Nuclear factor κB regulates gene expression of inducible nitricoxide synthase in human monocyte

AIM: To investigate whether gene expression of inducible nitric oxide synthase(iNOS)in human monocyte is modulated by nuclear factor κB(NF κB). METHODS: Human monocytes were cultured in medium with lipopolysaccharide(LPS)± pyrrolidine dithiocarbamate(PDTC)or in medium alone. One hour later, the nuclear protein of the monocytes was extracted for electrophoretic mobility shift assay (EMSA) and the monocyte coverslips were collected for immunofluorescence staining for NF κB p65 subunit. Ten hours later, RNA was extracted for RT PCR for iNOS. Twenty four hours later, whole cell extracts were used for Western blot and monocyte corverslips were collected for immunocytochemical staining. RESULTS: LPS increased the NF κB activation compared with that of the unstimulated cells. Ten hours later, the mRNA expression of iNOS increased and twenty four hours later the protein expression of iNOS increased markedly. The inhibition of NF κB activation by PDTC resulted in a significant decrease of the expressions of iNOS mRNA and protein hours later. The positive rate of nuclear staining of p65 and the DNA binding activity of NF κB significantly were correlated with the mRNA and protein expression of iNOS. CONCLUSION: NF κB regulates the gene expression of iNOS in human monocytes.