Asunto(s)
Osteólisis/fisiopatología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Artritis/complicaciones , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Diferenciación Celular , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiología , Osteoclastos/fisiología , Osteólisis/etiología , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Relación Estructura-ActividadRESUMEN
Mucopolysaccharidosis type VII (MPS VII) is a heritable lysosomal storage disease caused by a deficiency in beta-glucuronidase (GUSB) activity, leading to progressive accumulation of undegraded glycosaminoglycans in many tissues. Clinical features include growth and mental retardation, hearing and visual defects, shortened lifespan, and skeletal deformities. A murine model of MPS VII has been described that shares many of the manifestations of the human disease, including the skeletal dysplasia. In this study we describe abnormalities in the cellular morphology and function of osteoclasts and a localized defect in bone formation rate in the MPS VII mouse. Ultrastructural analysis revealed that MPS VII osteoclasts fail to form ruffled border membranes and many appeared to be detached from the bone surface. Following bone marrow transplantation, osteoclasts derived from wild-type donors showed normal morphology and were closely associated with the bone surface in MPS VII recipients. In vitro bone resorption assays demonstrated that MPS VII osteoclasts formed significantly smaller and fewer pits than those formed by osteoclasts derived from normal mice of the same strain. Although osteoclast morphology and function appeared to be abnormal in the MPS VII mouse, interleukin-1 (IL-1)-induced osteoclastogenesis in vivo was not affected. In addition to the osteoclast defects, MPS VII mice demonstrated a slower rate of bone matrix deposition in the epiphysis by in vivo calcein labeling experiments. These data suggest that abnormal morphology and function of MPS VII osteoclasts, combined with deficient matrix deposition, may contribute to the skeletal defects observed in this lysosomal storage disease.
Asunto(s)
Remodelación Ósea/fisiología , Resorción Ósea/patología , Mucopolisacaridosis VII/patología , Osteoclastos/patología , Animales , Modelos Animales de Enfermedad , Fémur/patología , Fluoresceínas , Colorantes Fluorescentes , Glucuronidasa/biosíntesis , Ratones , Ratones Mutantes , Osteoclastos/enzimologíaRESUMEN
The integrin alphavbeta3 has been shown to be tightly linked to progression of human melanoma. In this study, using two clones from the K1735 murine melanoma system, we investigated the role of alphavbeta3 in metastasis. The highly metastatic K1735M2 cells express the alphavbeta3 integrin, whereas the poorly metastatic K1735C23 cells do not. When transduced with the beta3 integrin subunit cDNA, the K1735C23 cells produced lung lesions and, in two animals, cardiac metastases, whereas the parental C23 cells did not. By contrast, transduction of the full-length beta3 integrin antisense DNA into the K1735M2 cells suppressed metastatic colonization. To specifically investigate the activation of beta3 integrin-mediated pathways, the beta3-positive and the beta3-negative K1735 cells were plated onto vitronectin, a major matrix molecule of both primary and metastatic melanomas. Tyr397 of FAK was phosphorylated several times higher in beta3-expressing K1735 melanoma cells than in beta3-negative cells. To determine whether phosphorylation of FAK was associated with K1735 melanoma motility, we expressed the FAK-related non-kinase (FRNK) in the highly metastatic K1735M2 cells. Exogenous expression of FRNK suppressed phosphorylation of FAK at Tyr397 and decreased the invasive ability of these cells. In addition, expression of a constitutively active mutant Src in poorly metastatic K1735C23 cells increased invasion in vitro; whereas expression of a kinase-inactive Src mutant suppressed invasion. Our results suggest that signals initiated by alphavbeta3 promote metastasis in K1735 melanoma cells through the phosphorylation of FAK and activation of Src.
