RESUMO
Osteoclasts are giant bone-digesting cells that harbor specialized lysosome-related organelles termed secretory lysosomes (SLs). SLs store cathepsin K and serve as a membrane precursor to the ruffled border, the osteoclast's 'resorptive apparatus'. Yet, the molecular composition and spatiotemporal organization of SLs remains incompletely understood. Here, using organelle-resolution proteomics, we identify member a2 of the solute carrier 37 family (Slc37a2) as a SL sugar transporter. We demonstrate in mice that Slc37a2 localizes to the SL limiting membrane and that these organelles adopt a hitherto unnoticed but dynamic tubular network in living osteoclasts that is required for bone digestion. Accordingly, mice lacking Slc37a2 accrue high bone mass owing to uncoupled bone metabolism and disturbances in SL export of monosaccharide sugars, a prerequisite for SL delivery to the bone-lining osteoclast plasma membrane. Thus, Slc37a2 is a physiological component of the osteoclast's unique secretory organelle and a potential therapeutic target for metabolic bone diseases.
Assuntos
Reabsorção Óssea , Osteoclastos , Camundongos , Animais , Osteoclastos/metabolismo , Transporte Biológico , Lisossomos/metabolismo , Osso e Ossos/metabolismo , Membrana Celular/metabolismo , Reabsorção Óssea/metabolismoRESUMO
Calcium/calmodulin-dependent protein kinase (CaMK) is a major down stream mediator of Ca(2+) signaling in a wide range of cellular functions, including ion channel and cell cycle regulation and neurotransmitter synthesis and release. Here we have investigated the role of the CaMK signaling pathway in osteoclast differentiation and bone resorption. We observed that the CaMKI, CaMKII gamma isoforms were present in both bone-marrow derived macrophages and RAW264.7 murine macrophage cell line, and that expression persisted during osteoclast differentiation in the presence of receptor activator of nuclear factor kappa B (NF-kappaB) ligand (RANKL). RANKL-induced differentiation was accompanied by increased cyclic AMP response element transcriptional activity, and ERK phosphorylation, which are both downstream targets of CaMK. Two selective inhibitors of CaMKs, KN-93 and KN-62, inhibited osteoclastogenesis in a time and concentration-dependent manner. This was accompanied by suppression of cathepsin K expression and osteoclastic bone resorption, which are markers for differentiated osteoclast function. KN-93 and KN-62 both inhibited RANKL-induced ERK phosphorylation and CREB transcriptional activity. These findings imply a role for CaMK in osteoclast differentiation and bone resorption.
Assuntos
Reabsorção Óssea/metabolismo , Sinalização do Cálcio , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Diferenciação Celular , Macrófagos/metabolismo , Osteoclastos/metabolismo , Ligante RANK/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Benzilaminas/farmacologia , Reabsorção Óssea/enzimologia , Reabsorção Óssea/genética , Reabsorção Óssea/prevenção & controle , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Catepsina K , Catepsinas/genética , Catepsinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Enzimológica da Expressão Gênica , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Sulfonamidas/farmacologia , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
The mechanisms by which erythemal UVB irradiation modulates systemic immune responses to antigens applied to non-irradiated sites are poorly understood. In this study, regulatory CD4+ T cells were identified in the skin-draining lymph nodes (SDLNs) of UVB-irradiated, but otherwise naive mice. A transgenic mouse strain (DO11.10) was utilized in which the majority of CD4+ T cells expressed the ovalbumin (OVA(323-339)) T-cell receptor. Thus, T-cell responses could be examined following erythemal UVB irradiation without further antigen sensitization. CD4+ T cells from the SDLNs of UVB-irradiated mice had significantly reduced capacity to respond to presentation of the OVA(323-339) peptide in vitro. Transfer of CD4+ T cells from the SDLNs of UVB-irradiated antigen-naive mice significantly reduced both OVA sensitization and contact hypersensitivity responses to an experimental hapten in the recipient mice. Depletion of CD4+CD25+ cells abrogated this UVB-suppressive effect in the in vitro proliferation assay. There was also a significant increase in the proportion of CD4+CD25+Foxp3+ cells in the SDLNs of UVB-irradiated mice. The potential of these regulatory cells poised to regulate responses to incoming antigens at distant non-irradiated sites broadens the biological impact of UVB irradiation of skin on immunity.
Assuntos
Formação de Anticorpos/fisiologia , Antígenos/imunologia , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/efeitos da radiação , Linfonodos/citologia , Raios Ultravioleta , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Dermatite de Contato/prevenção & controle , Relação Dose-Resposta à Radiação , Fatores de Transcrição Forkhead/metabolismo , Imunização , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/genética , Ovalbumina/imunologia , Ovalbumina/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de TempoRESUMO
UVB irradiation of the shaved dorsal skin of mice can cause both local and systemic suppression of contact hypersensitivity responses; the former demonstrated by administration of the sensitizing Ag/hapten to the irradiated site and the latter by its administration at least 72 h later to distal unirradiated sites. The immunological basis of systemic immunomodulation is not clear. When haptens (trinitrochlorobenzene, FITC) were administered to the shaved ventral skin 4 days after irradiation (8 kJ/m(2)) to the shaved dorsum of BALB/c mice, CD11c(+)/FITC(+) cells in the skin-draining lymph nodes from control and irradiated mice produced on a per cell basis similar levels of IL-12 and PGE(2) were phenotypically mature and efficient at presenting FITC to lymphocytes from FITC-sensitized mice. Ag presentation by FACS-sorted CD11c(+) lymph node cells isolated 4 days after UVB irradiation was as efficient as were cells from unirradiated mice at presentation in vitro of an OVA peptide (OVA(323-339)) to CD4(+) cells from OVA-TCR-transgenic DO11.10 mice. Further, IFN-gamma levels were increased in the cultures containing CD11c(+) cells from UVB-irradiated mice, suggesting that inflammation may precede downstream immunosuppression. These results suggest that the primary cause of reduced contact hypersensitivity responses in mice in which UV irradiation and the sensitizing Ag are applied to different sites several days apart must originate from cells other than CD11c(+) APCs that directly or by production of soluble mediators (IL-12, PGE(2)) affect cellular responses in the nodes of UVB-irradiated mice.