POST-TRANSCRIPTIONAL REGULATION OF INFLAMMATORY MEDIATORS

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DESCRIPTION (provided by applicant): Inflammation is the collective array of events within the host that occur in response to traumatic, infectious, toxic or autoimmune injury. Dysregulated inflammation is associated with human diseases such as arthritis, Alzheimer's disease and septic shock. The scope and duration of inflammatory reactions are dictated by host products called inflammatory mediators, whose precise spatial and temporal expression is critical in mounting an appropriate inflmammatory response. While significant progress has been made in understanding the transcriptional regulation of these mediators by microbial products, much less is known about post-transcriptional regulatory mechanisms. We recently discovered a MyD88-dependent pathway that stabilized interferon-gamma-induced cytokine and chemokine mRNAs. Several component members of this pathway, including MLK3 and MKK3, were identified by using a luciferase reporter construct bearing an AU-rich (ARE) sequence from 3'-untranslated region (UTR) of TNF. Here, we propose to identify additional unidentified factors involved in the post-transcriptional regulation of inflammatory mediators using a similar reporter construct-based screen. The goal of this project is to define and explore mechanisms underlying the stabilization of ARE-containing mRNAs. Our working hypothesis is that overexpression of certain signaling molecules can up-regulate reporter protein expression inside cells by enhancing its mRNA stability. We will: (1) screen a leukocyte cDNA library for genes capable of activating a luciferase reporter carrying ARE from TNF at its 3'-UTR, (2) define the molecular basis by which identified candidate genes affect the mRNA stability of inflammatory mediators, and (3) evaluate the role of one candidate gene in vitro and in vivo by testing inflammatory responses in mice deficient for these genes. The long term goal of this study is to gain insight into the pathological basis of inflammatory diseases as explained by derangements of physiological gene regulatory mechanisms and aid in the development of novel therapeutic strategies.