RESUMEN
OBJECTIVE: IĸB protein B cell lymphoma 3-encoded protein (BCL3) is a regulator of the NF-κB family of transcription factors. NF-κB signaling fundamentally influences the fate of bone-forming osteoblasts and bone-resorbing osteoclasts, but the role of BCL3 in bone biology has not been investigated. The objective of this study was to evaluate BCL3 in skeletal development, maintenance, and osteoarthritic pathology. METHODS: To assess the contribution of BCL3 to skeletal homeostasis, neonatal mice (n = 6-14) lacking BCL3 (Bcl3-/- ) and wild-type (WT) controls were characterized for bone phenotype and density. To reveal the contribution to bone phenotype by the osteoblast compartment in Bcl3-/- mice, transcriptomic analysis of early osteogenic differentiation and cellular function (n = 3-7) were assessed. Osteoclast differentiation and function in Bcl3-/- mice (n = 3-5) was assessed. Adult 20-week Bcl3-/- and WT mice bone phenotype, strength, and turnover were assessed. A destabilization of the medial meniscus model of osteoarthritic osteophytogenesis was used to understand adult bone formation in Bcl3-/- mice (n = 11-13). RESULTS: Evaluation of Bcl3-/- mice revealed congenitally increased bone density, long bone dwarfism, increased bone biomechanical strength, and altered bone turnover. Molecular and cellular characterization of mesenchymal precursors showed that Bcl3-/- cells displayed an accelerated osteogenic transcriptional profile that led to enhanced differentiation into osteoblasts with increased functional activity, which could be reversed with a mimetic peptide. In a model of osteoarthritis-induced osteophytogenesis, Bcl3-/- mice exhibited decreased pathological osteophyte formation (P < 0.05). CONCLUSION: Cumulatively, these findings demonstrate that BCL3 controls developmental mineralization to enable appropriate bone formation, whereas in a pathological setting, it contributes to skeletal pathology.
Asunto(s)
Proteínas del Linfoma 3 de Células B , Huesos , Osteogénesis , Animales , Ratones , Huesos/metabolismo , Densidad Ósea , Diferenciación Celular , FN-kappa B/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Proteínas del Linfoma 3 de Células B/genéticaRESUMEN
Paget's disease of bone (PDB) is characterized by focal increases in bone remodelling. Genome-wide association studies identified a susceptibility locus for PDB tagged by rs5742915, which is located within the PML gene. Here, we have assessed the candidacy of PML as the predisposing gene for PDB at this locus. We found that the PDB-risk allele of rs5742915 was associated with lower PML expression and that PML expression in blood cells from individuals with PDB was lower than in controls. The differentiation, survival and resorptive activity of osteoclasts prepared from Pml-/- mice was increased compared with wild type. Furthermore, the inhibitory effect of IFN-γ on osteoclast formation from Pml-/- was significantly blunted compared with wild type. Bone nodule formation was also increased in osteoblasts from Pml-/- mice when compared with wild type. Although microCT analysis of trabecular bone showed no differences between Pml-/- mice and wild type, bone histomorphometry showed that Pml-/- mice had high bone turnover with increased indices of bone resorption and increased mineral apposition rate. These data indicate that reduced expression of PML predisposes an individual to PDB and identify PML as a novel regulator of bone metabolism. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Resorción Ósea , Osteítis Deformante , Animales , Estudio de Asociación del Genoma Completo , Humanos , Ratones , Osteítis Deformante/genética , Osteoclastos/metabolismo , Osteogénesis , Proteína de la Leucemia PromielocíticaRESUMEN
Early onset familial Paget's disease of bone (EoPDB), familial expansile osteolysis, and expansile skeletal hyperphosphatasia are related disorders caused by insertion mutations in exon 1 of the TNFRSF11A gene, which encodes receptor activator of nuclear factor κB (RANK) protein. To understand the mechanisms underlying these disorders, we developed a mouse model carrying the 75dup27 mutation which causes EoPDB. Mice heterozygous for the mutation (Tnfrsf11a75dup27/- ) developed a PDB-like disorder with focal osteolytic lesions in the hind limbs with increasing age. Treatment of these mice with zoledronic acid completely prevented the development of lesions. Studies in vitro showed that RANK ligand (RANKL)-induced osteoclast formation and signaling was impaired in bone marrow cells from Tnfrsf11a75dup27/- animals, but that osteoclast survival was increased independent of RANKL stimulation. Surprisingly, Tnfrsf11a75dup27/75dup27 homozygotes had osteopetrosis at birth, with complete absence of osteoclasts. Bone marrow cells from these mice failed to form osteoclasts in response to RANKL and macrophage colony-stimulating factor (M-CSF) stimulation. This intriguing study has shown that in heterozygous form, the 75dup27 mutation causes focal osteolytic lesions in vivo reminiscent of the human disorder and extends osteoclast survival independently of RANKL signaling. In homozygous form, however, the mutation causes osteopetrosis due to failure of osteoclast formation and insensitivity to RANKL stimulation. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..
