RESUMO
Osteoclasts play a crucial role in osteolytic bone diseases, such as osteoporosis, rheumatoid arthritis, periodontitis, Paget's disease of bone and bone metastatic tumors. Therefore, controlling osteoclast differentiation and function has been considered a promising therapeutic strategy. Here, we show that necrostatin (Nec)-7, an inhibitor of programmed necrosis, strongly suppressed receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption, without compromising macrophage colony-stimulating factor (M-CSF)-supported survival and growth of osteoclast precursor cells. Accordingly, Nec-7 significantly decreased the levels of RANKL-induced osteoclastogenic marker genes, such as cathepsin K. Mechanistically, Nec-7 neither affected MAPK nor NF-κB activation; however, it strongly inhibited the RANKL receptor (RANK) to nuclear factor of activated T cells c1 (NFATc1) signaling. Lentiviral expression of RANK in bone marrow-derived macrophages significantly restored osteoclastogenesis and NFATc1 amplification in Nec-7-treated cells. In this study, we revealed that Nec-7-sensitive pathways are crucially involved in osteoclast formation and function. Investigation of the molecular mechanism(s) through which Nec-7 inhibits RANK-NFATc1 signaling axis may lead to the development of new therapeutic strategies for bone disease.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Animais , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Células Cultivadas , Feminino , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteoclastos/metabolismoRESUMO
Osteoclasts degrade bone matrix proteins via the secretion of lysosomal enzymes. However, the precise mechanisms by which lysosomal components are transported and fused to the bone-apposed plasma membrane, termed ruffled border membrane, remain elusive. Here, we identified coronin 1A as a negative regulator of exocytotic release of cathepsin K, one of the most important bone-degrading enzymes in osteoclasts. The modulation of coronin 1A expression did not alter osteoclast differentiation and extracellular acidification, but strongly affected the secretion of cathepsin K and osteoclast bone-resorption activity, suggesting the coronin 1A-mediated regulation of lysosomal trafficking and protease exocytosis. Further analyses suggested that coronin 1A prevented the lipidation-mediated sorting of the autophagy-related protein LC3 to the ruffled border and attenuated lysosome-plasma membrane fusion. In this process, the interactions between coronin 1A and actin were crucial. Collectively, our findings indicate that coronin 1A is a pivotal component that regulates lysosomal fusion and the secretion pathway in osteoclast-lineage cells and may provide a novel therapeutic target for bone diseases.
Assuntos
Reabsorção Óssea/metabolismo , Catepsina K/metabolismo , Lisossomos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Osteoclastos/metabolismo , Actinas/metabolismo , Animais , Reabsorção Óssea/diagnóstico por imagem , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Diferenciação Celular/genética , Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Osteoclastos/citologia , Ligação Proteica , Transporte Proteico , Ligante RANK/metabolismoRESUMO
BACKGROUND: Stimulation with antigen and IgE is known to activate NF-κB in mast cells. In the present research, we studied the role of NF-κB on cellular migration in mast cell-like RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) using the NF-κB inhibitor (-)-DHMEQ. METHODS: A Matrigel invasion chamber was used to evaluate cell migration. A PCR array was used to screen the expression of 84 key genes involved in cell migration. RESULTS: (-)-DHMEQ inhibited antigen/IgE-induced NF-κB activation and expressions of its target genes such as IL-6 and TNF-α. (-)-DHMEQ was found to inhibit in vitro invasion toward the antigen without any toxicity. We then looked for NF-κB-dependent genes that would be important for mast cell invasion using the PCR array. (-)-DHMEQ was found to lower the expression of matrix metalloproteinase (MMP)-2. The MMP inhibitor GM6001 also inhibited cellular invasion toward the antigen. These effects of (-)-DHMEQ were obtained in both RBL-2H3 cells and BMMCs. CONCLUSIONS: These findings indicate that (-)-DHMEQ suppressed mast cell migration via the inhibition of NF-κB-regulated MMP-2 expression.
Assuntos
Benzamidas/farmacologia , Movimento Celular/imunologia , Cicloexanonas/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Metaloproteinase 2 da Matriz/imunologia , NF-kappa B/imunologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Colágeno/farmacologia , Dipeptídeos/farmacologia , Combinação de Medicamentos , Ensaio de Desvio de Mobilidade Eletroforética , Interleucina-6/genética , Interleucina-6/imunologia , Laminina/farmacologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos , NF-kappa B/antagonistas & inibidores , Proteoglicanas/farmacologia , RNA/química , RNA/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
We previously reported that 9-methylstreptimidone, a piperidine compound isolated from a culture filtrate of Streptomyces, induces apoptosis selectively in adult T-cell leukemia cells. It was screened for a compound that inhibits LPS-induced NF-kappaB and NO production in mouse macrophages. However, 9-methystreptimidone is poorly obtained from the producing microorganism and difficult to synthesize. Therefore, in the present research, we studied the structure-activity relationship to look for new selective inhibitors. We found that the structure of the unsaturated hydrophobic portion of 9-methylstreptimidone was essential for the inhibition of LPS-induced NO production. Among the 9-methylstreptimidone-related compounds tested, (+/-)-4,alpha-diepi-streptovitacin A inhibited NO production in macrophage-like cells as potently as 9-methylstreptimidone and without cellular toxicity. Moreover, this compound selectively induced apoptosis in adult T-cell leukemia MT-1 cells.
