This is a submission made to the Council on Undergraduate Research for 2012's Posters On The Hill contest. The research was performed in the Collier lab at the University of Tennessee, led by Dr. Jason Collier and post-doctoral fellow Dr. Susan Burke. This abstract is a summary of a portion of a project, largely referred to as 'the iNOS story' by lab members, that involves various findings obtained through research in the lab. Work was done by using various promoter constructs for the inducible nitric oxide synthase (iNOS) gene in murine/rat beta-cell specific cell lines. We used various mutated iNOS promoters carrying mutations in the GAS (IFN-gamma activated site) element: a consensus mutation (cGAS), a GAS with a non-functional motif (GASm), and a construct carrying three tandem repeats of the cGAS mutation (3xGAS). On the background of these promoter constructs, which were adenovirally delivered to our beta-cell lines, we treated cells with various cytokines including interleukin 1-beta (IL-1beta), interferon gamma (IFN-gamma), and a combination of IL-1beta + IFN-gamma. Our findings prove a dual role for the signal transducer and activator of transcription 1 (STAT1) protein that, to date, has been confined to signaling of IFN-gamma alone. However, in our consensus GAS promoter construct, we see strong transcriptional activation by IL-1beta, a noncanonical signal transduction pathway. The pivotal interaction between the two distinct signal transduction pathways of the cytokines IFN-gamma and Il-1beta occurs through the involvement of STAT1. Below is a 250-word abstract submitted to CUR on October 18, 2011.
Abstract:
Type 1 diabetes mellitus is an autoimmune disease in which the insulin-producing pancreatic beta-cells are progressively destroyed. Secretion of pro-inflammatory cytokines, such as IL-1beta and gamma-IFN, from invading and resident immune cells coordinately alters gene expression patterns that lead to inflammation- based losses in pancreatic beta-cells. The iNOS gene is regulated by the NF-kB and signal transducer and activator of transcription (STAT) family of regulatory proteins. Normally, IL-1beta activates iNOS transcription through NF-kB while gamma-IFN controls gene regulation via STAT activation. To understand how these proteins regulate the iNOS gene, we made mutations within the regulatory DNA elements bound by NF-kB and STAT1 in the iNOS proximal gene promoter. Elimination of the NF-kB sites prevented cytokine-mediated induction of the iNOS gene by 80-90%, while mutations in the gamma activated sequences (GAS) blocked the response to gamma-IFN; the GAS mutations also diminished the IL-1beta response by 50%. A one nucleotide substitution changing the iNOS GAS to a consensus GAS was expected to enhance the response to gamma-IFN. Surprisingly, this gain-of-function mutation increased the sensitivity of the iNOS gene by 2-fold to IL-1beta with no concomitant increase in gamma-IFN-mediated transcription. To our knowledge, this is the first report of a dual role for the STAT1 transcription factor in regulating the iNOS gene through GAS elements. We conclude that NF-kB and STAT1 increase the expression of the iNOS gene requiring a heretofore undescribed dual function for STAT1, which has major implications for design of novel therapeutics to treat diabetes mellitus.
