RESUMEN
Osteoclasts are the cells primarily responsible for inflammation-induced bone loss, as is particularly seen in rheumatoid arthritis. Increasing evidence suggests that osteoclasts formed under homeostatic versus inflammatory conditions may differ in phenotype. While microRNA-29-3p family members (miR-29a-3p, miR-29b-3p, miR-29c-3p) promote the function of RANKL-induced osteoclasts, the role of miR-29-3p during inflammatory TNF-α-induced osteoclastogenesis is unknown. We used bulk RNA-seq, histology, qRT-PCR, reporter assays, and western blot analysis to examine bone marrow monocytic cell cultures and tissue from male mice in which the function of miR-29-3p family members was decreased by expression of a miR-29-3p tough decoy (TuD) competitive inhibitor in the myeloid lineage (LysM-cre). We found that RANKL-treated monocytic cells expressing the miR-29-3p TuD developed a hypercytokinemia/proinflammatory gene expression profile in vitro, which is associated with macrophages. These data support the concept that miR-29-3p suppresses macrophage lineage commitment and may have anti-inflammatory effects. In correlation, when miR-29-3p activity was decreased, TNF-α-induced osteoclast formation was accentuated in an in vivo model of localized osteolysis and in a cell-autonomous manner in vitro. Further, miR-29-3p targets mouse TNF receptor 1 (TNFR1/Tnfrsf1a), an evolutionarily conserved regulatory mechanism, which likely contributes to the increased TNF-α signaling sensitivity observed in the miR-29-3p decoy cells. Whereas our previous studies demonstrated that the miR-29-3p family promotes RANKL-induced bone resorption, the present work shows that miR-29-3p dampens TNF-α-induced osteoclastogenesis, indicating that miR-29-3p has pleiotropic effects in bone homeostasis and inflammatory osteolysis. Our data supports the concept that the knockdown of miR-29-3p activity could prime myeloid cells to respond to an inflammatory challenge and potentially shift lineage commitment toward macrophage, making the miR-29-3p family a potential therapeutic target for modulating inflammatory response.
Asunto(s)
Inflamación , MicroARNs , Osteoclastos , Osteólisis , Ligando RANK , MicroARNs/genética , MicroARNs/metabolismo , Animales , Osteólisis/genética , Osteólisis/patología , Osteólisis/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patología , Ratones , Ligando RANK/metabolismo , Ligando RANK/genética , Inflamación/genética , Inflamación/patología , Inflamación/metabolismo , Masculino , Macrófagos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Osteogénesis/genética , Ratones Endogámicos C57BL , Monocitos/metabolismoRESUMEN
Osteoclasts (OC) originate from either bone marrow (BM)-resident or circulating myeloid OC progenitors (OCP) expressing the receptor CX3CR1. Multiple lines of evidence argue that OCP in homeostasis and inflammation differ. We investigated the relative contributions of BM-resident and circulating OCP to osteoclastogenesis during homeostasis and fracture repair. Using CX3CR1-EGFP/TRAP tdTomato mice, we found CX3CR1 expression in mononuclear cells, but not in multinucleated TRAP+ OC. However, CX3CR1-expressing cells generated TRAP+ OC on bone within 5 d in CX3CR1CreERT2/Ai14 tdTomato reporter mice. To define the role that circulating cells play in osteoclastogenesis during homeostasis, we parabiosed TRAP tdTomato mice (CD45.2) on a C57BL/6 background with wild-type (WT) mice (CD45.1). Flow cytometry (CD45.1/45.2) demonstrated abundant blood cell mixing between parabionts after 2 wk. At 4 wk, there were numerous tdTomato+ OC in the femurs of TRAP tdTomato mice but almost none in WT mice. Similarly, cultured BM stimulated to form OC demonstrated multiple fluorescent OC in cell cultures from TRAP tdTomato mice, but not from WT mice. Finally, flow cytometry confirmed low-level engraftment of BM cells between parabionts but significant engraftment in the spleens. In contrast, during fracture repair, we found that circulating CX3CR1+ cells migrated to bone, lost expression of CX3CR1, and became OC. These data demonstrate that OCP, but not mature OC, express CX3CR1 during both homeostasis and fracture repair. We conclude that, in homeostasis mature OC derive predominantly from BM-resident OCP, whereas during fracture repair, circulating CX3CR1+ cells can become OC.
