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Biomedical Science Letters 2014, 20(1): 14~24 eISSN : 2288-7415

NF-κB Inhibitor Suppresses Hypoxia-induced Apoptosis of Mouse Pancreatic β-cell Line MIN6

Hyun Sook Koh and Jae Young Kim†

Department of Biological Science, Gachon University, Incheon 406-799, Korea

Hypoxia is one of the main reasons for islet apoptosis after transplantation as well as during isolation. In this study, we attempted to determine the potential usefulness of NF-κB inhibitor for suppression of hypoxia-induced β-cell apoptosis as well as the relationship between IP-10 induction and β-cell apoptosis in hypoxia. To accomplish this, we cultured the mouse pancreatic β-cell line MIN6 in hypoxia (1% O2). Among several examined chemokines, only IP-10 mRNA expression was induced under hypoxia, and this induced IP-10 expression was due to NF-κB activity. Since a previous study suggested that IP-10 mediates β-cell apoptosis, we measured hypoxia-induced IP-10 protein and examined the effect of anti-IP-10 neutralizing Ab on hypoxia-induced β-cell apoptosis. However, IP-10 protein was not detected, and anti-IP-10 neutralizing Ab did not rescue hypoxia-induced MIN6 apoptosis, indicating that there is no relationship between hypoxia-induced IP-10 mRNA expression and hypoxia-induced β-cell apoptosis. Since it was still not clear if NF-κB functions as an apoptotic or anti-apoptotic mediator in hypoxia-induced β-cell apoptosis, we examined possible involvement of NF-κB in hypoxia-induced β-cell apoptosis. Treatment with 1 μM NF-κB inhibitor suppressed hypoxia-induced apoptosis by more than 50%, while 10 μM AP-1 or 4 μM NF-AT inhibitor did not, indicating involvement of NF-κB in hypoxia-induced β-cell apoptosis. Overall, these results suggest that IP-10 is not involved in hypoxia-induced β-cell apoptosis, and that NF-κB inhibitor can be useful for ameliorating hypoxia-induced β-cell apoptosis. Key Words: NF-kappaB, Cell hypoxia, Insulin-secreting cells, Apoptosis, Chemokine CXCL10

INTRODUCTION

Pancreatic islet transplantation is one of the most effective

therapies for type 1 diabetes. However, islets suffer from hypoxia during their isolation and purification (Dionne et al., 1993; de Groot et al., 2003). Long term exposure of islets to hypoxia eventually impairs their secretory function and induces hypoxia-mediated β-cell apoptosis (Dionne et al., 1993; Giuliani et al., 2005). In addition, the majority of islets undergo apoptosis within the first several days of

transplantation due to hypoxic and/or pro-inflammatory chemokines/cytokines injury (Barshes et al., 2004). Acti- vation of iNOS and NO production (Ko et al., 2008) and/or increased ROS and subsequent activation of pro-apoptotic pathways have been suggested as mechanisms underlying hypoxia-induced pancreatic β-cell apoptosis (Ko et al., 2008; Moritz et al., 2002; Greijer AE and van der Wall, 2004). In addition to these mechanisms, induction of C/EBP homologous protein (CHOP), which is an endoplasmic reticulum stress-dependent pro-apoptotic transcription factor, has recently been suggested as a major factor for hypoxia-mediated β-cell apoptosis (Zheng et al., 2012). However, suppression of CHOP expression only partially rescued MIN6 cells from hypoxia-mediated apoptosis, suggesting the existence of alternative pathways (Zheng et al., 2012). Therefore, the underlying mechanisms of hypoxia-induced β-cell apoptosis are still not fully understood.

Original Article

*Received: March 16, 2014 / Revised: March 23, 2014 Accepted: March 27, 2014

†Corresponding author: Jae Young Kim. Department of Biological Science, Gachon University, Incheon 406-799, Korea. Tel: +82-32-820-4551, Fax: +82-32-820-4549 e-mail: jykim85@gachon.ac.kr ○C The Korean Society for Biomedical Laboratory Sciences. All rights reserved.

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In this study, we cultured the mouse pancreatic β-cell line, MIN6 under hypoxic conditions (1% O2). We found that the cells undergo apoptosis and over-express the CXC chemokine interferon-gamma inducible protein 10 (IP-10, CXCL10) mRNA over time under these conditions. IP-10 was recently reported to be an apoptotic mediator that induces pancreatic β-cell death by interacting with TLR4 instead of its typical receptor, CXCR3 (Schulthess et al., 2009).

