Cytokine-Mediated Regulation of Cholesterogenesis

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DESCRIPTION (provided by applicant): Squalene synthase (SQS) catalyzes the first reaction of the isoprenoid metabolic pathway committed to cholesterol biosynthesis and its activity regulates the flux of intermediates to sterols. SQS is regulated by sterols, lipopolysaccharide (LPS), and the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interlukin-1-beta (IL-1-beta). Sterol-mediated transcriptional regulation of SQS is well understood, but little is known about the mechanism of its regulation during the inflammatory response. We propose to test the hypothesis that LPS and cytokines regulate SQS mRNA transcription and stability and enzyme protein posttranslational modification, stability and subcellular localization. In Specific Aim 1 we will determine the effects of LPS and cytokines on hepatic SQS transcription and mRNA stability in Syrian hamsters fed with different cholesterogenic diets. We will also determine changes in SQS mRNA level and size during response to cytokines and the contribution of the specific cytokines to these processes. In Specific Aim 2 we propose to elucidate the molecular mechanisms responsible for SQS mRNA regulation by LPS and cytokines. This will be achieved, in cultured cells, by examining promoter sequence elements responsible for the transcriptional repression by cytokines, by identification of transcription factors involved in this suppression, and by the elucidation of cellular signaling pathways involved in the transcriptional regulation. In addition, sequences required for SQS mRNA destabilization by cytokines will be localized. In Specific Aim 3 we propose to elucidate the LPS-and cytokine-induced, post-translational mechanisms underlying the decrease in SQS enzymic activity. We will examine SQS protein destabilization, mechanisms for its degradation, post-translational modification by phosphorylation, and change in its subcellular localization in response to LPS and cytokines. Achievement of the three Specific Aims will contribute directly to our long-term goal to elucidate the importance of SQS regulation on isoprenoid metabolic flux. It will increase our understanding of sterol metabolism in acute phase response (APR) to infection and inflammation and the role of SQS regulation in this process. Finally, it will enhance our overall understanding of hepatic cholesterol production in normal and pathological situations.