Pancreatic deletion of the interleukin-1 receptor disrupts whole body glucose homeostasis and promotes islet ß-cell de-differentiation.

OBJECTIVE: Pancreatic tissue, and islets in particular, are enriched in expression of the interleukin-1 receptor type I (IL-1R). Because of this enrichment, islet ß-cells are exquisitely sensitive to the IL-1R ligands IL-1α and IL-1ß, suggesting that signaling through this pathway regulates health and function of islet ß-cells. METHODS: Herein, we report a targeted deletion of IL-1R in pancreatic tissue (IL-1RPdx1-/-) in C57BL/6J mice and in db/db mice on the C57 genetic background. Islet morphology, ß-cell transcription factor abundance, and expression of the de-differentiation marker Aldh1a3 were analyzed by immunofluorescent staining. Glucose and insulin tolerance tests were used to examine metabolic status of these genetic manipulations. Glucose-stimulated insulin secretion was evaluated in vivo and in isolated islets ex vivo by perifusion. RESULTS: Pancreatic deletion of IL-1R leads to impaired glucose tolerance, a phenotype that is exacerbated by age. Crossing the IL-1RPdx1-/- with db/db mice worsened glucose tolerance without altering body weight. There were no detectable alterations in insulin tolerance between IL-1RPdx1-/- mice and littermate controls. However, glucose-stimulated insulin secretion was reduced in islets isolated from IL-1RPdx1-/- relative to control islets. Insulin output in vivo after a glucose challenge was also markedly reduced in IL-1RPdx1-/- mice when compared with littermate controls. Pancreatic islets from IL-1RPdx1-/- mice displayed elevations in Aldh1a3, a marker of de-differentiation, and reduction in nuclear abundance of the ß-cell transcription factor MafA. Nkx6.1 abundance was unaltered. CONCLUSIONS: There is an important physiological role for pancreatic IL-1R to promote glucose homeostasis by suppressing expression of Aldh1a3, sustaining MafA abundance, and supporting glucose-stimulated insulin secretion in vivo.

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

Proinflammatory cytokines impact islet ß-cell mass and function by altering the transcriptional activity within pancreatic ß-cells, producing increases in intracellular nitric oxide abundance and the synthesis and secretion of immunomodulatory proteins such as chemokines. Herein, we report that IL-1ß, 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-1ß. Nitric oxide production, which is markedly elevated in pancreatic ß-cells exposed to IL-1ß, is a negative regulator of both glucose-stimulated insulin secretion and glucose-induced increases in intracellular calcium levels. By contrast, the IL-1ß-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-1ß were dependent on NF-κB transcriptional activity. We conclude that IL-1ß-induced transcriptional reprogramming via NF-κB reciprocally regulates chemokine and insulin secretion while also negatively regulating ß-cell proliferation. These findings are consistent with NF-κB as a major regulatory node controlling inflammation-associated alterations in islet ß-cell function and mass.

Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet ß-Cell Function.

Glucocorticoids signal through the glucocorticoid receptor (GR) and are administered clinically for a variety of situations, including inflammatory disorders, specific cancers, rheumatoid arthritis, and organ/tissue transplantation. However, glucocorticoid therapy is also associated with additional complications, including steroid-induced diabetes. We hypothesized that modification of the steroid backbone is one strategy to enhance the therapeutic potential of GR activation. Toward this goal, two commercially unavailable, thiobenzothiazole-containing derivatives of hydrocortisone (termed MS4 and MS6) were examined using 832/13 rat insulinoma cells as well as rodent and human islets. We found that MS4 had transrepression properties but lacked transactivation ability, whereas MS6 retained both transactivation and transrepression activities. In addition, MS4 and MS6 both displayed anti-inflammatory activity. Furthermore, MS4 displayed reduced impact on islet ß-cell function in both rodent and human islets. Similar to dexamethasone, MS6 promoted adipocyte development in vitro, whereas MS4 did not. Moreover, neither MS4 nor MS6 activated the Pck1 (Pepck) gene in primary rat hepatocytes. We conclude that modification of the functional groups attached to the D-ring of the hydrocortisone steroid molecule produces compounds with altered structure-function GR agonist activity with decreased impact on insulin secretion and reduced adipogenic potential but with preservation of anti-inflammatory activity.

CCL20 is elevated during obesity and differentially regulated by NF-κB subunits in pancreatic ß-cells.

Enhanced leukocytic infiltration into pancreatic islets contributes to inflammation-based diminutions in functional ß-cell mass. Insulitis (aka islet inflammation), which can be present in both T1DM and T2DM, is one factor influencing pancreatic ß-cell death and dysfunction. IL-1ß, an inflammatory mediator in both T1DM and T2DM, acutely (within 1h) induced expression of the CCL20 gene in rat and human islets and clonal ß-cell lines. Transcriptional induction of CCL20 required the p65 subunit of NF-κB to replace the p50 subunit at two functional κB sites within the CCL20 proximal gene promoter. The NF-κB p50 subunit prevents CCL20 gene expression during unstimulated conditions and overexpression of p50 reduces CCL20, but enhances cyclooxygenase-2 (COX-2), transcript accumulation after exposure to IL-1ß. We also identified differential recruitment of specific co-activator molecules to the CCL20 gene promoter, when compared with the CCL2 and COX2 genes, revealing distinct transcriptional requirements for individual NF-κB responsive genes. Moreover, IL-1ß, TNF-α and IFN-γ individually increased the expression of CCR6, the receptor for CCL20, on the surface of human neutrophils. We further found that the chemokine CCL20 is elevated in serum from both genetically obese db/db mice and in C57BL6/J mice fed a high-fat diet. Taken together, these results are consistent with a possible activation of the CCL20-CCR6 axis in diseases with inflammatory components. Thus, interfering with this signaling pathway, either at the level of NF-κB-mediated chemokine production, or downstream receptor activation, could be a potential therapeutic target to offset inflammation-associated tissue dysfunction in obesity and diabetes.

