RESUMEN
Deletion of c-Src, a ubiquitously expressed tyrosine kinase, results in osteoclast dysfunction and osteopetrosis, in which bones harden into "stone." In contrast, deletion of the genes encoding other members of the Src family kinase (SFK) fails to produce an osteopetrotic phenotype. This suggests that c-Src performs a unique function in the osteoclast that cannot be compensated for by other SFKs. We aimed to identify the molecular basis of this unique role in osteoclasts and bone resorption. We found that c-Src, Lyn, and Fyn were the most highly expressed SFKs in WT osteoclasts, whereas Hck, Lck, Blk, and Fgr displayed low levels of expression. Formation of the podosome belt, clusters of unique actin assemblies, was disrupted in src-/- osteoclasts; introduction of constitutively activated SFKs revealed that only c-Src and Fyn could restore this process. To identify the key structural domains responsible, we constructed chimeric Src-Hck and Src-Lyn constructs in which the unique, SH3, SH2, or catalytic domains had been swapped. We found that the Src unique, SH3, and kinase domains were each crucial to establish Src functionality. The SH2 domain could however be substituted with Lyn or Hck SH2 domains. Furthermore, we demonstrate that c-Src's functionality is, in part, derived from an SH3-proximal proline-rich domain interaction with c-Cbl, leading to phosphorylation of c-Cbl Tyr700. These data help clarify Src's unique functionality in the organization of the cytoskeleton in osteoclasts, required for efficient bone resorption and explain why c-Src cannot be replaced, in osteoclasts, by other SFKs.
Asunto(s)
Osteoclastos/metabolismo , Podosomas/metabolismo , Dominios Homologos src , Familia-src Quinasas/metabolismo , Animales , Resorción Ósea/genética , Resorción Ósea/metabolismo , Diferenciación Celular , Células HEK293 , Humanos , Ratones , Osteoclastos/citología , Familia-src Quinasas/genéticaRESUMEN
Lactation induces bone loss to provide sufficient calcium in the milk, a process that involves osteoclastic bone resorption but also osteocytes and perilacunar resorption. The exact mechanisms by which osteocytes contribute to bone loss remain elusive. Osteocytes express genes required in osteoclasts for bone resorption, including cathepsin K (Ctsk), and lactation elevates their expression. We show that Ctsk deletion in osteocytes prevented the increase in osteocyte lacunar area seen during lactation, as well as the effects of lactation to increase osteoclast numbers and decrease trabecular bone volume, cortical thickness and mechanical properties. In addition, Ctsk deletion in osteocytes increased bone Parathyroid Hormone related Peptide (PTHrP), prevented the decrease in serum Parathyroid Hormone (PTH) induced by lactation, but amplified the increase in serum 1,25(OH)2D. The net result of these changes is to maintain serum and milk calcium levels in the normal range, ensuring normal offspring skeletal development. Our studies confirm the fundamental role of osteocytic perilacunar remodeling in physiological states of lactation and provides genetic evidence that osteocyte-derived Ctsk contributes not only to osteocyte perilacunar remodeling, but also to the regulation of PTH, PTHrP, 1,25-Dyhydroxyvitamin D (1,25(OH)2D), osteoclastogenesis and bone loss in response to the high calcium demand associated with lactation.
