145: The erthyrocyte cytosolic 5′-nucleotidase III is a broad tissue IFN-stimulated gene that limits NF-κB-mediated inflammatory response

Abstract

Type-I interferon (IFN) has been shown to inhibit inflammatory processes such as NF- κ B activation and cytokine production through largely unknown mechanisms. We have found that pyrimidine 5′-nucleotidase (PN-I or NT5C3), previously designated as erythrocyte-specific gene product, is stimulated by IFN in a number of cell types, including normal, transformed, epithelial and fibroblast cells. Type-I IFN but not IFN- γ exerts dose-dependent stimulation of NT5C3 mRNA and protein. This activation requires Stat1/tyk2 factors and ISRE binding. NT5C3 was also induced by a number of viruses and pro-inflammatory cytokines. Ectopic over-expression of NT5C3 in HEK293 cells significantly reduced TNF- α -mediated NF- κ B reporter activity. IFN- α -mediated suppression of the chemokine IL-8 mRNA and protein expression in the IFN-sensitive HeLa cells is more pronounced with NT5C3 overexpression. Down-modulation of NT5C3 in HEK293 cells significantly increased both basal and TNF- α -induced NF- κ B reporter activation. In order to explore further mechanisms, we investigated upstream signaling components, and showed that NT5C3 reduced IKK β -mediated activation of NF- κ B reporter.- IKK β is a kinase required to phosphorylate and degrade NF-kB inhibitor (IK β ). Results were further confirmed with NT5C3 silencing showing enhancement of NF- κ B reporter activity in cells overexpressing wild-type IKK β . These activities were not seen in the case of IKK β (S177E) mutant experiments suggesting that NT5C3 may interfere with IKK β related activity such as its phosphorylation. Indeed, we found that NT5C3 could reduce phosphorylated levels of IKK β during TNF- α activation. Global gene expression profiling.-and further QPCR/WB confirmatory tests.- indicated that serine/threonine protein phosphatase (PPP2R1A), which dephosphorylates IKK β expression, was enhanced in NT5C3-expressing cells. IFN-mediated suppression of IL-8 expression is diminished in both NT5C3- or PPP2R1A-silenced cells. Overall, these results suggest that NT5C3 is broad ISG involved in dampening inflammation through changes in PPP2R1A/IKK β /NF- κ B network with reduction in pro-inflammatory cytokines highlighting a novel mode for the anti-inflammatory action of Type-I IFN.