Martine I. Darville

Regulation by cytokines of the inducible nitric oxide synthase promoter in insulin-producing cells

Summary Cytokines could contribute to beta-cell damage in Type I diabetes mellitus. The radical nitric oxide, generated by the inducible form of nitric oxide synthase (iNOS), is a potential mediator of cytokine-induced beta-cell dysfunction. In rat pancreatic islets and insulin-producing cell lines, interleukin-1β (IL-1β) induces expression of iNOS mRNA and increases NO production, an effect potentiated by interferon-γ (IFN-γ). In human islet cells both IL-1β and IFN-γ are required for iNOS expression. We have shown previously that both the transcription factors nuclear factor-kB (NF-kB) and interferon regulatory factor-1 (IRF-1) are activated by cytokines in rodent and human islets but there is no direct information on the regulation of the iNOS promoter in insulin-producing cells. We presently investigated the effects of cytokines on iNOS transcriptional regulation in both rat insulin-producing RINm5F cells and in primary FACS-purified rat beta cells. Transient transfection experiments with the 1.5-kb rat promoter region and 5 ′ deletants of it showed that a distal region extending up to –1002 bp, and containing a distal and a proximal nuclear factor-kB (NF-kB) binding site, a γ-interferon activated site (GAS) and two adjacent IFN-stimulated response elements (ISRE), is required for IL-1β induction and IFN-γ potentiation of iNOS activation. Site-mutation analysis showed that both the distal and proximal NF-kB and GAS are necessary for IL-1β-induced iNOS expression in RINm5F cells. In these cells IFN-γ potentiation is mostly mediated by GAS and ISRE, suggesting a role for the IFN-γ-induced transcription factors Stat1α (which binds GAS) and IRF-1 (which binds ISRE), which may cooperate with NF-kB induced by IL-1β for iNOS activation. In primary beta cells both NF-kB binding sites are required for IL-1β-induced iNOS promoter activation. In these cells IFN-γ neither increased IL-1β-induced iNOS promoter activity nor iNOS mRNA expression but it induced a twofold increase in NO production. The present results unveiled the nature of the promoter binding sites necessary for iNOS expression in rodent beta cells. This information could be relevant for the development of new strategies aimed at preventing cytokine-induced iNOS expression and consequent beta-cell damage. [Diabetologia (1998) 41: 1101–1108]

Gene conversion as a mechanism for antigenic variation in Trypanosomes

Expression of the gene coding for the trypanosome AnTat 1.1 surface antigen is linked to the duplicative transposition of a basic copy (BC) of this gene to an expression site. In two trypanosome clones successively derived from AnTat 1.1 (AnTat 1.10 and AnTat 1.1B) we found evidence that gene conversions are involved in the transformation of the AnTat 1.1 transposed element into the two new surface antigen coding sequences. Although the three resultant mRNAs--AnTat 1.1, 1.10, and 1.1B--are different, they still share large homologies. Two of them, AnTat 1.1 and 1.1B, code for surface coats that are indistinguishable by conventional serological techniques, whereas AnTat 1.10 has been found different by the same methods. The three genomic rearrangements involve two of the five members of the AnTat 1.1 gene family. These two members are both located in unstable telomeric regions similar to the expression site, each in a different orientation with respect to the DNA terminus. We have concluded that the duplicative transposition is achieved by a gene conversion that may affect variable lengths of the same silent genes, and that different members of the same surface antigen gene family can contribute to the diversification of the antigen repertoire.