Asunto(s)
Catepsina K/fisiología , Lactancia/fisiología , Osteocitos/fisiología , Osteoporosis/etiología , Hormona Paratiroidea/sangre , Animales , Remodelación Ósea/fisiología , Resorción Ósea/etiología , Resorción Ósea/prevención & control , Calcio/análisis , Catepsina K/deficiencia , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos C57BL , Osteogénesis , Osteoporosis/prevención & controlRESUMEN
The adhesion of osteoclasts (OCs) to bone and bone resorption require the assembly of specific F-actin adhesion structures, the podosomes, and their dense packing into a sealing zone. The OC-specific formation of the sealing zone requires the interaction of microtubule (MT) + ends with podosomes. Here, we deleted cofilin, a cortactin (CTTN)- and actin-binding protein highly expressed in OCs, to determine if it acts downstream of the MT-CTTN axis to regulate actin polymerization in podosomes. Conditional deletion of cofilin in OCs in mice, driven by the cathepsin K promoter (Ctsk-Cre), impaired bone resorption in vivo, increasing bone density. In vitro, OCs were not able to organize podosomes into peripheral belts. The MT network was disorganized, MT stability was decreased, and cell migration impaired. Active cofilin stabilizes MTs and allows podosome belt formation, whereas MT disruption deactivates cofilin via phosphorylation. Cofilin interacts with CTTN in podosomes and phosphorylation of either protein disrupts this interaction, which is critical for belt stabilization and for the maintenance of MT dynamic instability. Accordingly, active cofilin was required to rescue the OC cytoskeletal phenotype in vitro. These findings suggest that the patterning of podosomes into a sealing zone involves the dynamic interaction between cofilin, CTTN, and the MTs + ends. This interaction is critical for the functional organization of OCs and for bone resorption. © 2016 American Society for Bone and Mineral Research.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Resorción Ósea/metabolismo , Resorción Ósea/patología , Cortactina/metabolismo , Osteoclastos/metabolismo , Podosomas/metabolismo , Acetilación , Animales , Resorción Ósea/diagnóstico por imagen , Eliminación de Gen , Marcación de Gen , Histona Desacetilasa 6/metabolismo , Humanos , Masculino , Ratones , Microtúbulos/metabolismo , Fenotipo , Fosforilación , Unión Proteica , Microtomografía por Rayos XRESUMEN
Biphasic calcium phosphates (BCPs), consisting of hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP), exhibit good biocompatibility and osteoconductivity, maintaining a balance between resorption of the biomaterial and formation of new bone. We tested whether the chemical composition and/or the microstructure of BCPs affect osteoclasts (OCs) differentiation and/or their ability to crosstalk with osteoblasts (OBs). To this aim, OCs were cultured on BCPs with HA content of 5, 20 or 60% and their differentiation and activity were assessed. We found that OC differentiation is partially impaired by increased HA content, but not by the presence of micropores within BCP scaffolds, as indicated by TRAP staining and gene profile expression. We then investigated whether the biomaterial-induced changes in OC differentiation also affect their ability to crosstalk with OBs and regulate OB function. We found that BCPs with low percentage of HA favored the expression of positive coupling factors, including sphingosine-kinase 1 (SPHK1) and collagen triple helix repeat containing 1 (Cthrc1). In turn, the increase of these secreted coupling factors promotes OB differentiation and function. All together our studies suggest that the chemical composition of biomaterials affects not only the differentiation and activity of OCs but also their potential to locally regulate bone formation.
Asunto(s)
Hidroxiapatitas/química , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Animales , Comunicación Celular , Diferenciación Celular , Células Cultivadas , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Osteoblastos/citología , Osteoclastos/citología , Osteogénesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
Cell-cell fusion is an evolutionarily conserved process that leads to the formation of multinucleated myofibers, syncytiotrophoblasts and osteoclasts, allowing their respective functions. Although cell-cell fusion requires the presence of fusogenic membrane proteins and actin-dependent cytoskeletal reorganization, the precise machinery allowing cells to fuse is still poorly understood. Using an inducible knockout mouse model to generate dynamin 1- and 2-deficient primary osteoclast precursors and myoblasts, we found that fusion of both cell types requires dynamin. Osteoclast and myoblast cell-cell fusion involves the formation of actin-rich protrusions closely associated with clathrin-mediated endocytosis in the apposed cell. Furthermore, impairing endocytosis independently of dynamin also prevented cell-cell fusion. Since dynamin is involved in both the formation of actin-rich structures and in endocytosis, our results indicate that dynamin function is central to the osteoclast precursors and myoblasts fusion process, and point to an important role of endocytosis in cell-cell fusion.
