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

Cellular Physiology and Biochemistry  2019;52(1):141-155. Epub 2019 Feb 18. | https://doi.org/10.33594/000000010  

Annu Joseph, Lekshmy CR Nair, Betcy Susan Johnson, Philip Litto Thomas, Renjini Ambika Padmanabhan, Neethu Puthumadathil, Malini Laloraya

Abstract
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 Stat5bsilenced 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 H2O2Stat5bsilencing 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.

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