RESUMO
This study was designed to determine whether the use of acetylcholinesterase inhibitors (AChEIs), a group of drugs that stimulate acetylcholine receptors and are used to treat Alzheimer's disease (AD), is associated with osteoporosis protection and inhibition of osteoclast differentiation and function. Firstly, we examined the effects of AChEIs on RANKL-induced osteoclast differentiation and function with osteoclastogenesis and bone resorption assays. Next, we investigated the impacts of AChEIs on RANKL-induced nuclear factor κB and NFATc1 activation and expression of osteoclast marker proteins CA-2, CTSK and NFATc1, and dissected the MAPK signaling in osteoclasts in vitro by using luciferase assay and Western blot. Finally, we assessed the in vivo efficacy of AChEIs using an ovariectomy-induced osteoporosis mouse model, which was analyzed using microcomputed tomography, in vivo osteoclast and osteoblast parameters were assessed using histomorphometry. We found that Donepezil and Rivastigmine inhibited RANKL-induced osteoclastogenesis and impaired osteoclastic bone resorption. Moreover, AChEIs reduced the RANKL-induced transcription of Nfatc1, and expression of osteoclast marker genes to varying degrees (mainly Donepezil and Rivastigmine but not Galantamine). Furthermore, AChEIs variably inhibited RANKL-induced MAPK signaling accompanied by downregulation of AChE transcription. Finally, AChEIs protected against OVX-induced bone loss mainly by inhibiting osteoclast activity. Taken together, AChEIs (mainly Donepezil and Rivastigmine) exerted a positive effect on bone protection by inhibiting osteoclast function through MAPK and NFATc1 signaling pathways through downregulating AChE. Our findings have important clinical implications that elderly patients with dementia who are at risk of developing osteoporosis may potentially benefit from therapy with the AChEI drugs. Our study may influence drug choice in those patients with both AD and osteoporosis.
Assuntos
Reabsorção Óssea , Osteoporose , Camundongos , Animais , Feminino , Humanos , Osteogênese , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/uso terapêutico , Acetilcolinesterase , Rivastigmina/farmacologia , Rivastigmina/uso terapêutico , Donepezila/farmacologia , Donepezila/uso terapêutico , Microtomografia por Raio-X , Reabsorção Óssea/genética , Osteoclastos/metabolismo , Fatores de Transcrição , NF-kappa B/metabolismo , Osteoporose/etiologia , Ligante RANK/metabolismo , Fatores de Transcrição NFATC/metabolismo , Diferenciação Celular , Ovariectomia/efeitos adversosRESUMO
Longitudinal bone growth, achieved through endochondral ossification, is accomplished by a cartilaginous structure, the physis or growth plate, comprised of morphologically distinct zones related to chondrocyte function: resting, proliferating and hypertrophic zones. The resting zone is a stem cell-rich region that gives rise to the growth plate, and exhibits regenerative capabilities in response to injury. We discovered a FoxA2+group of long-term skeletal stem cells, situated at the top of resting zone, adjacent the secondary ossification center, distinct from the previously characterized PTHrP+ stem cells. Compared to PTHrP+ cells, FoxA2+ cells exhibit higher clonogenicity and longevity. FoxA2+ cells exhibit dual osteo-chondro-progenitor activity during early postnatal development (P0-P28) and chondrogenic potential beyond P28. When the growth plate is injured, FoxA2+ cells expand in response to trauma, and produce physeal cartilage for growth plate tissue regeneration.