Asunto(s)
Dinamina II/genética , Dinamina I/genética , Endocitosis/fisiología , Mioblastos/citología , Osteoclastos/citología , Animales , Resorción Ósea/genética , Resorción Ósea/metabolismo , Comunicación Celular/fisiología , Diferenciación Celular , Fusión Celular , Membrana Celular/metabolismo , Células Cultivadas , Endocitosis/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño , Células MadreRESUMEN
In osteoclasts (OCs) podosomes are organized in a belt, a feature critical for bone resorption. Although microtubules (MTs) promote the formation and stability of the belt, the MT and/or podosome molecules that mediate the interaction of the two systems are not identified. Because the growing "plus" ends of MTs point toward the podosome belt, plus-end tracking proteins (+TIPs) might regulate podosome patterning. Among the +TIPs, EB1 increased as OCs matured and was enriched in the podosome belt, and EB1-positive MTs targeted podosomes. Suppression of MT dynamic instability, displacement of EB1 from MT ends, or EB1 depletion resulted in the loss of the podosome belt. We identified cortactin as an Src-dependent interacting partner of EB1. Cortactin-deficient OCs presented a defective MT targeting to, and patterning of, podosomes and reduced bone resorption. Suppression of MT dynamic instability or EB1 depletion increased cortactin phosphorylation, decreasing its acetylation and affecting its interaction with EB1. Thus, dynamic MTs and podosomes interact to control bone resorption.
Asunto(s)
Estructuras de la Membrana Celular/metabolismo , Cortactina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Osteoclastos/metabolismo , Familia-src Quinasas/metabolismo , Acetilación , Actinas/metabolismo , Animales , Línea Celular , Células Cultivadas , Cortactina/genética , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Immunoblotting , Cinética , Ratones , Ratones Noqueados , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/genética , Osteoclastos/citología , Fosforilación , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Familia-src Quinasas/genéticaRESUMEN
Bone remodeling requires osteoclasts to generate and maintain an acidified resorption compartment between the apical membrane and the bone surface to solubilize hydroxyapatite crystals within the bone matrix. This acidification process requires (i) apical proton secretion by a vacuolar H(+)-ATPase, (ii) actin cytoskeleton reorganization into a podosome belt that forms a gasket to restrict lacunar acid leakage, and (iii) basolateral chloride uptake and bicarbonate extrusion by an anion exchanger to provide Cl(-) permissive for apical acid secretion while preventing cytoplasmic alkalinization. Here we show that osteoclast-targeted deletion in mice of solute carrier family 4 anion exchanger member 2 (Slc4a2) results in osteopetrosis. We further demonstrate a previously unrecognized consequence of SLC4A2 loss of function in the osteoclast: dysregulation of calpain-dependent podosome disassembly, leading to abnormal actin belt formation, cell spreading, and migration. Rescue of SLC4A2-deficient osteoclasts with functionally defined mutants of SLC4A2 indicates regulation of actin cytoskeletal reorganization by anion-exchange activity and intracellular pH, independent of SLC4A2's long N-terminal cytoplasmic domain. These data suggest that maintenance of intracellular pH in osteoclasts through anion exchange regulates the actin superstructures required for bone resorption.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Transporte de Anión/metabolismo , Antiportadores/metabolismo , Calpaína/metabolismo , Antiportadores de Cloruro-Bicarbonato/metabolismo , Osteoclastos/metabolismo , Animales , Proteínas de Transporte de Anión/deficiencia , Proteínas de Transporte de Anión/genética , Antiportadores/deficiencia , Antiportadores/genética , Células Cultivadas , Antiportadores de Cloruro-Bicarbonato/deficiencia , Antiportadores de Cloruro-Bicarbonato/genética , Concentración de Iones de Hidrógeno , Ratones , Ratones Noqueados , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Osteoclastos/patología , Osteopetrosis/genética , Osteopetrosis/metabolismo , Osteopetrosis/patología , Proteínas SLC4ARESUMEN
Cathepsin K (CTSK) is secreted by osteoclasts to degrade collagen and other matrix proteins during bone resorption. Global deletion of Ctsk in mice decreases bone resorption, leading to osteopetrosis, but also increases the bone formation rate (BFR). To understand how Ctsk deletion increases the BFR, we generated osteoclast- and osteoblast-targeted Ctsk knockout mice using floxed Ctsk alleles. Targeted ablation of Ctsk in hematopoietic cells, or specifically in osteoclasts and cells of the monocyte-osteoclast lineage, resulted in increased bone volume and BFR as well as osteoclast and osteoblast numbers. In contrast, targeted deletion of Ctsk in osteoblasts had no effect on bone resorption or BFR, demonstrating that the increased BFR is osteoclast dependent. Deletion of Ctsk in osteoclasts increased their sphingosine kinase 1 (Sphk1) expression. Conditioned media from Ctsk-deficient osteoclasts, which contained elevated levels of sphingosine-1-phosphate (S1P), increased alkaline phosphatase and mineralized nodules in osteoblast cultures. An S1P1,3 receptor antagonist inhibited these responses. Osteoblasts derived from mice with Ctsk-deficient osteoclasts had an increased RANKL/OPG ratio, providing a positive feedback loop that increased the number of osteoclasts. Our data provide genetic evidence that deletion of CTSK in osteoclasts enhances bone formation in vivo by increasing the generation of osteoclast-derived S1P.