Asunto(s)
Osteítis Deformante , Osteopetrosis , Animales , Homocigoto , Humanos , Ratones , Mutagénesis Insercional , Mutación , Osteítis Deformante/genética , Osteoclastos , Osteopetrosis/genética , Fenotipo , Ligando RANK/genética , Receptor Activador del Factor Nuclear kappa-B/genéticaRESUMEN
Lactation-induced bone loss occurs due to high calcium requirements for fetal growth but skeletal recovery is normally achieved promptly postweaning. Dietary protein is vital for fetus and mother but the effects of protein undernutrition on the maternal skeleton and skeletal muscles are largely unknown. We used mouse dams fed with normal (N, 20%) or low (L, 8%) protein diet during gestation and lactation and maintained on the same diets (NN, LL) or switched from low to normal (LN) during a 28 d skeletal restoration period post lactation. Skeletal muscle morphology and neuromuscular junction integrity was not different between any of the groups. However, dams fed the low protein diet showed extensive bone loss by the end of lactation, followed by full skeletal recovery in NN dams, partial recovery in LN and poor bone recovery in LL dams. Primary osteoblasts from low protein diet fed mice showed decreased in vitro bone formation and decreased osteogenic marker gene expression; promoter methylation analysis by pyrosequencing showed no differences in Bmpr1a, Ptch1, Sirt1, Osx, and Igf1r osteoregulators, while miR-26a, -34a, and -125b expression was found altered in low protein fed mice. Therefore, normal protein diet is indispensable for maternal musculoskeletal health during the reproductive period.
Asunto(s)
Fenómenos Fisiológicos Nutricionales de los Animales/fisiología , Enfermedades Óseas Metabólicas/fisiopatología , Dieta con Restricción de Proteínas , Lactancia/fisiología , Músculo Esquelético/fisiología , Reproducción/fisiología , Animales , Animales Recién Nacidos , Peso Corporal , Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones Transgénicos , MicroARNs/genética , Músculo Esquelético/metabolismo , Osteoblastos/metabolismo , Osteogénesis/genética , DesteteRESUMEN
In patients with postmenopausal osteoporosis low bone volume is associated with high bone marrow adipose tissue (MAT). Moreover, high MAT is associated with increased fracture risk. This suggests an interaction between MAT and bone turnover, however literature remains equivocal. Estrogen treatment decreases MAT, but the effect of raloxifene, a selective estrogen receptor modulator (SERM) registered for treatment of postmenopausal osteoporosis, on MAT is not known. The aim of this study is 1] to determine the effect of raloxifene on MAT and 2] to determine the relationship between MAT and bone turnover in patients with osteoporosis. Bone biopsies from the MORE trial were analyzed. The MORE trial investigated the effects of raloxifene 60 or 120mg per day versus placebo on bone metabolism and fracture incidence in patients with postmenopausal osteoporosis. We quantified MAT in iliac crest biopsies obtained at baseline and after 2years of treatment (n=53; age 68.2±6.2years). Raloxifene did not affect the change in MAT volume after 2years compared to baseline (placebo: 1.89±10.84%, raloxifene 60mg: 6.31±7.22%, raloxifene 120mg: -0.77±10.72%), nor affected change in mean adipocyte size (placebo: 1.45 (4.45) µm, raloxifene 60mg: 1.45 (4.35) µm, raloxifene 120mg: 0.81 (5.21) µm). Adipocyte number tended to decrease after placebo treatment (-9.92 (42.88) cells/mm2) and tended to increase during raloxifene 60mg treatment (13.27 (66.14) cells/mm2) while adipocyte number remained unchanged in the raloxifene 120mg group, compared to placebo (3.06 (39.80) cells/mm2, Kruskal-Wallis p=0.055, post hoc: placebo vs raloxifene 60mg p=0.017). MAT volume and adipocyte size were negatively associated with osteoclast number at baseline (R2=0.123, p=0.006 and R2=0.098, p=0.016 respectively). Furthermore adipocyte size was negatively associated with osteoid surface (R2=0.067, p=0.049). Finally, patients with vertebral fractures had higher MAT volume (50.82 (8.80)%) and larger adipocytes (55.75 (3.14) µm) compared to patients without fractures (45.58 (12.72)% p=0.032, 52.77 (3.73) µm p=0.004 respectively). In conclusion, raloxifene did not affect marrow adipose tissue, but tended to increase adipocyte number compared to placebo. At baseline MAT volume and adipocyte size were associated with bone resorption, and adipocyte size was associated with osteoid surface, suggesting an interaction between bone marrow adipocytes and bone turnover. In addition, we found that high MAT volume and larger adipocyte size are associated with prevalent vertebral fractures in postmenopausal women with osteoporosis, indicating that adipocyte size affects bone quality independent of bone volume.
