PTPN2, a candidate gene for type 1 diabetes, modulates interferon-gamma-induced pancreatic beta-cell apoptosis

Abstract

Objective: The pathogenesis of type 1 diabetes has a strong genetic component. Genome-wide association scans recently identified novel susceptibility genes including the phosphatases PTPN22 and PTPN2. We hypothesized that PTPN2 plays a direct role in beta-cell demise and assessed PTPN2 expression in human islets and rat primary and clonal beta-cells, besides evaluating its role in cytokine-induced signaling and beta-cell apoptosis.

Research design and methods: PTPN2 mRNA and protein expression was evaluated by real-time PCR and Western blot. Small interfering (si)RNAs were used to inhibit the expression of PTPN2 and downstream STAT1 in beta-cells, allowing the assessment of cell death after cytokine treatment.

Results: PTPN2 mRNA and protein are expressed in human islets and rat beta-cells and upregulated by cytokines. Transfection with PTPN2 siRNAs inhibited basal- and cytokine-induced PTPN2 expression in rat beta-cells and dispersed human islets cells. Decreased PTPN2 expression exacerbated interleukin (IL)-1beta + interferon (IFN)-gamma-induced beta-cell apoptosis and turned IFN-gamma alone into a proapoptotic signal. Inhibition of PTPN2 amplified IFN-gamma-induced STAT1 phosphorylation, whereas double knockdown of both PTPN2 and STAT1 protected beta-cells against cytokine-induced apoptosis, suggesting that STAT1 hyperactivation is responsible for the aggravation of cytokine-induced beta-cell death in PTPN2-deficient cells.

Conclusions: We identified a functional role for the type 1 diabetes candidate gene PTPN2 in modulating IFN-gamma signal transduction at the beta-cell level. PTPN2 regulates cytokine-induced apoptosis and may thereby contribute to the pathogenesis of type 1 diabetes.

FIG. 1.

Cytokines upregulate PTPN2 expression in primary fluorescence-activated cell–sorted rat β-cells, human islets, and INS-1E cells. A: Rat β-cells were cultured for 2 days and subsequently left untreated or treated with the combination of IL-1β (10 units/ml) + IFN-γ (100 units/ml) for 24 h. B, D, and E: Hand-picked human islets were cultured overnight and then left untreated or exposed to IL-1β (50 units/ml) + IFN-γ (1,000 units/ml) for 48 h. C: INS-1E cells were left untreated or treated with IL-1β (10 units/ml) + IFN-γ (100 units/ml) for 2, 6, and 24 h as indicated. A– C: PTPN2 mRNA expression was assayed by RT-PCR and normalized for the housekeeping gene GAPDH. D: PTPN2 and α-tubulin expression in human islets were evaluated by Western blot. E: Mean optical density measurements of PTPN2 Western blots corrected for protein loading by α-tubulin (representative figure in D). Results are means ± SE of three to five independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. untreated cells by Student's t test. NS, nonstimulated (untreated).

FIG. 2.

Effects of cytokine exposure on PTPN2 subcellular distribution. INS-1E cells were plated onto glass coverslips and left untreated or treated for 15 min with IFN-γ (100 units/ml), IL-1β (10 units/ml) + IFN-γ (100 units/ml), or TNF-α (1,000 units/ml) + IFN-γ (100 units/ml). Cells were then fixed and processed for immunofluorescence as described in research design and methods. The figure is representative of five independent experiments. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

A siRNA targeting PTPN2 inhibits basal and cytokine-induced PTPN2 expression in INS-1E cells. INS-1E cells were left untransfected (NT □) or transfected with 30 nmol/l of either a control siRNA (siCtrl, ▨) or a pool of siRNAs targeting PTPN2 (siPTPN2, ■). After 2 days of recovery, cells were left untreated, or treated for 24 h with IL-1β (100 units/ml), IFN-γ (100 units/ml), TNF-α (1,000 units/ml), IL-1β (10 units/ml) + IFN-γ (100 units/ml), or TNF-α (1,000 units/ml) + IFN-γ (100 units/ml). A: PTPN2 mRNA expression was assayed by RT-PCR and normalized for the housekeeping gene GAPDH. Results are means ± SE of four independent experiments. B: PTPN2 and α-tubulin protein expression were evaluated by Western blot. The results are representative of five independent experiments. C: Mean optical density measurements of PTPN2 Western blots, corrected for protein loading by α-tubulin. Results are means ± SE of five independent experiments; a: P < 0.001, b: P < 0.01, and c: P < 0.05 vs. untreated NT or untreated transfected with the same siRNA; d: P < 0.01 vs. untreated NT and siCtrl; e: P < 0.01 vs. IL-1β–treated NT and siCtrl; f: P < 0.01 vs. IFN-γ–treated NT and siCtrl; g: P < 0.01 vs. TNF-α–treated NT and siCtrl; h: P < 0.001 vs. IL-1β + INF-γ–treated NT and siCtrl, i: P < 0.01 vs. TNF-α + INF-γ–treated NT and siCtrl; ANOVA followed by Student's t test with Bonferroni correction.

