The FASEB Journal • Research Communication The stretch responsive microRNA miR-148a-3p is a novel repressor of IKBKB, NF-kB signaling, and inflammatory gene expression in human aortic valve cells Vishal Patel,* ,1 Katrina Carrion,* ,1 Andrew Hollands, † Andrew Hinton, ‡ Thomas Gallegos, § Jeffrey Dyo,* Roman Sasik, { Emma Leire, † Gary Hardiman, k,# Salah A. Mohamed,** Sanjay Nigam, § Charles C. King, ‡ Victor Nizet, † and Vishal Nigam* ,††,2 *Department of Pediatrics (Cardiology), † Department of Pediatrics and School of Pharmacy, ‡ Pediatrics Diabetes Research Center, § Departments of Pediatrics and Cellular and Molecular Medicine, and { Department of Medicine, University of California, San Diego, La Jolla, California, USA; k Computational Science Research Center and Biomedical Informatics Research Center, San Diego State University, San Diego, California, USA; # Department of Medicine, Medical University of South Carolina, Charleston, South Carolina, USA; **Department of Cardiac Surgery, University Clinic of Schleswig-Holstein, Campus Luebeck, Luebeck, Germany; and †† Rady Children’s Hospital, San Diego, California, USA ABSTRACT Bicuspid aortic valves calcify at a signifi- cantly higher rate than normal aortic valves, a process that involves increased inflammation. Because we have pre- viously found that bicuspid aortic valve experience greater stretch, we investigated the potential connection between stretch and inflammation in human aortic valve interstitial cells (AVICs). Microarray, quantitative PCR (qPCR), and protein assays performed on AVICs exposed to cyclic stretch showed that stretch was sufficient to increase expression of interleukin and metalloproteinase family members by more than 1.5-fold. Conditioned media from stretched AVICs was sufficient to activate leukocytes. microRNA sequencing and qPCR experiments demonstrated that miR-148a-3p was re- pressed in both stretched AVICs (43% repression) and, as a clinical correlate, human bicuspid aortic valves (63% re- duction). miR-148a-3p was found to be a novel repressor of IKBKB based on data from qPCR, luciferase, and Western blot experiments. Furthermore, increasing miR-148a-3p levels in AVICs was sufficient to decrease NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) sig- naling and NF-kB target gene expression. Our data dem- onstrate that stretch-mediated activation of inflammatory pathways is at least partly the result of stretch-repression of miR-148a-3p and a consequent failure to repress IKBKB. To our knowledge, we are the first to report that cyclic stretch of human AVICs activates inflammatory genes in a tissue-autonomous manner via a microRNA that regu- lates a central inflammatory pathway.—Patel, V., Carrion, K., Hollands, A., Hinton, A., Gallegos, T., Dyo, J., Sasik, R., Leire, E., Hardiman, G., Mohamed, S. A., Nigam, S., King, C. C., Nizet, V., Nigam V. The stretch responsive microRNA miR-148a-3p is a novel repressor of IKBKB, NF-kB signaling, and inflammatory gene expression in human aortic valve cells. FASEB J. 29, 000–000 (2015). www.fasebj.org Key Words: inflammation • NF-kB • aortic valve calcifica- tion • mechanotransduction STRETCH IS A MAJOR BIOMECHANICAL stimulus; a wide variety of cells in the body—such as cardiac, vascular, skeletal, skin, muscle, and gastrointestinal cells—are exposed to stretch. In particular, the response of cells in the heart and blood vessels to stretch is important because they are exposed to stretch every time the heart beats. Although stretch has been shown to activate NF-kB (nuclear factor kappa-light- chain-enhancer of activated B cells) signaling (1, 2), the mechanism is not understood. MicroRNAs (miRNAs) are small noncoding RNAs (20 to 24 base pairs long) that play important regulatory roles by binding to target mRNAs to promote degradation or block translation of their target mRNAs. One mechanism by which miRNAs can repress NF-kB signaling is by targeting IKBKB, which encodes IkB kinase b (IKKb) (3–5). IKKb phosphorylates IkB, resulting in dissociation of IkB from NF-kB that allows NF-kB trans- location to the nucleus. NF-kB signaling plays a crucial role in the inflammatory cascade. The purpose of this study was to determine if exposing human aortic valve interstitial cells (AVICs) to cyclic stretch activates inflammatory pathways and elucidate the mech- anisms involved. Aortic valve calcification/stenosis (AVC) is the third leading cause of adult heart disease (6) and the most common form of acquired valvular disease in Abbreviations: AVC, aortic valve calcification/stenosis; AVIC, aortic valve interstitial cell; BAV, bicuspid aortic valve; FDR, false discovery rate; IKKb,IkB kinase b; miRNA, microRNA; miRNA- Seq, miRNA sequencing; MMP, metalloproteinase; NF-kB, nu- clear factor kappa-light-chain-enhancer of activated B cells; PMA, phorbol-12-myristate-13-acetate; qPCR, quantitative PCR 1 These authors contributed equally to this study. 2 Correspondence: Department of Pediatrics (Cardiology), University of California San Diego, 9500 Gilman Drive Box 0731, La Jolla, CA 92093, USA. E-mail: vnigam@ucsd.edu doi: 10.1096/fj.14-257808 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 0892-6638/15/0029-0001 © FASEB 1 The FASEB Journal article fj.14-257808. Published online January 28, 2015.