Transcriptional Regulation of Nos2 via STAT5B Binding to Nos2 Gene Promoter Mediates Nitric Oxide Production: Relevance in β-Cell Maintenance


Annu Josepha,b    Lekshmy CR Naira    Betcy Susan Johnsona,b    Philip Litto Thomasa,b    

Renjini Ambika Padmanabhana    Neethu Puthumadathila    Malini Lalorayaa


aFemale Reproduction and Metabolic Syndromes Laboratory, Division of Molecular Reproduction, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, India, bResearch Scholar, Kerala University, Senate House Campus, Palayam, Thiruvananthapuram, India





Key Words

Type 1 diabetes • STAT5B • Transcriptional regulation • NOS2 • Nitric oxide • Hydrogen peroxide • Oxidative stress • Beta cell



Background/Aims: Type 1 Diabetes (T1D) involves autoimmune attack due to reduced regulatory T cells as an effect of mutant Stat5b(C1462A) in non-obese diabetic (NOD) mice, a T1D model resulting in pancreatic β-cell destruction. Although reactive oxygen species are considered to orchestrate the immune attack, the role of nitric oxide (·NO) still remains debatable. Since JAK-STAT pathway is known to induce Nos2, we investigated the role of STAT5B in nitric oxide generation and oxidative stress. Methods: In this study, we have used chromatin immunoprecipitation with STAT5B antibody to explore whether STAT5B binds Nos2 promoter. Using Stat5b gene silencing and overexpression models in MIN6 mouse pancreatic β-cell line we have assayed nitric oxide and its end products, superoxide levels, H2O2 levels, and expression of genes related to redox pathway by immunocytochemistry, biochemical assays, quantitative real time PCR and western blotting. Results: Our results prove that STAT5B binds to the candidate gamma-interferon-activated (GAS) element in Nos2 promoter thereby inducing Nos2 mRNA transcription resulting in NOS2 protein expression in MIN6, a mouse pancreatic β-cell line. Our findings are substantiated by reduced ·NO as well as nitric oxide end products (nitrate and nitrite), and increased superoxide production in Stat5b silenced MIN6 cells. Our results indicate that C1462A mutant STAT5B shows lack of ·NO generation ability. To detoxify excess superoxide as a consequence of lowered Nos2, an overexpressed SOD2 in Stat5b silenced cells results in increased H2O2 production. H2O2 metabolizing enzymes do not show upregulation upon Stat5b silencing, and thus oxidative stress is brought about by amassed H2O2. Stat5b silencing finally reduces AKT expression, a prosurvival signal. Conclusion: Our study enables us to conclude that β-cell stress is aggravated by the incapability of STAT5B to induce Nos2 resulting in H2O2 accumulation and the ensuing oxidative stress enhances β-cell damage.





Type 1 diabetes (T1D) is acknowledged to occur due to autoimmune mediated destruction of β-cells which secrete insulin [1]. The loss of β-cells has been ascribed to autoimmune attack as a consequence of 'insulitis', an inflammatory reaction due to infiltration by mononuclear cells [2]. The role of cytotoxic diabetogenic CD8+ and CD4+ cells [3] has been elucidated in the pathogenesis of T1D.  Peripheral tolerance is compromised in T1D due to defective FOXP3(+)CD4(+) regulatory T cells (Tregs) frequency or due to reduced FOXP3 expression [4]. T1D management revolves around developing methods to improve Treg number and function [5]. Reduced Treg numbers are seen in patients with Stat5b deficiency [6]. STAT5B is the maestro of Treg generation as increased FOXP3 expression under IL2-STAT5B axis is an important step in Treg generation, and this has a bearing on diabetes pathogenesis [7] since non obese diabetic (NOD) mice have a L327M mutant STAT5B with weaker DNA binding property [8].

It is important to note that STAT5 activity in β-cells affects susceptibility to experimentally-induced Type 1 and Type 2 diabetes [9]. Also, high levels of STAT5B protein compared to STAT5A are reported to be present in pancreatic β-cells [10] suggesting that STAT5B could have a direct role in β-cell fate. β-cell death is thought to be a consequence of oxidative stress since the intensity of autoimmune attack on β-cells is modulated by reactive oxygen species (ROS) which is critical to proinflammatory cytokine production, CD8+ T cell proliferation, CTL target lysis, and granzyme expression thereby regulating adaptive immune response [11]. Macrophage and T-cell generated superoxide in particular is essential for autoreactivity induction and T1D [12]. Macrophages infiltrate pancreatic β-cells and release ROS, superoxide and ·NO, to cause extensive damage [13] showing a close nexus of ROS and adaptive immunity. Out of the three isoforms of NOS in pancreatic islets, nNOS (neuronal NOS, NOS1) and iNOS (inducible NOS, NOS2) identified in pancreatic β-cells and eNOS (endothelial NOS, NOS3) in islet endothelial cells [14] are engaged in ·NO production. Elevated ·NO production by β-cell specific Nos2 (iNOS) overexpression causes β-cell degeneration leading to insulin dependent diabetes [15]. T1D onset is associated with increased plasma .NO [16]. High concentrations of proinflammatory cytokines (IL1β, TNFα and IFNγ) which induce nitrite and cGMP accumulation by human islets inhibits insulin secretion but conversely low concentration stimulates insulin secretion [17]. Reports of reduced ·NO in Type 2 diabetes (T2D) [18] as well as association of NOS2 polymorphism with T2DM [19] further complicates the scenario about the role of .NO in diabetes.

