© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 4076-4090 doi:10.1242/jcs.190009 RESEARCH ARTICLE Regulation of Hspb7 by MEF2 and AP-1: implications for Hspb7 in muscle atrophy Stephanie Wales Tobin1,2,3, Dabo Yang4, John Girgis4, Ali Farahzad1,2,3, Alexandre Blais4 and John C. McDermott1,2,3,5,* ABSTRACT 2000; Prado et al., 2009). In the ubiquitin proteasome pathway, the Mycocyte enhancer factor 2 (MEF2) and activator protein 1 (AP-1) forkhead box protein (FoxO) family of transcription factors transcription complexes have been individually implicated in activates muscle atrophy through induction of two E3 ubiquitin myogenesis, but their genetic interaction has not previously been ligases, MAFbx/atrogin-1 and MuRF1 (also known as TRIM63) addressed. Using MEF2A, c-Jun and Fra-1 chromatin (Sandri et al., 2004). Current treatment programs for muscle immunoprecipitation sequencing (ChIP-seq) data and predicted AP- atrophy include activating the serine/threonine protein kinase (Akt) 1 consensus motifs, we identified putative common MEF2 and AP-1 pathway, which induces muscle hypertrophy by inactivating FoxO target genes, several of which are implicated in regulating the actin proteins (Stitt et al., 2004). However, Akt can be inhibited by β β cytoskeleton. Because muscle atrophy results in remodelling or myostatin, a member of the transforming growth factor (TGF- ) degradation of the actin cytoskeleton, we characterized the superfamily (Trendelenburg et al., 2009), superseding Akt expression of putative MEF2 and AP-1 target genes (Dstn, Flnc, activation as a treatment option. A new antibody recently Hspb7, Lmod3 and Plekhh2) under atrophic conditions using characterized to bind to both members (A and B) of the dexamethasone (Dex) treatment in skeletal myoblasts. Heat shock myostatin/activin type II receptor (ActRII) induces hypertrophy in vivo protein b7 (Hspb7) was induced by Dex treatment and further in a muscle wasting model (Lach-Trifilieff et al., 2014). analyses revealed that loss of MEF2A using siRNA prevented Dex- Additionally, targeting ActRIIB in cancer cachexia models can regulated induction of Hspb7. Conversely, ectopic Fra-2 or c-Jun prevent atrophy, which results in prolonged survival without expression reduced Dex-mediated upregulation of Hspb7 whereas tumour manipulation (Zhou et al., 2010). AP-1 depletion enhanced Hspb7 expression. In vivo, expression of The autophagy pathway is an alternative mechanism of protein Hspb7 and other autophagy-related genes was upregulated in degradation that has also been implicated in muscle wasting. Foxo3, response to atrophic conditions in mice. Manipulation of Hspb7 unlike other members of the FoxO family, is able to regulate – levels in mice also impacted gross muscle mass. Collectively, these autophagy in addition to the ubiquitin proteasome pathway data indicate that MEF2 and AP-1 confer antagonistic regulation of (Mammucari et al., 2007; Zhao et al., 2007). Several possible Hspb7 gene expression in skeletal muscle, with implications for autophagy pathways have been identified in muscle, two of which autophagy and muscle atrophy. are macroautophagy and chaperone-mediated autophagy (CMA). Although both processes ultimately lead to protein degradation in KEY WORDS: Myogenesis, MEF2, AP-1, Autophagy the lysosome, they achieve this through different mechanisms. In CMA, heat shock cognate 70 (Hsc70) targets proteins directly to the INTRODUCTION lysosome (Chiang et al., 1989). Macroautophagy requires de novo Muscle atrophy is a phenomenon associated with reduced muscle synthesis of autophagosomes in a multistep process that involves fibre number and size caused by increased proteolysis and autophagy-related protein (Atg) family members. Autophagy is decreased protein synthesis (Romanick et al., 2013). In the required for muscle homeostasis, as demonstrated by the muscle elderly, muscle wasting is referred to as sarcopenia (Morley atrophy in mouse knockout models that lack proteins involved in et al., 2001); in patients with cancer, AIDS or other chronic autophagosome formation, such as Atg5 and Atg7 (Masiero et al., diseases, muscle atrophy is referred to as cachexia (Kotler, 2000). 