Asunto(s)
Movimiento Celular/fisiología , Melanoma/metabolismo , Receptores de Vitronectina/metabolismo , Animales , Antígenos CD/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Técnicas In Vitro , Integrina beta3 , Melanoma/secundario , Ratones , Ratones Endogámicos C3H , Trasplante de Neoplasias , Fosforilación , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/metabolismo , Tirosina/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
RANK, the receptor activator of NF-kappaB, and its ligand RANKL (initially termed TRANCE, also termed ODF and OPGL), are a TNF superfamily receptor-ligand pair that govern the development and function of osteoclasts, lymphoid tissue, and mammary epithelium. While TNF family cytokines share a common structural scaffold, individual receptor-ligand pairs associate with high specificity. Given the low level of amino acid conservation among members of the TNF superfamily, the means by which these molecules achieve specificity cannot be completely understood without knowledge of their three-dimensional structures. To determine the elements of RANKL that mediate RANK activation, we have crystallized the ectodomain of murine RANKL and solved its structure to a resolution of 2.6 A. RANKL self-associates as a homotrimer with four unique surface loops that distinguish it from other TNF family cytokines. Mutagenesis of selected residues in these loops significantly modulates RANK activation, as evidenced by in vitro osteoclastogenesis, thereby establishing their necessity in mediating the biological activities of RANKL. Such structural determinants of RANKL-RANK specificity may be of relevance in the pharmacologic design of compounds to ameliorate osteopenic disorders of bone.
Asunto(s)
Proteínas Portadoras/química , Glicoproteínas de Membrana/química , Factor de Necrosis Tumoral alfa/química , Animales , Proteínas Portadoras/genética , Cristalografía por Rayos X , Ligandos , Glicoproteínas de Membrana/genética , Ratones , Modelos Moleculares , Estructura Terciaria de Proteína , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
In most circumstances, NF-kappaB, which is essential for osteoclastogenesis, is activated following serine 32/36 phosphorylation of its cytosolic inhibitory protein, IkappaBalpha. In contrast to other cell types, IkappaBalpha, in bone marrow macrophages (BMMs), which are osteoclast precursors, is tyrosine-phosphorylated by c-Src kinase. To address the role of IkappaBalpha phosphorylation in osteoclastogenesis, we generated TAT fusion proteins containing wild-type IkappaBalpha (TAT-WT-IkappaB), IkappaBalpha lacking its NH(2)-terminal 45 amino acids (TAT-IkappaB(46-317)), and IkappaBalpha in which tyrosine residue 42, the c-Src target, is mutated into phenylalanine (TAT-IkappaB(Y42F)). TAT-IkappaB efficiently enters BMMs, and the NF-kappaB-inhibitory protein, once intracellular, is functional. While TAT-WT-IkappaB only slightly inhibits osteoclastogenesis, osteoclast recruitment is diminished >80% by TAT-IkappaB(46-317), an event mirrored by dentin resorption. The fact that TAT alone does not impact osteoclastogenesis, which also resumes following withdrawal of TAT-IkappaB(46-317), establishes that the mutant's anti-osteoclastogenic properties do not reflect toxicity. Affirming a functional role for IkappaB(Tyr(42)) in osteoclastogenesis, TAT-IkappaB(Y42F) is as efficient as TAT-IkappaB(46-317) in blocking osteoclast differentiation. Thus, dominant-negative IkappaBalpha constructs block osteoclastogenesis, and Tyr(42) is essential to the process, increasing the possibility that nonphosphorylatable forms of IkappaBalpha may be a means of preventing pathological bone loss.