RESUMEN
We found that protease-activated receptor 1 (PAR1) was transiently induced in cultured osteoclast precursor cells. Therefore, we examined the bone phenotype and response to resorptive stimuli of PAR1-deficient (knockout [KO]) mice. Bones and bone marrow-derived cells from PAR1 KO and wild-type (WT) mice were assessed using microcomputed tomography, histomorphometry, in vitro cultures, and RT-PCR. Osteoclastic responses to TNF-α (TNF) challenge in calvaria were analyzed with and without a specific neutralizing Ab to the Notch2-negative regulatory region (N2-NRR Ab). In vivo under homeostatic conditions, there were minimal differences in bone mass or bone cells between PAR1 KO and WT mice. However, PAR1 KO myeloid cells demonstrated enhanced osteoclastogenesis in response to receptor activator of NF-κB ligand (RANKL) or the combination of RANKL and TNF. Strikingly, in vivo osteoclastogenic responses of PAR1 KO mice to TNF were markedly enhanced. We found that N2-NRR Ab reduced TNF-induced osteoclastogenesis in PAR1 KO mice to WT levels without affecting WT responses. Similarly, in vitro N2-NRR Ab reduced RANKL-induced osteoclastogenesis in PAR1 KO cells to WT levels without altering WT responses. We conclude that PAR1 functions to limit Notch2 signaling in responses to RANKL and TNF and moderates osteoclastogenic response to these cytokines. This effect appears, at least in part, to be cell autonomous because enhanced osteoclastogenesis was seen in highly purified PAR1 KO osteoclast precursor cells. It is likely that this pathway is involved in regulating the response of bone to diseases associated with inflammatory signals.
Asunto(s)
Enfermedades Óseas/inmunología , Inflamación/inmunología , Osteoclastos/fisiología , Receptor Notch2/metabolismo , Receptor PAR-1/metabolismo , Animales , Anticuerpos Neutralizantes/metabolismo , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteogénesis/genética , Ligando RANK/metabolismo , Receptor Notch2/inmunología , Receptor PAR-1/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Notch receptors are determinants of cell fate and function and play a central role in skeletal development and bone remodeling. Hajdu Cheney syndrome, a disease characterized by osteoporosis and fractures, is associated with NOTCH2 mutations resulting in a truncated stable protein and gain-of-function. We created a mouse model reproducing the Hajdu Cheney syndrome by introducing a 6955CâT mutation in the Notch2 locus leading to a Q2319X change at the amino acid level. Notch2(Q2319X) heterozygous mutants were smaller and had shorter femurs than controls; and at 1 month of age they exhibited cancellous and cortical bone osteopenia. As the mice matured, cancellous bone volume was restored partially in male but not female mice, whereas cortical osteopenia persisted in both sexes. Cancellous bone histomorphometry revealed an increased number of osteoclasts and bone resorption, without a decrease in osteoblast number or bone formation. Osteoblast differentiation and function were not affected in Notch2(Q2319X) cells. The pre-osteoclast cell pool, osteoclast differentiation, and bone resorption in response to receptor activator of nuclear factor κB ligand in vitro were increased in Notch2(Q2319X) mutants. These effects were suppressed by the γ-secretase inhibitor LY450139. In conclusion, Notch2(Q2319X) mice exhibit cancellous and cortical bone osteopenia, enhanced osteoclastogenesis, and increased bone resorption.
