Undergraduate Summer Research Internship 2011 Research Paper

A One Nucleotide Substitution within the iNOS Gene Promoter Enhances Cross-talk Between Two Distinct Signaling Pathways
By: Barrett L. Updegraff Faculty Mentor: Dr. Jason Collier

In mammalian organisms, the pancreatic islets of Langerhans are comprised of various cell types that secrete polypeptide hormones, which control the storage and utilization of simple metabolic fuels (2). In type 1 diabetes mellitus (T1DM), an autoimmune mediated destruction of the insulin-producing pancreatic -cells produces insulin deficiency, requiring individuals to use insulin injections to control blood sugar levels. Much of the -cell destruction occurs through an autoimmune-mediated inflammatory response involving a number of signaling pathways that collectively result in transcriptional changes within the pancreatic -cells, leading to vicious feed-forward amplification of insulitis (1,3). This inflammatory response occurs when the resident and invading immune cells secrete pro-inflammatory cytokines, such as interleukin-1 (IL1) and gamma-interferon (-IFN). IL-1 and -IFN increase the expression of the inducible nitric oxide synthase (iNOS) gene, leading to micromolar amounts of nitric oxide production from endogenous b-cells, which initially impairs b-cell function and eventually leads to -cell death (4). A key transcriptional regulatory controlling the expression of the iNOS gene is NF-B (4).

Figure 1: Suggested model schematic of -IFN and IL-1 signaling pathways through the iNOS gene promoter as they relate to T1DM. Results Data collected in our lab indicates reproducible cross-talk between the NF-B signaling pathway and the -IFN signaling pathway. In the 832/13 rat insulinoma cell line iNOS gene promoter is a gamma-interferon activated site (GAS) consisting of nine nucleotides in which activated STAT1 dimers bind and enhance promoter activity in response to -IFN stimulation, upregulating the iNOS gene. However, a one nucleotide substitution within the GAS of the iNOS gene promoter elicits non-canonical synergy

between the two distinct pathways of IL-1 and -IFN signal transduction. This onenucleotide substitution creates a consensus GAS site (cGAS) that we hypothesized to be more sensitive to -IFN stimulation, but this substitution has negligible effects on enhancing promoter activity through -IFN stimulation alone. In the presence of another cytokine, interleukin-1 (IL-1), the relative promoter activity response is amplified to synergistic levels, suggesting enhanced cross-talk created by this one-nucleotide substitution (Fig.2a).

Figure 2: A) Proximal and distal B mutations on the background of a cGAS mutation. B) Wild-type RIN 832/13 iNOS promoter activity in response to cytokines and viral overexpression of p65 siRNA, p50 siRNA, and control Scramble siRNA. C) cGAS RIN 832/13 iNOS promoter activity in response to cytokines and viral overexpression of p65 siRNA, p50 siRNA, and control Scramble siRNA. D) Schematic representation of our cGAS-containing promoter construct with either proximal or distal B element mutations.

The canonical IL-1 signaling pathway is that of the NF-B pathway, in which treatment with IL-1 sets off a signaling cascade resulting in B dimers translocating to the nucleus and upregulating genes that contain B sites within their promoters (3). There are no studies to our knowledge that indicate that IL-1 can act through GAS sites. Our current study suggests cross-talk between these two pathways through recruitment of additional transcription factors and accessory proteins that are yet to be elucidated. On the background of our cGAS mutation, we adenovirally expressed siRNA to see if our mutation could rescue responses to IL-1. siRNA of interest was that of p65, p50, and STAT1. Our cGAS-containing iNOS promoter partially rescued promoter response to IL1 stimulation when siRNA for p65 was introduced via adenoviral cotransfection. siRNA for p65 alone completely demolishes promoter response to IL-1 stimulation, but on the background of our cGAS mutation, this response is partially restored (Fig. 2b and 2c). On the background of our cGAS promoter, as well as the wild-type iNOS gene promoter we mutated either the distal B element (dB) or the proximal B element (pB) and treated the cells with cytokines (Fig. 2a and 2d). The cGAS/pB double mutation rescued the response to IL-1 by roughly 75-fold and the IL-1 + -IFN by roughly 150-fold above the pB mutation alone. This data suggests cross-talk between our consensus GAS and B promoter elements. Discussion Taken together, conclusions can be made suggesting shared transcription factors, accessory proteins, and unorthodox dimeric configurations between the NF-b signaling pathway and the signal transducer and activator of transcription 1 (STAT1) signaling pathway. It is important to note that all of the results obtained in this study are essentially due to a one nucleotide substitution in a ~1kb promoter. Future research including chromatin immunoprecipitation assays (ChIP) will be required to elucidate binding of specific proteins in the context of the cell. To date, no lab has seen a valid role for the GAS site in IL-1 signal transduction, so binding will be essential to confirm our results and verify the proteins, transcription factors, and other accessory molecules involved in transcription. References 1. Eizirik, D. L. et al. (2009) The role of inflammation in insulitis and -cell loss in type 1 diabetes. Nat. Rev. Endocrinol. 5, 219-226 2. Steiner, D. J. et al. (2010) Pancreatic islet plasticity: interspecies comparison of islet architecture and composition. Islets. 2:3, 135-145 3. Viatour, P. et al. (2005) Review: phosphorylation of NF-B and IB proteins: implications in cancer and inflammation. TRENDS Biochem. Sci. 30, 1

4. Kleinert, H. et al. (2003) Regulation of expression of inducible nitric oxide synthase. J. Biol. Chem. 384, 1343-1364