Since prolactin (PRL) signals through JAK-STAT5 pathway and induces several genes including inducible nitric oxide synthase (iNOS/NOS2) [20] and PRL receptors are expressed by pancreatic β-cells [21], we explored whether STAT5B could regulate NOS2 expression in β-cells. Thus we analyzed Nos2 promoter region for presence of STAT5B binding site, conducted chromatin immunoprecipitation (ChIP) experiments to prove that STAT5B can bind Nos2 promoter and assessed the levels of Nos2 in PRL-stimulated MIN6 pancreatic β-cell line. Fluorescent dyes as well as fluorescent probes have been successfully in use for detection of different ROS and reactive nitrogen species (RNS) in cells, with negligible side-products. Non-fluorescent 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM DA) forms a fluorescent product, which is comparatively photostable and specific, on reaction with ·NO [22]. MitoSOX Red is a commercially available fluorescent probe for detection of mitochondrial superoxide [23]. Since these tools are specific in the detection of their products, we have used them for our study. We also assessed the impact of Stat5b knockdown or mutant Stat5b transfected, on the other enzymes involved in ROS and RNS regulation instrumental in causing oxidative stress.



Materials and Methods


Cell line, constructs and reagents

MIN6 cell pancreatic β-cell line (obtained from NCCS, Pune) was routinely maintained in DMEM containing 25mM glucose supplemented with 15% FBS, 2mM L-Glutamine, 0.1mM β-mercaptoethanol, 100U/mL penicillin, and 100µg/mL streptomycin. Medium was changed every 2 days and the cells were passaged when 80-85% confluent. All culture media and supplements were from Thermo Fisher Scientific, Waltham, MA, USA. Control siRNA (sc-37007) and Stat5b siRNA (sc-37011) were from Santa Cruz Biotechnology (SCBT), Dallas, TX, USA. Wild and mutant (C1462A mutation with weaker DNA binding ability) Stat5b constructs were made for transfection studies using pDsRed-Express-C1 vector (Clontech, Takara Bio, Japan). 500ng/mL PRL (Biovision Inc., San Francisco, USA) was used to stimulate Stat5b silenced/overexpressed MIN6 cells for 30 minutes. Anti-RNA polymerase II (ab5408, Abcam, Cambridge, UK) and anti-STAT5B (sc-1656X, SCBT, Dallas, TX, USA) were used in chromatin immunoprecipitation studies. Primary antibodies against actin (A2668, Sigma Aldrich, St. Louis USA), STAT5B (ab178941, Abcam, Cambridge, UK), and NOS2 (sc-651), SOD2 (sc-11407), catalase (sc-50508), AKT1/2/3 (sc-8312), BCL-xL (sc-7195) and ERK2 (sc-154) (SCBT, Dallas, TX, USA) were used for western blotting experiments.


siRNA-mediated silencing of Stat5b in MIN6 cells

The expression of Stat5b in MIN6 cells was silenced by transfection of siRNA targeting mouse Stat5b (100nM) in 40-50% confluent cells. The silencing was performed using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) reagent. Non-targeting control siRNA was used for control group. Lipofectamine-siRNA complex was added to each well and the plates were incubated at 37°C for 72 hours. It was further followed by 500ng/mL PRL stimulation for 30 minutes, and the cells were trypsinized and collected.


Wild and C1462A mutant Stat5b transfection into MIN6 cell line

Transfection of wild and mutant (NOD) Stat5b-DsRed constructs was done in MIN6 cells at 90-95% confluency in DMEM culture medium (composition detailed earlier). The constructs were transfected using Lipofectamine 3000 reagent and incubated for 48 hours followed by PRL stimulation. The cells were washed with PBS, fixed with 4% paraformaldehyde and observed under confocal microscope (Nikon, Tokyo, Japan).


Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) was done using EpiTect ChIP OneDay Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. MIN6 cells were cultured in T25 flasks until 80% confluency. The proteins were cross-linked to DNA using 1% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 10 minutes. The cells were then harvested and lysed using Lysis Buffer containing protease inhibitor cocktail and sonicated with Bioruptor® sonication device (Diagenode, Liege, Belgium) for the generation of chromatin fragments. The lysate was pelleted and supernatant pre-cleared with Protein A beads. Specific antibodies have to be used for the immunoprecipitation of chromatin fragments to which the protein of interest is bound. For this, precleared lysate was separated into three IP fractions, and incubated at 4°C overnight with mouse control IgG (Genei, Bangalore, India) as negative control, anti-RNA polymerase II as positive control or anti-STAT5B for the protein of interest. 1% of IP fraction was kept as input fraction. Since RNA polymerase II is present at the promoters of all actively transcribed genes, it was used as the positive control. The IP fractions containing protein of interest bound to the corresponding chromatin region were immunoprecipitated with Protein A beads. Genomic DNA was isolated and purified, followed by RT-PCR using EpiTect ChIP qPCR Primer Assay for mouse Ccnd2, a known STAT5B target, and Nos2, the region under investigation (Qiagen, Hilden, Germany).


Simultaneous detection of ·NO and superoxide by DAF-FM DA and MitoSOX Red

MIN6 cells after 72 hours of siRNA transfection (Stat5b/Control) or 48 hours of wild Stat5b-DsRed/C1462A mutant Stat5b-DsRed were washed, and the media replaced with phenol red-free DMEM containing 2mM L-glutamine and 10µM DAF-FM DA (D23842, Thermo Fisher Scientific, Waltham, MA, USA). After 30 minutes of incubation, cells were treated with 500ng/ml of PRL (Biovision Inc. USA) and further incubated at 37°C for 30 minutes. For siRNA treated samples, the cells were washed twice to remove excess probe and incubated with phenol red-free DMEM containing 5μM MitoSOX Red (M36008, Thermo Scientific) for 60 minutes. The cells were again rinsed with PBS and fixed with 4% PFA for 20 minutes at 4°C. This was followed by DAPI (4′,6-diamidino-2-phenylindole) staining and imaging with Nikon AIR Confocal microscope  using NIS Elements AR 4.00.04 software.


Measurement of NOx end products

The total NOx (nitrate + nitrite) and nitrite were measured using Nitrate/Nitrite colorimetric assay kit (Cayman Chemical, MI, USA). Nitrite was measured by addition of Griess reagent to 100µL sample directly. Since nitrate cannot be directly measured, it is converted to nitrite using nitrate reductase enzyme, followed by the addition of Griess Reagents. For total NOx assay, phenol-red free culture media used for growing MIN6 cells after treatment was analyzed. The samples and standards were added to each well of a 96-well mictrotitre plate followed by nitrate reductase enzyme and cofactors according to the manufacturer's instructions. After incubation for 2 hours, Griess reagent was added. The final azo product was measured at 540nm on Tecan Infinite 200 (Tecan, Männedorf, Switzerland).


Measurement of SOD activity

SOD activity was measured using SOD determination kit (Sigma-Aldrich, St. Louis, USA) which utilizes the Dojindo’s tetrazolium salt WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt) to produce a water-soluble formazan dye upon reduction with superoxide anion. The rate of reduction is inhibited by SOD and the residual dye is measured by colorimetry. Briefly, MIN6 cells after treatment were collected and proteins extracted in a non-denaturing buffer (20mM Tris-HCl, pH 8.0, 137mM NaCl, 10% glycerol, 1% NP-40, 2mM EDTA, protease inhibitor and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, USA)). Protein estimation was done and 25µg of each sample was used for the assay as per manufacturer’s instruction. The plate was incubated for 20 minutes and absorbance read at 450nm on iMark Microplate reader (Bio-Rad, Hercules, CA, USA).


Kinetics of H2O2 released from cells

The kinetics of H2O2 released from cells was done using Amplex® Red (10-acetyl-3, 7-dihydroxyphenoxazine) hydrogen peroxide/peroxidase assay kit (Thermo Fisher Scientific, Waltham, MA, USA) as per the kit protocol with minor modifications. Briefly, spent medium was removed from the wells of 96-well culture plate after 72 hours of siRNA treatment and Krebs-Ringer phosphate (KRPG) buffer added to the cells. This was followed by 100µL of reaction mixture containing 50µM Amplex® Red reagent and 0.1U/mL HRP in KRPG.  Amplex® Red reagent reacts with H2O2 to produce resorufin. Fluorescence (excitation at 530-560nm and emission detection at 590nm) was measured immediately, and subsequently measured every 30 minutes until 90 minutes on Tecan Infinite 200 (Tecan, Männedorf, Switzerland).


MTT Assay

For the assay, 10μL MTT reagent (TC-191, HiMedia Laboratories, India) was added to MIN6 cells in 90μL media without phenol red after 72 hours of siRNA treatment, to a final concentration of 0.5μg/μL, and incubated at 37°C for 2 hours under dark. After incubation, 100μL solubilisation buffer (prepared using 400μL of 1M HCl, 10mL Triton-X 100 and 90mL isopropanol) was added and incubated at room temperature for 1 hour. Absorbance was read at 570nm.