Double-Stranded RNA Cooperates with Interferon-γ and IL-1β to Induce Both Chemokine Expression and Nuclear Factor-κB-Dependent Apoptosis in Pancreatic β-Cells: Potential Mechanisms for Viral-Induced Insulitis and β-Cell Death in Type 1 Diabetes Mellitus

Viral infections may trigger the autoimmune assault leading to type 1 diabetes mellitus. Double-stranded RNA (dsRNA) is produced by many viruses during their replicative cycle. The dsRNA, tested as synthetic poly(IC) (PIC), in synergism with the proinflammatory cytokines interferon-gamma (IFN-gamma) and/or IL-1 beta, results in nitric oxide production, Fas expression, beta-cell dysfunction, and death. Activation of the transcription nuclear factor-kappa B (NF-kappa B) is required for PIC-induced inducible nitric oxide synthase expression in beta-cells, and we hypothesized that this transcription factor may also participate in PIC-induced Fas expression and beta-cell apoptosis. This hypothesis, and the possibility that PIC induces expression of additional chemokines and cytokines (previously reported as NF-kappa B dependent) in pancreatic beta-cells, was investigated in the present study. We observed that the PIC-responsive region in the Fas promoter is located between nucleotides -223 and -54. Site-directed mutations at the NF-kappa B and CCAAT/enhancer binding protein-binding sites prevented PIC-induced Fas promoter activity. Increased Fas promoter activity was paralleled by enhanced susceptibility of PIC + cytokine-treated beta-cells to apoptosis induced by Fas ligand. beta-Cell infection with the NF-kappa B inhibitor AdI kappa B((SA)2) prevented both necrosis and apoptosis induced by PIC + IL-1 beta or PIC + IFN-gamma. Messenger RNAs for several chemokines and one cytokine were induced by PIC, alone or in combination with IFN-gamma, in pancreatic beta-cells. These included IP-10, interferon-gamma-inducible protein-10, IL-15, macrophage chemoattractant protein-1, fractalkine, and macrophage inflammatory protein-3 alpha. There was not, however, induction of IL-1 beta expression. We propose that dsRNA, generated during a viral infection, may contribute for beta-cell demise by both inducing expression of chemokines and IL-15, putative contributors for the build-up of insulitis, and by synergizing with locally produced cytokines to induce beta-cell apoptosis. Activation of the transcription factor NF-kappa B plays a central role in at least part of the deleterious effects of dsRNA in pancreatic beta-cells.

NF-κB Is Required for Cytokine-Induced Manganese Superoxide Dismutase Expression in Insulin-Producing Cells1

Reactive oxygen species play an important role in the cytotoxic effect of inflammatory cytokines on pancreatic beta-cells in type 1 diabetes mellitus. The antioxidant enzyme manganese superoxide dismutase (MnSOD) is part of the cellular defenses against these deleterious radicals. MnSOD gene expression is induced by cytokines in insulin-producing cells, but the transcriptional regulation of MnSOD expression in these cells is not well understood. In this report, we investigated the transcriptional regulation by cytokines of the rat MnSOD gene in insulin-producing cells. By transient transfections with promoter-luciferase reporter constructs, we identified two interleukin (IL)-1beta-responsive elements, conferring each an additive 3-fold IL-1beta-induced transcriptional activity. The first is located in the promoter region, whereas the second is located in the second intron of the MnSOD gene. Interestingly, the intronic element is required for interferon-gamma-induced potentiation. Site-directed mutagenesis and band-shift assays showed that an NF-kappaB binding site in each region is necessary, but not sufficient, for transcriptional induction by IL-1beta. Our results suggest that NF-kappaB may cooperate with CCAAT/enhancer-binding protein factors in the promoter region and with octamer and Ets factors in the intronic region.

Extracellular signal-regulated kinase is essential for interleukin-1-induced and nuclear factor κB-mediated gene expression in insulin-producing INS-1E cells