NF-κB is a critical transcription factor involved in various biological functions such as cellular responses to inflammatory stress and cell survival and development (Lai et al., 2009). NF-κB is also capable of responding to hypoxia (Lai et al., 2009; Johansson et al., 2003). NF-κB is generally considered an anti-apoptotic factor, however, it sometimes exerts a pro-apoptotic function according to cell types or stimuli (Ortis et al., 2008; Radhakrishnan and Kamalakaran, 2006). Nevertheless, the role of NF-κB in hypoxic β-cell apoptosis is still not known.

The purpose of the present study is twofold: to determine the potential usefulness of NF-κB inhibitor for suppression of hypoxia-induced β-cell apoptosis and to test possible involvement of chemokine IP-10 in hypoxia-induced β-cell apoptosis.

MATERIALS AND METHODS

Reagents

Apocynin as an NADPH oxidase inhibitor, allopurinol as a xanthin oxidase inhibitor, TTFA (2-thenoyltrifluoroacetone) as a mitochondria electron transport chain complex II inhibitor and hydrogen peroxide (H2O2) were purchased from Sigma Aldrich Corp. (Sat. Louis, Mo, USA). Curcumin, NF-κB activation inhibitor (6-amino-4-(4-phenoxypheny- lethylamino) quinazoline) and NF-AT inhibitor were pur- chased from Sigma Aldrich Corp. (Sat. Louis, Mo, U.S.), Enzo Life Sciences Inc. (Plymouth Meeting, PA, USA) and Calbiochem/EMD Biosciences (San Diego, CA, USA), respectively. RPMI-1640 medium for cell culture was obtained from Welgene Inc. (Daegu, Korea). Fetal bovine serum (FBS) was obtained from Lonza Ltd. (Basel, Switzerland).

Cell culture

Mouse pancreatic β-cell line, MIN6, was a gift from Prof. HS Jeon (Lee Ghil Ya Cancer & Diabetes Institute) and was routinely cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 25 mM glucose, 15% heat-inactivated FBS, 1% Antibiotic-Antimycotic (Invitrogen Corp., Gibco BRL, MD, USA) at 37℃ in a 5% CO2 humidified incubator. For the low-oxygen experiment, cells were maintained in Heracell gas addition incubator (Heraeus Instruments GmbH, Hanau, Germany) with gas mixtures consisting of 1% O2, 5% CO2, and 94% N2 (referred to as 1% O2). To measure effects of reactive oxygen species (ROS) synthesis inhibitors on the intracellular ROS pro- duction in MIN6 cells, we used 100 μM apocynin, 100 μM TTFA or 100 μM allopurinol. Cells were pretreated with each inhibitor for 30 min before cultivation under 1% O2. After 18h-incubation, intracellular ROS level was measured by FACS analysis. To measure effects of transcription factor (TF) inhibitors on H2O2-induced up-regulation of IP-10 expression in MIN6 cells, 10 μM curcumin (for AP-1), 10 μM quinazoline (for NF-κB) were pretreated for 30 min and 4 μM NF-AT inhibitor was pretreated for 1 h before H2O2 treatment. After 1 h incubation with H2O2, cells were harvested and IP-10 expression was examined by RT-PCR analysis.

Cell viability analysis

Cell viability and proliferation were determined with Ez-Cytox Cell Viability Assay Kit (Daeil Lab Service Co., Seoul, Korea) based on the cleavage of the tetrazolium salt to water-soluble formazan by succinate-tetrazolium reductase. Briefly, cells were incubated in the presence of 1% O2 or 20% O2 for 24 h in 96 well plates and then incubated with 10 μl of Ez-Cytox solution in a final volume of 100 μl culture medium per well for 6 h in the 37℃ incubator. Then absorbance was measured using the ELISA Reader (μ-Quant, Bio-Tek Instruments, Winooski, VT, USA) at 450 nm.