RNA isolation, cDNA synthesis, real time PCR

Total RNA was isolated from MIN6 cells using miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol and quantitated using ND-1000 spectrophotometer (Nanodrop Technologies, Thermo Fisher Scientific, Waltham, MA, USA). cDNA was prepared from 150ng RNA employing SuperScript VILO cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) as per manufacturer’s instructions.  qRT-PCR primers were designed using ABI Primer Express software. Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) was used to perform quantitative real-time PCR in MicroAmp® Optical 384-Well Reaction Plates (Thermo Fisher Scientific, Waltham, MA, USA). PCR runs and fluorescence measurements were done in 7900 HT Fast Real Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using standard cycling conditions. Relative expression levels were calculated using ΔΔCt method with 18SrRNA as the endogenous control.


Preparation of total protein extracts

The cells after treatment were collected by trypsinization and total protein extracted in protein extraction buffer containing 50mM Tris-HCl (pH 8.0), 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate. Protein quantitation was performed using Lowry’s method (DC Protein Assay kit, Bio-Rad, Hercules, CA, USA) on iMark Microplate reader (Bio-Rad, Hercules, CA, USA).


SDS-PAGE and Western blot development

The samples were run in SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membrane was washed with PBST (pH 7.4, with 0.1% Tween 20), blocked with 3% gelatin or 5% BSA in PBST for 2 hours to prevent non-specific binding of antibodies. The membranes were then incubated with protein-specific primary antibody of varying dilutions overnight at 4°C. The antibodies used were 1:3000 actin, 1:4000 STAT5B, 1:500 NOS2, 1:1000 SOD2, 1:500 catalase, 1:500 AKT1/2/3, 1:500 BCL-xL, and 1:100 ERK2. This was followed by incubation of the membrane with peroxidase-conjugated goat anti rabbit secondary antibody (Sigma-Aldrich, St. Louis, MO, USA). The blots were developed based on color reaction by enhanced chemiluminescence detection method (Clarity Western ECL Substrate; Bio-Rad, Hercules, CA, USA). The blots were imaged using VersaDoc Imaging System (Bio-Rad, Hercules, CA, USA).


Statistical analysis

Data are expressed as mean ± SD and statistical analysis was performed by Student’s t-test or one-way analysis of variance (ANOVA), as appropriate, using the statistical software Microsoft Excel and GraphPad Prism 5. P-values of ≤0.05 were accepted as statistically significant.





Identification of Nos2 as a STAT5B target in MIN6 cells

Both Nos2 and Stat5b genes are located on chromosome 11, as identified from their chromosome ideograms at the site UCSC Genome Browser on Mouse Dec. 2011 (GRCm38/mm10) Assembly ( (Fig. 1A) [24]. Nos2 promoter has a candidate gamma-interferon-activated (GAS) element, the known STAT5-binding site [25] and the binding site AGTTTCTTGGAAATC identified by ChampionChiP Transcription Factor Search Portal based on DECipherment Of DNA Elements (DECODE), proprietary database of SABiosciences, Qiagen is shown in Fig. 1B.


Fig. 1. STAT5B is a transcriptional regulator of Nos2. A) Chromosome ideogram of mouse Stat5b and Nos2 on chromosome 11( B) Cartoon representation of the putative binding site of STAT5B on Nos2 promoter, as identified from ChampionChiP Transcription Factor Search Portal (SABiosciences). C) Chromatin immunoprecipitation in MIN6 cells identified Nos2 as a direct target of STAT5B. Lane 1 - Input DNA, lane 2 - mouse control IgG, lane 3 - anti-RNA polymerase II antibody, lane 4 - anti-STAT5B antibody, lane 5 - DNA ladder. The image is a representation of experiment done thrice independently. D) Bar graph representation of 2-ΔΔCt of Stat5b and Nos2 transcripts showing downregulation in prolactin-induced Stat5b siRNA treated versus control siRNA treated MIN6 cells, calculated with respect to untreated cells and 18SrRNA as the endogenous control. (n = 3). E) Western blots and F) densitometric analysis of STAT5B and NOS2, showing a downregulation in PRL-induced siRNA treated samples, with actin as the loading control (n = 3). P-value was calculated using Student's t-test.


Since the presence of a binding site does not confirm actual binding of STAT5B to Nos2 promoter, we performed ChIP on PRL-stimulated MIN6 cells with STAT5B antibody. The qRT-PCR of subsequently isolated STAT5B pulled-down genomic DNA showed amplification for Nos2, proving STAT5B binding to Nos2 promoter (Fig. 1C). Amplification of Ccnd2, a known target of STAT5B served as positive control. No detectable amount of DNA was amplified in DNA from IP fraction with mouse control IgG, thus eliminating the possibility of non-specific amplification.


