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

Abstract

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-1β and IFN-γ using rat islets and β cell lines. IL-1β induced the expression of the CXCL10 gene and promoter activity, whereas the combination of IL-1β plus IFN-γ was synergistic. Small interfering RNA–mediated suppression of NF-κB 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-γ. Furthermore, we found that a JAK1 inhibitor dose dependently reduced IFN-γ–controlled CXCL10 gene expression and promoter activity, concomitant with a decrease in STAT1 phosphorylation at Tyr701. We further discovered that, although the Tyr701 phosphorylation site is inducible (within 15 min of IFN-γ exposure), the Ser727 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-γ–mediated potentiation of CXCL10 gene expression, promoter activity, and secretion of protein. Moreover, the Ser727 phosphorylation was neither contingent on a functional Y701 site in β cells nor was it required for cytokine-mediated expression of the CXCL10 gene. We conclude that the synergism of IL-1β and IFN-γ to induce expression of the CXCL10 gene requires NF-κB, STAT1 phosphorylated at Tyr701, recruitment of coactivators, and acetylation of histones H3 and H4.

<b>The role of IFN-γ in regulating Nfkbiz expression in epidermal </b><b>keratinocytes </b>

“…These results suggest that JAK1 but not JAK2 is critical for Nfkbiz expression in the JAK/STAT pathway and that NF-κB is an important transcriptional factor to respond to an inflammatory stimulus such as IFN-γ as well as the constitutive expression of Nfkbiz in the resting keratinocytes. Our findings that both JAK/STAT and NF-κB pathways are important for the synergism are consistent with a previous report on CXCL10 (3). Although STAT3 is known to play a very important role in inducing Nfkbiz expression in immune cells among the STAT family (9,11,12), the central STAT molecule acting downstream of IFN-γ signaling is STAT1 or STAT2 rather than STAT3 (1,15).…”

“…IFN-γ synergistically induced Nfkbiz expression in concurrent stimulation with IL-1. It is known that IFN-γ plus IL-1 synergistically induce inflammatory cytokine expression in certain types of cell (3,21). Both NF-κB binding sites and ISREs in the promoter region of target genes are important for this synergism (3).…”

IL-1β reciprocally regulates chemokine and insulin secretion in pancreatic β-cells via NF-κB

“…With many discrete chemokines made in response to IL-1␤ and IFN␥ in pancreatic ␤-cells (8,44), the ␤-cell influence on tissue leukocytosis and overall pancreatic inflammatory responses may be underappreciated. It also appears that NF-B is the major regulatory factor controlling chemokine production in response to IL-1␤ (9,10,12), whereas STAT1 is the predominant transcription factor mediating the increased chemokine production by IFN␥ (9, 12). Furthermore, NF-B is the key determinant of the chemokine/insulin secretion ratio, suggesting that modulation of NF-B would be beneficial for improving islet function by suppressing inflammatory responses.…”

Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice

“…Because both macrophage and T-cell populations contribute to autoimmune-mediated destruction of pancreatic β-cells [48,49], we examined transcriptional readouts from CCL2 and CXCL10 gene promoters. Using 832/13 rat insulinoma cells, a clonal β-cell population [36], we found that promoter activity driven by CCL2 or CXCL10 genomic regions is increased by IL-1β [50,51]. However, overnight culture of 832/13 rat insulinoma cells with 100 μg/mL herbal extract prior to IL-1β exposure decreased the activity of the CCL2 promoter by 29% (Fig.…”