Apoptosis assay

Apoptotic cell death was assessed by Annexin V-FITC Apoptosis Detection (BD PharmingenTM, San Diego, CA,

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USA) using Annexin V (AV) staining coupled with pro- pidium iodide (PI). MIN6 cells were cultured in normoxia (20% O2) or hypoxia (1% O2) at various time points. And then MIN6 cells were harvested and were washed twice with PBS. After washing with binding buffer, resuspended cells in 1X binding buffer were Transferred in 5 ml culture tube at a concentration of 1 × 105 cells in 100 μl of the solution. Cells were mixed gently with 5 μl of Annexin V-FITC and 5 μl of PI and incubated at RT in the dark for 15 min. And 400 μl of binding buffer were added to each tube. Cells were immediately analyzed by FACS.

Measurement of ROS

DCFH-DA is able to diffuse through the cell membrane and become cleaved by intracellular esterases forming DCFH, which is in nonfluorescent form and is oxidized to fluorescent compound DCF by ROS. Following exposure to 20% or 1% O2 for 0~24 h, cells were loaded with 10 μM DCFH-DA for 15 min at 37℃ and viewed under the fluorescence microscope. ROS measurement was carried out on FACS flow cytometer (Beckman Coulter, Fullerton, CA, USA).

RT-PCR

Total RNA was extracted with Reverse Transcriptase M-MuLV (Bioprince, Atlanta, GA, USA) according to the manufacturer's instruction, and complementary DNA (cDNA) was synthesized from 2 μg of total RNA. Amplification of cDNA using PCR was performed by 30 cycles in a total reaction volume of 30 μl containing 0.5 unit of Taq poly- merase (TaKaRa Bio Inc., Otsu, Shiga, Japan) and 10 pico- mole of primers specific for mouse MCP-1 (forward primer: 5'-GGAAAAATGGATCCACACCTTGC-3', reverse primer: 5'-TCTCTTCCTCCACCACCATGCAG-3', MIP-2 (for- ward primer: 5'-TGGGTGGGATGTAGCTAGTTCC-3', reverse primer: 5'-AGTTTGCCTTGACCCTGAAGCC-3'), MIP-1α (forward primer: 5'-GAAGAGTCCCTCGATGT- GGCTA-3', reverse primer: 5'-CCCTTTTCTGTTCTGC- TGACAAG-3'), MIP-1β (forward primer: 5'-CCACAAT- AGCAGAGAAACAGCAAT-3', reverse primer: 5'-AAC- CCCGAGCAACACCATGAAG-3'), RANTES (forward primer: 5'-TCTTCTCTGGGTTGGCACACAC-3', reverse

primer: 5'-CCTCACCATCATCCTCACTGCA-3'), IP-10 (forward primer: 5'-CGCACCTCCACATAGCTTACAG-3', reverse primer: 5'-CCTATCCTGCCCA CGTGTTGAG-3'), and β-actin (forward primer: 5'-GTATGGAATCCTGTGG- CATCC-3', reverse primer: 5'-TACGCAGCTCAGTAAC- AGTCC-3'). Amplified PCR products were separated on a 2% agarose gel using loadingstar (Dynebio, Sungnam, Korea), and visualized under UV in gel documentation system.

ELISA

The supernatant of MIN6 cells cultured under hypoxia for 0~72 h or in the presence of H2O2 for 3 h was immediately frozen at -80℃. The levels of CXCL10/IP-10 protein were determined by mouse CXCL10/IP-10/CRG-2 immunoassay kit (R&D Systems, Minneapolis, USA) according to the manufacturer's instructions.

Prediction of NF-κB binding sites

To identify putative binding sites for AP-1, NF-AT, and NF-κB within the IP-10 promoter region, the TF binding site prediction program (Farré et al., 2003), Alggen Promo software, V3.0.2 (http://alggen.lsi.upc.es) was used with the default setting of 15% dissimilarity value.

Statistical analysis

Student's t tests were performed using statistical software SPSS 12.0. All data are mean ± SD from at least three different experiments. P<0.05 was considered statistically significant.

RESULTS

Hypoxia induced apoptosis of MIN6 cells

The effects of hypoxia (1% O2) on survival of the mouse pancreatic β-cell line, MIN6, were investigated. The majority of cells cultured under 1% O2 for 24 h displayed apoptotic morphology (Fig. 1A). Cell viability in the hypoxia group was approximately 40% of that in the normoxia (20% O2) group (Fig. 1B). FACS analysis of apoptotic MIN6 cells after staining with annexin-V-FITC revealed that apoptosis gradually increased over time, and that approxi-

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mately 70% of cells were undergoing apoptosis after 96 h of cultivation under hypoxia (Fig. 1C).

