Negative Auto-Regulation of Myostatin Expression is Mediated by Smad3 and MicroRNA-27 Craig McFarlane 1" , Anuradha Vajjala 2. , Harikumar Arigela 2.¤a , Sudarsanareddy Lokireddy 2¤b , XiaoJia Ge 2 , Sabeera Bonala 2 , Ravikumar Manickam 2 , Ravi Kambadur 1,2 , Mridula Sharma 3 * " 1 Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore, Singapore, 2 School of Biological Sciences, Nanyang Technological University, Singapore, Singapore, 3 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore Abstract Growth factors, such as myostatin (Mstn), play an important role in regulating post-natal myogenesis. In fact, loss of Mstn has been shown to result in increased post-natal muscle growth through enhanced satellite cell functionality; while elevated levels of Mstn result in dramatic skeletal muscle wasting through a mechanism involving reduced protein synthesis and increased ubiquitin-mediated protein degradation. Here we show that miR-27a/b plays an important role in feed back auto- regulation of Mstn and thus regulation of post-natal myogenesis. Sequence analysis of Mstn 39 UTR showed a single highly conserved miR-27a/b binding site and increased expression of miR-27a/b was correlated with decreased expression of Mstn and vice versa both in vitro and in mice in vivo. Moreover, we also show that Mstn gene expression was regulated by miR- 27a/b. Treatment with miR-27a/b-specific AntagomiRs resulted in increased Mstn expression, reduced myoblast proliferation, impaired satellite cell activation and induction of skeletal muscle atrophy that was rescued upon either blockade of, or complete absence of, Mstn. Consistent with this, miR-27a over expression resulted in reduced Mstn expression, skeletal muscle hypertrophy and an increase in the number of activated satellite cells, all features consistent with impaired Mstn function. Loss of Smad3 was associated with increased levels of Mstn, concomitant with decreased miR- 27a/b expression, which is consistent with impaired satellite cell function and muscular atrophy previously reported in Smad3-null mice. Interestingly, treatment with Mstn resulted in increased miR-27a/b expression, which was shown to be dependent on the activity of Smad3. These data highlight a novel auto-regulatory mechanism in which Mstn, via Smad3 signaling, regulates miR-27a/b and in turn its own expression. In support, Mstn-mediated inhibition of Mstn 39 UTR reporter activity was reversed upon miR-27a/b-specific AntagomiR transfection. Therefore, miR-27a/b, through negatively regulating Mstn, plays a role in promoting satellite cell activation, myoblast proliferation and preventing muscle wasting. Citation: McFarlane C, Vajjala A, Arigela H, Lokireddy S, Ge X, et al. (2014) Negative Auto-Regulation of Myostatin Expression is Mediated by Smad3 and MicroRNA-27. PLoS ONE 9(1): e87687. doi:10.1371/journal.pone.0087687 Editor: Se-Jin Lee, Johns Hopkins University School of Medicine, United States of America Received May 14, 2013; Accepted January 2, 2014; Published January 31, 2014 Copyright: ß 2014 McFarlane et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work performed in this manuscript was supported by the following grants: Biomedical Research Council (BMRC; M4070097.080) http://www.a-star. edu.sg/AboutASTAR/BiomedicalResearchCouncil/tabid/64/Default.aspx, National Research Foundation (NRF; M4092014.0S4 CRP) http://www.nrf.gov.sg/nrf/ default.aspx and intramural research funding (C08031) from Agency for Science, Technology and Research (A*STAR), Singapore http://www.a-star.edu.sg/Default. aspx. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bchmridu@nus.edu.sg . These authors contributed equally to this work. " CM and MS are joint senior authors on this work. ¤a Current address: Department of Genetics, The Hebrew University of Jerusalem, Jerusalem, Israel ¤b Current address: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, United States of America Introduction Myostatin (Mstn) is a secreted growth factor that belongs to the TGF-b super-family. Mstn is produced predominantly in skeletal muscle with lower levels of expression observed in white adipose tissue [1], heart [2] and mammary gland [3]. Analysis has revealed that Mstn is a profound negative regulator of muscle growth; while inactivation or mutation of the Mstn gene leads to increased skeletal muscle mass [1,4], enhanced myoblast proliferation [5], differentiation [6] and improved skeletal muscle regeneration [7], increased levels of Mstn result in severe cachectic-like muscle wasting [8–11]. The expression of Mstn is initially detected at embryonic day 9.5 in developing somites and continues to be detected postnataly in adult skeletal muscle fibers [1]. Furthermore, the mRNA expression of Mstn is developmentally regulated. While relatively abundant expression of Mstn is observed during fetal development, following birth the expression of Mstn rapidly decreases and remains quite low during postnatal development [12]. Previous work has also demonstrated that in adult skeletal muscle the expression of Mstn is greater in fast-twitch when compared to slow-twitch muscles [13,14] and thus is speculated to play a role in regulating muscle fiber type. Although we know that the expression of Mstn is regulated during myogenesis the exact mechanisms through which the abundance of Mstn is regulated remain to be fully determined. However, work from our lab has revealed that Mstn is transcriptionally regulated by the transcrip- tion factor MyoD through E-Box elements contained within the enhancer region of the Mstn gene [15]. In addition, further work PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e87687