title: p-SMAD2/3 and DICER promote pre-miR-21 processing during pressure overload-associated myocardial remodeling.
abstract: Transforming growth factor-β (TGF-β) induces miR-21 expression which contributes to fibrotic events in the left ventricle (LV) under pressure overload. SMAD effectors of TGF-β signaling interact with DROSHA to promote primary miR-21 processing into precursor miR-21 (pre-miR-21). We hypothesize that p-SMAD-2 and -3 also interact with DICER1 to regulate the processing of pre-miR-21 to mature miR-21 in cardiac fibroblasts under experimental and clinical pressure overload. The subjects of the study were mice undergoing transverse aortic constriction (TAC) and patients with aortic stenosis (AS). In vitro, NIH-3T3 fibroblasts transfected with pre-miR-21 responded to TGF-β1 stimulation by overexpressing miR-21. Overexpression and silencing of SMAD2/3 resulted in higher and lower production of mature miR-21, respectively. DICER1 co-precipitated along with SMAD2/3 and both proteins were up-regulated in the LV from TAC-mice. Pre-miR-21 was isolated bound to the DICER1 maturation complex. Immunofluorescence analysis revealed co-localization of p-SMAD2/3 and DICER1 in NIH-3T3 and mouse cardiac fibroblasts. DICER1-p-SMAD2/3 protein-protein interaction was confirmed by in situ proximity ligation assay. Myocardial up-regulation of DICER1 constituted a response to pressure overload in TAC-mice. DICER mRNA levels correlated directly with those of TGF-β1, SMAD2 and SMAD3. In the LV from AS patients, DICER mRNA was up-regulated and its transcript levels correlated directly with TGF-β1, SMAD2, and SMAD3. Our results support that p-SMAD2/3 interacts with DICER1 to promote pre-miR-21 processing to mature miR-21. This new TGFβ-dependent regulatory mechanism is involved in miR-21 overexpression in cultured fibroblasts, and in the pressure overloaded LV of mice and human patients.
authors.affiliation: Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Cantabria, Santander, Spain; Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: García
authors.affiliation: Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain; Servicio de Cirugía Cardiovascular, Hospital Universitario Marqués de Valdecilla, Santander, Spain.
authors.forename: J Francisco
authors.initials: JF
authors.lastname: Nistal
authors.affiliation: Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Cantabria, Santander, Spain; Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Merino
authors.affiliation: Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, CT, USA; Integrative Cell Signaling and Neurobiology of Metabolism Program, Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Nathan L
authors.initials: NL
authors.lastname: Price
authors.affiliation: Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, CT, USA; Integrative Cell Signaling and Neurobiology of Metabolism Program, Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: Carlos
authors.initials: C
authors.lastname: Fernández-Hernando
authors.affiliation: Programa de Enfermedades Cardiovasculares, Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, Pamplona, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Beaumont
authors.affiliation: Programa de Enfermedades Cardiovasculares, Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, Pamplona, Spain.
authors.forename: Arantxa
authors.initials: A
authors.lastname: González
authors.affiliation: Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Cantabria, Santander, Spain; Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain. Electronic address: hurlem@unican.es.
authors.forename: María A
authors.initials: MA
authors.lastname: Hurlé
authors.affiliation: Departamento de Fisiología y Farmacología, Facultad de Medicina, Universidad de Cantabria, Santander, Spain; Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain.
authors.forename: Ana V
authors.initials: AV
authors.lastname: Villar
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001024:Aortic Valve Stenosis / Q000378:metabolism*; D002478:Cells, Cultured; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding; D012323:RNA Processing, Post-Transcriptional; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism*; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D020257:Ventricular Remodeling
chemical_list: C531950:MIRN21 microRNA, mouse; D035683:MicroRNAs; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D016212:Transforming Growth Factor beta; C509781:Dicer1 protein, mouse; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases
keywords: DICER; Myocardial fibrosis; Pre-miR-21; Pressure overload; TGF-β; p-SMAD2/3