Hypoxia induced intracellular ROS production in MIN6 cells

Since hypoxia is known to induce intracellular ROS

Fig. 1. Hypoxia induced apoptosis of MIN6 cells. (A) Morphology of MIN6 cells cultured in normoxia (upper) and hypoxia (lower). (B)Cell viability of hypoxic MIN6 cells. Cells were incubated in the presence of 1% O2 or 20% O2 for 24 h and then cell viability was determined with Ez-Cytox cell viability assay kit. (C) Time course apoptosis of MIN6 cells in hypoxia. Apoptotic cell death of MIN6 cells grown under hypoxia for 96 h was assessed by FACS analysis using Annexin V (AV) staining coupled with propidium iodide (PI).

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production (Chandel et al., 2000; Kim et al., 2010), we examined intracellular ROS production in hypoxic MIN6 (Fig. 2). As shown in Fig. 2A, fluorescent microscopy revealed high levels of intracellular ROS production in hypoxic cells. Because the enzymes responsible for intra- cellular ROS production in hypoxic pancreatic β-cells are currently unknown, we investigated enzymes generating intracellular ROS in hypoxic cells using three different ROS synthesis inhibitors; TTFA, mitochondria electron transport chain complex II inhibitor, apocynin, NADPH oxidase inhibitor, and allopurinol, xanthine oxidase inhibitor. As shown in Fig. 3, all three inhibitors inhibited intracellular ROS production of hypoxic MIN6 cells, suggesting possible involvement of mitochondria electron transport chain com- plex II, NADPH oxidase, and xanthine oxidase in ROS production of hypoxic MIN6.

Hypoxia enhanced IP-10 mRNA expression in MIN6 cells

We next examined chemokine mRNA expression in hypoxic MIN6. As shown in Fig. 4A, IP-10 mRNA expression began to increase 2 h after cultivation in hypoxia. In contrast, while MIP-1α, MIP-1β, and RANTES were not detected (data not shown), expression of MCP-1 and MIP-2 disappeared over time (Fig. 4A). We tested whether IP-10 expression is also induced by H2O2, which is known to be the most stable cellular ROS (Li and Jackson, 2002). IP-10 mRNA expression increased significantly in response to treatment with H2O2 at levels of 0.1 μM (Fig. 4B). Since previous studies reported that IP-10 mediates β-cell apoptosis (Schulthess et al., 2009), it is worth testing whether hypoxia- or H2O2-induced IP-10 expression are associated with the hypoxia-induced β-cell apoptosis observed in the present study.

NF-κB inhibitor suppressed H2O2-induced IP-10 expression

Since ROS is known to induce activation of TFs such as NF-κB, AP-1, and NF-AT (Kim et al., 2008; Han et al., 2007;

Fig. 2. Hypoxia induced ROS production in MIN6 cells. (A) A representative fluorescence microscopic images, which show the fluorescence of DCF oxidized by intracellular ROS of MIN6 cellscultured in 20% O2 (upper) and 1% O2 (lower) for 18 h. After cultivation, the cells were incubated with 10 μM DCFH-DA for 15 min at 37℃ and assessed by fluorescence microscopy. (B) Mean fluorescence intensities (MFI) of DCF in MIN6 cells grownin 1% O2 for 24 hr were examined by FACS analysis.

Fig. 3. ROS synthesis inhibitors suppressed intracellular ROS production in MIN6 cells cultured in hypoxia. Intracellular ROSproduction was examined in MIN6 cells grown in 1% O2 for 18 h inthe presence of three different ROS synthesis inhibitors; Apocynin(apo, 100 μM), NADPH oxidase inhibitor; TTFA (100 μM), mito-chondria electron transport chain complex II inhibitor; Allopurinol(allo, 100 μM), xanthine oxidase inhibitor. After cultivation, the cells were incubated with 10 μM DCFH-DA for 15 min at 37℃and assessed by FACS analysis.