Asunto(s)
Tejido Adiposo/patología , Médula Ósea/patología , Remodelación Ósea/efectos de los fármacos , Osteoporosis Posmenopáusica/tratamiento farmacológico , Clorhidrato de Raloxifeno/uso terapéutico , Tejido Adiposo/efectos de los fármacos , Adiposidad/efectos de los fármacos , Anciano , Médula Ósea/efectos de los fármacos , Femenino , Humanos , Osteoporosis Posmenopáusica/complicaciones , Clorhidrato de Raloxifeno/farmacología , Fracturas de la Columna Vertebral/complicaciones , Fracturas de la Columna Vertebral/patologíaRESUMEN
OBJECTIVE: Cartilage destruction in osteoarthritis (OA) is mediated mainly by matrix metalloproteinases (MMPs) and ADAMTS. The therapeutic candidature of targeting aggrecanases has not yet been defined in joints in which spontaneous OA arises from genetic susceptibility, as in the case of the STR/Ort mouse, without a traumatic or load-induced etiology. In addition, we do not know the long-term effect of aggrecanase inhibition on bone. We undertook this study to assess the potential aggrecanase selectivity of a variant of tissue inhibitor of metalloproteinases 3 (TIMP-3), called [-1A]TIMP-3, on spontaneous OA development and bone formation in STR/Ort mice. METHODS: Using the background of STR/Ort mice, which develop spontaneous OA, we generated transgenic mice that overexpress [-1A]TIMP-3, either ubiquitously or conditionally in chondrocytes. [-1A]TIMP-3 has an extra alanine at the N-terminus that selectively inhibits ADAMTS but not MMPs. We analyzed a range of OA-related measures in all mice at age 40 weeks. RESULTS: Mice expressing high levels of [-1A]TIMP-3 were protected against development of OA, while those expressing low levels were not. Interestingly, we also found that high levels of [-1A]TIMP-3 transgene overexpression resulted in increased bone mass, particularly in females. This regulation of bone mass was at least partly direct, as adult mouse primary osteoblasts infected with [-1A]TIMP-3 in vitro showed elevated rates of mineralization. CONCLUSION: The results provide evidence that [-1A]TIMP-3-mediated inhibition of aggrecanases can protect against cartilage degradation in a naturally occurring mouse model of OA, and they highlight a novel role that aggrecanase inhibition may play in increased bone mass.
Asunto(s)
Densidad Ósea/genética , Cartílago Articular/enzimología , Endopeptidasas/metabolismo , Osteoartritis/enzimología , Inhibidores de Proteasas/metabolismo , Animales , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Transgénicos , Osteoartritis/genética , Inhibidor Tisular de Metaloproteinasa-3/metabolismoRESUMEN
Extracellular signal-regulated protein kinase 5 (ERK5) has been implicated during development and carcinogenesis. Nkx3.1-mediated Cre expression is a useful strategy to genetically manipulate the mouse prostate. While grossly normal at birth, we observed an unexpected phenotype of spinal protrusion in Nkx3.1:Cre;Erk5 fl/fl (Erk5 fl/fl) mice by ~6-8 weeks of age. X-ray, histological and micro CT (µCT) analyses showed that 100% of male and female Erk5 fl/fl mice had a severely deformed curved thoracic spine, with an associated loss of trabecular bone volume. Although sex-specific differences were observed, histomorphometry measurements revealed that both bone resorption and bone formation parameters were increased in male Erk5 fl/fl mice compared to wild type (WT) littermates. Osteopenia occurs where the rate of bone resorption exceeds that of bone formation, so we investigated the role of the osteoclast compartment. We found that treatment of RANKL-stimulated primary bone marrow-derived macrophage (BMDM) cultures with small molecule ERK5 pathway inhibitors increased osteoclast numbers. Furthermore, osteoclast numbers and expression of osteoclast marker genes were increased in parallel with reduced Erk5 expression in cultures generated from Erk5 fl/fl mice compared to WT mice. Collectively, these results reveal a novel role for Erk5 during bone maturation and homeostasis in vivo.
