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

Moore F, Colli ML, Cnop M, Esteve MI, Cardozo AK, Cunha DA, Bugliani M, Marchetti P, Eizirik DL - Diabetes (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Medicine, Université Libre de Bruxelles, Brussels, Belgium. fmoore@ulb.ac.be.

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.

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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.
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Figure 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.

Mentions: Taking into account that STAT1 is a substrate of PTPN2 in other cell types (13) and that it is an important mediator of cytokine-induced β-cell apoptosis (18), we next examined the effect of PTPN2 inhibition on the kinetics and magnitude of IFN-γ–induced STAT1 phosphorylation. STAT1 phosphorylation was highly induced after 15 min of IFN-γ treatment in both untransfected and siCtrl-transfected controls, slowly decreasing between 2 and 24 h (Fig. 5A and B). However, IFN-γ–induced STAT1 phosphorylation was markedly enhanced in cells lacking PTPN2, reaching a peak at 1–2 h and slowly decreasing afterward (Fig. 5A and B). We confirmed that the increased STAT1 phosphorylation in PTPN2-deficient cells was not due to an augmentation of total STAT1 content in these cells (Fig. 5A). Comparable results were observed for STAT3, another target of PTPN2 phosphatase activity, with clearly increased STAT3 phosphorylation in PTPN2-deficient cells (Fig. 5A and C). These data demonstrate that the phosphatase PTPN2 is a major modulator of IFN-γ–induced STAT1 and STAT3 activity in β-cells. We also evaluated the p42/44 MAPK (ERK), EGFR, and IRβ activation pathways, previously described as PTPN2 targets in other cell types (14,16,40). Neither ERK nor EGFR signaling pathways were affected after PTPN2 inhibition in INS-1E cells (Supplementary Fig. A5). On the other hand, there was an increase in IRβ phosphorylation after 30 min of cytokine treatment in PTPN2-inhibited INS-1E cells that lasted until 14 h (Supplementary Fig. A5).


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

Moore F, Colli ML, Cnop M, Esteve MI, Cardozo AK, Cunha DA, Bugliani M, Marchetti P, Eizirik DL - Diabetes (2009)

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.
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Figure 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.
Mentions: Taking into account that STAT1 is a substrate of PTPN2 in other cell types (13) and that it is an important mediator of cytokine-induced β-cell apoptosis (18), we next examined the effect of PTPN2 inhibition on the kinetics and magnitude of IFN-γ–induced STAT1 phosphorylation. STAT1 phosphorylation was highly induced after 15 min of IFN-γ treatment in both untransfected and siCtrl-transfected controls, slowly decreasing between 2 and 24 h (Fig. 5A and B). However, IFN-γ–induced STAT1 phosphorylation was markedly enhanced in cells lacking PTPN2, reaching a peak at 1–2 h and slowly decreasing afterward (Fig. 5A and B). We confirmed that the increased STAT1 phosphorylation in PTPN2-deficient cells was not due to an augmentation of total STAT1 content in these cells (Fig. 5A). Comparable results were observed for STAT3, another target of PTPN2 phosphatase activity, with clearly increased STAT3 phosphorylation in PTPN2-deficient cells (Fig. 5A and C). These data demonstrate that the phosphatase PTPN2 is a major modulator of IFN-γ–induced STAT1 and STAT3 activity in β-cells. We also evaluated the p42/44 MAPK (ERK), EGFR, and IRβ activation pathways, previously described as PTPN2 targets in other cell types (14,16,40). Neither ERK nor EGFR signaling pathways were affected after PTPN2 inhibition in INS-1E cells (Supplementary Fig. A5). On the other hand, there was an increase in IRβ phosphorylation after 30 min of cytokine treatment in PTPN2-inhibited INS-1E cells that lasted until 14 h (Supplementary Fig. A5).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Medicine, Université Libre de Bruxelles, Brussels, Belgium. fmoore@ulb.ac.be.

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.

Show MeSH
Related in: MedlinePlus