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 H2O2. Stat5bsilencing
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.