Asunto(s)
Enfermedades Óseas Metabólicas/fisiopatología , Resorción Ósea , Diferenciación Celular , Modelos Animales de Enfermedad , Síndrome de Hajdu-Cheney/genética , Osteoclastos/citología , Receptor Notch2/genética , Animales , Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/metabolismo , Femenino , Fémur/crecimiento & desarrollo , Fémur/metabolismo , Síndrome de Hajdu-Cheney/metabolismo , Síndrome de Hajdu-Cheney/fisiopatología , Humanos , Masculino , Ratones , Mutación Missense , Osteoclastos/metabolismo , Osteogénesis , Mutación Puntual , Receptor Notch2/metabolismoRESUMEN
Osteoclast differentiation is regulated by transcriptional, post-transcriptional, and post-translational mechanisms. MicroRNAs are fundamental post-transcriptional regulators of gene expression. The function of the miR-29 (a/b/c) family in cells of the osteoclast lineage is not well understood. In primary cultures of mouse bone marrow-derived macrophages, inhibition of miR-29a, -29b, or -29c diminished formation of TRAP (tartrate-resistant acid phosphatase-positive) multinucleated osteoclasts, and the osteoclasts were smaller. Quantitative RT-PCR showed that all miR-29 family members increased during osteoclast differentiation, in concert with mRNAs for the osteoclast markers Trap (Acp5) and cathepsin K. Similar regulation was observed in the monocytic cell line RAW264.7. In stably transduced RAW264.7 cell lines expressing an inducible miR-29 competitive inhibitor (sponge construct), miR-29 knockdown impaired osteoclastic commitment and migration of pre-osteoclasts. However, miR-29 knockdown did not affect cell viability, actin ring formation, or apoptosis in mature osteoclasts. To better understand how miR-29 regulates osteoclast function, we validated miR-29 target genes using Luciferase 3'-UTR reporter assays and specific miR-29 inhibitors. We demonstrated that miR-29 negatively regulates RNAs critical for cytoskeletal organization, including Cdc42 (cell division control protein 42) and Srgap2 (SLIT-ROBO Rho GTPase-activating protein 2). Moreover, miR-29 targets RNAs associated with the macrophage lineage: Gpr85 (G protein-coupled receptor 85), Nfia (nuclear factor I/A), and Cd93. In addition, Calcr (calcitonin receptor), which regulates osteoclast survival and resorption, is a novel miR-29 target. Thus, miR-29 is a positive regulator of osteoclast formation and targets RNAs important for cytoskeletal organization, commitment, and osteoclast function. We hypothesize that miR-29 controls the tempo and amplitude of osteoclast differentiation.
Asunto(s)
Movimiento Celular/fisiología , MicroARNs/metabolismo , Osteoclastos/metabolismo , Regiones no Traducidas 3'/fisiología , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular , Línea Celular , Supervivencia Celular/fisiología , Citoesqueleto/genética , Citoesqueleto/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD , Proteínas Activadoras de GTPasa , Técnicas de Silenciamiento del Gen , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , MicroARNs/genética , Osteoclastos/citología , Fosfatasa Ácida Tartratorresistente , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Osteoporosis is a chronic disease characterized by reduced bone mass and increased fracture risk, estimated to affect over 10 million people in the United States alone. Drugs used to treat bone loss often come with significant limitations and/or long-term safety concerns. Proteoglycan-4 (PRG4, also known as lubricin) is a mucin-like glycoprotein best known for its boundary lubricating function of articular cartilage. In more recent years, it has been shown that PRG4 has anti-inflammatory properties, contributes to the maintenance of subchondral bone integrity, and patients with PRG4 mutations are osteopenic. However, it remains unknown how PRG4 impacts mechanical and material properties of bone. Therefore, our objective was to perform a phenotyping study of bone in a Prg4 gene trap (GT) mouse (PRG4 deficient). We found that femurs of Prg4 GT mice have altered mechanical, structural, and material properties relative to wildtype littermates. Additionally, Prg4 GT mice have a greater number of calvarial osteoclasts than wildtype mice, but do not have a notable inflammatory serum profile. Finally, Prg4 GT mice do not have an altered rate of bone formation, and exogenous recombinant human PRG4 (rhPRG4) administration inhibited osteoclastogenesis in vitro, suggesting that the skeletal phenotype may be due to changes in bone resorption. Overall, this work demonstrates that PRG4 deficiency affects several integral properties of bone structure, mechanics, and skeletal cell activity, and provides the foundation and insight toward future work evaluating PRG4 as a potential therapeutic target in treating bone loss.