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Frossi et al., 2007; Tu et al., 2007), we tested possible in- volvement of these TFs in hypoxia-induced IP-10 expression in MIN6. To accomplish this, we screened possible binding sites for three TFs within the IP-10 promoter region using a TF binding site prediction program as described in Materials and Methods. As shown in Table 1, we found three putative binding sites for each TF within the proximal region (upstream 250 bp) of the IP-10 promoter. We next examined IP-10 mRNA expression in H2O2 treated MIN6 cells with

Table 1. Summary of computer-assisted search for the putative binding sites for selected TFs

Factor name Start position End position Dissimilarity String RE equally RE query

AP-1 -142 -132 14.433034 TGGGTCACAAT 0.24033 0.26511 AP-1 -74 -64 3.401138 TGAGTCATCTC 0.01431 0.01498 AP-1 -51 -41 14.735895 ATCAGGACTCA 0.24033 0.26511 NF-AT1 -214 -206 8.742852 GGAAAGTGA 0.13733 0.15943 NF-AT1 -158 -150 9.180322 GGAAATTCC 0.09155 0.09240 NF-AT1 -35 -27 4.012064 GGAAACTCT 0.04578 0.05098 NF-kB -160 -150 14.239265 AGGGAAATTCC 0.03052 0.03092 NF-kB -104 -94 14.570957 GGGGACTTCCC 0.02909 0.02391 NF-kB -35 -25 14.117924 GGAAACTCTTT 0.03052 0.03092

The putative binding sites were predicted by using Alggen Promo software, V 3.0.2 (http://alggen.lsi.upc.es). Factor name - transcription factor with its TRANSFAC (V 8.3) database accession number; Start and End - start and end positions of putative binding sequences, respectively; Dissimilarity - rate of dissimilarity (%) between the putative and consensus sequences; String - nucleotide sequence of potentialbinding site; Random Expectation (RE) - expected occurrences of the match in a random sequence of the same length as the query sequence according to the dissimilarity index (RE equally - equiprobability for the four nucleotides and RE query - nucleotide frequenciesas in the query sequence).

Fig. 4. Hypoxia enhanced IP-10 mRNA expression in MIN6 cells. (A) A representative RT-PCR data showing time course mRNA expression of several chemokines in MIN6 cells grown in 1% O2 for 0 to 24 h. (B) A representative RT-PCR data showing IP-10 expression in MIN6 cells treated with H2O2. The cells that had been maintained at 37℃ in 20% O2 were treated with varyingconcentrations of H2O2 as indicated above for 1h and then the cellswere harvested and examined by RT-PCR analysis.

Fig. 5. NF-κB inhibitor suppressed H2O2-induced up-regulationof IP-10 expression. A representative RT-PCR data showing IP-10expression in MIN6 cells were treated with TF inhibitors for 30 min or 1 h before treatment with 10 μM H2O2. AP-1, NF-AT and NF-κB were blocked with curcumin (Sigma, 10 μM, preincubationfor 30 min), a cell-permeable NF-AT inhibitor peptide (Calbiochem,4 μM, for 1 h) and quinazoline (Enzo Life Sciences Inc., 0.1~10 μM, for 30 min), respectively. The cells that had been maintained at 37℃ in 20% O2 in the presence or absence of the inhibitors were treated with 10 μM H2O2 for 1 h, harvested and then examinedby RT-PCR analysis.

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or without pretreatment of TF specific inhibitor. As shown in Fig. 5, the induction of IP-10 mRNA expression by H2O2 was significantly suppressed by pre- treatment with NF-κB inhibitor at levels of 1 μM. In contrast, AP-1 or NF-AT inhibitor did not suppress the IP-10 mRNA expression induced by H2O2 treatment. These results suggest that IP-10 expression was controlled through NF-κB pathway.

1Hypoxia-induced IP-10 expression was not involved in hypoxia-induced MIN6 apoptosis

Since β-cell apoptosis and IP-10 mRNA expression were induced by hypoxia in the present study, we examined possible involvement of IP-10 in hypoxia-induced β-cell apoptosis. To accomplish this, we measured IP-10 protein in culture supernatant of hypoxic MIN6 by ELISA. However, we did not detect any IP-10 protein from samples collected at various incubation time points (Fig. 6A). Similarly, we did not detect any IP-10 protein in culture supernatant of H2O2 treated MIN6 (Fig. 6B). Not surprisingly, anti-IP-10 neutralizing Ab could not rescue the hypoxia-induced MIN6 apoptosis (Fig. 6C). These results indicate that hypoxia-induced IP-10 mRNA expression is not involved in hypoxia-induced β-cell apoptosis.