Aims/hypothesisThe beta cell destruction and insulin deficiency that characterises type 1 diabetes mellitus is partially mediated by cytokines, such as IL-1β, and by nitric oxide (NO)-dependent and -independent effector mechanisms. IL-1β activates mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK), and the nuclear factor kappa B (NFκB) pathway. Both pathways are required for expression of the gene encoding inducible nitric oxide synthase (iNOS) and for IL-1β-mediated beta cell death. The molecular mechanisms by which these two pathways regulate beta cell Nos2 expression are currently unknown. Therefore, the aim of this study was to clarify the putative crosstalk between MAPK and NFκB activation in beta cells.Materials and methodsThe MAPKs ERK, p38 and JNK were inhibited by SB203580, PD98059 or Tat-JNK binding domain or by cells overexpressing the JNK binding domain. The effects of MAPK inhibition on IL-1β-induced iNOS production and kappa B inhibitor protein (IκB) degradation were examined by western blotting. NFκB DNA binding was investigated by electrophoretic mobility shift assay, while NFκB-induced gene transcription was evaluated by gene reporter assays.ResultsInhibition of the MAPKs did not affect IκB degradation or NFκB DNA binding. However, inhibition of ERK reduced NFκB-mediated Nos2 expression; serine 276 phosphorylation of the p65 unit of the NFκB complex seemed critical, as evaluated by amino acid mutation analysis.Conclusions/interpretationERK activity is required for NFκB-mediated transcription of Nos2 in insulin-producing INS-1E cells, indicating that ERK regulates Nos2 expression by increasing the transactivating capacity of NFκB. This may involve phosphorylation of Ser276 on p65 by an as yet unidentified kinase.

Use of Microarray Analysis to Unveil Transcription Factor and Gene Networks Contributing to β Cell Dysfunction and Apoptosis

Abstract: The β cell fate following immune‐mediated damage depends on an intricate pattern of dozens of genes up‐ or downregulated in parallel and/or sequentially. We are utilizing microarray analysis to clarify the pattern of gene expression in primary rat β cells exposed to the proapoptotic cytokines, IL‐1β and/or IFN‐γ. The picture emerging from these experiments is that β cells are not passive bystanders of their own destruction. On the contrary, β cells respond to damage by activating diverse networks of transcription factors and genes that may either lead to apoptosis or preserve viability. Of note, cytokine‐exposed β cells produce and release chemokines that may contribute to the homing and activation of T cells and macrophages during insulitis. Several of the effects of cytokines depend on the activation of the transcription factor, NF‐κB. NF‐κB blocking prevents cytokine‐induced β cell death, and characterization of NF‐κB‐dependent genes by microarray analysis indicated that this transcription factor controls diverse networks of transcription factors and effector genes that are relevant for maintenance of β cell differentiated status, cytosolic and ER calcium homeostasis, attraction of mononuclear cells, and apoptosis. Identification of this and additional “transcription factor networks” is being pursued by cluster analysis of gene expression in insulin‐producing cells exposed to cytokines for different time periods. Identification of complex gene patterns poses a formidable challenge, but is now technically feasible. These accumulating evidences may finally unveil the molecular mechanisms regulating the β cell “decision” to undergo or not apoptosis in early T1DM.

Double-Stranded Ribonucleic Acid (RNA) Induces β-Cell Fas Messenger RNA Expression and Increases Cytokine-Induced β-Cell Apoptosis1

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by progressive destruction of insulin-producing pancreatic beta-cells. Both viral infections and the cytokines interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) have been suggested as potential mediators of beta-cell death in early T1DM. We presently investigated whether the viral replicative intermediate double stranded RNA [here used as synthetic polyinosinic-polycytidylic acid (PIC)] modifies the effects of IL-1beta and IFN-gamma on gene expression and viability of rat pancreatic beta-cells. For this purpose, fluorescence-activated cell sorting-purified rat beta-cells were exposed for 6-16 h (study of gene expression by RT-PCR) or 6-9 days (study of viability by nuclear dyes) to PIC and/or IL-1beta and IFN-gamma. PIC increased the expression of Fas and Mn superoxide dismutase messenger RNAs by 5- to 10-fold. IL-1beta and a combination of PIC and IFN-gamma (but not PIC or IFN-gamma alone) induced expression of inducible nitric oxide (NO) synthase (iNOS) and consequent NO production. Induction of iNOS expression by PIC and IFN-gamma requires nuclear factor-kappaB activation, as suggested by transfection experiments with iNOS promoter-luciferase reporter constructs into primary beta-cells. Combinations of IL-1beta plus IFN-gamma, PIC plus IFN-gamma, or PIC plus IL-1beta induced a 2- to 3-fold increase in the number of apoptotic beta-cells. Blocking of iNOS activity significantly decreased PIC- plus IL-1beta-induced, but not PIC- plus IFN-gamma-induced, apoptosis. In conclusion, PIC alone or in combination with cytokines modifies the expression of several genes in pancreatic beta-cells. Two of these genes, Fas and iNOS, may contribute to beta-cell death. The transcription factor nuclear factor-kappaB is required for PIC-induced iNOS expression. PIC has an additive effect on cytokine-induced beta-cell death by both NO-dependent (in the case of IL-1beta) and NO-independent (in the case of IFN-gamma) mechanisms. These findings suggest that viral intermediates in synergism with local cytokine production may play an important role in beta-cell apoptosis in early T1DM.