Asunto(s)
Catepsina K/deficiencia , Lisofosfolípidos/metabolismo , Osteoclastos/enzimología , Osteogénesis/fisiología , Esfingosina/análogos & derivados , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Resorción Ósea/enzimología , Resorción Ósea/patología , Resorción Ósea/prevención & control , Catepsina K/antagonistas & inhibidores , Catepsina K/genética , Diferenciación Celular , Retroalimentación Fisiológica , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Osteoblastos/citología , Osteoblastos/enzimología , Osteoclastos/citología , Osteogénesis/genética , Osteoprotegerina/metabolismo , Ligando RANK/metabolismo , Esfingosina/metabolismoRESUMEN
Jansen metaphyseal chondrodysplasia (JMC) is caused by a constitutively activating mutation of the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (PTHR1) and is characterized by widening of the metaphyses, reduction of long bone length, and short stature. A transgenic mouse expressing this mutation under the collagen α1(II) promoter has been generated to investigate the mechanisms responsible for this chondrodysplasia. We recently identified zinc finger protein 521 (Zfp521) as a downstream target gene of PTHrP signaling. Interestingly, loss of Zfp521 from chondrocytes leads to reduced cell proliferation and increased differentiation in the growth plate. Thus, we hypothesized that specifically ablating Zfp521 from Jansen chondrocytes could sufficiently rescue the chondrodysplasia phenotype. Our results show that Zfp521 expression is up-regulated in Jansen mouse growth plate chondrocytes and that PTHR1 is required for Zfp521 expression. Its ablation from Jansen chondrocytes restored normal cell differentiation, thus initiating chondrocyte apoptosis at the chondro-osseous junction, leading to partial rescue of endochondral bone formation shown by proper bone length. This study provides the first genetic evidence that Zfp521 is required downstream of PTHR1 signaling to act on chondrocyte proliferation, differentiation, and cell death.
Asunto(s)
Placa de Crecimiento/crecimiento & desarrollo , Osteocondrodisplasias/metabolismo , Factores de Transcripción/metabolismo , Animales , Desarrollo Óseo , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Eliminación de Gen , Regulación de la Expresión Génica/fisiología , Genotipo , Placa de Crecimiento/metabolismo , Ratones , Ratones Noqueados , Fenotipo , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
Runx2 is indispensable for osteoblast lineage commitment and early differentiation but also blocks osteoblast maturation, thereby causing bone loss in Runx2 transgenic mice. Zinc finger protein 521 (Zfp521) antagonizes Runx2 in vivo. Eliminating one Zfp521 allele mitigates the cleidocranial dysplasia-like phenotype of newborn Runx2(+/-) mice, whereas overexpressing Zfp521 exacerbates it. Overexpressing Zfp521 also reverses the severe osteopenia of adult Runx2 transgenic mice. Zfp521 binds to both Runx2 and histone deacetylase 3 (HDAC3), promotes their association, and antagonizes Runx2 transcriptional activity in an HDAC3-dependent manner. Mutating the Zfp521 zinc finger domains 6 and 26 reduces the binding of Zfp521 to Runx2 and inhibition of Runx2 activity. These data provide evidence that Zfp521 antagonizes Runx2 in vivo and thereby regulates two stages of osteoblast development, early during mesenchymal cell lineage commitment and later during osteoblast maturation. Thus, the balance and molecular interplay between Zfp521 and Runx2 contribute to the control of osteoblast differentiation, skeletal development, and bone homeostasis.
