SREBP-2 Responsive miR Operon Regulates Lipid Metabolism Tae-Il Jeon, Ph.D. Division of Animal Science Chonnam National University Cholesterol • • • • Myelin – Brain Surfactant – Lung Lipoprotein & Bile acids – Liver Steroid hormone – Adrenal • • • • Barrier – Skin Vitamin D – Bone Sex hormone – Gonads Storage – Fat All Cell Membranes Three Sources of Cholesterol Acetyl CoA Coated Pit HMGCS LDL Receptor HMG CoA HMGCR Lipid Droplet Mevalonate Cholesterol LDL Endosome Lysosome Jeon and Osborne TEM 2012 Sterol Regulatory Element Binding Proteins (SREBPs) 1a 1c 1a SREBP-1 bHLH-Lz Gene/mRNA bHLH-Lz 1a Protein 1c bHLH-Lz 1c Protein 2 bHLH-Lz 2 Protein Acetyl CoA CO2 Acetyl CoA ACC Acetoacetyl CoA SREBP1 Malonyl CoA FAS Several enzymes SREBP2 Cholesterols Palmitate Several enzymes Triglycerides SREBPs Processing SREBP SCAP ER C Nucleus N INSIG N SRE Sterol C N N Golgi S1P S2P + - Sterol SREBP-2 (M) Membrane SREBP-2 (N) Nuclear SREBPs Maturation SREBP InsR SCAP ER C N C N Insig ? ? AKT Golgi S2P ? S1P S6K TORC1 Lipin RAPAMYCIN SREBP1c mRNA or precursor Lipin N SRE Nucleus Jeon and Osborne TEM 2012 Normal chow diet + 1% Cholesterol (Ch) Normal chow diet + Lovastatin/Ezetimibe (LE) Lovastatin inhibits cholesterol synthesis Ezetimibe inhibits cholesterol uptake SREBP-2 and target genes Ch LE SREBP-2 Hmgcr Liver miRNA profiles LE vs Ch miRNA mmu-miR-182 mmu-miR-470* mmu-miR-34b-3p mmu-miR-741 mmu-miR-877 mmu-miR-20b mmu-miR-297a* mmu-miR-19a mmu-miR-875-5p mmu-miR-33* mmu-miR-188-5p mmu-miR-467a* mmu-miR-195 mmu-miR-877* mmu-miR-126-5p mmu-miR-130b mmu-miR-101a mmu-miR-339-3p mmu-miR-301b mmu-miR-331-3p mmu-miR-135a* mmu-miR-185 mmu-miR-676 mmu-miR-15a mmu-miR-339-5p mmu-miR-425 mmu-miR-221 mmu-miR-215 mmu-miR-210 mmu-miR-455 Fold change 81.6 28.3 17.7 12.5 7.2 6.2 5.7 4.3 3.6 3.2 3.2 3.0 2.9 2.9 2.8 2.7 2.3 2.1 2.1 2.1 2.1 2.1 .50 .28 .26 .23 .20 .19 .10 .04 miR-182-96-183 cluster Fold Regulation 35 25 N Ch LE Fold Regulation 45 15 5 4 3 2 1 0 miR-183 miR-96 miR-182 75 65 55 45 35 25 15 5 4 3 2 1 0 miR-183 miR-96 miR-182 WT Tg-SREBP-1a Tg-SREBP-1c Tg-SREBP-2 SREBP-2 regulatory region Hmgcr 3F 3R mIR182 1 2F 2 3 2R 1F 1R Promoter region of miR-182-96-183 cluster 3F 3R 2F 2R 1F 1R mIR182 Luciferase pSyn-miR X pSyn-miR-MT1 pSyn-miR-MT2 Luciferase X Luciferase Relative Luciferas activity pSyn-miR-MT1,2 X X Luciferase SREBP-2 miR-183/96/182 miRNA target prediction FBXW7 INSIG2 SREBP processing ER miR-96 Chol LE N C INSIG Insig-2 SCAP SREBP Fbxw7 β-actin N C Golgi S2P 1.0 miR-182 Rel. RNA S1P N N N 1.0 Chol. 0.5 0.5 0 0 Insig-2a SRE Nucleus Insig-2 Fbxw7 SREBPs LE Fbxw7 A FBXW7 3’UTR Con 182 Fold Regulation 1.0 0.5 0.0 B Wt 3’ Mut INSIG-2 3’UTR Fold Regulation 1.2 1.0 0.8 0.6 0.4 0.2 0.0 miR-183/96/182 Insig-2 Fbxw7 Con 96 Wt 3’ Mut RPL10 Luciferase 3’UTR (INSIG-2) (FBXW7) Coordinate and reciprocal regulation of nSREBP-2 with miR-182 or Fbxw7 A Chol. 1 2 N 3 L/E 4 5 L/E Cholesterol 1 2 3 6 7 8 9 10 11 12 nSREBP-2 Fbxw7 C A-miR-Con. A-miR-182 Ch LE LE YY-1 1 2 3 4 5 6 7 8 9 nSREBP-2 β-actin FBXW7 YY-1 B Relative value * * β-actin Fbxw7 miR-182 nSREBP-2 FBXW7 overexpression rescues nSREBP-2 accumulation by miR-182 Sterols miR 182 S- S+ NC 182 F7 F7 P N SREBP-2 (FLAG) FBXW7 (GST) β-actin miR-183/96/182 SREBP-2 Fbxw7 Effect of ectopic FBXW7 on nSREBPs is sensitive to miR-182-binding sites in the FBXW7 3’UTR miR regulation of SREBP levels induces lipid biosynthesis pmol/ug Fatty acids * 1 2 3 pmol/ug Cholesterol ** 1 2 3 p SREBP-1 n FASN β-actin 1 Con 2 mIRs + + 3 - Sterols An SREBP-2 regulated miR operon regulates lipid metabolism C N INSIG SREBP ER SCAP C N Golgi S2P S1P N N N SRE miR-182 Nucleus miR-183 ACKNOLEDGEMENTS Seung-Soon Ryan Pete Kathy Functional Genomics Core facility (SBMRI) Christine C. Esau (Regulus Therapeutics) Tim Osborne Manuel Dong-Ju
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