Asunto(s)
Proteínas de Unión al ADN/química , Productos del Gen tat/química , Proteínas I-kappa B , Osteoclastos/metabolismo , Proteínas Recombinantes de Fusión/química , Tirosina/química , Animales , Células de la Médula Ósea/metabolismo , Resorción Ósea , Células Cultivadas , Citosol/metabolismo , Dentina/metabolismo , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Immunoblotting , Macrófagos/metabolismo , Ratones , Mutación , Inhibidor NF-kappaB alfa , Osteoclastos/fisiología , Fosforilación , Unión Proteica , Serina/química , Factores de TiempoRESUMEN
Osteoclastic bone resorption requires cell-matrix contact, an event mediated by the alpha v beta 3 integrin. The structural components of the integrin that mediate osteoclast function are, however, not in hand. To address this issue, we generated mice lacking the beta 3 integrin gene, which have dysfunctional osteoclasts. Here, we show the full rescue of beta 3(-/-) osteoclast function following expression of a full-length beta 3 integrin. In contrast, truncated beta 3, lacking a cytoplasmic domain (h beta 3c), is completely ineffective in restoring function to beta 3(-/-) osteoclasts. To identify the components of the beta 3 cytoplasmic domain regulating osteoclast function, we generated six point mutants known, in other circumstances, to mediate beta integrin signaling. Of the six, only the S(752)P substitution, which also characterizes a form of the human bleeding disorder Glanzmann's thrombasthenia, fails to rescue beta 3(-/-) osteoclasts or restore ligand-activated signaling in the form of c-src activation. Interestingly, the double mutation Y(747)F/Y(759)F, which disrupts platelet function, does not affect the osteoclast. Thus similarities and distinctions exist in the mechanisms by which the beta 3 integrin regulates platelets and osteoclasts.
Asunto(s)
Antígenos CD/genética , Resorción Ósea/genética , Integrinas/genética , Osteoclastos/metabolismo , Glicoproteínas de Membrana Plaquetaria/genética , Trombastenia/genética , Secuencia de Aminoácidos , Animales , Tamaño de la Célula , Citoesqueleto/patología , Integrina beta3 , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Osteoclastos/patología , Mutación Puntual , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Homología de Secuencia de Aminoácido , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
Integrins are implicated in cell adhesion, migration and homeostasis. An important feature is their ability to adopt different affinity states that can be regulated by a variety of intra- and extracellular factors. To study affinity modulation of the integrin ectodomain by extracellular factors, we produced a soluble recombinant form of mouse integrin alphavbeta3 in a mammalian expression system and isolated it to purity. We show that the two transmembrane truncated integrin subunits stably associate to form a functional receptor, soluble recombinant alphavbeta3. The affinity of this receptor for its ligands vitronectin, fibronectin and fibrinogen can be modulated by the divalent cations magnesium, calcium and manganese. Most importantly, we found that a cyclic RGD-peptide has a biphasic effect on rsalphavbeta3and native purified alphavbeta3, with an antagonistic phase at high concentrations, and an agonistic phase at low concentrations. This integrin superactivation by low antagonist concentrations is shown in binding of sralphavbeta3 to immobilized ligands by ELISA, and in adhesion of cells that express the chimaeric integrin ligand KISS31 to immobilized rsalphavbeta3 and native purified alphavbeta3. Our results indicate that low concentrations of the ligand mimetic cyclo-RGD can result in superactivation of the extracellular domain of integrin alphavbeta3 to a comparable level as activation by manganese.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Oligopéptidos/administración & dosificación , Receptores de Vitronectina/agonistas , Receptores de Vitronectina/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Modelos Biológicos , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genéticaRESUMEN
Expression of the alpha(v)beta(3) integrin by murine bone marrow macrophages is regulated by cytokines such as IL-4 and GM-CSF through transcriptional activation of the beta(3) subunit gene. To characterize the molecular mechanisms by which such regulation occurs, we isolated the murine beta(3) integrin promoter. To this end, we first cloned a full length beta(3) cDNA and used the 5'UTR and leader peptide coding sequence to identify genomic clones containing the beta(3) promoter region. The transcriptional start site, identified by primer extension and S1 nuclease assay, is 34 nt upstream of the translation initiation codon. A 1.1 kb fragment of the promoter region drives IL-4 responsive transcription in transiently transfected murine bone marrow macrophages. Deletion analysis of the beta(3) promoter indicates the IL-4 responsive element lies between -465 to -678 nt relative to the transcriptional start site. This promoter fragment contains two overlapping STAT consensus recognition sites and nuclear extracts from BMMs contain an IL-4-inducible DNA binding factor, identified by super shift analysis, as STAT-6. Furthermore, an oligonucleotide which includes the two STAT recognition sites residing in the IL-4 responsive region of the beta(3) promoter, competes for STAT-6 binding. Confirming IL-4 induction of the integrin subunit is specifically mediated by STAT-6, beta(3) mRNA is not enhanced in BMMs derived from STAT-6 deleted mice, which however, retain their capacity to respond to GM-CSF.