Asunto(s)
Desarrollo Óseo/fisiología , Remodelación Ósea/fisiología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Unión al ADN/metabolismo , Histona Desacetilasas/metabolismo , Osteogénesis/fisiología , Factores de Transcripción/genética , Fosfatasa Alcalina/metabolismo , Animales , Animales Recién Nacidos , Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/metabolismo , Huesos/anomalías , Huesos/embriología , Huesos/metabolismo , Huesos/patología , Calcificación Fisiológica/genética , Recuento de Células , Diferenciación Celular/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Proteínas de Unión al ADN/genética , Proteínas de la Matriz Extracelular/genética , Fracturas Espontáneas/genética , Expresión Génica/genética , Heterocigoto , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos , Ratones Transgénicos , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/genética , Osteopontina/genética , Osteoprotegerina/genética , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Ligando RANK/genética , Ratas , Elementos de Respuesta/fisiología , Factor de Transcripción Sp7 , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Dedos de Zinc/fisiologíaRESUMEN
In the growth plate, the interplay between parathyroid hormone-related peptide (PTHrP) and Indian hedgehog (Ihh) signaling tightly regulates chondrocyte proliferation and differentiation during longitudinal bone growth. We found that PTHrP increases the expression of Zfp521, a zinc finger transcriptional coregulator, in prehypertrophic chondrocytes. Mice with chondrocyte-targeted deletion of Zfp521 resembled PTHrP(-/-) and chondrocyte-specific PTHR1(-/-) mice, with decreased chondrocyte proliferation, early hypertrophic transition, and reduced growth plate thickness. Deleting Zfp521 increased expression of Runx2 and Runx2 target genes, and decreased Cyclin D1 and Bcl-2 expression while increasing Caspase-3 activation and apoptosis. Zfp521 associated with Runx2 in chondrocytes, antagonizing its activity via an HDAC4-dependent mechanism. PTHrP failed to upregulate Cyclin D1 and to antagonize Runx2, Ihh, and collagen X expression when Zfp521 was absent. Thus, Zfp521 is an important PTHrP target gene that regulates growth plate chondrocyte proliferation and differentiation.
Asunto(s)
Condrocitos/metabolismo , Placa de Crecimiento/patología , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrocitos/enzimología , Condrocitos/patología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Placa de Crecimiento/metabolismo , Histona Desacetilasas/metabolismo , Hipertrofia , Ratones , Ratones Noqueados , Osteogénesis/efectos de los fármacos , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
c-Cbl and Cbl-b are highly conserved adaptor proteins that participate in integrin signaling, regulating cytoskeletal organization, motility, and bone resorption. Deletion of both c-Cbl and Cbl-b in mice leads to embryonic lethality, indicating that the two proteins perform essential redundant functions. To examine the redundant actions of c-Cbl and Cbl-b in osteoclasts, we depleted c-Cbl in Cbl-b(-/-) osteoclasts by using a short hairpin RNA. Depleting both Cbl proteins disrupted both the podosome belt and the microtubule network and decreased bone-resorbing activity. Stabilizing the microtubules with paclitaxel or inhibiting histone deacetylase 6 (HDAC6), which destabilizes microtubules by deacetylating beta-tubulin, protected both the microtubule network and the podosome belt. Examination of the mechanism involved demonstrated that the conserved four-helix bundle of c-Cbl's tyrosine kinase binding domain bound to beta-tubulin, and both c-Cbl and Cbl-b displaced HDAC6. In addition to the effects on microtubules and the podosome belt, depleting both Cbls significantly increased the levels of the proapoptotic protein Bim and apoptosis relative to the levels induced by eliminating either protein alone. Thus, both c-Cbl and Cbl-b promote bone resorption via the stabilization of microtubules, allowing the formation of the podosome belt in osteoclasts, and by promoting osteoclast survival.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis , Histona Desacetilasas/metabolismo , Integrinas/metabolismo , Microtúbulos/metabolismo , Osteoclastos/enzimología , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Tubulina (Proteína)/metabolismo , Acetilación , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Biocatálisis , Resorción Ósea/metabolismo , Línea Celular , Citoprotección , Histona Desacetilasa 6 , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Proteínas de la Membrana/metabolismo , Osteoclastos/patología , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin's GTPase activity, and reduces Pyk2 Y402 phosphorylation in a GTPase-dependent manner, leading to decreased Src binding to Pyk2. Overexpressing dynamin decreased the macrophage colony-stimulating factor- and adhesion-induced phosphorylation of Pyk2 in osteoclastlike cells, suggesting that dynamin is likely to regulate Src-Pyk2 binding downstream of integrins and growth factor receptors with important cellular consequences. Furthermore, catalytically active Src promotes dynamin-Pyk2 association, and mutating specific Src-phosphorylated tyrosine residues in dynamin blunts the dynamin-induced decrease in Pyk2 phosphorylation. Thus, since Src binds to Pyk2 through its interaction with phospho-Y402, our results suggest that Src activates a negative-feedback loop downstream of integrin engagement and other stimuli by promoting both the binding of dynamin to Pyk2-containing complexes and the dynamin-dependent decrease in Pyk2 Y402 phosphorylation, ultimately leading to the dissociation of Src from Pyk2.