Asunto(s)
Proteína Quinasa 7 Activada por Mitógenos/fisiología , Osteoclastos/metabolismo , Columna Vertebral/anomalías , Animales , Resorción Ósea/genética , Hueso Esponjoso/anomalías , Catepsina K/biosíntesis , Recuento de Células , Femenino , Eliminación de Gen , Proteínas de Homeodominio/metabolismo , Integrasas/genética , Activación de Linfocitos , Masculino , Ratones , Ratones Transgénicos , Proteína Quinasa 7 Activada por Mitógenos/genética , Factores de Transcripción NFATC/biosíntesis , Osteogénesis/genética , Receptor Activador del Factor Nuclear kappa-B/biosíntesis , Factores de Transcripción/metabolismoRESUMEN
Micro-CT analysis has become the standard method for assessing bone volume and architecture in small animals. However, micro-CT does not allow the assessment of bone turnover parameters such as bone formation rate and osteoclast (OC) number and surface. For these crucial variables histomorphometric analysis is still an essential technique. Histomorphometry however, is time consuming and, especially in mouse bones, OCs can be difficult to detect. The main purpose of this study was to develop and validate a relatively easy and rapid method to measure static and dynamic bone histomorphometry parameters. Here we present the adaptation of established staining protocols and three novel open source image analysis packages: TrapHisto, OsteoidHisto and CalceinHisto that allow rapid, semi-automated analysis of histomorphometric bone resorption, osteoid, and calcein double labelling parameters respectively. These three programs are based on ImageJ, but use a relatively simple user interface that hides the underlying complexity of the image analysis.
Asunto(s)
Resorción Ósea/metabolismo , Programas Informáticos , Animales , Remodelación Ósea/fisiología , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoclastos/metabolismo , Osteogénesis/fisiologíaRESUMEN
Degenerative joint diseases such as osteoarthritis are characterised by aberrant region-specific bone formation and abnormal bone mineral content. A recent study suggested a role for the complement membrane attack complex in experimental models of osteoarthritis. Since CD59a is the principal regulator of the membrane attack complex in mice, we evaluated the impact of CD59a gene deletion upon maintenance of bone architecture. In vivo bone morphology analysis revealed that male CD59a-deficient mice have increased femur length and cortical bone volume, albeit with reduced bone mineral density. However, this phenomenon was not observed in female mice. Histomorphometric analysis of the trabecular bone showed increased rates of bone homeostasis, with both increased bone resorption and mineral apposition rate in CD59a-deficient male mice. When bone cells were studied in isolation, in vitro osteoclastogenesis was significantly increased in male CD59a-deficient mice, although osteoblast formation was not altered. Our data reveal, for the first time, that CD59a is a regulator of bone growth and homeostasis. CD59a ablation in male mice results in longer and wider bones, but with less density, which is likely a major contributing factor for their susceptibility to osteoarthritis. These findings increase our understanding of the role of complement regulation in degenerative arthritis.
Asunto(s)
Envejecimiento/patología , Huesos/patología , Antígenos CD59/metabolismo , Proteínas del Sistema Complemento/metabolismo , Eliminación de Gen , Caracteres Sexuales , Animales , Desarrollo Óseo , Células de la Médula Ósea/patología , Remodelación Ósea , Femenino , Masculino , Ratones Endogámicos C57BL , Tamaño de los Órganos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/patología , FenotipoRESUMEN
OBJECTIVE: Proteinase-activated receptor 2 (PAR2) deficiency protects against cartilage degradation in experimental osteoarthritis (OA). The wider impact of this pathway upon OA-associated pathologies such as osteophyte formation and pain is unknown. Herein, we investigated early temporal bone and cartilage changes in experimental OA in order to further elucidate the role of PAR2 in OA pathogenesis. METHODS: OA was induced in wild-type (WT) and PAR2-deficient (PAR2-/-) mice by destabilisation of the medial meniscus (DMM). Inflammation, cartilage degradation and bone changes were monitored using histology and microCT. In gene rescue experiments, PAR2-/- mice were intra-articularly injected with human PAR2 (hPAR2)-expressing adenovirus. Dynamic weight bearing was used as a surrogate of OA-related pain. RESULTS: Osteophytes formed within 7â days post-DMM in WT mice but osteosclerosis was only evident from 14â days post induction. Importantly, PAR2 was expressed in the proliferative/hypertrophic chondrocytes present within osteophytes. In PAR2-/- mice, osteophytes developed significantly less frequently but, when present, were smaller and of greater density; no osteosclerosis was observed in these mice up to day 28. The pattern of weight bearing was altered in PAR2-/- mice, suggesting reduced pain perception. The expression of hPAR2 in PAR2-/- mice recapitulated osteophyte formation and cartilage damage similar to that observed in WT mice. However, osteosclerosis was absent, consistent with lack of hPAR2 expression in subchondral bone. CONCLUSIONS: This study clearly demonstrates PAR2 plays a critical role, via chondrocytes, in osteophyte development and subchondral bone changes, which occur prior to PAR2-mediated cartilage damage. The latter likely occurs independently of OA-related bone changes.