Asunto(s)
Osteoclastos , Osteogénesis , Proteoglicanos , Animales , Osteogénesis/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Ratones , Humanos , Masculino , Ratones Endogámicos C57BL , Cráneo , Femenino , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Fémur/efectos de los fármacosRESUMEN
Matrix-producing osteoblasts and bone-resorbing osteoclasts maintain bone homeostasis. Osteoclasts are multinucleated, giant cells of hematopoietic origin formed by the fusion of mononuclear pre-osteoclasts derived from myeloid cells. Fusion-mediated giant cell formation is critical for osteoclast maturation; without it, bone resorption is inefficient. To understand how osteoclasts differ from other myeloid lineage cells, we previously compared global mRNA expression patterns in these cells and identified genes of unknown function predominantly expressed in osteoclasts, one of which is the d2 isoform of vacuolar (H(+)) ATPase (v-ATPase) V(0) domain (Atp6v0d2). Here we show that inactivation of Atp6v0d2 in mice results in markedly increased bone mass due to defective osteoclasts and enhanced bone formation. Atp6v0d2 deficiency did not affect differentiation or the v-ATPase activity of osteoclasts. Rather, Atp6v0d2 was required for efficient pre-osteoclast fusion. Increased bone formation was probably due to osteoblast-extrinsic factors, as Atp6v02 was not expressed in osteoblasts and their differentiation ex vivo was not altered in the absence of Atp6v02. Our results identify Atp6v0d2 as a regulator of osteoclast fusion and bone formation, and provide genetic data showing that it is possible to simultaneously inhibit osteoclast maturation and stimulate bone formation by therapeutically targeting the function of a single gene.
Asunto(s)
Osteoclastos/fisiología , Osteogénesis/fisiología , Bombas de Protones/genética , ATPasas de Translocación de Protón Vacuolares/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Fusión Celular , Células Cultivadas , Humanos , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Isoformas de Proteínas , Estructura Terciaria de Proteína , Ligando RANK/farmacología , ATPasas de Translocación de Protón Vacuolares/químicaRESUMEN
Osteoclasts are derived from myeloid lineage cells, and their differentiation is supported by various osteotropic factors, including the tumor necrosis factor (TNF) family member TNF-related activation-induced cytokine (TRANCE). Genetic deletion of TRANCE or its receptor, receptor activator of nuclear factor kappaB (RANK), results in severely osteopetrotic mice with no osteoclasts in their bones. TNF receptor-associated factor (TRAF) 6 is a key signaling adaptor for RANK, and its deficiency leads to similar osteopetrosis. Hence, the current paradigm holds that TRANCE-RANK interaction and subsequent signaling via TRAF6 are essential for the generation of functional osteoclasts. Surprisingly, we show that hematopoietic precursors from TRANCE-, RANK-, or TRAF6-null mice can become osteoclasts in vitro when they are stimulated with TNF-alpha in the presence of cofactors such as TGF-beta. We provide direct evidence against the current paradigm that the TRANCE-RANK-TRAF6 pathway is essential for osteoclast differentiation and suggest the potential existence of alternative routes for osteoclast differentiation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Osteoclastos/citología , Osteoclastos/metabolismo , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Proteínas Portadoras/genética , Cartilla de ADN , Eliminación de Gen , Técnicas Histológicas , Linfotoxina-alfa/farmacología , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor 6 Asociado a Receptor de TNF/deficiencia , Factor 6 Asociado a Receptor de TNF/genéticaRESUMEN
MicroRNAs (miRNAs) are a class of short RNA molecules that mediate the regulation of gene activity through interactions with target mRNAs and subsequent silencing of gene expression. It has become increasingly clear the miRNAs regulate many diverse aspects of bone biology, including bone formation and bone resorption processes. The role of miRNAs specifically in osteoclasts has been of recent investigation, due to clinical interest in discovering new paradigms to control excessive bone resorption, as is observed in multiple conditions including aging, estrogen deprivation, cancer metastases or glucocorticoid use. Therefore understanding the role that miRNAs play during osteoclastic differentiation is of critical importance. In this review, we highlight and discuss general aspects of miRNA function in osteoclasts, including exciting data demonstrating that miRNAs encapsulated in extracellular vesicles (EVs) either originating from osteoclasts, or signaling to osteoclast from divergent sites, have important roles in bone homeostasis.
