title: Rapid proteasomal elimination of 3-hydroxy-3-methylglutaryl-CoA reductase by interferon-γ in primary macrophages requires endogenous 25-hydroxycholesterol synthesis.
abstract: Interferons (IFNs) play a central role in immunity and emerging evidence suggests that IFN-signalling coordinately regulates sterol biosynthesis in macrophages, via Sterol Regulatory Element-Binding Protein (SREBP) dependent and independent pathways. However, the precise mechanisms and kinetic steps by which IFN controls sterol biosynthesis are as yet not fully understood. Here, we elucidate the molecular circuitry governing how IFN controls the first regulated step in the mevalonate-sterol pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), through the synthesis of 25-Hydroxycholesterol (25-HC) from cholesterol by the IFN-inducible Cholesterol-25-Hydroxylase (CH25H). We show for the first 30-min of IFN stimulation of macrophages the rate of de novo synthesis of the Ch25h transcript is markedly increased but by 120-min becomes transcriptionally curtailed, coincident with induction of the Activating Transcription Factor 3 (ATF3) repressor. We demonstrate ATF3 induction by Toll-like receptors is strictly dependent on IFN-signalling. While the SREBP-pathway dependent rates of de novo transcription of Hmgcr are relatively unchanged in the first 90-min of IFN treatment, we find HMGCR enzyme levels undergo a rapid proteasomal-mediated degradation, defining a previously unappreciated SREBP-independent mechanism for IFN-action. These events precede a sustained marked reduction in Hmgcr RNA levels involving SREBP-dependent mechanisms. We demonstrate that HMGCR proteasomal-degradation by IFN strictly requires the synthesis of endogenous 25-HC and functionally couples HMGCR to CH25H to coordinately suppress sterol biosynthesis. In conclusion, we quantitatively delineate proteomic and transcriptional levels of IFN-mediated control of HMGCR, the primary enzymatic step of the mevalonate-sterol biosynthesis pathway, providing a foundational framework for mathematically modelling the therapeutic outcome of immune-metabolic pathways.
authors.affiliation: Division of Infection and Pathway Medicine, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom.
authors.forename: Hongjin
authors.initials: H
authors.lastname: Lu
authors.affiliation: Division of Infection and Pathway Medicine, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom.
authors.forename: Simon
authors.initials: S
authors.lastname: Talbot
authors.affiliation: Division of Infection and Pathway Medicine, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom; SynthSys at Edinburgh University, The Kings Buildings, Edinburgh, United Kingdom.
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Robertson
authors.affiliation: Northern Ireland Centre for Stratified Medicine, University of Ulster, Altnagelvin Hospital Campus, Derry, Co Londonderry, Northern Ireland BT47 6SB, United Kingdom.
authors.forename: Steven
authors.initials: S
authors.lastname: Watterson
authors.affiliation: Division of Infection and Pathway Medicine, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom; SynthSys at Edinburgh University, The Kings Buildings, Edinburgh, United Kingdom.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Forster
authors.affiliation: Division of Infection and Pathway Medicine, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom.
authors.forename: Douglas
authors.initials: D
authors.lastname: Roy
authors.affiliation: Division of Infection and Pathway Medicine, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom; SynthSys at Edinburgh University, The Kings Buildings, Edinburgh, United Kingdom. Electronic address: p.ghazal@ed.ac.uk.
authors.forename: Peter
authors.initials: P
authors.lastname: Ghazal
mesh_terms: D051700:Activating Transcription Factor 3 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002478:Cells, Cultured; D006888:Hydroxycholesterols / Q000378:metabolism*; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology*; D007700:Kinetics; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D059748:Proteolysis / Q000187:drug effects; D040901:Proteomics; D012313:RNA / Q000096:biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism; D051780:Sterol Regulatory Element Binding Protein 1 / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D051700:Activating Transcription Factor 3; C103002:Atf3 protein, mouse; D006888:Hydroxycholesterols; D012333:RNA, Messenger; D051780:Sterol Regulatory Element Binding Protein 1; D012313:RNA; C007997:25-hydroxycholesterol; D007371:Interferon-gamma; D006903:Hydroxymethylglutaryl CoA Reductases; D013250:Steroid Hydroxylases; C507384:cholesterol 25-hydroxylase; D046988:Proteasome Endopeptidase Complex
keywords: 25-Hydroxycholesterol; CH25H; Cholesterol biosynthesis; Immunity; Infection; Macrophages