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Proinflammatory cytokines impact islet β-cell mass and function by altering the transcriptional activity within pancreatic β-cells, producing increases in intracellular nitric oxide abundance and the synthesis and secretion of immunomodulatory proteins such as chemokines. Herein, we report that IL-1β, a major mediator of inflammatory responses associated with diabetes development, coordinately and reciprocally regulates chemokine and insulin secretion. We discovered that NF-κB controls the increase in chemokine transcription and secretion as well as the decrease in both insulin secretion and proliferation in response to IL-1β. Nitric oxide production, which is markedly elevated in pancreatic β-cells exposed to IL-1β, is a negative regulator of both glucose-stimulated insulin secretion and glucose-induced increases in intracellular calcium levels. By contrast, the IL-1β-mediated production of the chemokines CCL2 and CCL20 was not influenced by either nitric oxide levels or glucose concentration. Instead, the synthesis and secretion of CCL2 and CCL20 in response to IL-1β were dependent on NF-κB transcriptional activity. We conclude that IL-1β-induced transcriptional reprogramming via NF-κB reciprocally regulates chemokine and insulin secretion while also negatively regulating β-cell proliferation. These findings are consistent with NF-κB as a major regulatory node controlling inflammation-associated alterations in islet β-cell function and mass., (Copyright © 2015 the American Physiological Society.)

Synthesis and secretion of immunomodulatory proteins, such as cytokines and chemokines, controls the inflammatory response within pancreatic islets. When this inflammation does not resolve, destruction of pancreatic islet β-cells leads to diabetes mellitus. Production of the soluble mediators of inflammation, such as TNF-α and IL-1β, from resident and invading immune cells, as well as directly from islet β-cells, is also associated with suboptimal islet transplantation outcomes. In this study, we found that IL-1β induces rapid increases in TNF-α mRNA in rat and human islets and the 832/13 clonal β-cell line. The surge in transcription of the TNF-α gene required the inhibitor of kappa B kinase beta (IκKβ), the p65 subunit of the NF-κB and a signal-specific recruitment of RNA polymerase II to the gene promoter. Of note was the increased intracellular production of TNF-α protein in a manner consistent with mRNA accumulation in response to IL-1β, but no detectable secretion of TNF-α into the media. Additionally, TNF-α specifically induces expression of CD11b, but not CD11c, on neutrophils, which could contribute to the inflammatory milieu and diabetes progression. We conclude that activation of the NF-κB pathway in pancreatic β-cells leads to rapid intracellular production of the pro-inflammatory TNF-α protein through a combination of specific histone covalent modifications and NF-κB signaling pathways., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The proinflammatory cytokines IL-1β and IFN-γ decrease functional islet β-cell mass in part through the increased expression of specific genes, such as inducible nitric oxide synthase (iNOS). Dysregulated iNOS protein accumulation leads to overproduction of nitric oxide, which induces DNA damage, impairs β-cell function, and ultimately diminishes cellular viability. However, the transcriptional mechanisms underlying cytokine-mediated expression of the iNOS gene are not completely understood. Herein, we demonstrated that individual mutations within the proximal and distal nuclear factor-κB sites impaired cytokine-mediated transcriptional activation. Surprisingly, mutating IFN-γ-activated site (GAS) elements in the iNOS gene promoter, which are classically responsive to IFN-γ, modulated transcriptional sensitivity to IL-1β. Transcriptional sensitivity to IL-1β was increased by generation of a consensus GAS element and decreased correspondingly with 1 or 2 nucleotide divergences from the consensus sequence. The nuclear factor-κB subunits p65 and p50 bound to the κB response elements in an IL-1β-dependent manner. IL-1β also promoted binding of serine-phosphorylated signal transducer and activator of transcription-1 (STAT1) (Ser727) but not tyrosine-phosphorylated STAT1 (Tyr701) to GAS elements. However, phosphorylation at Tyr701 was required for IFN-γ to potentiate the IL-1β response. Furthermore, coactivator p300 and coactivator arginine methyltransferase were recruited to the iNOS gene promoter with concomitant displacement of the coactivator CREB-binding protein in cells exposed to IL-1β. Moreover, these coordinated changes in factor recruitment were associated with alterations in acetylation, methylation, and phosphorylation of histone proteins. We conclude that p65 and STAT1 cooperate to control iNOS gene transcription in response to proinflammatory cytokines by a coactivator exchange mechanism. This increase in transcription is also associated with signal-specific chromatin remodeling that leads to RNA polymerase II recruitment and phosphorylation.

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 Tyr(701). We further discovered that, although the Tyr(701) phosphorylation site is inducible (within 15 min of IFN-γ 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-γ-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 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 Tyr(701), recruitment of coactivators, and acetylation of histones H3 and H4.

Release of pro-inflammatory cytokines from both resident and invading leukocytes within the pancreatic islets impacts the development of Type 1 diabetes mellitus. Synthesis and secretion of the chemokine CCL2 from pancreatic β-cells in response to pro-inflammatory signaling pathways influences immune cell recruitment into the pancreatic islets. Therefore, we investigated the positive and negative regulatory components controlling expression of the CCL2 gene using isolated rat islets and INS-1-derived β-cell lines. We discovered that activation of the CCL2 gene by IL-1β required the p65 subunit of NF-κB and was dependent on genomic response elements located in the -3.6 kb region of the proximal gene promoter. CCL2 gene transcription in response to IL-1β was blocked by pharmacological inhibition of the IKKβ and p38 MAPK pathways. The IL-1β-mediated increase in CCL2 secretion was also impaired by p38 MAPK inhibition and by glucocorticoids. Moreover, multiple synthetic glucocorticoids inhibited the IL-1β-stimulated induction of the CCL2 gene. Induction of the MAP Kinase Phosphatase-1 (MKP-1) gene by glucocorticoids or by adenoviral-mediated overexpression decreased p38 MAPK phosphorylation, which diminished CCL2 gene expression, promoter activity, and release of CCL2 protein. We conclude that glucocorticoid-mediated repression of IL-1β-induced CCL2 gene transcription and protein secretion occurs in part through the upregulation of the MKP-1 gene and subsequent deactivation of the p38 MAPK. Furthermore, the anti-inflammatory actions observed with MKP-1 overexpression were obtained without suppressing glucose-stimulated insulin secretion. Thus, MKP-1 is a possible target for anti-inflammatory therapeutic intervention with preservation of β-cell function.

The pro-inflammatory cytokine IL-1β leads to losses in functional β-cell mass in part by inducing the expression of genes that produce soluble mediators of inflammation, such as cyclooxygenase-2 (COX2). In the current study, we sought to understand what factors control the COX2 gene in response to IL-1β and how prostaglandins downstream of COX2 impact pro-inflammatory gene transcription in pancreatic β-cells. We analyzed COX2 gene expression in response to different maneuvers impacting NF-κB proteins. Also, we report alterations in the expression of COX2, EP-3 and EP-4 receptor genes by PGD(2) and PGE(2). Moreover, we examined whether PGD(2) and PGE(2) regulated NF-κB and interferon-gamma activated sequence (GAS) reporter gene activity. IL-1β-mediated induction of the COX2 gene requires the p65 and p50 subunits of NF-κB. In addition, PGD(2) and PGE(2) coordinately alter COX2 and EP receptor gene expression patterns and potentiate the cytokine-mediated transcriptional activity of promoters containing NF-κB or GAS response elements., (Copyright © 2011 Elsevier Inc. All rights reserved.)