Asunto(s)
Dinaminas/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Osteoclastos/fisiología , Familia-src Quinasas/metabolismo , Animales , Células Cultivadas , Dinaminas/genética , Quinasa 2 de Adhesión Focal/genética , Humanos , Factor Estimulante de Colonias de Macrófagos/metabolismo , Ratones , Osteoclastos/citología , Fosforilación , Unión Proteica , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tirosina/metabolismo , Familia-src Quinasas/genéticaRESUMEN
Cbl proteins are multifunctional adaptor molecules that modulate cellular activity by targeting the ubiquitylating system, endocytic complexes, and other effectors to a wide variety of regulatory proteins, especially activated receptor and nonreceptor tyrosine kinases. Cbl and Cbl-b perform unique functions in various cells, in addition to redundant functions that are required for embryonic development. We previously showed that eliminating Cbl impaired osteoclast motility, which modestly delayed embryonic bone development. We now report that Cbl-b(-/-) mice are osteopenic, because of increased bone resorption with little compensating increase in bone formation. In vitro bone-resorbing activity and differentiation of osteoclast-like cells (OCLs) were increased, as were some RANKL-induced signaling events (activation of NF-kappaB and the mitogen-activated protein kinases extracellular signal-regulated kinase [ERK] and p38), suggesting that specific RANKL-activated mechanisms contribute to the increased rate of differentiation and bone-resorbing activity. Re-expressing Cbl-b in Cbl-b(-/-) OCLs normalized the increased bone-resorbing activity and overexpressing Cbl-b in wildtype OCLs inhibited bone resorption. Cbl was without effect in either wildtype or Cbl-b(-/-) OCLs. Functional tyrosine kinase binding (TKB) and RING finger domains were required for the rescue by Cbl-b. Thus, both Cbl and Cbl-b perform regulatory functions in osteoclasts that are unique to one or the other protein (i.e., functions that cannot be compensated by the other homolog). One of Cbl-b's unique functions in osteoclasts is to downregulate bone resorption.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Enfermedades Óseas Metabólicas/metabolismo , Resorción Ósea/metabolismo , Osteoclastos/metabolismo , Proteínas Proto-Oncogénicas c-cbl , Animales , Desarrollo Óseo/genética , Enfermedades Óseas Metabólicas/genética , Resorción Ósea/genética , Diferenciación Celular/genética , Movimiento Celular , Embrión de Mamíferos/metabolismo , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Osteoclastos/patología , Estructura Terciaria de Proteína , Ligando RANK/genética , Ligando RANK/metabolismo , Transducción de Señal/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Obesity and osteoporosis are major health issues affecting millions of individuals. Transgenic mice overexpressing DeltaFosB, an activator protein-1 transcription factor, under the control of the enolase 2 (ENO2) promoter exhibit both an increase in bone density and a decrease in adipose mass. Here we demonstrate that DeltaFosB overexpression increases fatty-acid oxidation and energy expenditure, leading to a decrease in adipocyte size and adipose mass. In addition, the ENO2-DeltaFosB mice exhibit increased insulin sensitivity and glucose tolerance. Targeted overexpression of DeltaFosB in adipocytes using the adipocyte protein 2 promoter failed to induce changes in fat or in bone, showing that the effect on metabolic activity is not due to cell-autonomous effects of DeltaFosB within adipocytes. Detailed analysis of the ENO2-DeltaFosB mice demonstrated that energy expenditure was increased in muscle, independent of locomotor activity. These findings provide evidence that signaling downstream of DeltaFosB is a potential target for not only osteoporosis but also obesity and diabetes.