Assuntos
Lâmina de Crescimento , Proteína Relacionada ao Hormônio Paratireóideo , Cartilagem , Condrócitos , Fator 3-beta Nuclear de Hepatócito/metabolismo , Células-TroncoRESUMO
The tumor necrosis factor (TNF)-like core domain of receptor activator of nuclear factor-κB ligand (RANKL) is a functional domain critical for osteoclast differentiation. One of the missense mutations identified in patients with osteoclast-poor autosomal recessive osteopetrosis (ARO) is located in residue methionine 199 that is replaced with lysine (M199K) amid the TNF-like core domain. However, the structure-function relationship of this mutation is not clear. Sequence-based alignment revealed that the fragment containing human M199 is highly conserved and equivalent to M200 in rat. Using site-directed mutagenesis, we generated three recombinant RANKL mutants M200K/A/E (M200s) by replacing the methionine 200 with lysine (M200K), alanine (M200A), and glutamic acid (M200E), representative of distinct physical properties. TRAcP staining and bone pit assay showed that M200s failed to support osteoclast formation and bone resorption, accompanied by impaired osteoclast-related signal transduction. However, no antagonistic effect was found in M200s against wild-type rat RANKL. Analysis of the crystal structure of RANKL predicted that this methionine residue is located within the hydrophobic core of the protein, thus, likely to be crucial for protein folding and stability. Consistently, differential scanning fluorimetry analysis suggested that M200s were less stable. Western blot analysis analyses further revealed impaired RANKL trimerization by M200s. Furthermore, receptor-ligand binding assay displayed interrupted interaction of M200s to its intrinsic receptors. Collectively, our studies revealed the molecular basis of human M199-induced ARO and elucidated the indispensable role of rodent residue M200 (equivalent to human M199) for the RANKL function.
Assuntos
Mutação de Sentido Incorreto , Ligante RANK/genética , Animais , Reabsorção Óssea , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Osteoclastos/metabolismo , Osteogênese , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Ligante RANK/química , Ligante RANK/metabolismo , Células RAW 264.7 , Ratos , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Disuse osteoporosis can result from prolonged bed rest, paralysis, casts, braces, fractures and other conditions. Abaloparatide (ABL) is a PTHrP analog that increases bone density and strength by stimulating osteogenesis with limited effects on bone resorption. We examined skeletal responses to abaloparatide in young adult male rats with normal weight-bearing and with hindlimb unloading via a pelvic harness. Rats were allocated to four groups (10-12 per group): normal weight-bearing plus vehicle treatment (CON-VEH), normal weight-bearing plus ABL treatment (CON-ABL), hindlimb-unloading plus vehicle (HLU-VEH), or hindlimb-unloading plus ABL (HLU-ABL). Rats received ABL (25 µg/kg/day, s.c.) or vehicle throughout the 28-day unloading period and were then sacrificed, at which time HLU-VEH rats exhibited reduced bone formation and significant deficits in tibial, femoral, and vertebral bone mass compared with CON-VEH. ABL treatment increased serum osteocalcin in CON and HLU animals while having no effect on the osteoclast marker TRACP-5b. Longitudinal peripheral quantitative computed tomography (pQCT) indicated that ABL increased trabecular and cortical bone mass in the tibia. ABL was also associated with improved trabecular and cortical bone mass and architectural parameters at the femur, tibia, and vertebrae by µCT. Tibial histomorphometry indicated increased trabecular and endocortical bone formation with HLU-ABL versus HLU-VEH and with CON-ABL versus CON-VEH, and ABL was also associated with lower trabecular and endocortical osteoclast surfaces. Vertebral finite element analysis indicated higher ultimate load and stiffness for CON-ABL versus CON-VEH and for HLU-ABL versus HLU-VEH. In summary, ABL was associated with improved trabecular and cortical bone density and architecture in normal weight-bearing and hindlimb-unloaded rats, with higher bone formation and no difference in bone resorption. ABL was also associated with improved bone biomechanical parameters. These results provide rationale for investigating the ability of abaloparatide to prevent or treat disuse osteoporosis in humans.
