RESUMO
ApoE-null (ApoE-KO) mice fed a high-fat diet (HFD) develop atherosclerosis, due in part to activation of vascular inflammation by oxidized low-density lipoprotein. Since bone loss also occurs in these mice, we used them to investigate the impact of oxidized lipids on bone homeostasis and to search for underlying pathogenic pathways. Four-month-old female ApoE-KO mice fed a HFD for three months exhibited increased levels of oxidized lipids in bone, as well as decreased femoral and vertebral trabecular and cortical bone mass, compared with ApoE-KO mice on normal diet. Despite HFD-induced increase in expression of Alox15, a lipoxygenase that oxidizes LDL and promotes atherogenesis, global deletion of this gene failed to ameliorate the skeletal impact of HFD. Osteoblast number and function were dramatically reduced in trabecular and cortical bone of HFD-fed mice, whereas osteoclast number was modestly reduced only in trabecular bone, indicating that an imbalance in favor of osteoclasts was responsible for HFD-induced bone loss. These changes were associated with decreased osteoblast progenitors and increased monocyte/macrophages in the bone marrow as well as increased expression of IL-1ß, IL-6, and TNF. HFD also attenuated Wnt signaling as evidenced by reduced expression of Wnt target genes, and it decreased expression of pro-osteoblastogenic Wnt ligands. These results suggest that oxidized lipids decrease bone mass by increasing anti-osteoblastogenic inflammatory cytokines and decreasing pro-osteoblastogenic Wnt ligands.
Assuntos
Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/genética , Doenças Ósseas Metabólicas/genética , Osso e Ossos/imunologia , Osteogênese , Proteínas Wnt/genética , Absorciometria de Fóton , Animais , Araquidonato 12-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Western Blotting , Densidade Óssea , Doenças Ósseas Metabólicas/diagnóstico por imagem , Doenças Ósseas Metabólicas/imunologia , Doenças Ósseas Metabólicas/patologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Osso Esponjoso/diagnóstico por imagem , Osso Esponjoso/imunologia , Osso Esponjoso/metabolismo , Osso Esponjoso/patologia , Contagem de Células , Osso Cortical/efeitos dos fármacos , Osso Cortical/imunologia , Osso Cortical/metabolismo , Osso Cortical/patologia , Dieta Hiperlipídica , Feminino , Fêmur/diagnóstico por imagem , Fêmur/imunologia , Fêmur/metabolismo , Fêmur/patologia , Citometria de Fluxo , Separação Imunomagnética , Inflamação , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Lipoproteínas LDL/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Monócitos/imunologia , Osteoblastos/citologia , Osteoclastos/citologia , Porosidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/imunologia , Coluna Vertebral/metabolismo , Coluna Vertebral/patologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Bone mass declines with age but the mechanisms responsible remain unclear. Here we demonstrate that deletion of a conditional allele for Atg7, a gene essential for autophagy, from osteocytes caused low bone mass in 6-month-old male and female mice. Cancellous bone volume and cortical thickness were decreased, and cortical porosity increased, in conditional knock-out mice compared with control littermates. These changes were associated with low osteoclast number, osteoblast number, bone formation rate, and wall width in the cancellous bone of conditional knock-out mice. In addition, oxidative stress was higher in the bones of conditional knock-out mice as measured by reactive oxygen species levels in the bone marrow and by p66(shc) phosphorylation in L6 vertebra. Each of these changes has been previously demonstrated in the bones of old versus young adult mice. Thus, these results demonstrate that suppression of autophagy in osteocytes mimics, in many aspects, the impact of aging on the skeleton and suggest that a decline in autophagy with age may contribute to the low bone mass associated with aging.
