Translational Control of Rapid Endothelial Responses

University Of Utah, Salt Lake City UT

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Abstract

DESCRIPTION (provided by applicant): Endothelial cells (EC) rapidly transition from a quiescent to an activated phenotype in response to certain extracellular stimuli. The activated phenotype is crucial for host defense but, when uncontrolled, is implicated in producing diseases such as sepsis and atherosclerosis. New protein synthesis is required for EC to assume an activated phenotype. Some of these proteins, including the transcription factor JunB, appear before new gene transcription could have occurred. This project's overall hypothesis is that EC synthesize new proteins very shortly after stimulation through regulated translation of pre-existing stores of mRNA. Translational control, defined as a change in efficiency of mRNA utilization by the ribosomal apparatus, typically occurs at the step of translation initiation. General translational control is exerted by changing the activity of ribosomal proteins necessary to translate any mRNA thus affecting the translational efficiency of most transcripts in the cell. In contrast, selective controls affect only a subset of mRNAs that possess unique regulatory sites in their 5'- or 3'-untranslated regions (UTRs). EC activation produces selective changes in gene expression and selective translational controls are most likely to be used. We have used Translation State Array Analysis (TSAA) to identify instances of regulated translation in EC activated with thrombin or histamine. Approximately 10% of EC genes including transcription factors (JunB), signaling (PP2A), cell cycle (CDC34) and thromboregulatory proteins (PAl-l) are regulated through translational controls. JunB expression in activated EC was inhibited by p38 MAPK blockade. The main goal of this grant is to characterize the role of translational control in EC biology and the mechanisms used to selectively regulate translation of new proteins that produce the activated phenotype. This goal will be accomplished by pursuing the following four specific aims: 1. Define the contribution of translational regulation to altered gene expression in activated EC. 2. Identify and characterize regulatory sites in the 5'- and 3-UTRs of EC gene products under ' translational control. 3. Determine the site of translation initiation in mRNAs encoding EC gene products under translational control. 4. Determine the role of p38 MAPK signaling in regulating translational events in activated EC.