Asunto(s)
Resorción Ósea , MicroARNs , Biología , Diferenciación Celular , Humanos , MicroARNs/genética , OsteoclastosRESUMEN
The miR-29-3p family (miR-29a, miR-29b, miR-29c) of microRNAs is increased during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis. In vivo, activation of a miR-29-3p tough decoy inhibitor in Cre recombinase under the control of the lysozyme 2 promoter-expressing cells (myeloid lineage) resulted in mice displaying enhanced trabecular and cortical bone volume because of decreased bone resorption. Calcitonin receptor (Calcr) is a miR-29 target that negatively regulates bone resorption. CALCR was significantly increased in RANKL-treated miR-29-decoy osteoclasts, and these cells were more responsive to the inhibitory effect of calcitonin on osteoclast formation. Further, cathepsin K (Ctsk), which is critical for resorption, was decreased in miR-29-decoy cells. CALCR is a Gs-coupled receptor and its activation raises cAMP levels. In turn, cAMP suppresses cathepsin K, and cAMP levels were increased in miR-29-decoy cells. siRNA-mediated knock-down of Calcr in miR-29 decoy osteoclasts allowed recovery of cathepsin K levels in these cells. Overall, using a novel knockin tough decoy mouse model, we identified a new role for miR-29-3p in bone homeostasis. In RANKL-driven osteoclastogenesis, as seen in normal bone remodeling, miR-29-3p promotes resorption. Consequently, inhibition of miR-29-3p activity in the myeloid lineage leads to increased trabecular and cortical bone. Further, this study documents an interrelationship between CALCR and CTSK in osteoclastic bone resorption, which is modulated by miR-29-3p.
Asunto(s)
Huesos/metabolismo , Calcitonina/metabolismo , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Células Mieloides/metabolismo , Actinas/metabolismo , Alelos , Animales , Resorción Ósea , Hueso Esponjoso/efectos de los fármacos , Catepsina K/metabolismo , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Femenino , Homeostasis , Integrasas , Masculino , Ratones , Ratones Endogámicos C57BL , Muramidasa/química , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteogénesis/efectos de los fármacos , Precursores de Proteínas , Ligando RANK/metabolismo , Microtomografía por Rayos XRESUMEN
miRNAs play a vital role in post-transcriptional regulation of gene expression in osteoblasts and osteoclasts, and the miR-29 family is expressed in both lineages. Using mice globally expressing a miR-29-3p tough decoy, we demonstrated a modest 30-60% decrease all three miR-29-3p isoforms: miR-29a, miR-29b, and miR-29c. While the miR-29-3p decoy did not impact osteoclast number or function, the tough decoy decreased bone formation in growing mice, which led to decreased trabecular bone volume in mature animals. These data support previous in vitro studies suggesting that miR-29-3p is a positive regulator of osteoblast differentiation. In contrast, when mice were treated with intermittent parathyroid hormone (PTH1-34), inhibition of miR-29-3p augmented the effect of PTH on cortical bone anabolism, increased bone formation rate and osteoblast surface, and increased levels of Ctnnb1/ßcatenin mRNA, which is a miR-29 target. These findings highlight differences in the mechanisms controlling basal level bone formation and bone formation induced by intermittent PTH. Overall, the global miR-29-3p tough decoy model represents a modest loss-of-function, which could be a relevant tool for assessing the possible impact of systemically administered miR-29-3p inhibitors. Our studies provide a potential rationale for co-administration of PTH1-34 and miR-29-3p inhibitors, to boost bone formation in severely affected osteoporosis patients, particularly in the cortical compartment.
Asunto(s)
MicroARNs , Osteogénesis , Animales , Diferenciación Celular , Homeostasis , Humanos , Ratones , MicroARNs/genética , Osteoblastos , Hormona Paratiroidea/farmacología , Isoformas de ProteínasRESUMEN
Sexual dimorphism of the skeleton is well documented. At maturity, the male skeleton is typically larger and has a higher bone density than the female skeleton. However, the underlying mechanisms for these differences are not completely understood. In this study, we examined sexual dimorphism in the formation of osteoclasts between cells from female and male mice. We found that the number of osteoclasts in bones was greater in females. Similarly, in vitro osteoclast differentiation was accelerated in female osteoclast precursor (OCP) cells. To further characterize sex differences between female and male osteoclasts, we performed gene expression profiling of cultured, highly purified, murine bone marrow OCPs that had been treated for 3 days with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). We found that 125 genes were differentially regulated in a sex-dependent manner. In addition to genes that are contained on sex chromosomes, transcriptional sexual dimorphism was found to be mediated by genes involved in innate immune and inflammatory response pathways. Furthermore, the NF-κB-NFATc1 axis was activated earlier in female differentiating OCPs, which partially explains the differences in transcriptomic sexual dimorphism in these cells. Collectively, these findings identify multigenic sex-dependent intrinsic difference in differentiating OCPs, which results from an altered response to osteoclastogenic stimulation. In humans, these differences could contribute to the lower peak bone mass and increased risk of osteoporosis that females demonstrate relative to males. © 2021 American Society for Bone and Mineral Research (ASBMR).
