ABSTRACT
Cell death and survival are tightly controlled through the highly coordinated activation/inhibition of diverse signal transduction pathways to insure normal development and physiology. Imbalance between cell death and survival often leads to autoimmune diseases and cancer. Death receptors sense extracellular signals to induce caspase-mediated apoptosis. Acting upstream of CED-3 family proteases, such as caspase-3, Bcl-2 prevents apoptosis. Using short hairpin RNAs (shRNAs), we suppressed Bcl-2 expression in Jurkat T cells, and this increased TCR-triggered AICD and enhanced TNFR gene expression. Also, knockdown of Bcl-2 in Jurkat T cells suppressed the gene expression of FLIP, TNF receptor-associated factors 3 (TRAF3) and TRAF4. Furthermore, suppressed Bcl-2 expression increased caspase-3 and diminished nuclear factor kappa B (NF-κB) translocation.
ABSTRACT
Pancreatic ß-cells play a crucial role in glucose homeostasis, and the failure of these cells to function results in the development of type 1 diabetes (T1D). The MIN6 cell line, which closely resembles pancreatic ß-cells, was used to unravel the relationship between pancreatic ß-cell function and the antioxidant enzyme PRX-1. PRX-1 was knocked down in MIN6 cells using a shPRX-1 lentiviral construct, and a mixture of inflammatory cytokines was administered to challenge the MIN6 cells. Nitric oxide (NO) production and inducible NO synthase (iNOS) expression were elevated in shPRX-1 compared with the control. Also, shPRX-1 transduced cells showed higher levels of NF-κB nuclear translocation, suggesting that PRX-1 has a regulatory role in NF-κB nuclear translocation and iNOS expression. In correlation with NO levels, decreased anti-apoptotic gene Bcl-xl level and elevated pro-apoptotic gene Bim levels were observed in shPRX-1 cells compared with scramble, and cell viability decreased accordingly. A rescue experiment was performed subsequently using an iNOS inhibitor to confirm NO as the cause of cell death. Overall, the results of this study suggest possible protective roles of the antioxidant enzyme PRX-1 in the insulinoma cell line MIN6 and possibly in pancreatic ß-cells under T1D conditions.
Subject(s)
Cell Death/physiology , Cytokines/metabolism , Homeodomain Proteins/metabolism , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/physiology , Animals , Antioxidants/metabolism , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Cell Line, Tumor , Cell Survival/physiology , Membrane Proteins/metabolism , Mice , NF-kappa B/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/metabolism , Proto-Oncogene Proteins/metabolism , bcl-X Protein/metabolismABSTRACT
Type 1 diabetes (T1D) is caused by dysregulation of the immune system in the pancreatic islets, which eventually leads to insulin-producing pancreatic ß-cell death and destabilization of glucose homeostasis. One of the major characteristics of T1D pathogenesis is the production of inflammatory mediators by macrophages that result in destruction or damage of pancreatic ß-cells. In this study the inflammatory microenvironment of T1D was simulated with RAW264.7 cells and MIN6 cells, acting as macrophages and pancreatic ß-cells respectably. In this setting, peroxiredoxin-1, an anti-oxidant enzyme was knocked down to observe its functions in the pathogenesis of T1D. RAW264.7 cells were primed with lipopolysaccharide and co-cultured with MIN6 cells while PRX-1 was knocked down in one or both cell types. Our results suggest that hindrance of PRX-1 activity or the deficiency of this enzyme in inflammatory conditions negatively affects pancreatic ß-cell survival. The observed decrease in viability of MIN6 cells seems to be caused by nitric oxide production. Additionally, it seems that PRX-1 affects previously reported protective activity of IL-6 in pancreatic ß cells as well. These results signify new, undiscovered roles for PRX-1 in inflammatory conditions and may contribute toward our understanding of autoimmunity.