Assuntos
Densidade Óssea , Reabsorção Óssea , Animais , Reabsorção Óssea/tratamento farmacológico , Elevação dos Membros Posteriores , Masculino , Osteogênese , Proteína Relacionada ao Hormônio Paratireóideo , Ratos , Microtomografia por Raio-XRESUMO
Osteoporosis and Alzheimer's disease (AD) are common chronic degenerative disorders which are strongly associated with advanced age. We have previously demonstrated that amyloid beta peptide (Aß), one of the pathological hallmarks of AD, accumulated abnormally in osteoporotic bone specimens in addition to having an activation effect on osteoclast (Bone 2014,61:164-75). However, the underlying molecular mechanisms remain unclear. Activation of NF-κB, extracellular signal-regulated kinase (ERK) phosphorylates, and calcium oscillation signaling pathways by receptor activator NF-κB ligand (RANKL) plays a pivotal role in osteoclast activation. Targeting this signaling to modulate osteoclast function has been a promising strategy for osteoclast-related diseases. In this study, we investigated the effects of Aß on RANKL-induced osteoclast signaling pathways in vitro. In mouse bone marrow monocytes (BMMs), Aß exerted no effect on RANKL-induced osteoclastogenesis but promoted osteoclastic bone resorption. In molecular levels, Aß enhanced NF-κB activity and IκB-α degradation, activated ERK phosphorylation and stimulated calcium oscillation, thus leading to upregulation of NFAT-c1 expression during osteoclast activation. Taken together, our data demonstrate that Aß enhances RANKL-induced osteoclast activation through IκB-α degradation, ERK phosphorylation, and calcium oscillation signaling pathways and that Aß may be a promising agent in the treatment of osteoclast-related disease such as osteoporosis.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Cálcio/metabolismo , Ligante RANK/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Regulação para Cima/efeitos dos fármacosRESUMO
Morc3, a member of a highly conserved nuclear matrix protein super-family plays an important part in chromatin remodeling, DNA repair, epigenetic regulation and cellular senescence. However, its role in bone homeostasis is not known. In the present study, a phenotype-driven ENU mouse mutagenesis screen revealed that Morc3(mut +/-) mice exhibit reduced cortical area and thickness with increased cortical porosity. Morc3(mut +/-) mice displayed reduced osteoclast numbers and surface per bone surface as well as osteocyte numbers, concomitant with altered gene expressions such as Rankl/Opg and Sost in ex vivo long bones. In vitro experiments revealed a significant increase in the number of Sca-1(+)/c-kit(+) haematopoietic stem cells (HSCs), and a significant reduction in senescence associated ß-galactosidase activity in bone marrow macrophages (BMMs). In addition, we observed a decrease in osteoclastogenesis and bone resorption accompanied by upregulation of STAT1 expression in osteoclast lineage cells. Strikingly, Morc3 protein localization within the nuclear membrane was shifted to the cytoplasm in Morc3(mut +/-) osteoclasts. Further, Morc3(mut +/-) mice displayed increased osteoblast differentiation and altered gene expression. Collectively, our data show that Morc3 is a previously unreported regulator of cortical bone homeostasis and haematopoietic stem cells niche, accompanied by altered bone cell differentiation.
Assuntos
Adenosina Trifosfatases/genética , Osso e Ossos/anormalidades , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/citologia , Mutação , Osteoblastos/citologia , Proteínas Adaptadoras de Transdução de Sinal , Adenosina Trifosfatases/metabolismo , Animais , Osso e Ossos/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Células Cultivadas , Senescência Celular , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Glicoproteínas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Fator de Transcrição STAT1/metabolismo , Nicho de Células-TroncoRESUMO
Osteolytic bone diseases are commonly presented with enhanced osteoclast formation and bone resorption. Sesquiterpene lactone natural compounds have been found to possess anti-inflammatory and immune-modulation effects. Here, we identified three germacrane sesquiterpenes using computer-based virtual screening for the structural similarity with sesquiterpene lactone, parthenolide. We showed that natural germacrane sesquiterpene compounds A, B, and C inhibit osteoclast formation and bone resorption in a dose-dependent manner, with relative potency compound A > compound C > compound B based on their equimolar concentrations. Mechanistic studies by Luciferase reporter gene assay and Western blot analysis showed that germacrane sesquiterpene compound A inhibits RANKL-induced activation of NF-κB and IκBα degradation. This study reveals that natural germacrane sesquiterpene compounds are inhibitors for osteoclast formation and bone resorption, and provides evidence that naturally-occurring compounds might be beneficial as alternative medicine for the prevention and treatment of osteolysis.