Asunto(s)
Artritis Experimental/patología , Huesos/patología , Cartílago Articular/patología , Osteoartritis/patología , Receptor PAR-2/metabolismo , Animales , Artralgia/etiología , Artralgia/patología , Artritis Experimental/etiología , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Osteoartritis/etiología , Osteocitos/metabolismoRESUMEN
INTRODUCTION: Inflammatory joint diseases such as rheumatoid arthritis are associated with local bone erosions and systemic bone loss, mediated by increased osteoclastic activity. The receptor activator of nuclear factor (NF) κB ligand (RANKL) plays a key role in mediating inflammation-induced bone loss, whereas tumour necrosis factor (TNF) plays a central role in the inflammatory process. Here we tested whether a recently identified class of small molecule inhibitors of RANKL signalling (ABD compounds) also affect TNF signalling and whether these compounds inhibit inflammation in an animal model of rheumatoid arthritis. METHODS: The inhibitory effects of the ABD compounds on TNF-induced signalling were tested in mouse macrophage cultures by western blotting and in an NFκB luciferase-reporter cell line. The anti-inflammatory effects of the compounds were tested in the mouse collagen-induced arthritis model of rheumatoid arthritis. RESULTS: The ABD compounds ABD328 and ABD345 both inhibited TNF-induced activation of the NFκB pathway and the extracellular signal-regulated kinase (ERK) and Jun kinase (JNK) mitogen activated protein kinases (MAPKs). When tested in the mouse collagen-induced arthritis model of rheumatoid arthritis, the compounds suppressed inflammatory arthritis, inhibited joint destruction and prevented systemic bone loss. Furthermore, one of the compounds (ABD328) showed oral activity. CONCLUSIONS: Here we describe a novel class of small molecule compounds that inhibit both RANKL- and TNF-induced NFκB and MAPK signalling in osteoclasts and macrophages, and inflammation and bone destruction in a mouse model of rheumatoid arthritis. These novel compounds therefore represent a promising new class of treatments for inflammatory diseases, such as rheumatoid arthritis.
Asunto(s)
Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Compuestos de Bifenilo/farmacología , Resorción Ósea/metabolismo , Hexanonas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/efectos de los fármacos , Ligando RANK/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de Necrosis Tumoral/metabolismo , Animales , Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Resorción Ósea/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , FN-kappa B/inmunología , FN-kappa B/metabolismo , Ligando RANK/inmunología , Ligando RANK/metabolismo , Factores de Necrosis Tumoral/inmunologíaRESUMEN
Several studies have shown that in contrast to osteoporosis (OP), osteoarthritis (OA) is characterized by high bone mineral density (BMD). Bone strength not only depends on mineral content as determined by dual X-ray absorptiometry (DXA), but also on bone microarchitecture. We studied intertrochanteric bone from normal controls and OA and OP patients by bone histomorphometry (BHM) and microcomputed tomography (µCT) as well as DXA in order to first, test the differences between OA and OP comparing both groups to healthy controls, second, to assess variations between three different skeletal sites in controls and third, to determine the level of agreement between µCT, BHM, and DXA. Analysis was performed on 115 samples from OA and OP patients, and controls. We found significant differences between OA and OP samples in structural parameters and in the osteoid fraction (p < 0.05). The majority of the intra-skeletal differences were shown between lumbar spine and femoral head samples (p < 0.05). Significant agreements were found between µCT and BHM and DXA (r = 0.32-0.45, p < 0.05). Our findings suggest differences in intertrochanteric bone between OA and OP, the age-related intra-skeletal variations and a correlation between microscopic and macroscopic bone evaluation methods.