Asunto(s)
Antígenos CD/genética , Interleucina-4/metabolismo , Glicoproteínas de Membrana Plaquetaria/genética , Regiones Promotoras Genéticas , Transactivadores/metabolismo , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células de la Médula Ósea/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Clonación Molecular , ADN Complementario/metabolismo , Fémur/metabolismo , Eliminación de Gen , Biblioteca de Genes , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Integrina beta3 , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Modelos Genéticos , Datos de Secuencia Molecular , Elementos de Respuesta , Factor de Transcripción STAT6 , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Tibia/metabolismo , Transcripción Genética , Activación Transcripcional , Transfección , Regulación hacia ArribaRESUMEN
Receptor activator of nuclear factor-kappa B ligand [RANK ligand (RANK-L)] stimulates mature osteoclasts to resorb bone, a process associated with NF-kappa B activation. RANK-L also prompts macrophages to develop the osteoclast phenotype. Although NF-kappa B is essential for osteoclast differentiation, it is not known whether RANK-L activates this transcription complex in osteoclast precursors. We report that RANK-L rapidly induces NF-kappa B activation in both authentic osteoclast precursors, namely bone marrow macrophages, and RAW 264.7 cells, a murine macrophage line also capable of RANK-L-mediated osteoclastogenesis. Supershift studies reveal the RANK-L-induced DNA binding moiety contains p50/p65, the most common NF-kappa B complex. Subcellular translocation of p50 and p65 subunits is confirmed by Western blots and immunofluorescence analysis. RANK-L activates NF-kappa B in both bone marrow macrophages and RAW 264.7 cells by serine phosphorylation of I kappa B alpha within 5 min, resulting in rapid I kappa B alpha degradation and resynthesis. Attesting to function, RANK-L treatment of RAW 264.7 cells transiently transfected with a plasmid containing NF-kappa B consensus elements linked to luciferase greatly enhances reporter activity. Our data suggest that activation of the NF-kappa B pathway is an integral component of RANK-L-induced osteoclast differentiation.
Asunto(s)
Proteínas Portadoras/farmacología , Glicoproteínas de Membrana/farmacología , FN-kappa B/fisiología , Osteoclastos/metabolismo , Células Madre/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Dimerización , Genes Reporteros/efectos de los fármacos , Proteínas I-kappa B/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , FN-kappa B/genética , Osteoclastos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Serina/metabolismo , Células Madre/efectos de los fármacosRESUMEN
The differentiation of cells of the monocytic lineage into mature osteoclasts (OC) is specifically induced by the tumor necrosis factor-related factor, RANKL (receptor activator of NF-kappaB ligand; also known as OPGL, ODF, or TRANCE). Because inhibition of osteoclastogenesis is one of the main mechanisms by which estrogen (E2) prevents bone loss, it is likely that E2 may regulate either the production of, or the target cell responsiveness to RANKL. We found that E2 decreases the differentiation into OC of both murine bone marrow monocytes and RAW 264.7 cells, a monocytic line, by down-regulating the activation of Jun N-terminal kinase 1 (JNK1). Diminished JNK1 activity results in decreased nuclear levels of the key osteoclastogenic transcription factors, c-Fos and c-Jun, and lower binding of these transcriptional inducers to DNA. Thus, one novel mechanism by which E2 down-regulates osteoclastogenesis is by decreasing the responsiveness of OC precursors to RANKL.