Assuntos
Fêmur/metabolismo , Vértebras Lombares/metabolismo , Osteócitos/fisiologia , Envelhecimento , Animais , Autofagia , Proteína 7 Relacionada à Autofagia , Densidade Óssea , Diferenciação Celular , Células Cultivadas , Feminino , Fêmur/diagnóstico por imagem , Fêmur/patologia , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Estresse Oxidativo , Radiografia , Espécies Reativas de Oxigênio/metabolismoRESUMO
PURPOSE: Mitofusin 2 (Mfn2) is one of two mitofusins involved in regulating mitochondrial size, shape and function, including mitophagy, an important cellular mechanism to limit oxidative stress. Reduced expression of Mfn2 has been associated with impaired osteoblast differentiation and function and a reduction in the number of viable osteocytes in bone. We hypothesized that the genetic absence of Mfn2 in these cells would increase their susceptibility to aging-associated metabolic stress, leading to a progressive impairment in skeletal homeostasis over time. METHODS: Mfn2 was selectively deleted in vivo at three different stages of osteoblast lineage commitment by crossing mice in which the Mfn2 gene was floxed with transgenic mice expressing Cre under the control of the promoter for Osterix (OSX), collagen1a1, or DMP1 (Dentin Matrix Acidic Phosphoprotein 1). RESULTS: Mice in which Mfn2 was deleted using DMP1-cre demonstrated a progressive and dramatic decline in bone mineral density (BMD) beginning at 10 weeks of age (n = 5 for each sex and each genotype from age 10 to 20 weeks). By 15 weeks, there was evidence for a functional decline in muscle performance as assessed using a rotarod apparatus (n = 3; 2 males/ 1 female for each genotype), accompanied by a decline in lean body mass. A marked reduction in trabecular bone mass was evident on bone histomorphometry, and biomechanical testing at 25 weeks (k/o: 2 male/1 female, control 2 male/2 female) revealed severely impaired femur strength. Extensive regional myofiber atrophy and degeneration was observed on skeletal muscle histology. Electron microscopy showed progressive disruption of cellular architecture, with disorganized sarcomeres and a bloated mitochondrial reticulum. There was also evidence of neurodegeneration within the ventral horn and roots of the lumbar spinal cord, which was accompanied by myelin loss and myofiber atrophy. Deletion of Mfn2 using OSX-cre or Col1a1-cre did not result in a musculoskeletal phenotype. Where possible, male and female animals were analyzed separately, but small numbers of animals in each group limited statistical power. For other outcomes, where sex was not considered, small sample sizes might still limit the strength of the observation. CONCLUSION: Despite known functional overlap of Mfn1 and Mfn2 in some tissues, and their co-expression in bone, muscle and spinal cord, deletion of Mfn2 using the 8 kB DMP1 promoter uncovered an important non-redundant role for Mfn2 in maintaining the neuromuscular/bone axis.
Assuntos
Densidade Óssea , GTP Fosfo-Hidrolases , Animais , Feminino , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Masculino , Camundongos , Densidade Óssea/genética , Densidade Óssea/fisiologia , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Osso e Ossos/patologia , Osso e Ossos/metabolismo , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Osteoblastos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genéticaRESUMO
Craniotubular dysplasia, Ikegawa type (OMIM #619727) denotes the autosomal recessive skeletal disease identified in 2021 featuring blindness acquired in childhood. Five young members of four Indian families harbored a homozygous indel within TMEM53 (OMIM *619722), the gene that encodes transmembrane protein 53 (TMEM53). When intact, TMEM53 spans the nuclear envelope of osteoprogenitor cells, dampens BMP-SMAD signaling, and thereby slows bone formation. Consequently, defective TMEM53 accelerates osteogenesis. Herein, an American boy is compound heterozygous for a novel deletion and a novel missense mutation within TMEM53. His vision and sensorineural hearing became impaired. Radiographic survey revealed diploic thickening of his skull, broad calvarial and facial bones, skeletal modeling errors, vertebral body flattening, wide ribs, and osteopenia of expanded bones. DXA areal bone density (gm/cm2) Z-scores were low. His optic, auditory, and spinal canals were narrow. Mineral metabolism was intact. Serum alkaline phosphatase and osteocalcin levels were normal yet CTX was high. Iliac crest histomorphometry documented accelerated bone formation. His acute vision loss briefly improved following prednisone administration, optic canal decompression, and optic nerve sheath fenestration, but then progressed despite further surgeries and zoledronate treatment aimed to suppress bone turnover. Next generation sequencing of genes associated with elevated skeletal mass, including from high bone turnover, did not suggest an etiology. Whole genome sequencing then revealed within TMEM53: i) a paternally transmitted 54-base deletion, which included the mRNA splice acceptor site for exon 2 as well as 31 bases of exonic sequence (c. 62-23_92del), and ii) a maternally transmitted missense variant (c.650C > T, p.Ser217Leu: NM_024587.4/NP_078863.2) which is extremely rare in gnomAD (frequency = 0.000036), replaces Ser217 highly conserved across species, and is scored as damaging by SIFT and Mutation Taster. We call this new osteopathy TMEM53 craniotubular dysplasia.
