bone and gonadal fat were similar between genotypes. Interestingly, the high dose effect in uterus was enhanced (+ 26%) in C451A compared to WT mice. In conclusion, loss of mERα signaling reduces the sensitivity to physiological E2 treatment in both non-reproductive tissues and uterus. Furthermore, the E2 effect after high-dose treatment in uterus is enhanced in the absence of mERα, suggesting a protective effect of mERα signaling in this tissue against supraphysiological E2 levels.
Assuntos
Estradiol , Receptor alfa de Estrogênio , Feminino , Camundongos , Animais , Humanos , Receptor alfa de Estrogênio/genética , Estradiol/farmacologia , Osso e Ossos , Transdução de Sinais , Densidade Óssea , Útero , OvariectomiaRESUMO
BACKGROUND: Global sclerostin inhibition reduces fracture risk efficiently but has been associated with cardiovascular side effects. The strongest genetic signal for circulating sclerostin is in the B4GALNT3 gene region, but the causal gene is unknown. B4GALNT3 expresses the enzyme beta-1,4-N-acetylgalactosaminyltransferase 3 that transfers N-acetylgalactosamine onto N-acetylglucosaminebeta-benzyl on protein epitopes (LDN-glycosylation). METHODS: To determine if B4GALNT3 is the causal gene, B4galnt3-/- mice were developed and serum levels of total sclerostin and LDN-glycosylated sclerostin were analysed and mechanistic studies were performed in osteoblast-like cells. Mendelian randomization was used to determine causal associations. FINDINGS: B4galnt3-/- mice had higher circulating sclerostin levels, establishing B4GALNT3 as a causal gene for circulating sclerostin levels, and lower bone mass. However, serum levels of LDN-glycosylated sclerostin were lower in B4galnt3-/- mice. B4galnt3 and Sost were co-expressed in osteoblast-lineage cells. Overexpression of B4GALNT3 increased while silencing of B4GALNT3 decreased the levels of LDN-glycosylated sclerostin in osteoblast-like cells. Mendelian randomization demonstrated that higher circulating sclerostin levels, genetically predicted by variants in the B4GALNT3 gene, were causally associated with lower BMD and higher risk of fractures but not with higher risk of myocardial infarction or stroke. Glucocorticoid treatment reduced B4galnt3 expression in bone and increased circulating sclerostin levels and this may contribute to the observed glucocorticoid-induced bone loss. INTERPRETATION: B4GALNT3 is a key factor for bone physiology via regulation of LDN-glycosylation of sclerostin. We propose that B4GALNT3-mediated LDN-glycosylation of sclerostin may be a bone-specific osteoporosis target, separating the anti-fracture effect of global sclerostin inhibition, from indicated cardiovascular side effects. FUNDING: Found in acknowledgements.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Densidade Óssea , N-Acetilgalactosaminiltransferases , Animais , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Osso e Ossos , Densidade Óssea/genética , Glucocorticoides/farmacologia , Glicosilação , HumanosRESUMO
Estrogen receptor alpha (ERα) signaling has beneficial skeletal effects in males. ERα signaling also affects other tissues, and to find bone-specific treatments, more knowledge regarding tissue-specific ERα signaling is needed. ERα is subjected to posttranslational modifications, including phosphorylation, which can influence ERα function in a tissue-specific manner. To determine the importance of phosphorylation site S122 (corresponding to human ERα site S118) for the skeleton and other tissues, male mice with a S122A mutation were used. Total areal bone mineral density was similar between gonadal intact S122A and WT littermates followed up to 12 months of age, and weights of estrogen-responsive organs normalized for body weight were unchanged between S122A and WT males at both 3 and 12 months of age. Interestingly, 12-month-old S122A males had decreased body weight compared to WT. To investigate if site S122 affects the estrogen response in bone and other tissues, 12-week-old S122A and WT males were orchidectomized (orx) and treated with estradiol (E2) or placebo pellets for four weeks. E2 increased cortical thickness in tibia in both orx WT (+ 60%, p < 0.001) and S122A (+ 45%, p < 0.001) males. However, the E2 effect on cortical thickness was significantly decreased in orx S122A compared to WT mice (- 24%, p < 0.05). In contrast, E2 affected trabecular bone and organ weights similarly in orx S122A and WT males. Thus, ERα phosphorylation site S122 is required for a normal E2 response specifically in cortical bone in male mice, a finding that may have implications for development of future treatments against male osteoporosis.
Assuntos
Receptor alfa de Estrogênio , Estrogênios , Humanos , Camundongos , Masculino , Animais , Criança , Lactente , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Fosforilação , Estrogênios/farmacologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/metabolismo , Estradiol , Peso CorporalRESUMO
Among patients with knee osteoarthritis (OA), postmenopausal women are over-represented. The purpose of this study was to determine whether deficiency of female sex steroids affects OA progression and to evaluate the protective effect of treatment with a physiological dose of 17ß-estradiol (E2) on OA progression using a murine model. Ovariectomy (OVX) of female mice was used to mimic a postmenopausal state. OVX or sham-operated mice underwent surgery for destabilization of the medial meniscus (DMM) to induce OA. E2 was administered in a pulsed manner for 2 and 8 weeks. OVX of OA mice did not influence the cartilage phenotype or synovial thickness, while both cortical and trabecular subchondral bone mineral density (BMD) decreased after OVX compared with sham-operated mice at 8 weeks post-DMM surgery. Additionally, OVX mice displayed decreased motor activity, reduced threshold of pain sensitivity, and increased number of T cells in the inguinal lymph nodes compared to sham-operated mice 2 weeks after OA induction. Eight weeks of treatment with E2 prevented cartilage damage and thickening of the synovium in OVX OA mice. The motor activity was improved after E2 replacement at the 2 weeks time point, which was also associated with lower pain sensitivity in the OA paw. E2 treatment protected against OVX-induced loss of subchondral trabecular bone. The number of T cells in the inguinal lymph nodes was reduced by E2 treatment after 8 weeks. This study demonstrates that treatment with a physiological dose of E2 exerts a protective role by reducing OA symptoms.