Gene activation and re-expression of a Trypanosoma brucei variant surface glycoprotein.

The expression of the Trypanosoma brucei variant surface glycoprotein AnTat 1.1 proceeds by a mechanism that transfers a duplicated gene copy into a new genomic environment, the so‐called expression site, where it will be expressed. We have isolated a genomic fragment containing the region spanning the expression site‐transposon junction, and the 5′ half of the coding sequence. Comparing this DNA segment with its template copy (basic copy) allowed us to identify the exact breaking point and indicated a base sequence which could be involved in initiating the transposition event. Sequencing data also indicated that the co‐transposed segment 5′ to the coding sequence is 430 bp in length. The extreme 5′ end of the mRNA is derived from a region in the expression site not immediately adjacent to the transposed DNA segment. This particular sequence exists in multiple copies in the genome and is common to the mRNA of all variant surface glycoproteins so far analysed.

BCL-6: a possible missing link for anti-inflammatory PPAR-δ signalling in pancreatic beta cells

Aims/hypothesisInflammatory mediators contribute to pancreatic beta cell death in type 1 diabetes. Beta cells respond to cytokine exposure by activating gene networks that alter cellular metabolism, induce chemokine release (thereby increasing insulitis), and cause apoptosis. We have previously shown by microarray analysis that exposure of INS-1E cells to IL-1β + IFN-γ induces the transcription factor peroxisome proliferator-activated receptor (Ppar)-δ and several of its target genes. PPAR-δ controls cellular lipid metabolism and is a major regulator of inflammatory responses. We therefore examined the role of PPAR-δ in cytokine-treated beta cells.Materials and methodsPrimary beta cells that had been purified by fluorescence-activated cell sorting and INS-1E cells were cultured in the presence of the cytokines TNF-α, IL-1β, or IL-1β + IFN-γ, or the synthetic PPAR-δ agonist GW501516. Gene expression was analysed by real-time PCR. PPAR-δ, monocyte chemoattractant protein (MCP-1, now known as CCL2) promoter and NF-κB activity were determined by luciferase reporter assays.ResultsExposure of primary beta cells or INS-1E cells to cytokines induced Ppar-δ mRNA expression and PPAR-δ-dependent CD36, lipoprotein lipase, acyl CoA synthetase and adipophilin mRNAs. Cytokines and the PPAR-δ agonist GW501516 also activated a PPAR-δ response element reporter in beta cells. Unlike immune cells, neither INS-1E nor beta cells expressed the transcriptional repressor B-cell lymphoma-6 (BCL-6). As a consequence, PPAR-δ activation by GW501516 did not decrease cytokine-induced Mcp-1 promoter activation or mRNA expression, as reported for macrophages. Transient transfection with a BCL-6 expression vector markedly reduced Mcp-1 promoter and NF-κB activities in beta cells.Conclusions/interpretationCytokines activate the PPAR-δ gene network in beta cells. This network does not, however, regulate the pro-inflammatory response to cytokines because beta cells lack constitutive BCL-6 expression. This may render beta cells particularly susceptible to propagating inflammation in type 1 diabetes.