Asunto(s)
Absorciometría de Fotón , Huesos/diagnóstico por imagen , Osteoartritis/diagnóstico por imagen , Osteoporosis/diagnóstico por imagen , Microtomografía por Rayos X , Anciano , Anciano de 80 o más Años , Densidad Ósea , Huesos/patología , Estudios de Casos y Controles , Femenino , Cabeza Femoral/diagnóstico por imagen , Cabeza Femoral/patología , Humanos , Vértebras Lumbares/diagnóstico por imagen , Vértebras Lumbares/patología , Masculino , Persona de Mediana Edad , Osteoartritis/patología , Osteoporosis/patologíaRESUMEN
Endothelial nitric oxide synthase (eNOS) has long been held responsible for NO production by mechanically stimulated osteoblasts, but this has recently been disputed. We investigated whether one of the three known NOS isoforms is essential for NO production by mechanically stimulated osteoblasts in vitro and revisited the bone phenotype of the eNOS-/- mouse. Osteoblasts, obtained as outgrowths from mouse calvaria or long bones of wild-type (WT), eNOS-/-, inducible NOS-/- (iNOS-/-), or neuronal NOS-/- (nNOS-/-) mice, were subjected to mechanical stimulation by means of pulsating fluid flow (PFF); and NO production was determined. Tibiae and femora from 8-week-old mice were subjected to µCT and three-point bending tests. Deletion of single NOS isoforms did not lead to significant upregulation of alternate isoforms in cultured osteoblasts from WT, eNOS-/-, iNOS-/-, or nNOS-/- mice. Expression of eNOS mRNA in osteoblasts was below our detection limit, and no differences in growth between WT and eNOS-/- osteoblasts were found. PFF increased NO production by approximately fourfold in WT and eNOS-/- osteoblasts and significantly stimulated NO production in iNOS-/- and nNOS-/- osteoblasts. Tibiae and femora from WT and eNOS-/- mice showed no difference in bone volume and architecture or in mechanical parameters. Our data suggest that mechanical stimuli can enhance NO production by cultured osteoblasts singly deficient for each known NOS isoform and that lack of eNOS does not significantly affect bone mass and strength at 8 weeks of age. Our data challenge the notion that eNOS is a key effector of mechanically induced bone maintenance.
Asunto(s)
Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/biosíntesis , Osteoblastos/metabolismo , Animales , Huesos/diagnóstico por imagen , Huesos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Mecánico , Tomografía Computarizada por Rayos XRESUMEN
We previously described a novel series of biphenyl carboxylic acid derivatives which have potent antiresorptive effects in vitro and in vivo and do not affect osteoblast function. However, none of the previous compounds showed oral activity, probably because they were esters, which would be expected to be metabolized very rapidly. Here, we tested whether derivatives where the ester link was replaced by a ketone link were orally active. Compounds were tested in murine osteoclast and osteoblast cultures and in the mouse ovariectomy (OVX) model of osteoporosis. The ketones were at least as potent at inhibiting osteoclast formation and RANKL signaling in vitro as the esters and did not inhibit osteoblast differentiation or function. The basic ketone-linked compound ABD68 was only partially able to inhibit OVX-induced bone loss at an oral dose of 20 mg/kg daily. Substitutions on the phenyl rings increased the potency of the compounds in vitro and may prevent metabolism of the compounds in vivo. The most promising derivative, ABD328, completely prevented OVX-induced bone loss when administered by intraperitoneal injection at 3 mg/kg daily. Furthermore, ABD328 was also able to fully prevent OVX-induced bone loss when given orally at 10 mg/kg daily. The results indicate that biphenyl carboxylates like ABD328 are oral candidate drugs for the treatment of diseases characterized by increased bone resorption, such as postmenopausal osteoporosis.
Asunto(s)
Compuestos de Bifenilo/farmacología , Conservadores de la Densidad Ósea/farmacología , Resorción Ósea/prevención & control , Administración Oral , Animales , Compuestos de Bifenilo/administración & dosificación , Compuestos de Bifenilo/uso terapéutico , Conservadores de la Densidad Ósea/administración & dosificación , Conservadores de la Densidad Ósea/uso terapéutico , Diferenciación Celular , Células Cultivadas , Femenino , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoporosis/tratamiento farmacológico , Osteoporosis/prevención & control , Ovariectomía , Ligando RANK/metabolismoRESUMEN
Biphenylketones were identified as novel inhibitors of NFkappaB activation. Structure-activity studies led to the identification of compound 4c, which had good potency against osteoclasts (IC50=0.8 microM), showed oral activity, and was able to completely prevent inflammation and bone loss in vivo.
