Abstract
Cytokines contribute to pancreatic islet inflammation, leading to impaired glucose homeostasis and diabetic diseases. A plethora of data shows that proinflammatory cytokines are produced in pancreatic islets by infiltrating mononuclear immune cells. Here, we show that pancreatic islet α cells and β cells express tumor necrosis factor-α (TNF-α) and other cytokines capable of promoting islet inflammation when exposed to interleukin-1β (IL-1β). Cytokine expression by β cells was dependent on calcineurin (CN)/nuclear factor of activated T cells (NFAT) and MAPK signaling. NFAT associated with the TNF-α promoter in response to stimuli and synergistically activated promoter activity with ATF2 and c-Jun. In contrast, the β-cell-specific transcriptional activator MafA could repress NFAT-mediated TNF-α gene expression whenever C/EBP-β was bound to the promoter. NFAT differentially regulated the TNF-α gene depending upon the expression and MAPK-dependent activation of interacting basic leucine zipper partners in β cells. Both p38 and JNK were required for induction of TNF-α mRNA and protein expression. Collectively, the data show that glucose and IL-1β can activate signaling pathways, which control induction and repression of cytokines in pancreatic endocrine cells. Thus, by these mechanisms, pancreatic β cells themselves may contribute to islet inflammation and their own immunological destruction in the pathogenesis of diabetes.
Highlights
Cytokines are small secreted or membrane-bound signaling proteins classically known for their roles in regulating immunity, inflammation, and hematopoiesis [1]
We postulated that IL-1 induces expression of cytokines in islet endocrine cells that may contribute to or exacerbate islet inflammation
The results show that high glucose enhances acute induction of TNF-␣ mRNA expression in islet endocrine cells and that the response is rapidly reversed within 4 h of IL-1 treatment
Summary
MIN6 and INS-1 cells were cultured in DMEM and RPMI 1640 medium (Invitrogen), respectively, with 10% heat-inactivated fetal bovine serum (FBS), 10 mM HEPES, pH 7.4, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 mM -mercaptoethanol and penicillin (100 units/ml), and streptomycin (100 g/ ml) at 37 °C in 95% air, 5% CO2). ␣TC1 cells and RAW264.7 were maintained in DMEM with the supplements described above. ␣TC1 cells were supplemented with 1ϫ nonessential amino acids (Sigma). ␣TC1 cells and RAW264.7 were maintained in DMEM with the supplements described above. Antibodies were as follows: c-Jun, ATF2, c-Maf, C/EBP-, NeuroD1 (BETA2), PDX-1, and NFATc2 (Santa Cruz Biotechnology); phospho-ERK1/2 (Thr-202/Tyr-204) (Sigma); phospho-p38 (Thr-180/Tyr-182) and phospho-JNK (Thr-183/ Tyr-185) (Cell Signaling); PE-TNF-␣ (Pharmingen). Expression vectors for ATF2, c-Jun, C/EBP-, and MafA were described previously [36, 40]. NFATc2, dnNFAT, and dnNFAT mutant expression vectors were provided by Chi-Wing Chow (Albert Einstein College of Medicine). Vectors expressing green fluorescent protein (GFP) and mouse MafA shRNA were obtained from OriGene. The adenovirus harboring short hairpin (sh) RNA for C/EBP- was provided by Jed Friedman (University of Colorado School of Medicine). The C/EBP- shRNA was transduced by 3 ϫ 1011 plaque-forming units of adenovirus per 1.5 ϫ 106 cells (2 ϫ 105 multiplicity of infection) 48 h prior to cell treatments.
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