RESUMO
Tumor necrosis factor (TNF)-α exerts its biological function via TNF type 1 and type 2 receptors (TNFR1 and TNFR2). We have previously reported that bone resorption induced by lipopolysaccharide (LPS) in TNFR2-deficient mice is accelerated compared to that in wild-type (WT) mice. Although these results suggested that TNFR2 might have a protective role in bone resorption, we could not exclude the possibility that TNFR2 has no role in bone resorption. To clarify the role of TNFR2, we developed a TNF-α-induced bone resorption model using cholesterol-bearing pullulan nanogel as a TNF-α carrier to minimize the influence of inflammatory cytokines other than TNF-α. Injections of human TNF-α (hTNF), an agonist of mouse TNFR1, stimulated bone resorption lacunae on the calvariae in WT mice, but mouse TNF-α (mTNF), an agonist of both mouse TNFR1 and TNFR2, could not. To eliminate the possibility that the TNFR1 agonistic effects of hTNF were stronger than those of mTNF, we used the same model in TNFR2-deficient mice. Injection of mTNF resulted in clear bone resorption lacunae to the same extent observed after using hTNF in the TNFR2-deficient mice. Histomorphometric analysis of osteoclast number supported the observed changes in bone resorption lacunae. These data suggest that TNFR2 has a protective role in TNF-α-induced bone resorption.
Assuntos
Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Crânio/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/genética , Glucanos/química , Humanos , Camundongos , Camundongos Mutantes , Nanogéis , Polietilenoglicóis/química , Polietilenoimina/química , Receptores Tipo I de Fatores de Necrose Tumoral/agonistas , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Crânio/citologia , Crânio/metabolismoRESUMO
Activating receptor activator of NF-kappaB (RANK) and TNF receptor (TNFR) promote osteoclast differentiation. A critical ligand contact site on the TNFR is partly conserved in RANK. Surface plasmon resonance studies showed that a peptide (WP9QY) that mimics this TNFR contact site and inhibits TNF-alpha-induced activity bound to RANK ligand (RANKL). Changing a single residue predicted to play an important role in the interaction reduced the binding significantly. WP9QY, but not the altered control peptide, inhibited the RANKL-induced activation of RANK-dependent signaling in RAW 264.7 cells but had no effect on M-CSF-induced activation of some of the same signaling events. WP9QY but not the control peptide also prevented RANKL-induced bone resorption and osteoclastogenesis, even when TNFRs were absent or blocked. In vivo, where both RANKL and TNF-alpha promote osteoclastogenesis, osteoclast activity, and bone loss, WP9QY prevented the increased osteoclastogenesis and bone loss induced in mice by ovariectomy or low dietary calcium, in the latter case in both wild-type and TNFR double-knockout mice. These results suggest that a peptide that mimics a TNFR ligand contact site blocks bone resorption by interfering with recruitment and activation of osteoclasts by both RANKL and TNF.
Assuntos
Reabsorção Óssea , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Peptídeos/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Cálcio da Dieta , Proteínas de Transporte/química , Linhagem Celular , Células Cultivadas , Feminino , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Vértebras Lombares/anatomia & histologia , Vértebras Lombares/patologia , Masculino , Glicoproteínas de Membrana/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Dados de Sequência Molecular , Osteoclastos/citologia , Osteoclastos/fisiologia , Osteoprotegerina , Ovariectomia , Peptídeos/química , Peptídeos/genética , Conformação Proteica , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/genética , Alinhamento de Sequência , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The alternative NF-kappaB pathway consists predominantly of NF-kappaB-inducing kinase (NIK), IkappaB kinase alpha (IKKalpha), p100/p52, and RelB. The hallmark of the alternative NF-kappaB signaling is the processing of p100 into p52 through NIK, thus allowing the binding of p52 and RelB. The physiologic relevance of alternative NF-kappaB activation in bone biology, however, is not well understood. To elucidate the role of the alternative pathway in bone homeostasis, we first analyzed alymphoplasic (aly/aly) mice, which have a defective NIK and are unable to process p100, resulting in the absence of p52. We observed increased bone mineral density (BMD) and bone volume, indicating an osteopetrotic phenotype. These mice also have a significant defect in RANKL-induced osteoclastogenesis in vitro and in vivo. NF-kappaB DNA-binding assays revealed reduced activity of RelA, RelB, and p50 and no binding activity of p52 in aly/aly osteoclast nuclear extracts after RANKL stimulation. To determine the role of p100 itself without the influence of a concomitant lack of p52, we used p100(-/-) mice, which specifically lack the p100 inhibitor but still express p52. p100(-/-) mice have an osteopenic phenotype owing to the increased osteoclast and decreased osteoblast numbers that was rescued by the deletion of one allele of the relB gene. Deletion of both allele of relB resulted in a significantly increased bone mass owing to decreased osteoclast activity and increased osteoblast numbers compared with wild-type (WT) controls, revealing a hitherto unknown role for RelB in bone formation. Our data suggest a pivotal role of the alternative NF-kappaB pathway, especially of the inhibitory role of p100, in both basal and stimulated osteoclastogenesis and the importance of RelB in both bone formation and resorption.