RESUMO
Endogenous glucocorticoids (GCs) and inflammatory cytokines contribute to the age-associated loss of bone mass and strength, but the molecular mechanisms responsible for their deleterious effects on the aging skeleton are unclear. Based on evidence that oxidative stress is a causal mechanism of the insulin resistance produced by either one of these two agents, we tested the hypothesis that their adverse skeletal effects also result from increased oxidative stress. We report that administration of prednisolone to mice increased reactive oxygen species (ROS) and the phosphorylation of p66(shc) (an amplifier of H(2)O(2) generation in mitochondria) in bone. Dexamethasone (Dex) and TNFα had a similar effect on osteoblastic cells in vitro. The generation of ROS by Dex and TNFα required PKCß/p66(shc) signaling and was responsible for the activation of JNK and induction of apoptosis by both agents. The activity of Forkhead box O (FoxO) transcription factors was also increased in response to ROS; however, FoxO activation opposed apoptosis induced by Dex and TNFα. In addition, both agents suppressed Akt phosphorylation as well as Wnt-induced proliferation and osteoblast differentiation. However, the inhibitory actions on Wnt signaling were independent of PKCß/p66(shc). Instead, they were mediated by inhibition of Akt and stimulation of FoxOs. These results demonstrate that ROS-induced activation of a PKCß/p66(shc)/JNK signaling cascade is responsible for the pro-apoptotic effects of Dex and TNFα on osteoblastic cells. Moreover, modulation of Akt and FoxOs by GCs and TNFα are cell-autonomous mechanisms of Wnt/ß-catenin antagonism contributing to the adverse effects of GC excess and inflammatory cytokines on bone alike.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Osteoblastos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Prednisolona/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Adaptadoras da Sinalização Shc/genética , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Fator de Necrose Tumoral alfa/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: Bisphosphonates decrease bone resorption and are commonly used to treat or prevent osteoporosis. However, the effect of bisphosphonates on their target cells remains enigmatic, since in patients benefiting from therapy, little change, if any, has been observed in the number of osteoclasts, which are the cells responsible for bone resorption. METHODS: We examined 51 bone-biopsy specimens obtained after a 3-year, double-blind, randomized, placebo-controlled, dose-ranging trial of oral alendronate to prevent bone resorption among healthy postmenopausal women 40 through 59 years of age. The patients were assigned to one of five groups: those receiving placebo for 3 years; alendronate at a dose of 1, 5, or 10 mg per day for 3 years; or alendronate at a dose of 20 mg per day for 2 years, followed by placebo for 1 year. Formalin-fixed, undecalcified planar sections were assessed by bone histomorphometric methods. RESULTS: The number of osteoclasts was increased by a factor of 2.6 in patients receiving 10 mg of alendronate per day for 3 years as compared with the placebo group (P<0.01). Moreover, the number of osteoclasts increased as the cumulative dose of the drug increased (r=0.50, P<0.001). Twenty-seven percent of these osteoclasts were giant cells with pyknotic nuclei that were adjacent to superficial resorption cavities. Furthermore, giant, hypernucleated, detached osteoclasts with 20 to 40 nuclei were found after alendronate treatment had been discontinued for 1 year. Of these large cells, 20 to 37% were apoptotic, according to both their morphologic features and positive findings from in situ end labeling. CONCLUSIONS: Long-term alendronate treatment is associated with an increase in the number of osteoclasts, which include distinctive giant, hypernucleated, detached osteoclasts that are undergoing protracted apoptosis.