Asunto(s)
Osteoclastos , Caracteres Sexuales , Animales , Células de la Médula Ósea , Diferenciación Celular , Células Cultivadas , Femenino , Factor Estimulante de Colonias de Macrófagos , Masculino , Ratones , Factores de Transcripción NFATC , Osteogénesis , Ligando RANKRESUMEN
CD55 is a glycosylphosphatidylinositol (GPI)-anchored protein that regulates complement-mediated and innate and adaptive immune responses. Although CD55 is expressed in various cell types in the bone marrow, its role in bone has not been investigated. In the current study, trabecular bone volume measured by µCT in the femurs of CD55KO female mice was increased compared to wild type (WT). Paradoxically, osteoclast number was increased in CD55KO with no differences in osteoblast parameters. Osteoclasts from CD55KO mice exhibited abnormal actin-ring formation and reduced bone-resorbing activity. Moreover, macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) treatment failed to activate Rac guanosine triphosphatase (GTPase) in CD55KO bone marrow macrophage (BMM) cells. In addition, apoptotic caspases activity was enhanced in CD55KO, which led to the poor survival of mature osteoclasts. Our results imply that CD55KO mice have increased bone mass due to defective osteoclast resorbing activity resulting from reduced Rac activity in osteoclasts. We conclude that CD55 plays an important role in the survival and bone-resorption activity of osteoclasts through regulation of Rac activity. © 2019 American Society for Bone and Mineral Research.
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Resorción Ósea , Osteoclastos , Animales , Células de la Médula Ósea , Diferenciación Celular , Femenino , Factor Estimulante de Colonias de Macrófagos , Ratones , Osteoblastos , Ligando RANK , Transducción de SeñalRESUMEN
The Rac1-specific guanosine triphosphatase (GTPase)-activating protein Slit-Robo GAP2 (Srgap2) is dramatically upregulated during RANKL-induced osteoclastogenesis. Srgap2 interacts with the cell membrane to locally inhibit activity of Rac1. In this study, we determined the role of Srgap2 in the myeloid lineage on bone homeostasis and the osteoclastic response to TNFα treatment. The bone phenotype of mice specifically lacking Srgap2 in the myeloid lineage (Srgap2 f/f :LysM-Cre; Srgap2 conditional knockout [cKO]) was investigated using histomorphometric analysis, in vitro cultures and Western blot analysis. Similar methods were used to determine the impact of TNFα challenge on osteoclast formation in Srgap2 cKO mice. Bone parameters in male Srgap2 cKO mice were unaffected. However, female cKO mice displayed higher trabecular bone volume due to increased osteoblast surface and bone formation rate, whereas osteoclastic parameters were unaltered. In vitro, cells from Srgap2 cKO had strongly enhanced Rac1 activation, but RANKL-induced osteoclast formation was unaffected. In contrast, conditioned medium from Srgap2 cKO osteoclasts promoted osteoblast differentiation and had increased levels of the bone anabolic clastokine SLIT3, providing a possible mechanism for increased bone formation in vivo. Rac1 is rapidly activated by the inflammatory cytokine TNFα. Supracalvarial injection of TNFα caused an augmented osteoclastic response in Srgap2 cKO mice. In vitro, cells from Srgap2 cKO mice displayed increased osteoclast formation in response to TNFα. We conclude that Srgap2 plays a prominent role in limiting osteoclastogenesis during inflammation through Rac1, and restricts expression of the paracrine clastokine SLIT3, a positive regulator of bone formation. © 2019 American Society for Bone and Mineral Research.
