Effect of S-nitrosylation induced by IL-1β and IFN-γ on DNA binding activity of cAMP response element binding protein in fibroblast-like synoviocytes

AIM:To investigate the effect of S-nitrosylation induced by recombinant interleukin-1β (rIL 1β) and recombinant interferon-γ (rIFN-γ) on DNA binding activity of cAMP response element binding protein (CREB) in fibroblast-like synoviocytes (FLSs).METHODS:(1) FLSs were incubated with rIL-1β and rIFN-γ in the presence or absence of inducible nitric oxide synthase inhibitor aminoguanidine (AG) for 12 h.The supernatant of the cell culture was collected to determine the contents of nitric oxide (NO).The total proteins prepared from each group[only the total proteins prepared from rIL-1β+ rIFN-γ group was reacted with dithiothreitol (DTT) for 15 min in vitro]were subjected to the biotin switch assay,and the level of S-nitrosylation was determined by Western blotting.(2) FLSs were incubated with rIL-1β,rIL-1β+ rIFN-γ and AG respectively for 12 h.The nuclear extracts from each group were prepared.The nuclear extracts from each group were subjected to electrophoresis mobility shift assay to analyze the DNA binding activity of CREB (only the nuclear extracts from rIL-1β+ rIFN-γ group was reacted with DTT for 15 min in vitro before assaying).RESULTS:rIL-1β plus rIFN-γ increased the production of NO and the level of S-nitrosylation,which was inhibited by AG.Administration of DTT in the total proteins reversed the induction of S-nitrosylation by rIL 1β and rIFN-γ.Co-incubation with rIL-1β and rIFN-γ inhibited the CREB activity induced by rIL-1β alone,which was reversed by AG.Administration of DTT in the nuclear extracts reversed the effect of co-incubation of the cytokines.CONCLUSION:Co-incubation with rIL-1β and rIFN-γ may increase the level of S-nitrosylation through inducing the production of endogenous NO,leading to reversible thiols modification of CREB and inhibit the DNA binding activity of CREB in FLSs.