Asunto(s)
Proteínas Portadoras/metabolismo , División Celular/efectos de los fármacos , Regulación hacia Abajo , Estradiol/farmacología , Glicoproteínas de Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Osteoclastos/efectos de los fármacos , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Sondas de ADN , Activación Enzimática , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Osteoclastos/citología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ligando RANK , Receptor Activador del Factor Nuclear kappa-BRESUMEN
While TNF-alpha is pivotal to the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are unknown. We find that a pure population of murine osteoclast precursors fails to undergo osteoclastogenesis when treated with TNF-alpha alone. In contrast, the cytokine dramatically stimulates differentiation in macrophages primed by less than one percent of the amount of RANKL (ligand for the receptor activator of NF-kappaB) required to induce osteoclast formation. Mirroring their synergistic effects on osteoclast differentiation, TNF-alpha and RANKL markedly potentiate NF-kappaB and stress-activated protein kinase/c-Jun NH(2)-terminal kinase activity, two signaling pathways essential for osteoclastogenesis. In vivo administration of TNF-alpha prompts robust osteoclast formation in chimeric animals in which ss-galactosidase positive, TNF-responsive macrophages develop within a TNF-nonresponsive stromal environment. Thus, while TNF-alpha alone does not induce osteoclastogenesis, it does so both in vitro and in vivo by directly targeting macrophages within a stromal environment that expresses permissive levels of RANKL. Given the minuscule amount of RANKL sufficient to synergize with TNF-alpha to promote osteoclastogenesis, TNF-alpha appears to be a more convenient target in arresting inflammatory osteolysis.
Asunto(s)
Proteínas Portadoras/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Glicoproteínas de Membrana/farmacología , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Trasplante de Médula Ósea , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Histocitoquímica , Proteínas Quinasas JNK Activadas por Mitógenos , Macrófagos/enzimología , Macrófagos/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Osteoclastos/enzimología , Osteoclastos/metabolismo , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/enzimología , Células Madre/metabolismoRESUMEN
Integrin-mediated cell-matrix interactions play important roles in regulating cell function. Since transforming growth factor-beta (TGF-beta) modulates many osteoblast activities, we hypothesized that the growth factor acts in part by modulating integrin expression. TGF-beta increased cell adhesion to vitronectin and up-regulated the surface level of alpha(v)beta(5) via increasing beta(5) protein synthesis by a transcriptional mechanism. Promoter activity analysis demonstrated that a TGF-beta-responsive element resides between nucleotides -63 and -44. Electrophoretic mobility shift assay and immunoprecipitation/Western studies indicated that the nuclear complex formed using the -66/-42 oligonucleotide contained both Sp1/Sp3 and Smad proteins. Since nuclear Sp1/Sp3 levels were not altered, whereas Smad levels were increased by TGF-beta, we investigated the roles of Smad proteins in the up-regulation of beta(5) gene activation. Co-transfection of cells with beta(5) promoter reporter construct and expression vectors for Smad3, Smad4, and Sp1 increased the stimulatory effect of TGF-beta. Furthermore, expression of dominant negative Smad3 or Smad4 in cells decreased or abolished the stimulation of beta(5) promoter activity by TGF-beta. Smad4 mutant also inhibited the up-regulation of surface beta(5) level by TGF-beta. Thus, TGF-beta increases expression of the integrin beta(5) gene by mechanisms involving Sp1/Sp3 and Smad transcription factors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Integrinas/genética , Receptores de Vitronectina , Transducción de Señal/efectos de los fármacos , Factor de Transcripción Sp1/metabolismo , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/efectos de los fármacos , Animales , Secuencia de Bases , Adhesión Celular/efectos de los fármacos , Línea Celular , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Integrinas/metabolismo , Ratones , Datos de Secuencia Molecular , Mutación/genética , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta/genética , Proteína smad3 , Proteína Smad4 , Factor de Transcripción Sp3 , Transactivadores/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta2 , Vitronectina/metabolismoRESUMEN
The potent osteoclastogenic agent, tumor necrosis factor-alpha (TNF), exerts its biological effects via two receptors, namely TNF receptors 1 (p55r) and 2 (p75r), each present on osteoclast precursors. Thus, we asked if p55r and p75r differentially impact the osteoclastogenic process. Marrow derived from mice expressing only p55r generates substantially more osteoclasts, in the basal state, than does wild type, while marrow expressing only p75r, produces substantially fewer. Reflecting its preferential activation of p55r, exogenous TNF stimulates osteoclast formation by p55r(+/+)p75r(-/-), but not p55r(-/-)p75r(+/+), marrow. Consistent with the fact that NF-kappaB is essential for osteoclastogenesis, this transcription complex is activated, relative to wild type, in p55r(+/+)p75r(-/-) osteoclast precursors and suppressed in those expressing only p75r. Because p55r enhances, and p75r suppresses, osteoclastogenesis, we asked if their principal ligands, namely soluble and membrane-residing TNF, respectively, differentially impact basal osteoclast recruitment. We find, in contrast to the significant level of osteoclast formation in wild type marrow, osteoclastogenesis by that derived from mice expressing membrane, but not soluble, TNF, is negligible. Thus, optimal therapeutic inhibition of bone resorption may entail selective TNF receptor modulation and/or arrested cleavage of membrane TNF to its soluble form.
