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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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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.
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Figure 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.

Mentions: To evaluate the role of STAT1 in the exacerbation of cytokine-induced β-cell apoptosis observed after PTPN2 inhibition, we additionally interfered with STAT1 in a double knockdown approach. We first confirmed by Western blot that both PTPN2 and STAT1 siRNAs adequately inhibited their respective targets without affecting the expression of the other protein and also that the double transfection of PTPN2 and STAT1 siRNAs potently inhibited both target proteins (Fig. 6A). As previously shown (Fig. 4), IL-1β + IFN-γ or TNF-α + IFN-γ induced apoptosis to a similar degree in both control conditions, and PTPN2 inhibition exacerbated cytokine-induced apoptosis, also rendering treatment with IFN-γ alone toxic to the cells (Fig. 6B). Inhibition of STAT1 protected the cells against IL-1β + IFN-γ- and TNF-α + IFN-γ–induced apoptosis (by 56 and 67%, respectively). STAT1 knockdown abrogated the proapoptotic effect of PTPN2 inhibition in cells exposed to IFN-γ or to combinations of cytokines (Fig. 6B). These results were confirmed using a second siRNA targeting PTPN2 expression (Supplementary Fig. A6) and suggest that increased STAT1 activity contributes to the aggravation of cytokine-induced apoptosis in PTPN2-deficient β-cells.


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)

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.
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Figure 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.
Mentions: To evaluate the role of STAT1 in the exacerbation of cytokine-induced β-cell apoptosis observed after PTPN2 inhibition, we additionally interfered with STAT1 in a double knockdown approach. We first confirmed by Western blot that both PTPN2 and STAT1 siRNAs adequately inhibited their respective targets without affecting the expression of the other protein and also that the double transfection of PTPN2 and STAT1 siRNAs potently inhibited both target proteins (Fig. 6A). As previously shown (Fig. 4), IL-1β + IFN-γ or TNF-α + IFN-γ induced apoptosis to a similar degree in both control conditions, and PTPN2 inhibition exacerbated cytokine-induced apoptosis, also rendering treatment with IFN-γ alone toxic to the cells (Fig. 6B). Inhibition of STAT1 protected the cells against IL-1β + IFN-γ- and TNF-α + IFN-γ–induced apoptosis (by 56 and 67%, respectively). STAT1 knockdown abrogated the proapoptotic effect of PTPN2 inhibition in cells exposed to IFN-γ or to combinations of cytokines (Fig. 6B). These results were confirmed using a second siRNA targeting PTPN2 expression (Supplementary Fig. A6) and suggest that increased STAT1 activity contributes to the aggravation of cytokine-induced apoptosis in PTPN2-deficient β-cells.

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