RESUMO
BACKGROUND AND AIMS: Guillain-Barré syndrome (GBS) is a rare, acute neuropathy characterized by ascending muscle weakness. Age, axonal GBS variants, and antecedent Campylobacter jejuni infection are associated with severe GBS, but the detailed mechanisms of nerve damage are only partly explored. Pro-inflammatory myeloid cells express NADPH oxidases (NOX) that generate tissue-toxic reactive oxygen species (ROS) that are implicated in neurodegenerative diseases. This study analyzed the impact of variants of the gene encoding the functional NOX subunit CYBA (p22phox ) on acute severity, axonal damage, and recovery in adult GBS patients. METHODS: Extracted DNA from 121 patients was genotyped for allelic variation at rs1049254 and rs4673 within CYBA using real-time quantitative polymerase chain reaction. Serum neurofilament light chain was quantified by single molecule array. Patients were followed for severity and motor function recovery for up to 13 years. RESULTS: CYBA genotypes linked to reduced formation of ROS, i.e. rs1049254/G and rs4673/A, were significantly associated with unassisted ventilation, shorter time to normalization of serum neurofilament light chain and shorter time to regained motor function. Residual disability at follow-up was confined to patients carrying CYBA alleles associated with high formation of ROS. INTERPRETATION: These findings implicate NOX-derived ROS in GBS pathophysiology and CYBA alleles as biomarkers of severity.
Assuntos
Síndrome de Guillain-Barré , Adulto , Humanos , Alelos , Biomarcadores , Síndrome de Guillain-Barré/genética , Síndrome de Guillain-Barré/fisiopatologia , NADPH Oxidases/genética , Espécies Reativas de Oxigênio , Gravidade do PacienteRESUMO
BACKGROUND: Accumulating evidence supports the efficacy of administering natalizumab (NZ) with extended-interval dosing (EID) in patients with relapsing-remitting multiple sclerosis (RRMS). OBJECTIVES: We switched NZ dosing from 4-week to 6-week intervals in patients with RRMS, and investigated the effect on serum neurofilament light chain (sNfL) concentrations. METHODS: We included two cohorts of patients with RRMS treated with NZ: one received the standard-interval dosing (4 weeks) at baseline, and were switched to 6-week intervals (EID4-6, N = 45). The other cohort received EID (5- or 6-week intervals) both at baseline and during follow-up (EID5/6, N = 25). Serum samples were collected in the EID4-6 cohort at every NZ infusion, for 12 months. The primary outcome was the change in sNfL concentrations after switching to EID. RESULTS: The baseline mean sNfL concentration in the EID4-6 cohort was 10.5 ng/L (standard deviation (SD) = 6.1), and it remained unchanged at 12 months. Moreover, individual sNfL concentrations did not change significantly after extending the NZ dosing intervals. In addition, the EID4-6 and EID5/6 cohorts had similar baseline sNfL concentrations. CONCLUSION: We concluded that extending the NZ dosing interval did not increase axonal damage, as determined with sNfL, in patients with RRMS.
Assuntos
Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Estudos de Coortes , Humanos , Filamentos Intermediários , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Natalizumab , Proteínas de NeurofilamentosRESUMO
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
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
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
Females are, in general, more insulin sensitive than males. To investigate whether this is a direct effect of sex-steroids (SS) in white adipose tissue (WAT), we developed a male mouse model overexpressing the aromatase enzyme, converting testosterone (T) to estradiol (E2), specifically in WAT (Ap2-arom mice). Adipose tissue E2 levels were increased while circulating SS levels were unaffected in male Ap2-arom mice. Importantly, male Ap2-arom mice were more insulin sensitive compared with WT mice and exhibited increased serum adiponectin levels and upregulated expression of Glut4 and Irs1 in WAT. The expression of markers of macrophages and immune cell infiltration was markedly decreased in WAT of male Ap2-arom mice. The adipogenesis was enhanced in male Ap2-arom mice, supported by elevated Pparg expression in WAT and enhanced differentiation of preadipocyte into mature adipocytes. In summary, increased adipose tissue aromatase activity reduces adipose tissue inflammation and improves insulin sensitivity in male mice. We propose that estrogen increases insulin sensitivity via a local effect in WAT on adiponectin expression, adipose tissue inflammation, and adipogenesis.