Transcription of the gene encoding TNF-α is increased by IL-1ß in rat and human islets and ß-cell lines.

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 ß-cells leads to diabetes mellitus. Production of the soluble mediators of inflammation, such as TNF-α and IL-1ß, from resident and invading immune cells, as well as directly from islet ß-cells, is also associated with suboptimal islet transplantation outcomes. In this study, we found that IL-1ß induces rapid increases in TNF-α mRNA in rat and human islets and the 832/13 clonal ß-cell line. The surge in transcription of the TNF-α gene required the inhibitor of kappa B kinase beta (IκKß), 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-1ß, 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 ß-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.

NF-κB and STAT1 control CXCL1 and CXCL2 gene transcription.

Diabetes mellitus results from immune cell invasion into pancreatic islets of Langerhans, eventually leading to selective destruction of the insulin-producing ß-cells. How this process is initiated is not well understood. In this study, we investigated the regulation of the CXCL1 and CXCL2 genes, which encode proteins that promote migration of CXCR2(+) cells, such as neutrophils, toward secreting tissue. Herein, we found that IL-1ß markedly enhanced the expression of the CXCL1 and CXCL2 genes in rat islets and ß-cell lines, which resulted in increased secretion of each of these proteins. CXCL1 and CXCL2 also stimulated the expression of specific integrin proteins on the surface of human neutrophils. Mutation of a consensus NF-κB genomic sequence present in both gene promoters reduced the ability of IL-1ß to promote transcription. In addition, IL-1ß induced binding of the p65 and p50 subunits of NF-κB to these consensus κB regulatory elements as well as to additional κB sites located near the core promoter regions of each gene. Additionally, serine-phosphorylated STAT1 bound to the promoters of the CXCL1 and CXCL2 genes. We further found that IL-1ß induced specific posttranslational modifications to histone H3 in a time frame congruent with transcription factor binding and transcript accumulation. We conclude that IL-1ß-mediated regulation of the CXCL1 and CXCL2 genes in pancreatic ß-cells requires stimulus-induced changes in histone chemical modifications, recruitment of the NF-κB and STAT1 transcription factors to genomic regulatory sequences within the proximal gene promoters, and increases in phosphorylated forms of RNA polymerase II.

Regulation of iNOS gene transcription by IL-1ß and IFN-γ requires a coactivator exchange mechanism.

The proinflammatory cytokines IL-1ß and IFN-γ decrease functional islet ß-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 ß-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-1ß. Transcriptional sensitivity to IL-1ß 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-1ß-dependent manner. IL-1ß 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-1ß 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-1ß. 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.

Synergistic expression of the CXCL10 gene in response to IL-1ß 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-1ß and IFN-γ using rat islets and ß cell lines. IL-1ß induced the expression of the CXCL10 gene and promoter activity, whereas the combination of IL-1ß 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 ß cells nor was it required for cytokine-mediated expression of the CXCL10 gene. We conclude that the synergism of IL-1ß 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.

Regulation of the CCL2 gene in pancreatic ß-cells by IL-1ß and glucocorticoids: role of MKP-1.

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 ß-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 ß-cell lines. We discovered that activation of the CCL2 gene by IL-1ß 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-1ß was blocked by pharmacological inhibition of the IKKß and p38 MAPK pathways. The IL-1ß-mediated increase in CCL2 secretion was also impaired by p38 MAPK inhibition and by glucocorticoids. Moreover, multiple synthetic glucocorticoids inhibited the IL-1ß-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-1ß-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 ß-cell function.

Genistein inhibits tamoxifen effects on cell proliferation and cell cycle arrest in T47D breast cancer cells.

Tamoxifen is an antiestrogen used in the treatment of estrogen receptor-positive breast cancer in postmenopausal women. It functions by competitively inhibiting the estrogen receptor and inducing apoptosis and G1 cell cycle arrest. Genistein is a soy phytoestrogen that inhibits breast cancer cell growth in vitro at doses of 10 microM or above. At lower doses genistein may stimulate cell growth and entry into the cell cycle. We hypothesized that treatment with low-dose genistein would reverse the inhibitory effects of tamoxifen in estrogen-receptor-positive breast cancer cells. Cell cycle kinetics and cell proliferation in T47-D human breast cancer cells were examined after exposure to genistein and tamoxifen in a low-estrogen environment designed to mimic a post-menopausal state. Cell proliferation was assessed by a colorimetric assay. Cell cycle kinetics were determined by flow cytometry. Tamoxifen caused G1 arrest and a decrease in proliferation. Genistein reversed the inhibitory effects of tamoxifen on both proliferation and G1 arrest. Thus low-dose genistein was able to inhibit the therapeutic effects of tamoxifen in this postmenopausal model of breast cancer.