NF-κB inhibitor suppressed hypoxia-induced MIN6 apoptosis

Since the major question regarding whether NF-κB functions as an apoptotic or anti-apoptotic mediator in hypoxia-induced β-cell apoptosis remained, we examined possible involvement of NF-κB in hypoxia-induced β-cell apoptosis. As shown in Fig. 7, treatment with 1 μM NF-κB

Fig. 6. Hypoxia or hydrogen peroxide does not induce IP-10 secretion. (A) IP-10 secretion was measured in supernatants of MIN6 cells cultured in 1% O2 for 0 to 72 h by ELISA. Recom- binant mouse IP-10 was used for a positive control while DW was used for a negative control. (B) IP-10 secretion was measured in supernatants of MIN6 cells treated with various concentrations of H2O2 was measured by ELISA. (C) An apoptosis of MIN6 cells cultured in 20% O2 or 1% O2 for 48 h with or without anti-IP-10 Ab treatment was measured by FACS analysis.

Fig. 7. NF-κB inhibitor suppresses hypoxia-induced apoptosis of MIN6 cells. MIN6 cells were treated with TF inhibitors for 30 min or 1 h before culturing under hypoxia. AP-1, NF-AT and NF-κB were blocked with 10 μM curcumin, 4 μM NF-AT inhibitor peptide and 0.1 or 1 μM quinazoline, respectively. Then cells weremaintained in 20% O2 or 1% O2 for 24 h. Apoptotic cell death was assessed by FACS analysis using Annexin V staining coupled with propidium iodide. *P<0.05

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inhibitor suppressed the hypoxia-induced apoptosis by more than 50%. In contrast, 10 μM AP-1 or 4 μM NF-AT inhibitor did not suppress hypoxia-induced β-cell apoptosis to any extent (Fig. 7). Although NF-κB inhibitor did not completely rescue cells from hypoxia-induced apoptosis, these results suggest that NF-κB is one of the main mediators for hypoxia-induced β-cell apoptosis.

DISCUSSION

Hypoxia is one of the main causes of islet apoptosis after

transplantation. In general, hypoxia-inducible factor (HIF-1) is considered to play a major role in response to hypoxia (Cummins and Taylor, 2005) and it has been suggested that it is involved in hypoxia-induced β-cell apoptosis (Ko et al., 2008). However, it was recently suggested that HIF-1 is required to change β-cell metabolism for islet survival and revascularization in hypoxic environments (Cantley et al., 2010; Stokes et al., 2013). Furthermore, it was reported that HIF-1 is not associated with hypoxic β-cell apoptosis (Zheng et al., 2012). NF-κB plays a protective role in TNF-α-induced pancreatic β-cell apoptosis (Chang et al., 2003). Similarly, the NF-κB-induced anti-apoptotic gene, A20, suppressed TNF-induced β-cell death (Liuwantara et al., 2006). In addition, β-cells expressing a non-degradable form of IκBα were less sensitive to TNF-α and IFN-γ-mediated cell death (Kim et al., 2007). Human islets over-expressing the NF-κB subunit, c-Rel, were recently shown to be able to escape from cytokine-induced caspase3 activation and cell death (Mokhtari et al., 2009). Taken together, the results of these studies suggest an anti-apoptotic function of NF-κB. However, opposing results have also been reported. Specifically, islets over-expressing IκBα super-repressor escaped from IL-1β- or IFN-γ-induced islet dysfunction and cell death (Giannoukakis et al., 2000; Heimberg et al., 2001). Similarly, islets expressing degradation-resistant IκB were resistant to the toxic effects of IL-1β or IFN-γ (Eldor et al., 2006). These findings supported a possible pro-apoptotic role of NF-κB in β-cells. Recently, islet cells exposed to hypoxia were found to display NF-κB activation, up-regulation of genes related to apoptosis and stress response, as well as down-regulation

of genes associated with transcription and inflammation (Lai et al., 2009). These results suggest a possible relationship between hypoxia-induced NF-κB activation and hypoxia-induced β-cell apoptosis.

