RESUMO
SHIP is an SH2-containing inositol-5-phosphatase expressed in hematopoietic cells. It hydrolyzes the PI3K product PI(3,4,5)P(3) and blunts the PI3K-initiated signaling pathway. Although the PI3K/Akt pathway has been shown to be important for osteoclastogenesis, the molecular events involved in osteoclast differentiation have not been revealed. We demonstrate that Akt induces osteoclast differentiation through regulating the GSK3ß/NFATc1 signaling cascade. Inhibition of the PI3K by LY294002 reduces formation of osteoclasts and attenuates the expression of NFATc1, but not that of c-Fos. Conversely, overexpression of Akt in bone marrow-derived macrophages (BMMs) strongly induced NFATc1 expression without affecting c-Fos expression, suggesting that PI3K/Akt-mediated NFATc1 induction is independent of c-Fos during RANKL-induced osteoclastogenesis. In addition, we found that overexpression of Akt enhances formation of an inactive form of GSK3ß (phospho-GSK3ß) and nuclear localization of NFATc1, and that overexpression of a constitutively active form of GSK3ß attenuates osteoclast formation through downregulation of NFATc1. Furthermore, BMMs from SHIP knockout mice show the increased expression levels of phospho-Akt and phospho-GSK3ß, as well as the enhanced osteoclastogenesis, compared with wild type. However, overexpression of a constitutively active form of GSK3ß attenuates RANKL-induced osteoclast differentiation from SHIP-deficient BMMs. Our data suggest that the PI3K/Akt/GSK3ß/NFATc1 signaling axis plays an important role in RANKL-induced osteoclastogenesis.
Assuntos
Diferenciação Celular/imunologia , Quinase 3 da Glicogênio Sintase/imunologia , Fatores de Transcrição NFATC/imunologia , Osteoclastos/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/imunologia , Transporte Ativo do Núcleo Celular , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Núcleo Celular/genética , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Inositol Polifosfato 5-Fosfatases , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/imunologia , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/genética , Fosforilação/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK/genética , Ligante RANK/imunologia , Ligante RANK/metabolismo , Transdução de Sinais/genéticaRESUMO
NFATc1 (nuclear factor of activated T-cells c1), a key transcription factor, plays a role in regulating expression of osteoclast-specific downstream target genes such as TRAP (tartrate-resistant acid phosphatase) and OSCAR (osteoclast-associated receptor). It has been shown that RANKL [receptor activator of NF-κB (nuclear factor κB) ligand] induces NFATc1 expression during osteoclastogenesis at a transcriptional level. In the present study, we demonstrate that RANKL increases NFATc1 protein levels by post-translational modification. RANKL stimulates NFATc1 acetylation via HATs (histone acetyltransferases), such as p300 and PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor], thereby stabilizing NFATc1 proteins. PCAF physically interacts with NFATc1 and directly induces NFATc1 acetylation and stability, subsequently increasing the transcriptional activity of NFATc1. In addition, RANKL-mediated NFATc1 acetylation is increased by the HDAC (histone deacetylase) inhibitors sodium butyrate and scriptaid. Overexpression of HDAC5 reduces RANKL- or PCAF-mediated NFATc1 acetylation, stability and transactivation activity, suggesting that the balance between HAT and HDAC activities might play a role in the regulation of NFATc1 levels. Furthermore, RANKL and p300 induce PCAF acetylation and stability, thereby enhancing the transcriptional activity of NFATc1. Down-regulation of PCAF by siRNA (small interfering RNA) decreases NFATc1 acetylation and stability, as well as RANKL-induced osteoclastogenesis. Taken together, the results of the present study demonstrate that RANKL induces HAT-mediated NFATc1 acetylation and stability, and subsequently increases the transcriptional activity of NFATc1 during osteoclast differentiation.