RESUMO
We have previously shown that the transcription factor FOXO1 is elevated in conditions with high levels of bone resorption. To investigate the role of FOXO1 in the formation of osteoclasts, we examined mice with lineage-specific deletion of FOXO1 in osteoclast precursors and by knockdown of FOXO1 with small interfering RNA. The receptor activator for NF-κB ligand (RANKL), a principal bone-resorbing factor, induced FOXO1 expression and nuclear localization 2 d after stimulation in bone marrow macrophages and RAW264.7 osteoclast precursors. RANKL-induced osteoclast formation and osteoclast activity was reduced in half in vivo and in vitro with lineage-specific FOXO1 deletion (LyzM.Cre(+)FOXO1(L/L)) compared with matched controls (LyzM.Cre(-)FOXO1(L/L)). Similar results were obtained by knockdown of FOXO1 in RAW264.7 cells. Moreover, FOXO1-mediated osteoclast formation was linked to regulation of NFATc1 nuclear localization and expression as well as a number of downstream factors, including dendritic cell-specific transmembrane protein, ATP6vod2, cathepsin K, and integrin αv. Lastly, FOXO1 deletion reduced M-CSF-induced RANK expression and migration of osteoclast precursors. In the present study, we provide evidence that FOXO1 plays a direct role in osteoclast formation by mediating the effect of RANKL on NFATc1 and several downstream effectors. This is likely to be significant because FOXO1 and RANKL are elevated in osteolytic conditions.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Macrófagos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Ligante RANK/farmacologia , Animais , Western Blotting , Catepsina K/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Expressão Gênica/efeitos dos fármacos , Integrina alfa5/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Microscopia de Fluorescência , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Interferência de RNA , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
GW182 binds to Argonaute (AGO) proteins and has a central role in miRNA-mediated gene silencing. Using lentiviral shRNA-induced GW182 knockdown in HEK293 cells, this study identifies a new role of GW182 in regulating miRNA stability. Stably knocking down GW182 or its paralogue TNRC6B reduces transfected miRNA-mimic half-lives. Replenishment of GW182 family proteins, as well as one of its domain Δ12, significantly restores the stability of transfected miRNA-mimic. GW182 knockdown reduces miRNA secretion via secretory exosomes. Targeted siRNA screening identifies a 3'-5' exoribonuclease complex responsible for the miRNA degradation only when GW182 is knocked down. Immunoprecipitation further confirms that the presence of GW182 in the RISC complex is critical in protecting Argonaute-bound miRNA.