Stat5b knockdown MIN6 samples exhibit decreased Nos2 levels

In order to assess the impact of Stat5b silencing on NOS promoter, MIN6 cells treated with 100nM of Stat5b siRNA or control siRNA for 72 hours were used for further studies. Significant knockdown of Stat5b (p<0.008) was confirmed by analyzing its relative transcript levels, with 18SrRNA as the endogenous control (Fig. 1D). The real time transcript level expression of Nos2 was found to reduce in Stat5b silenced MIN6 cells, with a fold change of 6.813 down regulation (p <0.01) (Fig. 1D) with no significant change in Nos1 levels, thus confirming the ChIP results. Stat5b siRNA-treated samples showed a highly significant decrease in immunopositive band intensity in STAT5B (p<0.001) and NOS2 (p<0.001) proteins on western blots with actin as loading control (Figure 1E) corroborating our real time data. Graphical representation is shown in Fig. 1F after normalization with actin. Since untreated samples showed STAT5B and NOS2 expression similar to control siRNA treated samples, further experiments were performed on control versus Stat5b siRNA treated MIN6 cells.


.NO production is regulated by STAT5B levels in MIN6 cells

Techniques for simultaneous detection of .NO and superoxide without interference have been previously reported [22]. Non-fluorescent DAF-FM DA detects .NO by forming fluorescent benzotriazole on reaction with .NO which is excited at 495 nm [26]. MitoSOX Red is a fluorogenic dye which selectively gets oxidised by mitochondrial superoxide and emits red fluorescence. Reduced green fluorescence and increased red fluorescence was detected by confocal imaging in PRL stimulated Stat5b knock down cells compared to PRL stimulated control siRNA treated cells or untreated cells suggesting decreased .NO and increased superoxide in Stat5b knock down cells (Fig. 2A).


Fig. 2. Stat5b silencing inhibits ·NO production. A) Confocal imaging of nitric oxide accumulation in control, Stat5b siRNA treated and untreated MIN6 cells using DAF-FM DA. After 72 hours siRNA transfection, cells were treated with 10µM DAF-FM DA for 30 minutes followed by treatment with PRL, incubated for further 30 minutes, washed with PBS, incubated with 5μM MitoSOX Red for 60 minutes, stained with DAPI and imaged at 358nm, 495 nm and 510 nm excitation. B) & C) Representation of the concentration of total nitric oxide end products (nitrate + nitrite) and nitrite released from Stat5b siRNA treated versus control siRNA treated MIN6 cells, respectively. P-value was calculated using Student's t-test on GraphPad Prism 5.0 software (n = 3).


.NO end products are diminished in Stat5b siRNA treated samples

Since our confocal results indicated a decrease in ·NO levels in cells with reduced STAT5B (Fig. 2A), we analyzed the release of ·NO end products (nitrate and nitrite) in MIN6 cells. The total nitrate plus nitrite levels were found to be significantly lower in Stat5b knockdown samples at 16.88 ± 2.652 µM, compared to control siRNA-treated samples at 35.42 ± 3.146µM (n=3 in all groups, p = 0.0065) (Fig. 2B). Nitrite levels were also correspondingly reduced in the Stat5b siRNA treated samples at 3.533 ± 1.102 µM compared to the control samples at 7.000 ± 2.433 µM (n = 3) (Fig. 2C).


C1462A(L327M) mutant Stat5b is incapable of activating Nos2 promoter

The effect of STAT5B overexpression on ·NO levels was checked by confocal microscopy on transfecting MIN6 cells with wild-type and C1462A mutant Stat5b in DsRed vector followed by PRL stimulation. The cells transfected with wild-type Stat5b showed a substantial increase in NO levels, as observed by the increased fluorescence of DAF-FM DA, while mutant STAT5B-transfected cells did not show any variation compared to empty vector-transfected and non-transfected cells (Fig. 3), due to the inability of the mutant STAT5B to bind strongly to Nos2 promoter.


Fig. 3. C1462A(L327M) mutant Stat5b is unable to generate ·NO. Confocal imaging of nitric oxide accumulation in wild and mutant Stat5b-DsRed plasmid transfected MIN6 cells using DAF-FM DA. Increased nitric oxide levels were found on overexpression of STAT5B (wStat5b-DsRed) while no significant change was observed in mutant STAT5B (mStat5b-DsRed) with weaker DNA binding ability, compared to empty vector transfected and untransfected cells. The cells were treated with 10µM DAF-FM DA for 30 minutes followed by treatment with PRL, incubated for further 30 minutes, and imaged at 358 nm, 488 nm and 568 nm excitation. Nuclei were stained with DAPI.