Asunto(s)
Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Artritis/tratamiento farmacológico , Derivados del Benceno/química , Derivados del Benceno/uso terapéutico , Cetonas/química , Cetonas/uso terapéutico , Osteoporosis/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Artritis/inducido químicamente , Derivados del Benceno/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Femenino , Inflamación/tratamiento farmacológico , Cetonas/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , FN-kappa B/inmunología , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Hidrocarburos Policíclicos Aromáticos/química , Hidrocarburos Policíclicos Aromáticos/farmacología , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Runx1 is highly expressed in chondroprogenitor and osteoprogenitor cells and in vitro experiments suggest that Runx1 is important in the early stages of osteoblast and chondrocyte differentiation. However, because Runx1 knockout mice are early embryonic lethal due to failure of hematopoiesis, the role of Runx1 in skeletogenesis remains unclear. We studied the role of Runx1 in skeletal development using a Runx1 reversible knockout mouse model. By crossing with Tie2-Cre deletor mice, Runx1 expression was selectively rescued in the endothelial and hematopoietic systems but not in the skeleton. Although Runx1(Re/Re) embryos survived until birth and had a generally normal skeleton, the development of mineralization in the sternum and some skull elements was significantly disrupted. In contrast to wild-type embryos, the sternum of E17.5 Runx1(Re/Re) embryos showed high levels of Sox-9 and collagen type II expression and lack of development of hypertrophic chondrocytes. In situ hybridization analysis demonstrated that, in contrast to the vertebrae and long bones, the sternum of wild-type embryos expresses high levels of Runx1, but not Runx2, the master regulator of skeletogenesis. Thus, although Runx1 is not essential for major skeletal development, it does play an essential role in the development of the sternum and some skull elements.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Esternón/metabolismo , Animales , Huesos/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Ratones , Ratones Noqueados , Esternón/embriologíaRESUMEN
Three polymorphisms (-1997G/T; -1663IndelT and +1245G/T) have been identified in the 5' flank of COL1A1 gene that are associated with osteoporosis but the underlying mechanism is unclear. Here we investigated the functional effects of these variants on COL1A1 transcription. Transcription was 2-fold higher with the osteoporosis-associated G-del-T haplotype compared with the common G-Ins-G haplotype. Gel shift assays showed that the region surrounding the -1663IndelT polymorphism recognized a complex of proteins essential for osteoblast differentiation and function including Nmp4 and Osterix, and the osteoporosis-associated -1663delT allele had increased binding affinity for this complex. Chromatin immunoprecipitation assays confirmed that the region flanking -1663insdelT bound a complex of proteins including Osterix and Nmp4 and also showed evidence of recruitment of Nmp4 to the Sp1 binding site in intron 1. Further studies showed that haplotype G-del-T had higher binding affinity for RNA polymerase II, consistent with increased transcription of the G-del-T allele and there was a significant inverse association between carriage of G-del-T and bone mineral density (BMD) in a cohort of 3270 Caucasian women. We conclude that common polymorphic variants in the 5' flank of COLIA1 regulate transcription by affecting DNA-protein interactions and that increased levels of transcription correlate with reduced BMD values in vivo. This is consistent with a model whereby increased COL1A1 transcription predisposes to osteoporosis, probably by increasing production of the alpha 1 chain and disrupting the normal ratio of collagen type 1 alpha 1 and alpha 2 chains.
Asunto(s)
Colágeno Tipo I/genética , Predisposición Genética a la Enfermedad , Intrones , Osteoporosis/genética , Polimorfismo Genético , Regiones Promotoras Genéticas , Transcripción Genética , Secuencia de Aminoácidos , Densidad Ósea , Línea Celular Tumoral , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Femenino , Humanos , Datos de Secuencia Molecular , Osteoporosis/metabolismo , Osteoporosis/fisiopatología , Unión Proteica , Factor de Transcripción Sp7 , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Bisphosphonates are widely used in the treatment of osteoporosis, but they inhibit bone formation and blunt the anabolic effect of PTH. Here we describe a novel series of compounds that have potent antiresorptive effects in vitro and in vivo that do not adversely affect osteoblast function. The effects of the compounds on osteoclast formation and survival were studied on mouse osteoclasts generated from bone marrow macrophages and on osteoblast function using primary mouse calvarial osteoblast cultures and bone nodule cultures. Studies were performed in vivo using sham-operated or ovariectomized mice. The most potent compound tested was ABD350, a halogen-substituted derivative of the parent compound ABD56 in which the labile ester bond was replaced by a reduced ketone link, with IC50 osteoclast formation at a concentration of 1.3 microm. All compounds inhibited receptor activator of nuclear factor-kappaB ligand-induced inhibitor of nuclear factor kappaB phosphorylation and caused osteoclast apoptosis but no inhibitory effects on osteoblast function were observed at concentrations of up to 20 microm. ABD350 prevented ovariectomy-induced bone loss when given ip (5 mg/kg.d), whereas ABD56 was only partially effective at this dose. In contrast to the bisphosphonate alendronate, ABD350 had no inhibitory effect on PTH-induced bone formation in ovariectomized mice. In conclusion, the biphenyl carboxylic acid derivatives like ABD350 represent a new class of antiresorptive drugs that inhibit osteoclast activity but have no significant inhibitory effects on osteoblast activity in vitro or PTH-induced bone formation in vivo.
