title: Repression of let-7 by transforming growth factor-β1-induced Lin28 upregulates collagen expression in glomerular mesangial cells under diabetic conditions.
abstract: Accumulation of mesangial extracellular matrix (ECM) proteins such as collagen type 1-α2 (Col1a2) and collagen type 4-α1 (Col4a1) is a key feature of diabetic nephropathy (DN). Transforming growth factor (TGF)-β1 plays important roles in ECM accumulation in DN, and evidence shows a mediatory role for microRNAs. In the present study, we found that microRNA let-7 family members (let-7b/c/d/g/i) were downregulated in TGF-β-treated mouse mesangial cells (MMCs) along with upregulation of Col1a2 and Col4a1. Ectopic expression of let-7b in TGF-β-treated MMCs attenuated Col1a2 and Col4a1 upregulation. Conversely, let-7b inhibitors increased Col1a2 and Col4a1 levels. Cotransfection of MMCs with mouse Col1a2 or Col4a1 3'-untranslated region luciferase constructs and let-7b inhibitors increased luciferase activity. However, constructs with let-7 target site mutations were unresponsive to TGF-β. TGF-β-induced 3'-untranslated region activity was attenuated by let-7b mimics, suggesting that Col1a2 and Col4a1 are direct targets of let-7b. In addition, Lin28b, a negative regulator of let-7 biogenesis, was upregulated in TGF-β-treated MMCs. Luciferase assays showed that the Lin28b promoter containing the Smad-binding element (SBE) responded to TGF-β, which was abolished in constructs without SBE. Chromatin immunoprecipitation assays showed TGF-β-induced enrichment of Smad2/3 at the Lin28b promoter, together suggesting that Lin28b is transcriptionally induced by TGF-β through SBE. Furthermore, let-7b levels were decreased, whereas Lin28b, Col1a2, and Col4a1 levels were increased, in glomeruli of diabetic mice compared with nondiabetic control mice, demonstrating the in vivo relevance of this Lin28/let-7/collagen axis. These results identify Lin28 as a new TGF-β target gene and suggest a novel role for the Lin28/let-7 pathway in controlling TGF-β-induced collagen accumulation in DN.
authors.affiliation: Department of Internal Medicine, College of Medicine, Yonsei University, Seoul, Republic of Korea; and Division of Molecular Diabetes Research, Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, California.
authors.forename: Jung Tak
authors.initials: JT
authors.lastname: Park
authors.affiliation: Division of Molecular Diabetes Research, Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, California.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Kato
authors.affiliation: Division of Molecular Diabetes Research, Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, California.
authors.forename: Linda
authors.initials: L
authors.lastname: Lanting
authors.affiliation: Division of Molecular Diabetes Research, Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, California.
authors.forename: Nancy
authors.initials: N
authors.lastname: Castro
authors.affiliation: Department of Internal Medicine, College of Medicine, Yonsei University, Seoul, Republic of Korea; and.
authors.forename: Bo Young
authors.initials: BY
authors.lastname: Nam
authors.affiliation: Division of Molecular Diabetes Research, Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, California.
authors.forename: Mei
authors.initials: M
authors.lastname: Wang
authors.affiliation: Department of Internal Medicine, College of Medicine, Yonsei University, Seoul, Republic of Korea; and.
authors.forename: Shin-Wook
authors.initials: SW
authors.lastname: Kang
authors.affiliation: Division of Molecular Diabetes Research, Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, California RNatarajan@coh.org.
authors.forename: Rama
authors.initials: R
authors.lastname: Natarajan
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism*; D024141:Collagen Type IV / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D003928:Diabetic Nephropathies / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005355:Fibrosis; D006801:Humans; D050527:Mesangial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D034622:RNA Interference; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D013311:Streptozocin; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D015854:Up-Regulation
chemical_list: D020413:3' Untranslated Regions; D024042:Collagen Type I; D024141:Collagen Type IV; C488352:Lin-28 protein, mouse; D035683:MicroRNAs; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1; C542687:mirnlet7 microRNA, mouse; D013311:Streptozocin
keywords: Lin28b; diabetic nephropathy; let-7; microRNA; transforming growth factor-β