Differential SOD2 expression in Stat5b siRNA treated MIN6 cells

Lowered .NO levels in condition with nonfunctional STAT5B could result in superoxide accumulation since ·NO would not be available to react with it. Hence we studied the downstream events as a consequence of this reduced ·NO. Superoxide anion (O2.-) metabolizing Sod1, Sod2 and Sod3 transcripts were analyzed in Stat5b siRNA treated versus control siRNA treated MIN6 cells by qRT-PCR. The relative transcript levels of Sod1 and Sod3 did not show significant variation between control and Stat5b knockdown samples (Fig. 4A) while the relative expression of Sod2, the mitochondrial form of SOD, showed 2.06 fold upregulation (p = 0.023) in Stat5b knockdown samples (Fig. 4A). This difference in SOD2 was corroborated in our western blot data (p = 0.017) (Fig. 4B-C).


Fig. 4. Impact of Stat5b silencing on SOD2 and H2O2. A) Bar graph representation of increased relative transcript levels (2-ΔΔCt) of Sod1, Sod2 and Sod3 in PRL induced Stat5b siRNA treated MIN6 cells versus control samples, with 18SrRNA as endogenous control (n = 3). B) Western blot and C) densitometric analysis of SOD2 denoting an increase in protein levels in Stat5b siRNA treated samples (n = 3). The bands were normalized using actin in Fig. 1E. D) Bar graph represents the effect of Stat5b siRNA treatment on percentage inhibition activity of SOD on WST-1, showing an increase in SOD activity in Stat5b siRNA treated samples (n = 3). P-value was calculated using Student's t-test on GraphPad Prism 5.0 software. E) Representation of detection of H2O2 released from cultured MIN6 cells treated with Stat5b siRNA versus control siRNA over a period of 0 to 90 minutes. The values show mean and SD of fluorescence from 3 replicates and indicates higher amounts of H2O2 released from Stat5b knockdown samples.


Increased SOD activity is evident in Stat5b silenced MIN6 cells

To assess whether the change in SOD2 expression is reflected in its enzymic activity, the inhibition activity of total SOD on WST-1 to produce a water soluble formazan dye was measured. The percentage inhibition activity of SOD was found to be 5.167% ±2.309 in control and 21.33% ±3.617 in Stat5b silenced samples with respect to untreated samples (n = 3 in each group, p = 0.0028) (Fig. 4D).


Kinetics of H2O2 released show an increase in Stat5b siRNA treated cells

H2O2 levels were measured using Amplex Red reagent and HRP, and the fluorescence was measured every 30 minutes for 90 minutes. The kinetics of H2O2 released from the cells showed a substantial increase in fluorescence in Stat5b knockdown samples compared to control samples, indicating that the treatment has caused an increase in H2O2 levels after Stat5b knockdown (Fig. 4E) thus reiterating that SOD2 overexpression in Stat5b silenced cells translates to increased H2O2 production.


No increase in H2O2 metabolizing enzymes in Stat5b silenced cells

Real time transcript levels (Fig. 5A), as well as western blot (Fig. 5B-C) of the primary H2O2 detoxification enzyme catalase (CAT) did not show a significant difference between control and Stat5b siRNA treated MIN6 cell samples. The same was reflected in the transcript levels of Gpx2 and Gpx4, while the transcript levels of mitochondrial Gpx1 showed a fold change reduction of 0.58 (p = 0.024) (Fig. 5D), indicating that the enzymes present in the β-cells are not enough for the conversion of excess H2O2 produced to harmless O2, leading to its accumulation and induction of cellular stress.


Fig. 5. Status of H2O2 metabolizing enzymes and AKT upon Stat5b silencing. A) qRT-PCR graphs showing the relative expression of H2O2 metabolizing enzyme Cat in control and Stat5b siRNA treated MIN6 cells with 18SrRNA as endogenous control. The transcript levels do not show significant difference between control and treated samples (n = 3). B) Western blot and C) densitometric analysis of catalase (n = 3) corroborate the Cat qRT-PCR results. D) qRT-PCR graphs showing the relative expression of H2O2 metabolizing enzymes Gpx1, Gpx2 and Gpx4 in control and Stat5b siRNA treated MIN6 cells with 18SrRNA as endogenous control (n = 3). Except for Gpx1, the transcript levels do not show significant difference between control and treated samples. E) Western blot of prosurvival molecules AKT1/2/3, BCL-xL and ERK2. AKT1/2/3 and BCL-xL shows decreased levels in Stat5b siRNA treated samples compared to control siRNA treated samples, also represented by densitometric analysis of the bands (F and G), while ERK2 (H) did not show significant difference (n = 3). The bands were normalized using actin in Fig. 1E. I) Representation of percentage viability of control and Stat5b siRNA treated cells, as identified by MTT assay. Stat5b siRNA treated cells show decreased cell viability compared to the control siRNA treated cells (n = 3).