Asunto(s)
Densidad Ósea/genética , Metabolismo Energético/efectos de los fármacos , Insulina/farmacología , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-fos/genética , Animales , Ingestión de Energía , Ácidos Grasos/metabolismo , Ratones , Obesidad/genética , Tamaño de los Órganos/genética , Osteocalcina/metabolismo , Fosfopiruvato Hidratasa/genética , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-fos/deficienciaRESUMEN
Zfp521, a 30 C2H2 Kruppel-like zinc finger protein, is expressed at high levels at the periphery of early mesenchymal condensations prefiguring skeletal elements and in all developing bones in the perichondrium and periosteum, in osteoblast precursors and osteocytes, and in chondroblast precursors and growth plate prehypertrophic chondrocytes. Zfp521 expression in cultured mesenchymal cells is decreased by BMP-2 and increased by PTHrP, which promote and antagonize osteoblast differentiation, respectively. In vitro, Zfp521 overexpression reduces the expression of several downstream osteoblast marker genes and antagonizes osteoblast differentiation. Zfp521 binds Runx2 and represses its transcriptional activity, and Runx2 dose-dependently rescues Zfp521's inhibition of osteoblast differentiation. In contrast, osteocalcin promoter-targeted overexpression of Zfp521 in osteoblasts in vivo results in increased bone formation and bone mass. We propose that Zfp521 regulates the rate of osteoblast differentiation and bone formation during development and in the mature skeleton, in part by antagonizing Runx2.
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Diferenciación Celular/fisiología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Unión al ADN/metabolismo , Osteoblastos/citología , Osteogénesis/fisiología , Factores de Transcripción/metabolismo , Animales , Northern Blotting , Células Cultivadas , Proteínas de Unión al ADN/genética , Técnica del Anticuerpo Fluorescente , Hibridación in Situ , Técnicas In Vitro , Proteína del Locus del Complejo MDS1 y EV11 , Ratones , Ratones Transgénicos , Osteoblastos/metabolismo , Proto-Oncogenes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , TransfecciónRESUMEN
INTRODUCTION: Activator protein (AP)-1 family members play important roles in the development and maintenance of the adult skeleton. Transgenic mice that overexpress the naturally occurring DeltaFosB splice variant of FosB develop severe osteosclerosis. Translation of Deltafosb mRNA produces both DeltaFosB and a further truncated isoform (Delta2DeltaFosB) that lacks known transactivation domains but, like DeltaFosB, induces increased expression of osteoblast marker genes. MATERIALS AND METHODS: To test Delta2DeltaFosB's ability to induce bone formation in vivo, we generated transgenic mice that overexpress only Delta2DeltaFosB using the enolase 2 (ENO2) promoter-driven bitransgenic Tet-Off system. RESULTS: Despite Delta2DeltaFosB's failure to induce transcription of an AP-1 reporter gene, the transgenic mice exhibited both the bone and the fat phenotypes seen in the ENO2-DeltaFosB mice. Both DeltaFosB and Delta2DeltaFosB activated the BMP-responsive Xvent-luc reporter gene and increased Smad1 expression. Delta2DeltaFosB enhanced BMP-induced Smad1 phosphorylation and the translocation of phospho-Smad1 (pSmad1) to the nucleus more efficiently than DeltaFosB and showed a reduced induction of inhibitory Smad6 expression. CONCLUSIONS: DeltaFosB's AP-1 transactivating function is not needed to induce increased bone formation, and Delta2DeltaFosB may act, at least in part, by increasing Smad1 expression, phosphorylation, and translocation to the nucleus.
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Osteoclastos/metabolismo , Osteosclerosis/genética , Isoformas de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-fos/fisiología , Proteína Smad1/metabolismo , Factor de Transcripción AP-1/fisiología , Empalme Alternativo , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/genéticaRESUMEN
The protein tyrosine kinase Pyk2 is highly expressed in osteoclasts, where it is primarily localized in podosomes. Deletion of Pyk2 in mice leads to mild osteopetrosis due to impairment in osteoclast function. Pyk2-null osteoclasts were unable to transform podosome clusters into a podosome belt at the cell periphery; instead of a sealing zone only small actin rings were formed, resulting in impaired bone resorption. Furthermore, in Pyk2-null osteoclasts, Rho activity was enhanced while microtubule acetylation and stability were significantly reduced. Rescue experiments by ectopic expression of wild-type or a variety of Pyk2 mutants in osteoclasts from Pyk2(-/-) mice have shown that the FAT domain of Pyk2 is essential for podosome belt and sealing zone formation as well as for bone resorption. These experiments underscore an important role of Pyk2 in microtubule-dependent podosome organization, bone resorption, and other osteoclast functions.
