Application of small interfering RNA for inhibition of lipopolysaccharide-induced osteoclast formation and cytokine stimulation.
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.
Fahid
Farshid S
FS
Division of Endodontology, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030-1715, USA.
Jiang
Jin
J
Zhu
Qiang
Q
Zhang
Cuiping
C
Filbert
Elizabeth
E
Safavi
Kamran E
KE
Spångberg
Larz S W
LS
D000818:Animals; D056657:Cathepsin K; D002403:Cathepsins / Q000096:biosynthesis / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors*; D051379:Mice; D009000:Monocytes / Q000187:drug effects; D050778:NFATC Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics; D010010:Osteoclasts / Q000187:drug effects*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
D016207:Cytokines; D015850:Interleukin-6; D008070:Lipopolysaccharides; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D002403:Cathepsins; D056657:Cathepsin K; C534132:Ctsk protein, mouse