RESUMO
RNA interference (RNAi) is a unique and powerful tool used for the study of gene function by suppressing its expression. Nuclear factor of activated T cells (NFATc1) is the most strongly induced transcription factor mediated by receptor activator for nuclear factor kappa B ligand stimulation and has shown to be a key regulator of osteoclastogenesis. To determine the application of small interfering RNA (siRNA) for inhibition of lipopolysaccharide (LPS)-induced cytokine stimulation and osteoclast formation, murine monocyte, RAW 264.7 cells as well as differentiated osteoclasts were transfected with NFATc1-specific siRNA and then stimulated with 100 ng/mL LPS. By using real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay, we confirmed that monocytes whose NFATc1 protein expression was silenced by using RNAi produced lower levels of inflammatory cytokines, fewer numbers evolved into mature osteoclasts, and osteoclasts expressed lower levels of osteoclast-specific gene markers such as tartrate-resistant acid phosphatase and cathepsin K. These results suggested that RNAi could be used to modulate the effects of LPS stimulation.
Assuntos
Citocinas/biossíntese , Lipopolissacarídeos/antagonistas & inibidores , Fatores de Transcrição NFATC/antagonistas & inibidores , Osteoclastos/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Animais , Catepsina K , Catepsinas/biossíntese , Catepsinas/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Camundongos , Monócitos/efeitos dos fármacos , Fatores de Transcrição NFATC/genética , Interferência de RNA , Transfecção , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
Lipopolysaccharide (LPS) in the outer layers of Gram-negative bacteria plays an important role in initiating and sustaining periapical lesions. To understand the mechanisms of osteoclastic bone resorption in periapical lesions induced by LPS, we stimulated osteoclast precursors, RAW 264.7 cells with LPS. LPS stimulated osteoclastogenesis when osteoclast precursors were primed with activator for NF-kappaB ligand (RANKL) as little as 24 h. By employing real-time PCR analysis, we have confirmed that osteoclast-like cells stimulated by LPS express high level of osteoclast-specific gene markers such as TRAP, cathepsin K, and calcitonin receptor. These results suggest that bone-resportive action by LPS is partially independent of RANKL.