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Densidad Ósea/fisiología , Quinasa 2 de Adhesión Focal/metabolismo , Microtúbulos/metabolismo , Osteoclastos/ultraestructura , Animales , Resorción Ósea/metabolismo , Resorción Ósea/patología , Células Cultivadas , Quinasa 2 de Adhesión Focal/genética , Ratones , Ratones Noqueados , Osteoclastos/metabolismo , Osteopetrosis/metabolismo , Osteopetrosis/patología , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Osteoclast motility is thought to depend on rapid podosome assembly and disassembly. Both mu-calpain and m-calpain, which promote the formation and disassembly of focal adhesions, were observed in the podosome belt of osteoclasts. Calpain inhibitors disrupted the podosome belt, blocked the constitutive cleavage of the calpain substrates filamin A, talin, and Pyk2, which are enriched in the podosome belt, induced osteoclast retraction, and reduced osteoclast motility and bone resorption. The motility and resorbing activity of mu-calpain(-/-) osteoclast-like cells were also reduced, indicating that mu-calpain is required for normal osteoclast activity. Histomorphometric analysis of tibias from mu-calpain(-/-) mice revealed increased osteoclast numbers and decreased trabecular bone volume that was apparent at 10 weeks but not at 5 weeks of age. In vitro studies suggested that the increased osteoclast number in the mu-calpain(-/-) bones resulted from increased osteoclast survival, not increased osteoclast formation. Calcitonin disrupted the podosome ring, induced osteoclast retraction, and reduced osteoclast motility and bone resorption in a manner similar to the effects of calpain inhibitors and had no further effect on these parameters when added to osteoclasts pretreated with calpain inhibitors. Calcitonin inhibited the constitutive cleavage of a fluorogenic calpain substrate and transiently blocked the constitutive cleavage of filamin A, talin, and Pyk2 by a protein kinase C-dependent mechanism, demonstrating that calcitonin induces the inhibition of calpain in osteoclasts. These results indicate that calpain activity is required for normal osteoclast activity and suggest that calcitonin inhibits osteoclast bone resorbing activity in part by down-regulating calpain activity.
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Calcitonina/fisiología , Calpaína/fisiología , Movimiento Celular/fisiología , Osteoclastos/fisiología , Animales , Enfermedades Óseas Metabólicas/fisiopatología , Resorción Ósea/fisiopatología , Calpaína/genética , Supervivencia Celular , Regulación hacia Abajo , Humanos , Ratones , ConejosRESUMEN
Podosomes are highly dynamic actin-containing adhesion structures found in osteoclasts, macrophages, and Rous sarcoma virus (RSV)-transformed fibroblasts. After integrin engagement, Pyk2 recruits Src and the adaptor protein Cbl, forming a molecular signaling complex that is critical for cell migration, and deletion of any molecule in this complex disrupts podosome ring formation and/or decreases osteoclast migration. Dynamin, a GTPase essential for endocytosis, is also involved in actin cytoskeleton remodeling and is localized to podosomes where it has a role in actin turnover. We found that dynamin colocalizes with Cbl in the actin-rich podosome belt of osteoclasts and that dynamin forms a complex with Cbl in osteoclasts and when overexpressed in 293VnR or SYF cells. The association of dynamin with Cbl in osteoclasts was decreased by Src tyrosine kinase activity and we found that destabilization of the dynamin-Cbl complex involves the recruitment of Src through the proline-rich domain of Cbl. Overexpression of dynamin increased osteoclast bone resorbing activity and migration, whereas overexpression of dynK44A decreased osteoclast resorption and migration. These studies suggest that dynamin, Cbl, and Src coordinately participate in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton, leading to changes in osteoclast adhesion, migration, and resorption.