In the present study, we showed that NF-κB inhibitor significantly inhibited hypoxia-induced β-cell apoptosis for the first time. However, NF-κB inhibitor did not completely block hypoxia-induced apoptosis, indicating the existence of additional pathways for apoptosis. Nevertheless, these findings suggest that NF-κB is one of the main transcription factors involved in hypoxic β-cell apoptosis. Although it has been reported that hypoxic β-cells underwent apoptosis and that intracellular ROS levels were increased in the cells (Li and Jackson, 2002), most studies of the effects of oxidative stress on β-cells used exogenous H2O2 and a substantial relationship between endogenous ROS and hypoxic β-cell death has not yet been investigated. In the present study, we determined that NADPH oxidase, xanthine oxidase, and mitochondria electron transport chain complex II are all responsible for hypoxia-induced intracellular ROS production for the first time. Since NADPH oxidase (Satoh et al., 2005) and xanthine oxidase (Desco et al., 2002) are known to play a important role in development of oxidative stress in diabetes, suppressing these enzymes effectively inhibits islet cell apoptosis and the process of diabetes (Yuan et al., 2010; Pacher et al., 2006).

Although there is little or no information available regarding chemokine expression in hypoxic β-cells, here we report significant up-regulation of IP-10 mRNA expression in hypoxic MIN6. Previous studies using IL-1β, TNF-α, and IFN-γ treated MIN6 cells revealed up-regulation of IP-10 and MCP-1, with up-regulated levels that were greater than MIN6 deficient NF-κB signals, suggesting possible involvement of NF-κB in cytokine-induced chemokine expression (Baker et al., 2003). Although various TFs such as NF-κB (Liu et al., 2005), IRF1/2 (Buttmann et al., 2007), IRF3 (Lu et al., 2011), and p48/STAT-1α (Majumder et al., 1998) are known to be involved in the regulation of IP-10 expression, we found that NF-κB is involved in control of IP-10 expression in MIN6 cells cultured under oxidative stress. Similarly, studies of skin flbroblasts (Villagomez et al., 2004), intestinal epithelial cells (Yeruva et al., 2008),

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hepatocytes (Zhou et al., 2010) and adipocytes (Krinninger et al., 2011) revealed NF-κB-mediated regulation of IP-10 expression.

IP-10 was recently reported to be capable of inducing β-cell apoptosis (Schulthess et al., 2009). Specifically, islets isolated from diabetic patients were found to highly express IP-10 mRNA and secrete IP-10 proteins. In addition, treat- ment of isolated islets with IP-10 led to reduced β-cell viability, insulin mRNA expression and secretion. The interaction between IP-10 and TLR4 instead of its typical receptor CXCR3, and subsequent production of an apoptosis signal were suggested as the underlying mechanisms for these reduction (Schulthess et al., 2009). This report led us to speculate the possible involvement of hypoxia-induced IP-10 expression in hypoxia-induced β-cell apoptosis. However, we did not detect any IP-10 protein in the present study, despite up-regulation of IP-10 mRNA in hypoxic MIN6. Although it is currently unclear why IP-10 protein was not produced in hypoxic MIN6, inactivation of trans- lation initiation factor eIF2a in response to ER stress and subsequent inhibition of translation of most of the mRNA within the cells may be responsible for the absence of IP-10 protein (Fonseca et al., 2009; Volchuk and Ron, 2010). Regardless of reason, it is clear that IP-10 was not involved in hypoxia-induced β-cells apoptosis in the present study. Since IP-10, which is capable of recruiting activated T cells and macrophages into inflamed tissues (Rotondi et al., 2007), is expressed in islet β-cells of type 1 diabetic patients (Tanaka et al., 2009; Aida et al., 2011), inhibition of IP-10 expression using NF-κB inhibitor may help suppress the progress of diabetes. Furthermore, since NF-κB is a redox-sensitive TF (Foncea et al., 2000; Lavie, 2005) and a major upstream regulator of iNOS (Jia et al., 2013), which is another candidate mediator responsible for hypoxia-induced β-cell apoptosis, we suggest potential use of NF-κB inhibitor to ameliorate hypoxia-induced β-cell apoptosis.

Acknowledgements We thank professor Hee Sook Jeon for generously

providing MIN6 cells. This research was supported by a grant (2009K001561) from Korea Biotech R&D Group of Next-generation growth engine project of the Ministry of

Education, Science and Technology, Republic of Korea.

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