Stat5b silencing affects cell viability and prosurvival signals

Since our data shows that STAT5B controls ·NO and superoxide production, we wanted to see whether it affects cell viability. The yellow tetrazolium dye 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) is converted to purple formazan in the presence of cellular oxidoreductase enzymes present in viable cells. This product is then solubilised using acidified isopropanol to form a coloured solution the absorbance of which can then be read at 570nm. Percentage cell viability was calculated after experiment using the formula (Absorbance of control or Stat5b siRNA treated sample/ Absorbance of untreated sample) *100. Control siRNA treated cells showed 98.65% ± 9.54 viability while viability of Stat5b knockdown cells decreased to 65.78% ± 6.31 (n = 3 in all groups, p = 0.0076) [Fig. 5, I]. This prompted us to look at the pro-survival pathway molecules. Upon Stat5b silencing a significant down-regulation was seen in AKT (p<0.01) (Fig. 5, E-F) and a reduction in BCL-xL (Fig. 5, E,G) while no change was seen in ERK2 levels (Fig. 5, E,H). Thus our results indicate that molecules regulating pro-survival pathway which are known to be under NO regulation are controlled by STAT5B.





Dysregulation of STAT5B, located near the Idd4 Type 1 diabetes susceptibility interval, has been suggested to be involved in susceptibility to diabetes [8, 9]. Our work clearly establishes that STAT5B is a transcriptional regulator of Nos2 (iNOS).  It is known that cytokines like IFN-γ promotes Nos2 transcription via interferon response factor (IRF1) [27], and PRL also behaves like a cytokine inducing Nos2 expression through JAK-STAT pathway [28]. Although presence of a candidate gamma-interferon-activated (GAS) element which is the known STAT5-binding site had been reported much earlier in the Nos2 promoter and ·NO production modulated by STAT1α binding to GAS element under bacterial lipopolysaccharide (LPS) and interferon (IFN)-gamma stimulation [25], our study for the first time to our knowledge proved a direct STAT5B binding to Nos2 promoter and regulating NO production. In our study Nos2, a putative target of STAT5B, was proved to be a bona fide target by CHIP experiments which showed that STAT5B binds to Nos2 promoter (Fig. 1C). Our finding of down regulation of Nos2 expression (Fig. 1D-F) and corresponding decreased levels of NO and its products nitrate and nitrite, and elevated superoxide levels in Stat5b knockdown MIN6 cells, reiterate that NO production is under STAT5B control (Fig. 2). Since Nos2 has been found to be a direct target of STAT5B and C1462A(L327M) mutant STAT5B has a weaker DNA binding ability in non obese diabetic mouse (NOD) compared to the wild-type [7], we looked at the effect of STAT5B overexpression on ·NO levels. Thus, further authentication comes from the lack of NO production in C1462A (L327M) mutant Stat5b transfected β-cells in contrast to increased NO generation in wild type Stat5b transfected β-cells (Fig. 3).

β-cell destruction in T1D is thought to be due to the inability to generate regulatory T-cell because of the incapability to induce FOXP3 expression as a consequence of defective STAT5B [6, 7]. ·NO has been shown to be involved in immune response [29] especially in induction of CD4+CD25+ Foxp3 regulatory T cells christened NO-Tregs [30]. Thus our finding of a direct regulation of Nos2 transcription by STAT5B would potentiate the failure to generate Tregs in cases where Stat5b is defective. Our finding gains strength from the reports of association of lowered ·NO production in type 2 diabetes and diabetic nephropathy [18, 31] as well as increased β-cell apoptosis in iNOS (-/-) mice [32]. It also appears that ˙NO production would represent an autoregulatory loop for STAT5B activation as NOS2 and ˙NO are known to augment IL-2 production [33, 34].

During oxidative metabolism, 0.4 to 4% of the oxygen gets converted to superoxide anion. ·NO can interact with superoxide radicals forming peroxynitrite, and is the only known biological molecule which can prevail over superoxide dismutase (SOD) for taking up superoxide under pathological conditions [35]. Due to decreased STAT5B activation on account of Stat5b silencing, β-cells have reduced Nos2 expression which causes decreased ·NO and its end products in our data which would result in more amounts of superoxide radicals available for SOD to convert to hydrogen peroxide (H2O2). Since only the mitochondrial SOD2 is upregulated in our study while SOD1 and SOD3 remain unchanged (Fig. 4A-C), we postulate that the surge in O2.radicals is contained in the mitochondria itself by enhanced SOD2 activity leading to increased H2O2 in Stat5b silenced MIN6 β-cells (Fig. 4E).