Assuntos
Apoptose/efeitos dos fármacos , Cicloeximida/análogos & derivados , Leucemia de Células T/tratamento farmacológico , Óxido Nítrico/metabolismo , Piperidonas/farmacologia , Animais , Células Cultivadas , Cicloeximida/química , Cicloeximida/farmacologia , Humanos , Células Jurkat , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Piperidonas/química , Relação Estrutura-AtividadeRESUMO
NF-κB is a transcription factor for the immune activation and tissue stability, but excess activation of NF-κB often causes inflammation and cancer. An NF-κB component RelB is involved in B-cell maturation and autoimmunity. In the present research we studied the role of the RelB DNA binding domain on cellular stability and importin affinity. We prepared a RelB protein mutated at Arg141 to Ala and Tyr142 to Ala (AA mutant) having no DNA binding activity. The stability of this mutant protein was greatly reduced compared with that of the wild-type protein. We also constructed a nuclear localization signal-inactivated mutant of RelB, and found that this mutant was also unstable in the cells. Thus, RelB destabilization was caused by the loss of DNA binding possibly because of the change in cellular localization. The mutation also decreased the affinity to importin-α5 decreasing the nuclear localization. Our newly discovered NF-κB inhibitor (-)-DHMEQ binds to a specific Cys residue in RelB to inhibit DNA binding and also decreased the stability and importin affinity. These findings would indicate that the DNA binding activity of this transcription factor is a crucial for its stability and intracellular localization.
Assuntos
Carioferinas/metabolismo , Fator de Transcrição RelB/metabolismo , Alanina/química , Alanina/genética , Sequência de Aminoácidos , Arginina/química , Arginina/genética , Benzamidas/farmacologia , Sítios de Ligação , Núcleo Celular/metabolismo , Cicloexanonas/farmacologia , Células HeLa , Humanos , Carioferinas/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Sinais de Localização Nuclear , Estabilidade Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição RelB/química , Fator de Transcrição RelB/genética , Tirosina/química , Tirosina/genéticaRESUMO
OBJECTIVE: We have previously synthesized a novel piperidine compound, 3-[(dodecylthiocarbonyl)methyl]glutarimide (DTCM-glutarimide), that inhibits LPS-induced NO production, and in the present research we studied further the anti-inflammatory activity of DTCM-glutarimide in a macrophage cell line and in mice bearing transplanted hearts. MATERIALS AND METHODS: Mouse macrophage-like RAW264.7 cells were employed for the evaluation of cellular inflammatory activity. DTCM-glutarimide was synthesized in our laboratory. The AP-1 activity was measured by nuclear translocation and phosphorylation. For the heart transplantation experiment, male C57BL/6 (H-2b) and BALB/c (H-2d) mice were used as donor and recipient, respectively. DTCM-glutarimide was administered intraperitoneally. RESULTS: DTCM-glutarimide inhibited the LPS-induced expression of iNOS and COX-2 in macrophages; but, unexpectedly, it did not inhibit LPS-induced NF-κB activation. Instead, it inhibited the nuclear translocation of both c-Jun and c-Fos. It also inhibited LPS-induced c-Jun phosphorylation. Moreover, it inhibited the mixed lymphocyte reaction in primary cultures of mouse spleen cells; and furthermore, in mice it prolonged the graft survival in heart transplantation experiments. CONCLUSION: The novel piperidine compound, DTCM-glutarimide, was found to be a new inhibitor of macrophage activation, inhibiting AP-1 activity. It also inhibited graft rejection in mice, and thus may be a candidate for an anti-inflammatory agent.
Assuntos
Anti-Inflamatórios/farmacologia , Rejeição de Enxerto/prevenção & controle , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Piperidonas/química , Piperidonas/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/efeitos dos fármacos , Sobrevivência de Enxerto/imunologia , Transplante de Coração/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estrutura Molecular , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
The amino-epoxyquinols 6a and 6b were synthesized as soluble derivatives of an NF-κB inhibitor DHMEQ (1). In spite of the opposite configuration from 1, 6b rather than 6a affected the deactivation of NF-κB, based on NO secretion and MALDI-TOF MS analysis. It was indicated that 6b inhibited the activation by different manner from that of 1.
Assuntos
Benzamidas/química , Cicloexanonas/química , Compostos de Epóxi/química , Hidroquinonas/química , NF-kappa B/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Cicloexanonas/síntese química , Cicloexanonas/farmacologia , Compostos de Epóxi/síntese química , Compostos de Epóxi/farmacologia , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Previously, we designed and synthesized a potent NF-kappaB inhibitor, DHMEQ. Although DHMEQ showed potent anti-inflammatory and anticancer activities in animals, its molecular target has not been elucidated. In the present study, its target protein was found to be p65 and other Rel homology proteins. We found that (-)-DHMEQ bound to p65 covalently with a 1:1 stoichiometry by conducting SPR and MALDI-TOF MS analyses. MS analysis of the chymotrypsin-digested peptide suggested the binding of (-)-DHMEQ to a Cys residue. Formation of Cys/(-)-DHMEQ adduct in the protein was supported by chemical synthesis of the adduct. Substitution of specific Cys in p65 and other Rel homology proteins resulted in the loss of (-)-DHMEQ binding. (-)-DHMEQ is the first NF-kappaB inhibitor that was proven to bind to the specific Cys by chemical methodology. These findings may explain the highly selective inhibition of NF-kappaB and the low toxic effect of (-)-DHMEQ in cells and animals.