Assuntos
Alendronato/farmacologia , Apoptose/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacologia , Remodelação Óssea/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Fosfatase Ácida/análise , Adulto , Alendronato/uso terapêutico , Biópsia , Conservadores da Densidade Óssea/uso terapêutico , Osso e Ossos/citologia , Osso e Ossos/patologia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Humanos , Isoenzimas/análise , Modelos Lineares , Pessoa de Meia-Idade , Osteoclastos/química , Osteoclastos/citologia , Osteoclastos/patologia , Pós-Menopausa , Fosfatase Ácida Resistente a TartaratoRESUMO
In 2003, we briefly reported the remarkable osteopathy of a 12-year-old boy who at age two months began fracturing his limbs with subsequent hyperplastic callus formation and expansion and fusion of appendicular bones. By age ten years he had coalesced his lumbosacral spine, pelvis, femurs, and leg and foot bones as a single structure. Computed tomography of expanded bone revealed a thin cortical shell, diminished irregular trabeculae, and cystic areas. Histopathology featured foci of woven bone, densely packed osteocytes, cartilage, fibrovascular tissue, and massive fat deposition in the marrow space lacking hematogenous precursor cells. Bone turnover markers indicated accelerated remodeling and the few radiographically assessable appendicular bones improved during brief adherence to alendronate therapy. Following puberty, serum multiplex biomarker profiling confirmed accelerated bone turnover. At age 23 years, macrospecimens from leg amputation revealed ossification along capsular tissue together with hyaline cartilage degeneration. Concurrently, the life-long course of this same disorder was delineated in an unrelated woman until her death at age 51 years. Both patients demonstrated the radiographic hallmarks and harbored the heterozygous point mutation (c.-14C>T) in the 5'-UTR of IFITM5 associated with osteogenesis imperfecta type V (OI-V). Herein, we detail the clinical, radiological, histopathological, biochemical, and molecular findings and discuss the etiology and pathogenesis of this extraordinary osteopathy that we call coalescing expansile skeletal disease.
Assuntos
Osteogênese Imperfeita , Regiões 5' não Traduzidas , Adulto , Osso e Ossos , Criança , Feminino , Humanos , Lactente , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mutação/genética , Osteogênese Imperfeita/diagnóstico por imagem , Osteogênese Imperfeita/genética , Adulto JovemRESUMO
Autosomal dominant hypophosphatemic rickets (ADHR) is remarkable among the hypophosphatemic rickets syndromes for its variable age of presentation and periods of quiescence during which serum phosphate and fibroblast growth factor 23 (FGF 23) levels are normal without therapy. In contrast, hypophosphatemia in X-linked hypophosphatemic rickets (XLH) manifests soon after birth and requires lifelong therapy. This suggests that there are environmental factors which can alter FGF 23 activity in ADHR but not in XLH. We present an adult with ADHR in whom resolution of hypophosphatemia was achieved by correcting iron deficiency without the need for phosphate supplementation. Serial iron and FGF 23 levels revealed an inverse relationship (r=-0.79, p<0.04). All patients with ADHR who present with hypophosphatemia and worsening symptoms should be screened for iron deficiency. If iron deficiency is detected, therapy with a combination of calcitriol and iron supplementation should be considered without phosphate supplementation.
Assuntos
Raquitismo Hipofosfatêmico Familiar , Hipofosfatemia , Adulto , Calcitriol , Raquitismo Hipofosfatêmico Familiar/complicações , Raquitismo Hipofosfatêmico Familiar/tratamento farmacológico , Raquitismo Hipofosfatêmico Familiar/genética , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos , Humanos , Ferro , FosfatosAssuntos
Difosfonatos/uso terapêutico , Glucocorticoides/efeitos adversos , Osteoporose/prevenção & controle , Asma/tratamento farmacológico , Densidade Óssea , Doenças Ósseas , Cálcio/uso terapêutico , Feminino , Fraturas Ósseas/prevenção & controle , Humanos , Pessoa de Meia-Idade , Osteoporose/induzido quimicamente , Guias de Prática Clínica como Assunto , Prednisona/efeitos adversos , Fatores de Risco , Vitamina D/uso terapêuticoRESUMO
Receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for osteoclast differentiation, and hormones and cytokines that stimulate bone resorption increase RANKL expression in stromal/osteoblastic cells. We have previously shown that PTH and 1,25-dihydroxyvitamin D(3) control murine RANKL gene expression in vitro, in part, via an evolutionarily conserved transcriptional enhancer, designated the distal control region (DCR), located 76 kb upstream from the transcription start site. Herein we describe the phenotype of mice lacking this enhancer. Deletion of the DCR reduced PTH and 1,25-dihydroxyvitamin D(3) stimulation of RANKL mRNA and osteoclast formation in primary bone marrow cultures as well as stimulation of RANKL mRNA in bone. DCR deletion also reduced basal RANKL mRNA levels in bone, thymus, and spleen. Moreover, mice lacking the DCR exhibited increased bone mass and strength. The increase in bone mass was due to reduced osteoclast and osteoblast formation leading to a low rate of bone remodeling similar to that observed in humans and mice with hypoparathyroidism. These findings demonstrate that hormonal control of RANKL expression via the DCR is a critical determinant of the rate of bone remodeling.
