LIGHT/IFN-γ triggers β cells apoptosis via NF-κB/Bcl2-dependent mitochondrial pathway

Abstract

LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN-γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ-induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ - induced pancreatic beta cell destruction. LIGHT and IFN-γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V(+) cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF-κB activation, the combination of LIGHT and IFN-γ caused an obvious decrease in expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, but an increase in expression of the pro-apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF-κB activation and Bak expression, and peri-insulitis in non-obese diabetes mice. Inhibition of NF-κB activation with the specific NF-κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl-xL down-regulation and Bax up-regulation, and led to a significant increase in LIGHT- and IFN-γ-treated cell viability. Moreover, cleaved caspase-9, -3, and PARP (poly (ADP-ribose) polymerase) were observed after LIGHT and IFN-γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT- and IFNγ-induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN-γ induces beta cells apoptosis via an NF-κB/Bcl2-dependent mitochondrial pathway.

Keywords: LIGHT; NF-κB; apoptosis; mitochondrial stress; pancreatic beta cell.

Figure 1

LIGHT and IFN‐γ synergistically inhibit beta cell viability. (A) MIN6 cells (3 × 10⁴/well) were seeded in 96‐well microtiter plates and treated with different concentrations of LIGHT in the presence of 100 ng/ml IFN‐γ for 48 h. Treatment with 100 ng/ml IFN‐γ plus 10 ng/ml TNF‐α was regarded as positive control. (B) MIN6 cells, or (C) islet cells of NOD mice with (3 × 10⁴/well) were seeded in 96‐well microtiter plates and treated with IFN‐γ (100 ng/ml) or LIGHT (5 μg/ml) alone or in combination for 48 h. Cell viability of the aforementioned groups was measured by MTT assay. The OD value was detected at 490 nm. The OD value at 490 nm of the untreated cells was set to 100%. Results are expressed as means ± SEM. *P < 0.05 and **P < 0.01. All experiments were repeated at least three times.

Figure 2

The combination of LIGHT and IFN‐γ treatment induces MIN6 cell apoptosis. (A) Cells were treated with IFN‐γ (100 ng/ml) and LIGHT (5 μg/ml) in combination for 0, 24 and 48 h, and were photographed under a phase contrast microscope. Magnification, 100×. (B) Cells were treated with media, IFN‐γ (100 ng/ml) or LIGHT (5 μg/ml) alone, or in combination for 24 and 48 h. Cells were double stained with Annexin V‐FITC and 7‐AAD and the percentage of apoptotic cells (Annexin V⁺ and 7‐AAD ⁻ cells) was determined by flow cytometry. Data shown are representative of two‐independent experiments.

Figure 3

Increased release of cytochrome c and alterations in Bcl‐2 family members expression in LIGHT and IFN‐γ‐induced apoptosis of MIN6 cells. Cells were treated with LIGHT (5 μg/ml) and IFN‐γ (100 ng/ml) in combination for the indicated times. Cells were harvested and cytoplasmic protein was extracted. (A) Cytochrome c release was evaluated by Western blot. Cytochrome oxidase subunit IV (COX4), located exclusively in the mitochondria, was used here to confirm whether the cytoplasmic protein fractions included mitochondrial proteins. (B) The expression of Bcl‐2, Bcl‐xL, Bak and Bax was measured by Western blot. Equal protein loading in each lane was confirmed by probing the blots with anti‐β‐actin antibody. Data are representative of two‐independent experiments. (C) The expression of Bak in primary islets of 20‐week‐old female LIGHT ^+/+ and LIGHT ^−/− NOD mice was measured by immunohistochemisty. Magnification, 200×.

Figure 4

NF‐κB activation is involved in beta cell destruction. (A) and (B) MIN6 cells were treated with LIGHT (5 μg/ml) or IFN‐γ (100 ng/ml) alone, or in combination for the indicated times. Cytoplasmic NF‐κB p65 levels were determined by Western blot. Equal protein loading in each lane was confirmed by probing the blots with anti‐β‐actin antibody. (C) MIN6 cells were treated with LIGHT (5 μg/ml) and IFN‐γ (100 ng/ml) in combination for indicated times. The nuclear localization of NF‐κB p65 was determined by confocal laser scanning microscopy. Nuclei were stained with Hoechst 33258 solution (5 μg/ml). (D) The expression of NF‐κB p65 in primary islets of 20‐week‐old female LIGHT ^+/+ and LIGHT ^−/− NOD mice was measured by immunohistochemistry. Magnification, 200×. (E) The nuclear localization of NF‐κB p65 in LIGHT ^+/+ NOD mice with (peri)insulitis was further determined by confocal laser scanning microscopy. Nuclei were stained with Hoechst 33258 solution (5 μg/ml).

Figure 5

Beta cell destruction is NF‐κB/Bcl‐2 pathway dependent. (A) After pretreatment with or without PDTC (50 μM), an NF‐κB inhibitor, for 1 h, MIN6 cells were treated with a combination of IFN‐γ and TNF‐α for 12 h. The expression of cytoplasmic NF‐κB p65, Bcl‐xL, and Bax was determined by Western blot. Equal protein loading in all lanes was confirmed by probing the blots with anti‐β‐actin antibody. (B) After pretreatment with or without PDTC (50 μM) for 1 h, MIN6 cells were treated with or without the LIGHT and IFN‐γ combination for 48 h. Cell viability was measured by MTT assay. (C) Reverse expression of p65 and Bcl‐2 was observed in primary islets in NOD mice with (peri) insulitis by immunohistochemisty. [Left and right represent different fields in 2 NOD mice with (peri) insulitis]. Magnification, 200×. Data are expressed as mean ± SEM. *P < 0.05. Data are representative of two‐independent experiments.

Figure 6

LIGHT and IFNγ‐induced MIN6 cells apoptosis is caspase‐dependent. Cells were treated with IFN‐γ (100 ng/ml) and LIGHT (5 μg/ml) in combination for 0.5, 1, 12 and 24 h. The expression of caspase‐3, ‐9, cleaved caspase‐3, ‐9 (A), and cleaved PARP (B) was measured by Western blot. Equal protein loading in all lanes was assessed by probing the blots with β‐actin antibody. (C) After pretreatment with or without Z‐VAD‐FMK (50 μM), a broad range caspase inhibitor, for 1 h, cells were treated with IFN‐γ and LIGHT in combination for 48 h. Cells were double stained with annexin V and 7‐AAD and then analysed by flow cytometry. Data are representative of three independent experiments.

Figure 7

Blockage of LIGHT activity by soluble receptor fusion proteins reverses LIGHT/IFN‐γ synergism‐mediated MIN6 cell apoptosis. After preincubation with soluble receptor fusion proteins HVEM‐Ig or LTβR‐Ig or N66F‐Ig (a negative control, which is an Fc fusion protein that does not interfere with LTβR/HVEM/LIGHT interactions), cells were treated with IFN‐γ and LIGHT in combination for 48 h. Cells were double stained with annexin V and 7‐AAD and then analysed by flow cytometry. Data are representative of two‐independent experiments.

Figure 8

NF‐κB/Mitochondria pathway is involved in beta cells apoptosis induced by LIGHT in the presence of IFN‐γ. LIGHT signalling activates NF‐κB‐mediated mitochondrial pathway via the regulation of Bcl‐2 family member's expression, followed with mitochondrial permeabilization, and cytochrome c release, and caspase‐9 activation. Caspase‐9 then activates caspase‐3. The signal from caspase‐3 is transmitted to PARP and then leads to cells apoptosis.