Asunto(s)
Antígenos CD/fisiología , División Celular/fisiología , Osteoclastos/citología , Receptores del Factor de Necrosis Tumoral/fisiología , Animales , Antígenos CD/genética , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , Osteoclastos/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis TumoralAsunto(s)
Virus del Sarampión , Osteítis Deformante/virología , Receptores Citoplasmáticos y Nucleares , Resorción Ósea/fisiopatología , Proteínas Portadoras/metabolismo , Citocinas/farmacología , Glicoproteínas/metabolismo , Humanos , Factor Estimulante de Colonias de Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteítis Deformante/patología , Osteoclastos/inmunología , Osteoclastos/virología , Osteoprotegerina , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Necrosis Tumoral , Transducción de SeñalRESUMEN
Long-term administration of pharmacological doses of glucocorticoids inhibits bone formation and results in osteoporosis. Since integrin-mediated cell-matrix interactions are essential for osteoblast function, we hypothesized that the detrimental effect of glucocorticoids on bone derived, at least in part, from decreased integrin-matrix interactions. Because alphavbeta3 and alphavbeta5 integrins can interact with several bone matrix proteins, we analyzed the effects of dexamethasone (Dex) on the expression of these integrins in normal human osteoblastic cells. We found adhesion of these cells to osteopontin and vitronectin to be dependent on alphavbeta3 and alphavbeta5, respectively; this ligand specificity was not altered by Dex. The effects of Dex on the adhesion of human osteoblastic cells to osteopontin and vitronectin were biphasic with an increase after 2 days, followed by a decrease after 8 days of treatment. Consistently, surface alphavbeta3 and alphavbeta5 integrins, which were increased after 2 days of Dex treatment, were decreased after 8 days. Similarly, total cellular alphav, beta3, and beta5 proteins, which were increased by Dex early in the culture, were diminished after 8 days. Metabolic labeling studies indicated that Dex exhibited biphasic regulation on the biosynthesis of alphavbeta5, with stimulation observed during the second day of treatment, followed by inhibition during the 8th day of exposure. By contrast, the biosynthesis of alphavbeta3 was inhibited by Dex on day 1 and remained inhibited on day 8. Analysis of the mRNA indicated that alphav and beta5 levels were increased by Dex during early exposure (1-3 days), followed by inhibition after prolonged exposure (>/=7 days). By contrast, Dex decreased beta3 mRNA level at all the time points analyzed. Consistently, Dex decreased beta3 promoter activity after 1 day and persisted over 8-day period. By contrast, Dex stimulated beta5 promoter activity after 1 or 2 days but had no effect after 8 days. To further evaluate mechanism(s) leading to the decreased integrin expression after prolonged Dex treatment, mRNA stability was analyzed. Dex was found to accelerate the degradation of alphav, beta3 and beta5 mRNA after an 8-day treatment. Thus, the regulation of alphavbeta3 was dependent on transcription and posttranscriptional events whereas the expression of alphavbeta5 was dependent mainly on posttranscriptional events after prolonged Dex treatment. In conclusion, Dex exhibited time-dependent regulation on the expression of alphavbeta3 and alphavbeta5 integrins in normal human osteoblastic cells. Short-term exposure to Dex increased the levels of alphavbeta3 and alphavbeta5 on the surface and cell adhesion to osteopontin and vitronectin whereas long-term exposure to Dex decreased the expression of both integrins and inhibited the cell adhesion to matrix proteins.