Assuntos
Densidade Óssea/genética , Remodelação Óssea/genética , Elementos Facilitadores Genéticos , Deleção de Genes , Mutagênese Sítio-Dirigida , Ligante RANK/genética , Animais , Osso e Ossos/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Tecido Linfoide/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hormônio Paratireóideo/farmacologia , Ligante RANK/metabolismoRESUMO
Atherosclerosis and osteoporosis are epidemiologically linked and oxidation specific epitopes (OSEs), such as phosphocholine (PC) of oxidized phospholipids (PC-OxPL) and malondialdehyde (MDA), are pathogenic in both. The proatherogenic effects of OSEs are opposed by innate immune antibodies. Here we show that high-fat diet (HFD)-induced bone loss is attenuated in mice expressing a single chain variable region fragment of the IgM E06 (E06-scFv) that neutralizes PC-OxPL, by increasing osteoblast number and stimulating bone formation. Similarly, HFD-induced bone loss is attenuated in mice expressing IK17-scFv, which neutralizes MDA. Notably, E06-scFv also increases bone mass in mice fed a normal diet. Moreover, the levels of anti-PC IgM decrease in aged mice. We conclude that OSEs, whether produced chronically or increased by HFD, restrain bone formation, and that diminished defense against OSEs may contribute to age-related bone loss. Anti-OSEs, therefore, may represent a novel therapeutic approach against osteoporosis and atherosclerosis simultaneously.
Assuntos
Epitopos/imunologia , Imunoglobulina M/imunologia , Osteogênese/imunologia , Osteoporose/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Epitopos/genética , Epitopos/metabolismo , Imunoglobulina M/genética , Lipoproteínas LDL/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/imunologia , Osteogênese/genética , Osteoporose/etiologia , Osteoporose/genética , Oxirredução , Anticorpos de Cadeia Única/genéticaRESUMO
Since the initial demonstration of the phenomenon in murine and human bone sections approximately 10 yr ago, appreciation of the biologic significance of osteoblast apoptosis has contributed greatly not only to understanding the regulation of osteoblast number during physiologic bone remodeling, but also the pathogenesis of metabolic bone diseases and the pharmacology of some of the drugs used for their treatment. It is now appreciated that all major regulators of bone metabolism including bone morphogenetic proteins (BMPs), Wnts, other growth factors and cytokines, integrins, estrogens, androgens, glucocorticoids, PTH and PTH-related protein (PTHrP), immobilization, and the oxidative stress associated with aging contribute to the regulation of osteoblast and osteocyte life span by modulating apoptosis. Moreover, osteocyte apoptosis has emerged as an important regulator of remodeling on the bone surface and a critical determinant of bone strength, independently of bone mass. The detection of apoptotic osteoblasts in bone sections remains challenging because apoptosis represents only a tiny fraction of the life span of osteoblasts, not unlike a 6-mo-long terminal illness in the life of a 75-yr-old human. Importantly, the phenomenon is 50 times less common in human bone biopsies because human osteoblasts live longer and are fewer in number. Be that as it may, well-controlled assays of apoptosis can yield accurate and reproducible estimates of the prevalence of the event, particularly in rodents where there is an abundance of osteoblasts for inspection. In this perspective, we focus on the biological significance of the phenomenon for understanding basic bone biology and the pathogenesis and treatment of metabolic bone diseases and discuss limitations of existing techniques for quantifying osteoblast apoptosis in human biopsies and their methodologic pitfalls.
Assuntos
Apoptose , Osteoblastos/citologia , Osteócitos/citologia , Envelhecimento/fisiologia , Animais , Hormônios/metabolismo , Humanos , Osteoblastos/metabolismo , Osteócitos/metabolismo , Estresse OxidativoRESUMO
Glucocorticoids depress bone formation by inhibiting osteoblastogenesis and increasing osteoblast apoptosis. However, the role of bone resorption in the initial rapid phase of bone loss characteristic of glucocorticoid-induced osteoporosis is unexplained, and the reason for the efficacy of bisphosphonates in this condition remains unknown. We report that in murine osteoclast cultures, glucocorticoids prolonged the baseline survival of osteoclasts and antagonized bisphosphonate-induced caspase activation and apoptosis by a glucocorticoid receptor-mediated action. Consistent with the in vitro evidence, in a murine model of glucocorticoid-induced osteoporosis, the number of cancellous osteoclasts increased, even though osteoclast progenitor number was reduced. Moreover, in mice receiving both glucocorticoids and bisphosphonates, the expected proapoptotic effect of bisphosphonates on osteoclasts was abrogated, as evidenced by maintenance of osteoclast numbers and, additionally, loss of bone density. In contrast, bisphosphonate administration prevented glucocorticoid-induced osteoblast apoptosis. These results indicate that the early loss of bone with glucocorticoid excess is caused by extension of the life span of pre-existing osteoclasts, an effect not preventable by bisphosphonates. Therefore, the early beneficial effects of these agents must be due, in part, to prolonging the life span of osteoblasts.