Assuntos
Produtos Biológicos/farmacologia , Reabsorção Óssea/metabolismo , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Sesquiterpenos de Germacrano/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Macrófagos , Camundongos , Inibidor de NF-kappaB alfa , Proteólise/efeitos dos fármacosRESUMO
INTRODUCTION: Structural alterations in intra-articular and subchondral compartments are hallmarks of osteoarthritis, a degenerative disease that causes pain and disability in the aging population. Protein kinase C delta (PKC-δ) plays versatile functions in cell growth and differentiation, but its role in the articular cartilage and subchondral bone is not known. METHODS: Histological analysis including alcian blue, safranin O staining and fluorochrome labeling were used to reveal structural alterations at the articular cartilage surface and bone-cartilage interface in PKC-δ knockout (KO) mice. The morphology and organization of chondrocytes were studied using confocal microscopy. Glycosaminoglycan content was studied by micromass culture of chondrocytes of PKC-δ KO mice. RESULTS: We uncovered atypical structural demarcation between articular cartilage and subchondral bone of PKC-δ KO mice. Histology analyses revealed a thickening of the articular cartilage and calcified bone-cartilage interface, and decreased safranin O staining accompanied by an increase in the number of hypertrophic chondrocytes in the articular cartilage of PKC-δ KO mice. Interestingly, loss of demarcation between articular cartilage and bone was concomitant with irregular chondrocyte morphology and arrangement. Consistently, in vivo calcein labeling assay showed an increased intensity of calcein labeling in the interface of the growth plate and metaphysis in PKC-δ KO mice. Furthermore, in vitro culture of chondrocyte micromass showed a decreased alcian blue staining of chondrocyte micromass in the PKC-δ KO mice, indicative of a reduced level of glycosaminoglycan production. CONCLUSIONS: Our data imply a role for PKC-δ in the osteochondral plasticity of the interface between articular cartilage and the osteochondral junction.
Assuntos
Osso e Ossos/metabolismo , Cartilagem Articular/metabolismo , Osteoartrite/metabolismo , Proteína Quinase C-delta/metabolismo , Animais , Osso e Ossos/patologia , Cartilagem Articular/patologia , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Glicosaminoglicanos/metabolismo , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Osteoartrite/genética , Proteína Quinase C-delta/genética , Coloração e Rotulagem/métodosRESUMO
Bone remodeling is intrinsically regulated by cell signaling molecules. The Protein Kinase C (PKC) family of serine/threonine kinases is involved in multiple signaling pathways including cell proliferation, differentiation, apoptosis and osteoclast biology. However, the precise involvement of individual PKC isoforms in the regulation of osteoclast formation and bone homeostasis remains unclear. Here, we identify PKC-δ as the major PKC isoform expressed among all PKCs in osteoclasts; including classical PKCs (-α, -ß and -γ), novel PKCs (-δ, -ε, -η and -θ) and atypical PKCs (-ι/λ and -ζ). Interestingly, pharmacological inhibition and genetic ablation of PKC-δ impairs osteoclastic bone resorption in vitro. Moreover, disruption of PKC-δ activity protects against LPS-induced osteolysis in mice, with osteoclasts accumulating on the bone surface failing to resorb bone. Treatment with the PKC-δ inhibitor Rottlerin, blocks LPS-induced bone resorption in mice. Consistently, PKC-δ deficient mice exhibit increased trabeculae bone containing residual cartilage matrix, indicative of an osteoclast-rich osteopetrosis phenotype. Cultured ex vivo osteoclasts derived from PKC-δ null mice exhibit decreased CTX-1 levels and MARKS phosphorylation, with enhanced formation rates. This is accompanied by elevated gene expression levels of cathepsin K and PKC -α, -γ and -ε, as well as altered signaling of pERK and pcSrc416/527 upon RANKL-induction, possibly to compensate for the defects in bone resorption. Collectively, our data indicate that PKC-δ is an intrinsic regulator of osteoclast formation and bone resorption and thus is a potential therapeutic target for pathological osteolysis.