Asunto(s)
Alendronato/farmacología , Benzoatos/farmacología , Compuestos de Bifenilo/farmacología , Osteoblastos/citología , Osteoclastos/citología , Osteogénesis/efectos de los fármacos , Hormona Paratiroidea/farmacología , Animales , Animales Recién Nacidos , Células de la Médula Ósea/citología , Resorción Ósea/etiología , Femenino , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Ovariectomía/efectos adversos , CráneoRESUMEN
We have previously shown that the nitrosylated flurbiprofen derivative HCT1026 inhibits bone resorption, both in vivo and in vitro, and that its mechanism of action is independent of nitric oxide release and prostaglandin synthesis inhibition. Here we describe the effects of HCT1026 on osteoclast formation, activity, survival and cell signalling in vitro. HCT1026 strongly inhibited osteoclast formation, activity and survival in murine osteoclast cultures, whereas macrophages and osteoblasts were unaffected. HCT1026 induced osteoclast apoptosis, and this was partially prevented by increasing the concentration of receptor activator of nuclear factor kappa B ligand (RANKL). This suggests that HCT1026 inhibits bone resorption by inhibiting the effects of RANKL. In agreement with this we found that HCT1026 inhibited RANKL-induced activation of the nuclear factor kappa B (NFkappaB) and extracellular signal-regulated kinase (ERK) pathways in both osteoclast and macrophage cultures, whereas its parent compound flurbiprofen did not. In addition, HCT1026 also inhibited tumor necrosis factor (TNF)-, interleukin-1 (IL1)- and LPS-induced signalling, but not macrophage colony stimulating factor induced signalling. The pathways that are inhibited by HCT1026 all share a similar kinase complex upstream of the NFkappaB and ERK pathways, and this is the most likely target for the actions of HCT1026. Although the rationale for the modification of flurbiprofen with a nitric oxide donor group was to prevent gastro-intestinal toxicity, the resulting compound HCT1026 gained unexpected additional cytokine-inhibitory properties. As RANKL, TNF and IL1 are all important mediators of inflammation and joint destruction, compounds like HCT1026 could represent a novel class of anti-inflammatory compounds.
Asunto(s)
Citocinas/antagonistas & inhibidores , Citocinas/farmacología , Flurbiprofeno/análogos & derivados , Compuestos Nitrosos/química , Transducción de Señal/efectos de los fármacos , Actinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Resorción Ósea/metabolismo , Resorción Ósea/patología , Citocinas/metabolismo , Activación Enzimática/efectos de los fármacos , Flurbiprofeno/química , Flurbiprofeno/farmacología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoclastos/patología , Ligando RANK/metabolismoRESUMEN
Bisphosphonates are widely used for the treatment of bone diseases associated with increased osteoclastic bone resorption. Bisphosphonates are known to inhibit biochemical markers of bone formation in vivo, but it is unclear to what extent this is a consequence of osteoclast inhibition or a direct inhibitory effect on cells of the osteoblast lineage. In order to investigate this issue, we studied the effects of various bisphosphonates on osteoblast growth and differentiation in vitro. The aminobisphosphonates pamidronate and alendronate inhibited osteoblast growth, caused osteoblast apoptosis, and inhibited protein prenylation in osteoblasts in a dose-dependent manner over the concentration range 20-100 microM. Further studies showed that alendronate in a dose of 0.1 mg/kg inhibited protein prenylation in calvarial osteoblasts in vivo, indicating that alendronate can be taken up by osteoblasts in sufficient amounts to inhibit protein prenylation at clinically relevant doses. Pamidronate and alendronate inhibited bone nodule formation at concentrations 10-fold lower than those required to inhibit osteoblast growth. These effects were not observed with non-nitrogen-containing bisphosphonates or with other inhibitors of protein prenylation and were only partially reversed by cotreatment with a fourfold molar excess of ss-glycerol phosphate. We conclude that aminobisphosphonates cause osteoblast apoptosis in vitro at micromolar concentrations and inhibit osteoblast differentiation at nanomolar concentrations by mechanisms that are independent of effects on protein prenylation and may be due in part to inhibition of mineralization. While these results need to be interpreted with caution because of uncertainty about the concentrations of bisphosphonates that osteoblasts are exposed to in vivo, our studies clearly demonstrate that bisphosphonates exert strong inhibitory effects on cells of the osteoblast lineage at similar concentrations to those that cause osteoclast inhibition. This raises the possibility that inhibition of bone formation by bisphosphonates may be due in part to a direct inhibitory effect on cells of the osteoblast lineage.