Asunto(s)
Dexametasona/farmacología , Glucocorticoides/farmacología , Cadenas beta de Integrinas , Integrinas/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Receptores de Vitronectina/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Adhesión Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Integrina alfaV , Integrina beta3 , Integrinas/genética , Osteoblastos/citología , Osteopontina , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Regiones Promotoras Genéticas , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Vitronectina/genética , Sialoglicoproteínas/metabolismo , Vitronectina/metabolismoRESUMEN
Murine osteoclast precursors and osteoblasts express the integrin alpha(v)beta(5), the appearance of which on the cell surface is controlled by the beta(5), and not the alpha(v), subunit. Here, we show that a 173-base pair proximal region of the beta(5) promoter mediates beta(5) basal transcription in macrophage (osteoclast precursor)-like and osteoblast-like cells. DNase I footprinting reveal four regions (FP1-FP4) within the 173-base pair region, protected by macrophage nuclear extracts. In contrast, osteoblast nuclear extracts protect only FP1, FP2, and FP3. FP1, FP2, and FP3 bind Sp1 and Sp3 from both macrophage and osteoblast nuclear extracts. FP4 does not bind osteoblast proteins but binds PU.1 from macrophages. Transfection studies show that FP1 and FP2 Sp1/Sp3 sites act as enhancers in both MC3T3-E1 (osteoblast-like) and J774 (macrophage-like) cell lines, whereas the FP3 Sp1/Sp3 site serves as a silencer. Mutation of the FP2 Sp1/Sp3 site totally abolishes promoter activity in J774 cells, with only partial reduction in MC3T3-E1 cells. Finally, we demonstrate that PU.1 acts as a beta(5) silencer in J774 cells but plays no role in MC3T3-E1 cells. Thus, three Sp1/Sp3 sites regulate beta(5) gene expression in macrophages and osteoblast-like cells, with each element exhibiting cell-type and/or activation-suppression specificity.
Asunto(s)
Cadenas beta de Integrinas , Integrinas/genética , Macrófagos/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Diferenciación Celular/genética , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Integrinas/biosíntesis , Macrófagos/citología , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Osteoblastos/citología , Osteoclastos/citología , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3 , Células Madre/citología , Células Madre/metabolismo , Transactivadores/metabolismoRESUMEN
Osteoclasts express the alphavbeta3 integrin, an adhesion receptor that has been implicated in bone resorption and that is therefore a potential therapeutic target. To assess the role of this heterodimer in skeletal development in vivo, we engineered mice in which the gene for the beta3 integrin subunit was deleted. Bone marrow macrophages derived from these mutants differentiate in vitro into numerous osteoclasts, thus establishing that alphavbeta3 is not necessary for osteoclast recruitment. Furthermore, the closely related integrin, alphavbeta5, does not substitute for alphavbeta3 during cytokine stimulation or authentic osteoclastogenesis. beta3 knockout mice, but not their heterozygous littermates, develop histologically and radiographically evident osteosclerosis with age. Despite their increased bone mass, beta3-null mice contain 3.5-fold more osteoclasts than do heterozygotes. These mutant osteoclasts are, however, dysfunctional, as evidenced by their reduced ability to resorb whale dentin in vitro and the significant hypocalcemia seen in the knockout mice. The resorptive defect in beta3-deficient osteoclasts may reflect absence of matrix-derived intracellular signals, since their cytoskeleton is distinctly abnormal and they fail to spread in vitro, to form actin rings ex vivo, or to form normal ruffled membranes in vivo. Thus, although it is not required for osteoclastogenesis, the integrin alphavbeta3 is essential for normal osteoclast function.
