Synergistic Expression of the CXCL10 Gene in Response to IL-1β and IFN-γ Involves NF-κB, Phosphorylation of STAT1 at Tyr701, and Acetylation of Histones H3 and H4

The CXCL10 gene encodes a peptide that chemoattracts a variety of leukocytes associated with type 1 and type 2 diabetes. The present study was undertaken to determine the molecular mechanisms required for expression of the CXCL10 gene in response to IL-1 beta and IFN-gamma using rat islets and beta cell lines. IL-1 beta induced the expression of the CXCL10 gene and promoter activity, whereas the combination of IL-1 beta plus IFN-gamma was synergistic. Small interfering RNA-mediated suppression of NF-kappa B p65 markedly inhibited the ability of cytokines to induce the expression of the CXCL10 gene, whereas targeting STAT1 only diminished the synergy provided by IFN-gamma. Furthermore, we found that a JAK1 inhibitor dose dependently reduced IFN-gamma-controlled CXCL10 gene expression and promoter activity, concomitant with a decrease in STAT1 phosphorylation at Tyr(701). We further discovered that, although the Tyr(701) phosphorylation site is inducible (within 15 min of IFN-gamma exposure), the Ser(727) site within STAT1 is constitutively phosphorylated. Thus, we generated single-mutant STAT1 Y701F and double-mutant STAT1 Y701F/S727A adenoviruses. Using these recombinant adenoviruses, we determined that overexpression of either the single- or double-mutant STAT1 decreased the IFN-gamma-mediated potentiation of CXCL10 gene expression, promoter activity, and secretion of protein. Moreover, the Ser(727) phosphorylation was neither contingent on a functional Y701 site in beta cells nor was it required for cytokine-mediated expression of the CXCL10 gene. We conclude that the synergism of IL-1 beta and IFN-gamma to induce expression of the CXCL10 gene requires NF-kappa B, STAT1 phosphorylated at Tyr(701), recruitment of coactivators, and acetylation of histones H3 and H4.