Assuntos
Alendronato/farmacologia , Glucocorticoides/farmacologia , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/patologia , Sobrevivência Celular/efeitos dos fármacos , Dexametasona/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoporose/induzido quimicamente , Osteoporose/patologia , Prednisolona/farmacologiaRESUMO
Two mouse strains, AKR/J and SAMP6, were assessed longitudinally for bone mineral density of the spine. They displayed very different time courses of bone accrual, with the SAMP6 strain reaching a plateau for vertebral BMD at 3 months, whereas AKR/J mice continued to increase spine BMD for at least 8 months. Among 253 F(2) progeny of an AKR/JxSAMP6 cross, at 4 months of age, the BMD variance was 5-6% of the mean, vs. 15% for weight. Variance increased with age for every parameter measured, and was generally higher among males. The ratio of 6-month/4-month spine BMDs, termed DeltasBMD, had a normal distribution with 5.7% variance, and was largely independent of spine BMD (R=-0.23) or body weight (R=-0.12) at maturity. Heritability of the DeltasBMD trait was calculated at 0.59. Genetic mapping identified two significant loci, both distinct from those observed for BMD at maturity--implying that different genes regulate skeletal growth vs. remodeling. A locus on the X chromosome, replicated in two mouse F(2) populations (P<10(-4) for combined discovery and confirmation), affects age-dependent BMD change for both spine and the full skeleton. Its position agrees with a very narrow region identified by association mapping for effects on lumbar bone density in postmenopausal women [Parsons CA, Mroczkowski HJ, McGuigan FE, Albagha OM, Manolagas S, Reid DM, et al. Interspecies synteny mapping identifies a quantitative trait locus for bone mineral density on human chromosome Xp22. Hum Mol Genet 2005;14:3141-8]. A second locus, on chromosome 7, was observed in only one cross. Single-nucleotide polymorphisms (SNPs) are highly clustered near these loci, distinguishing the parental strains over only limited spans.
Assuntos
Densidade Óssea/genética , Locos de Características Quantitativas , Coluna Vertebral/crescimento & desenvolvimento , Cromossomo X/genética , Absorciometria de Fóton , Fatores Etários , Animais , Predisposição Genética para Doença , Genótipo , Humanos , Camundongos , Polimorfismo de Nucleotídeo Único , Fatores Sexuais , Especificidade da EspécieRESUMO
In search of the sequence of pathogenic events leading to glucocorticoid-induced osteonecrosis, we determined the molecular, biomechanical, cellular, and vascular changes in the femur of C57BL/6 mice receiving prednisolone for 14, 28, or 42 days. The femoral head, but not the distal femur, of mice treated for 14 days showed a decrease in the expression of the hypoxia-inducible factor (Hif)-1α and vascular endothelial growth factor (VEGF), the number of osteoblasts, and bone formation rate and strength and showed an increase in osteoclasts. These changes were accompanied by conversion of the normal dendritic vasculature to pools of edema as detected by magnetic resonance imaging, providing robust diagnostic evidence of early osteonecrosis. At that time point, there were no detectable changes in bone density, cortical or cancellous bone architecture, midshaft or distal cancellous bone, or osteocyte apoptosis. In mice treated for 28 days, femoral head cancellous density, cortical width, and trabecular thickness decreased, and by 42 days the femoral heads had full-depth cortical penetrations and cancellous tissue osteonecrosis. These results indicate that the femoral head is a particularly sensitive anatomical site to the adverse effects of glucocorticoid excess on bone and that decreases of Hif-1α and VEGF expression, bone vascularity, and strength precede the loss of bone mass and microarchitectural deterioration, thus rendering the femoral head vulnerable to collapse.