Assuntos
Tecido Adiposo Branco/metabolismo , Aromatase/genética , Estradiol/metabolismo , Resistência à Insulina/genética , Testosterona/metabolismo , Adipócitos , Adipogenia/genética , Adiponectina/metabolismo , Tecido Adiposo Branco/imunologia , Animais , Técnicas de Introdução de Genes , Transportador de Glucose Tipo 4/metabolismo , Inflamação , Proteínas Substratos do Receptor de Insulina/metabolismo , Macrófagos/imunologia , Masculino , Camundongos , PPAR gama/metabolismo , Regulação para CimaRESUMO
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
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
BACKGROUND: Teriflunomide is a disease modifying treatment (DMT) approved for relapsing-remitting multiple sclerosis (RRMS) in adults and children. It reduces lymphocyte proliferation by inhibiting the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) and thereby the pyrimidine synthesis. Although most DMTs in multiple sclerosis (MS) modulate or inhibit the immune system in the periphery, the efficacy may improve if the agent also targets immune activity within the central nervous system (CNS), acts as a neuro-protective and enhances neuro-regeneration. The objective of this study was to determine the passage of teriflunomide over the blood-cerebrospinal fluid barrier (BCSFB). METHODS: Plasma and cerebrospinal fluid (CSF) teriflunomide concentrations were determined at steady state in 12 patients with RRMS, treated with oral teriflunomide 14 mg once daily. Included patients were all clinically stable without relapse or disability worsening within 6 months prior from baseline and were on no other immune modulating or immunosuppressive drugs. RESULTS: The mean teriflunomide concentrations in plasma and CSF were 38775 (SEM ± 7256) ng/mL and 68 (SEM ± 15) ng/mL, respectively. The passage over the BCSFB was 0.17 % (SEM ± 0.01). While no correlation was found between the function of the BCSFB assessed with the albumin ratio and the CSF teriflunomide concentration, the CSF and plasma teriflunomide concentrations were highly correlated (rs = 0.90, < 0.0001). CONCLUSIONS: Further studies are warranted to determine if the obtained CSF teriflunomide concentration reflects that in the CNS and is able to influence inflammatory and degenerative processes within the CNS.
Assuntos
Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Adulto , Criança , Humanos , Crotonatos/uso terapêutico , Imunossupressores/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/induzido quimicamente , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Toluidinas/uso terapêuticoRESUMO
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
Selective estrogen receptor modulators (SERMs) act as estrogen receptor (ER) agonists or antagonists in a tissue-specific manner. ERs exert effects via nuclear actions but can also utilize membrane-initiated signaling pathways. To determine if membrane-initiated ERα (mERα) signaling affects SERM action in a tissue-specific manner, C451A mice, lacking mERα signaling due to a mutation at palmitoylation site C451, were treated with Lasofoxifene (Las), Bazedoxifene (Bza), or estradiol (E2), and various tissues were evaluated. Las and Bza treatment increased uterine weight to a similar extent in C451A and control mice, demonstrating mERα-independent uterine SERM effects, while the E2 effect on the uterus was predominantly mERα-dependent. Las and Bza treatment increased both trabecular and cortical bone mass in controls to a similar degree as E2, while both SERM and E2 treatment effects were absent in C451A mice. This demonstrates that SERM effects, similar to E2 effects, in the skeleton are mERα-dependent. Both Las and E2 treatment decreased thymus weight in controls, while neither treatment affected the thymus in C451A mice, demonstrating mERα-dependent SERM and E2 effects in this tissue. Interestingly, both SERM and E2 treatments decreased the total body fat percent in C451A mice, demonstrating the ability of these treatments to affect fat tissue in the absence of functional mERα signaling. In conclusion, mERα signaling can modulate SERM responses in a tissue-specific manner. This novel knowledge increases the understanding of the mechanisms behind SERM effects and may thereby facilitate the development of new improved SERMs.
Assuntos
Receptor alfa de Estrogênio , Moduladores Seletivos de Receptor Estrogênico , Animais , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Feminino , Camundongos , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Transdução de SinaisRESUMO
Obesity has previously been thought to protect bone since high body weight and body mass index are associated with high bone mass. However, some more recent studies suggest that increased adiposity negatively impacts bone mass. Here, we aimed to test whether acute loss of adipose tissue, via adipocyte apoptosis, alters bone mass in age-related obese mice. Adipocyte apoptosis was induced in obese male FAT-ATTAC mice through AP20187 dimerizer-mediated activation of caspase 8 selectively in adipocytes. In a short-term experiment, dimerizer was administered to 5.5 month-old mice that were terminated 2 weeks later. At termination, the total fat mass weighed 58% less in dimerizer-treated mice compared with vehicle-treated controls, but bone mass did not differ. To allow for the detection of long-term effects, we used 9-month-old mice that were terminated six weeks after dimerizer administration. In this experiment, the total fat mass weighed less (- 68%) in the dimerizer-treated mice than in the controls, yet neither bone mass nor biomechanical properties differed between groups. Our findings show that adipose tissue loss, despite the reduced mechanical loading, does not affect bone in age-related obese mice. Future studies are needed to test whether adipose tissue loss is beneficial during more severe obesity.