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Resorción Ósea , Proteínas Activadoras de GTPasa , Osteogénesis , Animales , Huesos , Diferenciación Celular , Femenino , Proteínas Activadoras de GTPasa/fisiología , Masculino , Proteínas de la Membrana , Ratones , Neuropéptidos , Osteoclastos , Ligando RANK , Proteína de Unión al GTP rac1RESUMEN
PURPOSE OF REVIEW: Our goal is to comprehensively review the most recent reports of microRNA (miRNA) regulation of osteoclastogenesis. We highlight validated miRNA-target interactions and their place in the signaling networks controlling osteoclast differentiation and function. RECENT FINDINGS: Using unbiased approaches to identify miRNAs of interest and reporter-3'UTR assays to validate interactions, recent studies have elucidated the impact of specific miRNA-mRNA interactions during in vitro osteoclastogenesis. There has been a focus on signaling mediators downstream of the RANK and CSF1R signaling, and genes essential for differentiation and function. For example, several miRNAs directly and indirectly target the master osteoclast transcription factor, Nfatc1 (e.g. miR-124 and miR-214) and Rho-GTPases, Cdc42 and Rac1 (e.g. miR-29 family). SUMMARY: Validating miRNA expression patterns, targets, and impact in osteoclasts and other skeletal cells is critical for understanding basic bone biology and for fulfilling the therapeutic potential of miRNA-based strategies in the treatment bone diseases.
RESUMEN
Due to deleterious side effects of currently available medications, the search for novel, safe, and effective preventive agents for improving bone health in aging continues and is urgently needed. This study aimed to determine whether dietary blackcurrants (BC), an anthocyanin-rich berry, can improve bone mass in a mouse model of age-related bone loss. Thirty-five female C57BL/6J mice, 3 months old (n = 20) and 18 months old (n = 15), were randomized to consume either a standard chow diet or a standard chow diet with 1% (w/w) BC for four months. Dual-energy X-ray absorptiometry, Micro computed tomography (µCT), and histomorphometric analyses were conducted to assess bone parameters on femurs. Biochemical assays were conducted to determine bone resorption, antioxidant activity, and inflammation in humerus homogenates. Trabecular bone volume (BV/TV) was significantly lower in aged mice compared to young mice (young control, 3.7 ± 0.4% vs aged control, 1.5 ± 0.5%, mean ± SEM (standard error of mean), p < 0.01; young BC, 5.3 ± 0.6% vs aged BC, 1.1 ± 0.3%, p < 0.001). µCT analysis revealed that BC supplementation increased trabecular BV/TV in young mice by 43.2% (p < 0.05) compared to controls. Histomorphometric analysis revealed a 50% increase, though this effect was not statistically significant (p = 0.07). The osteoblast surface increased by 82.5% in aged mice with BC compared to controls (p < 0.01). In humerus homogenates of young mice, BC consumption reduced C-telopeptide of type I collagen by 12.4% (p < 0.05) and increased glutathione peroxidase by 96.4% (p < 0.05). In humerus homogenates of aged mice, BC consumption increased catalase by 12% (p = 0.09). Aged mice had significantly elevated concentrations of tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine contributing to bone resorption, which was reduced by 43.3% with BC consumption (p = 0.06). These results suggest that early consumption of BC may protect from aging-associated bone loss.
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Envejecimiento , Hueso Esponjoso/metabolismo , Ribes/química , Absorciometría de Fotón , Animales , Antocianinas/farmacología , Densidad Ósea , Resorción Ósea/prevención & control , Dieta , Modelos Animales de Enfermedad , Femenino , Glutatión Peroxidasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteoporosis/prevención & control , Distribución Aleatoria , Factor de Necrosis Tumoral alfa/metabolismo , Microtomografía por Rayos XRESUMEN
Coupling is the process that links bone resorption to bone formation in a temporally and spatially coordinated manner within the remodeling cycle. Several lines of evidence point to the critical roles of osteoclast-derived coupling factors in the regulation of osteoblast performance. Here, we used a fractionated secretomic approach and identified the axon-guidance molecule SLIT3 as a clastokine that stimulated osteoblast migration and proliferation by activating ß-catenin. SLIT3 also inhibited bone resorption by suppressing osteoclast differentiation in an autocrine manner. Mice deficient in Slit3 or its receptor, Robo1, exhibited osteopenic phenotypes due to a decrease in bone formation and increase in bone resorption. Mice lacking Slit3 specifically in osteoclasts had low bone mass, whereas mice with either neuron-specific Slit3 deletion or osteoblast-specific Slit3 deletion had normal bone mass, thereby indicating the importance of SLIT3 as a local determinant of bone metabolism. In postmenopausal women, higher circulating SLIT3 levels were associated with increased bone mass. Notably, injection of a truncated recombinant SLIT3 markedly rescued bone loss after an ovariectomy. Thus, these results indicate that SLIT3 plays an osteoprotective role by synchronously stimulating bone formation and inhibiting bone resorption, making it a potential therapeutic target for metabolic bone diseases.