Assuntos
Necrose da Cabeça do Fêmur/induzido quimicamente , Cabeça do Fêmur/efeitos dos fármacos , Glucocorticoides/efeitos adversos , Osteonecrose/induzido quimicamente , Animais , Apoptose/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Cabeça do Fêmur/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Osteogênese/efeitos dos fármacosRESUMO
Decreased cortical thickness and increased cortical porosity are the key anatomic changes responsible for osteoporotic fractures in elderly women and men. The cellular basis of these changes is unbalanced endosteal and intracortical osteonal remodeling by the osteoclasts and osteoblasts that comprise the basic multicellular units (BMUs). Like humans, mice lose cortical bone with age, but unlike humans, this loss occurs in the face of sex steroid sufficiency. Mice are therefore an ideal model to dissect age-specific osteoporotic mechanisms. Nevertheless, lack of evidence for endosteal or intracortical remodeling in mice has raised questions about their translational relevance. We show herein that administration of the antiosteoclastogenic cytokine osteoprotegerin to Swiss Webster mice ablated not only osteoclasts, but also endosteal bone formation, demonstrating the occurrence of BMU-based endosteal remodeling. Femoral cortical thickness decreased in aged male and female C57BL/6J mice, as well as F1 hybrids of C57BL/6J and BALB/cBy mice. This decrease was greater in C57BL/6J mice, indicating a genetic influence. Moreover, endosteal remodeling became unbalanced because of increased osteoclast and decreased osteoblast numbers. The porosity of the femoral cortex increased with age but was much higher in females of both strains. Notably, the increased cortical porosity resulted from de novo intracortical remodeling by osteon-like structures. Age-dependent cortical bone loss was associated with increased osteocyte DNA damage, cellular senescence, the senescence-associated secretory phenotype, and increased levels of RANKL. The demonstration of unbalanced endosteal and intracortical remodeling in old mice validates the relevance of this animal model to involutional osteoporosis in humans.
Assuntos
Envelhecimento/patologia , Remodelação Óssea , Porosidade , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Animais , Osteoblastos/citologia , Osteoclastos/citologiaRESUMO
UNLABELLED: Mechanical stimulation of cultured osteocytic cells attenuates their apoptosis. We report here that, conversely, reduced mechanical forces in the murine model of unloading by tail suspension increases the prevalence of osteocyte apoptosis, followed by bone resorption and loss of mineral and strength. INTRODUCTION: Mechanical loading is critical for the maintenance of bone mass; weightlessness, as with reduced physical activity in old age, bed rest, or space flight, invariably leads to bone loss. However, the cellular and molecular mechanisms responsible for these phenomena are poorly understood. Based on our earlier findings that physiologic levels of mechanical strain prevent apoptosis of osteocytic cells in vitro, we examined here whether, conversely, reduced mechanical forces increase the prevalence of osteocyte apoptosis in vivo and whether this event is linked to bone loss. MATERIALS AND METHODS: Swiss Webster mice or OG2-11beta-hydroxysteroid dehydrogenase type 2 (OG2-11beta-HSD2) transgenic mice and wildtype littermates were tail-suspended or kept under ambulatory conditions. Static and dynamic histomorphometry and osteocyte and osteoblast apoptosis by in situ end-labeling (ISEL) were assessed in lumbar vertebra; spinal BMD was measured by DXA; and bone strength was measured by vertebral compression. RESULTS: We show that within 3 days of tail suspension, mice exhibited an increased incidence of osteocyte apoptosis in both trabecular and cortical bone. This change was followed 2 weeks later by increased osteoclast number and cortical porosity, reduced trabecular and cortical width, and decreased spinal BMD and vertebral strength. Importantly, whereas in ambulatory animals, apoptotic osteocytes were randomly distributed, in unloaded mice, apoptotic osteocytes were preferentially sequestered in endosteal cortical bone--the site that was subsequently resorbed. The effect of unloading on osteocyte apoptosis and bone resorption was reproduced in transgenic mice in which osteocytes are refractory to glucocorticoid action, indicating that stress-induced hypercortisolemia cannot account for these effects. CONCLUSIONS: We conclude that diminished mechanical forces eliminate signals that maintain osteocyte viability, thereby leading to apoptosis. Dying osteocytes in turn become the beacons for osteoclast recruitment to the vicinity and the resulting increase in bone resorption and bone loss.