Assuntos
Adiposidade , Osso e Ossos/patologia , Adipócitos/patologia , Animais , Apoptose , Biomarcadores/sangue , Fenômenos Biomecânicos , Células da Medula Óssea/patologia , Remodelação Óssea , Contagem de Linfócitos , Camundongos Transgênicos , Tamanho do Órgão , Baço/patologiaRESUMO
Apart from the role of sex steroids in reproduction, sex steroids are also important regulators of the immune system. 17ß-estradiol (E2) represses T and B cell development, but augments B cell function, possibly explaining the different nature of immune responses in men and women. Both E2 and selective estrogen receptors modulators (SERM) act via estrogen receptors (ER). Activating functions (AF)-1 and 2 of the ER bind to coregulators and thus influence target gene transcription and subsequent cellular response to ER activation. The importance of ERαAF-1 and AF-2 in the immunomodulatory effects of E2/SERM has previously not been reported. Thus, detailed studies of T and B lymphopoiesis were performed in ovariectomized E2-, lasofoxifene- or raloxifene-treated mice lacking either AF-1 or AF-2 domains of ERα, and their wild-type littermate controls. Immune cell phenotypes were analyzed with flow cytometry. All E2 and SERM-mediated inhibitory effects on thymus cellularity and thymic T cell development were clearly dependent on both ERαAFs. Interestingly, divergent roles of ERαAF-1 and ERαAF-2 in E2 and SERM-mediated modulation of bone marrow B lymphopoiesis were found. In contrast to E2, effects of lasofoxifene on early B cells did not require functional ERαAF-2, while ERαAF-1 was indispensable. Raloxifene reduced early B cells partly independent of both ERαAF-1 and ERαAF-2. Results from this study increase the understanding of the impact of ER modulation on the immune system, which can be useful in the clarification of the molecular actions of SERMs and in the development of new SERM.
Assuntos
Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Linfopoese/genética , Ativação Transcricional/genética , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Linfopoese/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Domínios Proteicos/genética , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
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.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Hormônios Esteroides Gonadais/sangue , Espectrometria de Massas em Tandem/métodos , Androstenodiona/análise , Androstenodiona/sangue , Animais , Desidroepiandrosterona/análise , Desidroepiandrosterona/sangue , Di-Hidrotestosterona/análise , Di-Hidrotestosterona/sangue , Estradiol/análise , Estradiol/sangue , Estrona/análise , Estrona/sangue , Feminino , Hormônios Esteroides Gonadais/análise , Camundongos , Progesterona/análise , Progesterona/sangue , Ratos , Testosterona/análise , Testosterona/sangueRESUMO
The gut microbiota (GM) modulates the hosts metabolism and immune system. Probiotic bacteria are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host and can alter the composition of the GM. Germ-free mice have increased bone mass associated with reduced bone resorption indicating that the GM also regulates bone mass. Ovariectomy (ovx) results in bone loss associated with altered immune status. The purpose of this study was to determine if probiotic treatment protects mice from ovx-induced bone loss. Mice were treated with either a single Lactobacillus (L) strain, L. paracasei DSM13434 (L. para) or a mixture of three strains, L. paracasei DSM13434, L. plantarum DSM 15312 and DSM 15313 (L. mix) given in the drinking water during 6 weeks, starting two weeks before ovx. Both the L. para and the L. mix treatment protected mice from ovx-induced cortical bone loss and bone resorption. Cortical bone mineral content was higher in both L. para and L. mix treated ovx mice compared to vehicle (veh) treated ovx mice. Serum levels of the resorption marker C-terminal telopeptides and the urinary fractional excretion of calcium were increased by ovx in the veh treated but not in the L. para or the L. mix treated mice. Probiotic treatment reduced the expression of the two inflammatory cytokines, TNFα and IL-1ß, and increased the expression of OPG, a potent inhibitor of osteoclastogenesis, in cortical bone of ovx mice. In addition, ovx decreased the frequency of regulatory T cells in bone marrow of veh treated but not probiotic treated mice. In conclusion, treatment with L. para or the L. mix prevents ovx-induced cortical bone loss. Our findings indicate that these probiotic treatments alter the immune status in bone resulting in attenuated bone resorption in ovx mice.
Assuntos
Reabsorção Óssea/etiologia , Reabsorção Óssea/prevenção & controle , Ovariectomia/efeitos adversos , Probióticos/uso terapêutico , Animais , Biomarcadores/sangue , Peso Corporal/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Reabsorção Óssea/sangue , Reabsorção Óssea/tratamento farmacológico , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Citocinas/metabolismo , Feminino , Mediadores da Inflamação/metabolismo , Lactobacillus/efeitos dos fármacos , Lactobacillus/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Minerais/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Osteoprotegerina/metabolismo , Probióticos/farmacologia , Ligante RANK/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
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.
Assuntos
Fraturas Ósseas/metabolismo , Fraturas Ósseas/prevenção & controle , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Envelhecimento/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Células Cultivadas , Suscetibilidade a Doenças , Fraturas Ósseas/genética , Fraturas Ósseas/patologia , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , Tamanho do Órgão/genética , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Osteócitos/patologia , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteoprotegerina/metabolismo , Ligante RANK/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Crânio/patologia , Proteínas Wnt/deficiênciaRESUMO
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.