Pancreatic β-cells are also highly susceptible to oxidative stress as antioxidant enzymes, CAT) and glutathione peroxidase (GPX), the enzymes responsible for deactivating H2O2 to oxygen and water, are considerably less in pancreatic islets compared to other tissues (only 5% of liver), and hence islets are more sensitive to cellular damage [36]. Of further concern is that pancreatic β-cells are incapable of upregulating the antioxidant enzymes during cellular stress and over-expression of catalase can help them combat H2O2 induced toxicity [37]. This inapt adaptation is also seen in our data where no corresponding increase occurs in H2O2 metabolizing enzymes, catalase and GPX isoforms, on Stat5b silencing. On the contrary, the situation is worsened by decreased Gpx1 mRNA expression upon Stat5b silencing (Fig. 5A-D). An overactive Sod2 coupled to a repressed Gpx1 would result in excessive mitochondrial H2O2 levels which would result in oxidative stress resulting in β-cell destruction. The resulting oxidative stress further would inactivate STAT5 as reported earlier [38] thus setting off another regulatory route and block STAT5B mediated Nos2 induction. Thus it appears that increased oxidative stress coupled to decreased·NO bioavailability affects β-cell survival.

PRL is also known to promote β-cell survival by increasing the expression of anti-apoptotic protein BCL-xL via JAK2/STAT5 pathway [39]. PRL mediated STAT5 activation is also shown to regulate AKT1 expression by binding to a unique promoter in Akt1 and propagating survival signals and evading apoptosis [40].·NO on the other hand is known to attenuate phosphatidylinositol 3-kinase (PI3K)/Akt survival pathway in insulin-producing RINm5F cells [41] and has been reported to be involved in AKT activation [42]. Thus our results of STAT5B boosting·NO production by binding to Nos2 promoter enable us to postulate that STAT5B transcriptionally activates Nos2 and maintains AKT and BCL-xL levels (Fig. 5E-H), thereby functioning as a major regulator of β-cell maintenance, also indicated by the reduced viability in Stat5b knockdown cells (Fig. 5I). This is further strengthened by the recent report of differentiation of stem cells into functional β-cells by·NO induced epigenetic modification of PDX1 (Pancreatic and duodenal homeobox 1) important for pancreatic development during embryogenesis [43].





Taken together, our results enable us to postulate that STAT5B is a key player in pancreatic β-cell maintenance. STAT5B upregulates Nos2 expression by binding to its promoter and this culminates in efficient removal of superoxide by NO produced thereby reducing the H2O2 load of β-cell which suffers from inapt H2O2 detoxification capacity.  The reduced H2O2 load in β-cell under STAT5B signaling does not allow occurrence of oxidative stress. Reduced NO from physiological levels and increased superoxide and H2O2 levels under conditions of STAT5B insufficiency can cause decreased in Tregs on one hand, and decreased pro-survival molecule AKT on the other. The proposed involvement of STAT5B in the governance of oxidative stress important for β-cell maintenance is represented in Fig. 6. Thus transcriptional regulation of Nos2 by STAT5B in addition to its control of FOXP3 places STAT5B as a central figure responsible for β-cell maintenance and hence critical to protection from β-cell stress.


Fig. 6. Cartoon depicting the proposed role of STAT5B in regulation of oxidative stress in MIN6 β-cell line. The boxed area illustrates our hypothesis on regulation of nitric oxide by STAT5B. Binding of STAT5B to Nos2 promoter results in transcriptional activation of NOS2 and increased nitric oxide synthesis. This can augment prosurvival pathways by regulating AKT and BCL-xL expression and enables β-cell survival. This is in addition to the existing knowledge that STAT5B-regulated FOXP3+ T-regulatory cells (Tregs) and NO.-regulated FOXP3- Tregs promote immune tolerance, and nitric oxide can cause epigenetic modification of PDX1 enabling β-cell differentiation. When there is a dearth of STAT5B, Nos2 activation is decreased with a diminished production of nitric oxide and its end products. At the same time, more superoxide anions from electron transport chain are available for SOD to be converted to H2O2. With no corresponding increase in H2O2 metabolizing enzymes, the surge in H2O2 results in β-cell stress.





ML was supported by a Grant from Board of Research in Nuclear Sciences (BRNS) (#37(1)/14/58/2014-BRNS/2180) and Rajiv Gandhi Centre for Biotechnology-Department of Biotechnology (RGCB-DBT) core funds. AJ was supported by a Research Fellowship from Council for Scientific and Industrial Research (09/716(0130)/2010-EMR-I), PLT by a Faculty Improvement  program of University Grants Commission (FIP/12th Plan/KLMG026 TF-14), BSJ by a Research Fellowship from Department of Science & Technology (DST/INSPIRE Fellowship/2015/1F150361), RAP was supported by an Research Associateship from Science & Engineering Research Board (SERB) DST (SB/SO/AS -016 / 2014) while LCR was supported by the BRNS grant.

ML was involved in project conception/supervision, experiment/Fig. 6 designing and manuscript review. AJ designed experiments/Fig. 6, executed experiments in Fig. 1-5, analyzed data, wrote manuscript. AJ, BSJ, RAP contributed to Fig. 1C.  AJ, BSJ, PLT, LCR contributed to Fig. 2A and 5I. LCR and NP assisted AJ for Figures 1E, 2B-C and 3.



Disclosure Statement


The authors declare no competing financial interests.





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