Asunto(s)
Antígenos CD/genética , Resorción Ósea/genética , Cadenas beta de Integrinas , Osteoclastos/citología , Osteosclerosis/genética , Glicoproteínas de Membrana Plaquetaria/genética , Animales , Células de la Médula Ósea/citología , Diferenciación Celular , Movimiento Celular , Matriz Extracelular/metabolismo , Fémur/citología , Integrina beta3 , Integrinas/metabolismo , Macrófagos/citología , Ratones , Ratones Noqueados , Osteoclastos/metabolismo , Transducción de SeñalRESUMEN
Early osteoclast precursors, in the form of murine bone marrow macrophages (BMMs), while expressing no detectable alpha(v)beta3 integrin, contain abundant alpha(v)beta5 and attach to matrix in an alpha(v) integrin-dependent manner. Furthermore, alpha(v)beta5 expression by osteoclast precursors progressively falls as they assume the resorptive phenotype. We find the osteoclastogenic agent, tumor necrosis factor-alpha, (TNF) down-regulates alpha(v)beta5 expression by BMMS via attenuation of beta5 messenger RNA (mRNA) t1/2. Using BMMs from TNF receptor knockout mice we establish the p55 receptor transmits the beta5 suppressive effect. The functional implications of TNF-mediated alpha(v)beta5 down-regulation are underscored by the capacity of an alpha(v) inhibitory peptide mimetic to prevent spreading by BMMs expressing abundant alpha(v)beta5 while failing to impact those in which the integrin has been diminished by TNF. Finally, beta5 mRNA in BMMs of wild-type mice administered lipopolysaccharide (LPS) progressively falls with time of in vivo treatment. Alternatively, beta5 mRNA does not decline in BMMs of LPS-treated mice lacking both TNF receptors, documenting down-regulation of the beta5 integrin subunit, in vivo, is mediated by TNF. Thus, matrix attachment of osteoclast precursors and mature osteoclasts are governed by distinct alpha(v) integrins which are differentially regulated by specific cytokines.
Asunto(s)
Regulación de la Expresión Génica/genética , Integrinas/biosíntesis , Integrinas/genética , Osteoclastos/metabolismo , Receptores de Vitronectina , Factor de Necrosis Tumoral alfa/fisiología , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Northern Blotting , Diferenciación Celular/efectos de los fármacos , Regulación hacia Abajo , Técnicas In Vitro , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Transgénicos , Osteoclastos/ultraestructura , Osteogénesis/genética , Osteogénesis/fisiología , Pruebas de Precipitina , ARN Mensajero/biosíntesis , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Transcripción Genética/genética , Factor de Necrosis Tumoral alfa/agonistasRESUMEN
The ruffled membrane, the resorptive organelle of the osteoclast, is generated by fusion of intracytoplasmic acidifying vesicles with the plasma membrane, an event analogous to regulated exocytosis. While the ruffled membrane is essential to the bone resorptive process, the mechanisms governing its generation are unknown. However, regulated exocytosis is mediated, in part, by isoforms of the Rab3 subset of Rab GTPases. Because of similarities between exocytosis and ruffled membrane formation, we asked if Rab3 proteins are expressed by osteoclasts or their precursors, and if so, are these molecules regulated by agents known to prompt the osteoclast phenotype? We find murine osteoclast precursors, in the form of bone marrow macrophages (BMMs), express at least two Rab3 isoforms, namely A and B/C, which are individually enhanced by a variety of hematopoietic cytokines. Consistent with the osteoclastogenic properties of a number of these cytokines, differentiation of BMMs into osteoclasts, in vitro, is associated with increased expression of both isoforms, particularly Rab3B/C. Finally, Rab3B/C localizes with the avian osteoclast H+ATPase (vacuolar proton pump) and pp60c-src, both intracellularly and within acidifying vesicles derived largely from the ruffled membrane. Thus, expression of specific rab3 proteins, an event which may control formation of the osteoclast ruffled membrane, is modulated by cytokines during osteoclastogenesis.