Assuntos
Apoptose , Reabsorção Óssea/patologia , Osteoclastos/citologia , Osteócitos/patologia , Ausência de Peso/efeitos adversos , Animais , Densidade Óssea , Feminino , Camundongos , Camundongos Transgênicos , Osteoclastos/patologiaRESUMO
Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1(flox/flox) mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2(-/-) mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4- to 6-week-old DKO mice at all sites when measured by dual-energy X-ray absorptiometry (DXA) and serum cross-linked C-telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4- to 6-week-old DKO animals. In 14- to 16-week-old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once-daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism.
Assuntos
Envelhecimento , Deleção de Genes , Neuropeptídeos , Osteoblastos , Osteoclastos , Osteopetrose , Proteínas rac de Ligação ao GTP , Proteínas rac1 de Ligação ao GTP , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Contagem de Células , Humanos , Camundongos , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteopetrose/genética , Osteopetrose/metabolismo , Osteopetrose/patologia , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína RAC2 de Ligação ao GTPRESUMO
Because osteoblasts and marrow adipocytes are derived from a common mesenchymal progenitor, increased adipogenesis may occur at the expense of osteoblasts, leading to bone loss. Our previous in vitro studies indicated that activation of the proadipogenic transcription factor peroxisome proliferator-activated receptor isoform gamma 2 with rosiglitazone suppressed osteoblast differentiation. Here, we show that 5-month-old Swiss-Webster mice receiving rosiglitazone for 28 d exhibited bone loss associated with an increase in marrow adipocytes, a decrease in the ratio of osteoblasts to osteoclasts, a reduction in bone formation rate, and a reduction in wall width--an index of the amount of bone formed by each team of osteoblasts. Rosiglitazone had no effect on the number of early osteoblast or osteoclast progenitors, or on osteoblast life span, but decreased the expression of the key osteoblastogenic transcription factors Runx2 and Osterix in cultures of marrow-derived mesenchymal progenitors. These effects were associated with diversion of bipotential progenitors from the osteoblast to the adipocyte lineage, and suppression of the differentiation of monopotential osteoblast progenitors. However, rosiglitazone had no effect on osteoblastic cells at later stages of differentiation. Hence, rosiglitazone attenuates osteoblast differentiation and thereby reduces bone formation rate in vivo, leading to bone loss. These findings provide a mechanistic explanation for the recent evidence that peroxisome proliferator-activated receptor isoform gamma activation is a negative regulator of bone mass and suggest that the increased production of oxidized fatty acids with age may indeed be an important mechanism for age-related osteoporosis in humans.
Assuntos
Osso e Ossos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Osteoblastos/citologia , Tiazolidinedionas/farmacologia , Adipócitos/metabolismo , Envelhecimento , Animais , Osso e Ossos/efeitos dos fármacos , Densitometria , Ácidos Graxos/metabolismo , Hipoglicemiantes/farmacologia , Ligantes , Mesoderma/metabolismo , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoporose , PPAR gama/metabolismo , Rosiglitazona , Fator de Transcrição Sp7 , Células-Tronco , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
Adiponectin or adipocyte complement-related protein of 30 kDa (Acrp30) is a circulating protein produced exclusively in adipocytes. Circulating Acrp30 levels have been associated with insulin sensitivity in adult mice and humans, yet the Acrp30 profile over the lifespan and its hormonal regulation in vivo have not been previously described. Hence, we set forth to determine whether hormonal and metabolic changes associated with sexual maturation, reproduction, aging, and calorie restriction affect Acrp30. In mice, Acrp30 levels increase during sexual maturation by 4-fold in males and 10-fold in females. Neonatal castration (CX) allows Acrp30 of adults to reach female levels. CX in adults does not lead to female Acrp30 levels unless glucocorticoid exposure is elevated simultaneously by implant. Ovariectomy of infant mice does not interfere with the pubertal rise of Acrp30. However, ovariectomy in adults increases Acrp30. Estrogen suppressed Acrp30 in mice and 3T3-L1 adipocytes. In parallel to changes in estrogen action, Acrp30 decreased in late gestation but increased in both calorie-restricted and old (anovulatory) mice. The reduction of Acrp30 in lactating dams is consistent with a suppressive effect of prolactin and a stimulating effect of bromocriptine. In summary, Acrp30 levels in serum are under complex hormonal control and may play a key role in determining systemic insulin sensitivity under the respective conditions.