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Comunicación Autocrina , Resorción Ósea/metabolismo , Proteínas de la Membrana/metabolismo , Osteoclastos/metabolismo , Osteogénesis , Animales , Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/metabolismo , Enfermedades Óseas Metabólicas/patología , Resorción Ósea/genética , Resorción Ósea/patología , Diferenciación Celular , Femenino , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/patología , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteínas RoundaboutRESUMEN
CREM belongs to the ATF/CREB family of basic leucine zipper transcription factors. We previously showed that PTH induces ICER (inducible cAMP early repressor) in osteoblasts. ICER proteins, which are transcribed from the P2 promoter of the Crem gene, act as transcriptional attenuators. The objective of this study was to determine whether the Crem gene plays a role in the response of bone to intermittent PTH. Adult Crem knockout (KO) and wild type (WT) male mice were given daily subcutaneous injections of vehicle or hPTH(1-34) (160 mug/kg) for 10 days. Bone mineral content and density (BMC and BMD, respectively) were measured in femur and tibia by dual energy X-ray absorptiometry (DEXA). Bone morphometry was analyzed by X-ray computed microtomography (microCT) and histomorphometry. Serum bone turnover markers were measured. In vitro osteoclast formation assays were performed in bone marrow cultures treated with PTH or the combination of RANKL and M-CSF. KO mice had slightly higher basal bone mass than wild type mice. PTH treatment increased tibial BMC and BMD to a greater extent in WT mice compared to KO mice. PTH increased both cortical area and trabecular bone area in WT but not in KO femurs. PTH increased the bone formation rate and percent osteoblast surface to the same extent in femurs of WT and KO mice but increased osteoclast parameters and calvarial porosity to a greater extent in KO mice. PTH increased serum osteocalcin levels to the same extent in WT and KO mice. PTH-induced osteoclast formation was 2-fold greater in bone marrow cultures from KO mice. Collectively, our data suggest that the CREM deficiency in mice alters the response of bone to intermittent PTH treatment such that osteoclastogenesis is increased. Crem gene may specify the anabolic response to intermittent PTH treatment by restraining PTH-induced osteoclastogenesis.
Asunto(s)
Huesos/efectos de los fármacos , Huesos/metabolismo , Modulador del Elemento de Respuesta al AMP Cíclico/deficiencia , Hormona Paratiroidea/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Animales , Densidad Ósea/efectos de los fármacos , Resorción Ósea/etiología , Células Cultivadas , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Esquema de Medicación , Inyecciones Subcutáneas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacosRESUMEN
Osteogenesis imperfecta (OI) is a disease caused by defects in type I collagen production that results in brittle bones. While the pathology is mainly caused by defects in the osteoblast lineage, there is also elevated bone resorption by osteoclasts resulting in high bone turnover in severe forms of the disease. Osteoclasts originate from hematopoietic myeloid cells, however changes in hematopoiesis have not been previously documented in OI. In this study, we evaluated hematopoietic lineage distribution and osteoclast progenitor cell frequency in bone marrow, spleen and peripheral blood of osteogenesis imperfecta murine (OIM) mice, a model of severe OI. We found splenomegaly in all ages examined, and expansion of myeloid lineage cells (CD11b+) in bone marrow and spleen of 7-9week old male OIM animals. OIM spleens also showed an increased frequency of purified osteoclast progenitors. This phenotype is suggestive of chronic inflammation. Isolated osteoclast precursors from both spleen and bone marrow formed osteoclasts more rapidly than wild-type controls. We found that serum TNFα levels were increased in OIM, as was IL1α in OIM females. We targeted inflammation therapeutically by treating growing animals with murine TNFR2:Fc, a compound that blocks TNFα activity. Anti-TNFα treatment marginally decreased spleen mass in OIM females, but failed to reduce bone resorption, or improve bone parameters or fracture rate in OIM animals. We have demonstrated that OIM mice have changes in their hematopoietic system, and form osteoclasts more rapidly even in the absence of OI osteoblast signals, however therapy targeting TNFα did not improve disease parameters.