1. Micro-managing smooth muscle phenotype: a role for microRNA in the Vasculature. Brandi Davis, Aaron Hilyard, Peter Nguyen, Giorgio Lagna and Akiko Hata Tufts Medical Center, Department of Biochemistry Abstract Smooth muscle microRNA microRNA expression in SMC Vascular smooth muscle cells (VSMC), unlike the majority of differentiated cell types, retain phenotypic plasticity throughout development. In response to extracellular cues, VSMC switch between a contractile, non-proliferative state and a synthetic, migratory, and proliferative phenotype. This plasticity allows VSMC to perform the seemingly disparate and highly specialized functions of contractility and vessel repair. Deregulation of phenotype switching is thought to underlie multiple vascular disorders including hypertension, atherosclerosis, and restenosis. Our lab and others have begun to elucidate the molecular signaling components of VSMC phenotype switching. Platelet Derived Growth Factor (PDGF) strongly promotes the de-differentiation of smooth muscle cells, a process characterized by reduced expression of contractile genes and increased proliferation. Conversely, Transforming Growth Factor Beta (TGF-ß) and its family member Bone Morphogenetic Protein (BMP) promote a differentiated, contractile smooth muscle cell phenotype. The precise mechanism of signal transduction which gives rise to these physiological changes are not completely understood. We hypothesized that growth factor modulation of microRNA levels could play a critical role in the regulation of VSMC phenotype. MicroRNAs are a newly discovered class of short (21-24nt) non-coding RNAs which post transcriptionally regulate the expression of target genes. Although only recently identified, it is clear that microRNAs are intimately involved in normal development as well as pathological processes. To address this question, we isolated, cloned and sequenced the microRNA population of primary human Pulmonary Artery Smooth Muscle cells (PASMC) treated with vehicle, BMP or PDGF. BMP treatment dramatically enriched the level of miR-21 expressed in PASMC. Interestingly, we have shown that miR-21 is up-regulated post-transcriptionally by BMP and TGF-ß through a novel interaction of the smads with the microRNA processing complex. Furthermore, experimental up- and down-regulation of miR-21 showed miR-21 is required for BMP induced smooth muscle cell differentiation. Additionally, we identified miR-221 as rapidly transcriptionally induced by PDGF treatment. Experimental modulation of miR-221 indicates miR-221 serves an opposing role to miR-21 in that it promotes the de-differentiation of vascular smooth muscle cells. Together these results indicate microRNAs play a critical role in the regulation of smooth muscle phenotype. Given their small size, microRNA are an excellent target for therapeutic modulation. Our results suggest that either increased miR-21 or inhibition of miR-221 may be useful in the treatment of vascular disorders induced by smooth muscle cell de-differentiation. BMP/TGF- β PDGF Synthetic Contractile Isolate ~100ug total RNA from Control, BMP, & PDGF treated PASM 15% polyacrylamide denaturing gel to isolate 10-30nt fraction Ligate 3’ linker 5’p OH 3’ 5’rApp /3ddC/3’ isolate 30-45nt fraction 5’p /3ddC/3’ Ligate 5’ linker isolate 50-60nt fraction RT PCR of small RNAs OH3’ cDNA PCR amplify small RNA cDNA Isolate, concatamerize and clone into vector. Sequence Clones ~100/condition The relative number of clones approximates the abundance of miR present in each condition TopoTA miR-21 targets PDCD4 to promote SMC differentiation +BMP4 miR-21 over-expressed~ 2.5X Relative mRNA levels normalized to GAPDH SMA Relative mRNA levels normalized to GAPDH PDCD4 Relative mRNA levels normalized to GAPDH miR-21 is post-transcriptionally regulated by BMP signaling BMP/ TGF- β Pri-miR Pre-miR miR-221 targets c-kit & p27 to promote SMC de-differentiation PDGF promotes transcription of miR-221 Relative mRNA levels normalized to GAPDH Davis, et al. Nature 2008 Contractile proteins Proliferation Migration Contractile proteins Proliferation Migration Deregulated vSMC phenotype switching plays a critical role in the development of vascular disorders including Hypertension, Atherosclerosis, Restenosis & Asthma. A better understanding of this process will provide novel therapeutic avenues for the treatment of human disease. Relative mRNA levels normalized to U6 Relative mRNA levels normalized to U6 microRNAs are a newly discovered class of short, 19-24nt, non-coding RNAs which repress gene expression through imperfect binding to sites within the 3’UTR of target genes In order to identify miR important for SMC differentiation, the small RNA fraction of PASM was cloned and sequenced. miR function was assessed by knockdown followed by SMA IF. Over expression of miR-21 increases the expression of contractile smooth muscle gene SMA as well as calponin and SM22 (data not shown). We identified Programmed Cell Death Protein 4 (PDCD4) as a key target of miR-21 in VSMC. Knockdown of PDCD4 by siRNA mimics the effect of miR-21 over-expression, confirming its role as a repressor of contractile SMC gene expression. Over expression of miR-221 inhibits the expression of contractile smooth muscle genes as well as the miR-221 targets c-kit and p27. miR-221 promotes SMC proliferation through down-regulation of p27. Interestingly, we identified a novel role for c-kit in SMC: through the regulation of myocardin, c-kit promotes expression of contractile smooth muscle genes. Mature miR-221 is rapidly induced by PDGF simulation in PASM. Analysis of the miR-221 intermediates suggests miR-221 is transcriptionally regulated by down-stream components of the PDGF signaling cascade. Mature and pre-miR21 are rapidly induced following BMP treatment without an increase in the primary transcript. We identify smads as required for induction of miR-21 and through a series of interaction and processing studies show Smad association with Drosha promotes the pri- to pre- processing step for a subset of miR. This is represents a novel role for smads in the control of gene expression. BMP miR-21 PDCD4 Contractile Gene Expression Smad-dependent miR processing PDGF miR-221 c-kit Contractile Gene Expression transcription p27 myocardin proliferation Model of miR-mediated SMC phenotype switching smad Drosha p68 DGCR8