FIG. 4.

siRNA-mediated PTPN2 inhibition exacerbates cytokine-induced apoptosis in INS-1E cells, primary rat β-cells, and dispersed human islets, independently of NO production. INS-1E cells were transfected and treated as described in Fig. 3. Apoptosis was evaluated after 24 h using HO/PI staining (A) and a Cell Death Detection ELISAplus kit (B). C: INS-1E cells were transfected and treated as described in Fig. 3, and nitrite concentrations in supernatants were measured as described in research design and methods (D). Primary fluorescence-activated cell–sorted rat β-cells were cultured for 2 days and then transfected as described in Fig. 3. After 2 days of recovery, cells were left untreated (NT) or treated for 72 h with IFN-γ (100 units/ml), IL-1β (10 units/ml) + IFN-γ (100 units/ml), or TNF-α (1,000 units/ml) + IFN-γ (100 units/ml). E and F: Dispersed human islets were left untransfected, or transfected with 30 nmol/l of siCtrl or human siPTPN2 #1 or #2 and cultured for a 48-h recovery period. Cells were then treated with IL-1β (50 units/ml) + IFN-γ (1,000 units/ml) for 48 h when PTPN2 and α-tubulin were evaluated by Western blot (E) and 96 h when apoptosis was evaluated by HO/PI staining (F). Results are means ± SE of four experiments; a: P < 0.001 and b: P < 0.05 vs. untreated NT or untreated transfected with the same siRNA; c: P < 0.001 vs. IFN-γ–treated NT and siCtrl, d: P < 0.001 and e: P < 0.05 vs. IL-1β + INF-γ–treated NT and siCtrl, f: P < 0.001 and g: P < 0.05 vs. TNF-α + INF-γ–treated NT and siCtrl; ANOVA followed by Student's t test with Bonferroni correction.

FIG. 5.

PTPN2 inhibition increases IFN-γ–induced STAT1 and STAT3 phosphorylation. INS-1E cells were left untransfected (NT) or transfected with 30 nmol/l of either a control siRNA (siCtrl) or with a pool of siRNAs targeting PTPN2 (siPTPN2). After 2 days of recovery, cells were left untreated or treated with IFN-γ (100 units/ml) for 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h. A: phospho-STAT1, total STAT1, phospho-STAT3, total STAT3, PTPN2, and α-tubulin proteins were evaluated by Western blot. These results are representative of five independent experiments. B and C: Mean optical density measurements of phospho-STAT1 (B) and phospho-STAT3 (C) Western blots corrected for protein loading by α-tubulin. Results are means ± SE of five independent experiments; **P < 0.01 and ***P < 0.001 vs. NT and siCtrl at the same time point, ANOVA followed by Student's t test with Bonferroni correction.

FIG. 6.

Double knockdown of PTPN2 and STAT1 protects INS-1E cells from cytokine-induced apoptosis. INS-1E cells were left untransfected (NT) or were transfected with 60 nmol/l of a control siRNA (siCtrl), or with 30 nmol/l of either a pool of siRNAs targeting PTPN2 (siPTPN2), or a siRNA targeting STAT1 (siSTAT1), or double transfected with 30 nmol/l of both siPTPN2 and siSTAT1. After 2 days of recovery, cells were left untreated, or treated for 24 h with IFN-γ (100 units/ml), IL-1β (10 units/ml) + IFN-γ (100 units/ml), or TNF-α (1,000 units/ml) + IFN-γ (100 units/ml) as indicated. A: Expression of STAT1, PTPN2, and α-tubulin proteins were evaluated by Western blot. The results are representative of three independent experiments. B: Apoptosis was evaluated using HO/PI staining. Results are means ± SE of four independent experiments; a: P < 0.001 vs. untreated NT or untreated transfected with the same siRNA; b: P < 0.001 vs. IFN-γ–treated NT and siCtrl; c: P < 0.001 vs. IFN-γ–treated siSTAT1 and siPTPN2 + siSTAT1; d: P < 0.001 vs. IL-1β + INF-γ–treated NT and siCtrl; e: P < 0.001 vs. IL-1β + INF-γ–treated siSTAT1 and siPTPN2 + siSTAT1; f: P < 0.001 vs. TNF-α + INF-γ–treated NT and siCtrl; g: P < 0.001 vs. TNF-α + INF-γ–treated siSTAT1 and siPTPN2 + siSTAT1; ANOVA followed by Student's t test with Bonferroni correction.