2009; Raben et al., 2008). Aged muscle shows decreased autophagy Improving or maintaining muscle mass in these populations has a and therefore build-up of protein aggregates (Demontis and profound impact on the ‘health span’ of individuals. For example, Perrimon, 2010). LC3B (Map1lc3b) is an Atg protein that there is evidence that cachexia in cancer patients directly affects the provides a useful readout for autophagy because it is post- time to tumour progression and disease recurrence (Kadar et al., translationally modified as it becomes part of the autophagosome (Kabeya et al., 2000). First, pro-LC3B is cleaved by Atg4 to form 1Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, cytosolic LC3B-I. Atg7 then lipidates LC3B-I to form LC3B-II, Canada M3J 1P3. 2Muscle Health Research Centre (MHRC), York University, 4700 which can form part of the autophagosome. Using samples from Keele Street, Toronto, Ontario, Canada M3J 1P3. 3Centre for Research in Biomolecular Interactions (CRBI), 4700 Keele Street, Toronto, Ontario, Canada M3J various atrophic mouse models, LC3B was shown to be strongly 1P3. 4Ottawa Institute of Systems Biology, University of Ottawa, Health Sciences upregulated (Lecker et al., 2004). Furthermore, Foxo3 can directly Campus, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5. 5Centre for regulate several autophagy-related genes, including that encoding Research in Mass Spectrometry (CRMS), York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3. LC3B (Mammucari et al., 2007; Zhao et al., 2007). A form of autophagy termed chaperone-assisted selective autophagy (CASA) *Author for correspondence ( [email protected]) merges the chaperone-mediated and macroautophagy pathways. In J.C.M., 0000-0001-9696-8929 CASA, Hsc70 forms a complex with Bag3, Hspb8 and E3 ubiquitin ligase CHIP to identify protein aggregates and target them to the Received 29 March 2016; Accepted 8 September 2016 autophagosome (Arndt et al., 2010). Journal of Cell Science 4076 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4076-4090 doi:10.1242/jcs.190009 Myocyte-enhancer factor 2 (MEF2) is a member of the MADS- contained 6507. To determine the percentage of shared binding sites box family of transcription factors found in many tissues, including across these datasets we used a MEF2A-centric analysis (Fig. 1A). skeletal and cardiac muscle (Edmondson et al., 1994; Lin et al., From this analysis we observed that the majority of MEF2A binding 1997). MEF2 functions in a homo- or heterodimer complex with sites (69%) were independent of c-Jun or Fra-1 recruitment, yet 17% four different MEF2 isoforms in vertebrates (MEF2A–MEF2D), of MEF2A-bound DNA also contained c-Jun recruitment and 12% which bind to the consensus sequence [C/TTA(A/T)4TAG/A]. contained both c-Jun and Fra-1. Fra-1 and MEF2A alone shared few Previously we have shown that MEF2A can target a shared subset of binding sites (2%). genes in C2C12 myoblasts, an in vitro model of skeletal myogenesis, Functional roles for shared MEF2A and AP-1 binding sites were and in primary cardiomyocytes (Wales et al., 2014). Gene ontology identified using the Genomics Regions Enrichment of Analysis (GO) analysis contained terms enriched for actin cytoskeleton Tool (GREAT), which revealed enriched GO terms for biological organization and actin filament-based processes. In addition, many processes, the top ten of which are depicted in Fig. 1B. The GO of these cytoskeletal MEF2 target genes were enriched for activator terms were ranked by binomial raw P-value; the number of genes protein 1 (AP-1) cis elements, suggesting the possibility of within each GO term is indicated. DNA enriched for MEF2A-alone combinatorial control. AP-1, consisting of Jun homodimer or Jun– was associated with the better-known functions of MEF2 such as Fos family heterodimers, recognizes the consensus sequence TGAG/ actin-filament-based processes and skeletal muscle tissue CTCA (Angel et al., 1987). Global analysis of myoblast development, but also with cell development (blue). There were determination protein (MyoD) target genes in skeletal myoblasts no GO terms identified for MEF2A and Fra-1. However, MEF2A likewise showed that AP-1 motifs are prominent in neighbouring and c-Jun had GO terms for striated muscle development, vascular sequences (Blais et al., 2005; Cao et al., 2010). Neighbouring MEF2 development and heart morphogenesis (purple). The complete list and AP-1 cis elements have recently been shown to be enriched in of GO terms is given in Supplementary Dataset S1. Fewer GO terms macrophages and neurons (Ma and Telese, 2015; Nagy et al., 2013). were enriched for genes associated with all three factors and these Several AP-1 subunits have been implicated in myogenesis. In were related to the actin cytoskeleton and negative regulation of particular, c-Jun antagonizes MyoD transcriptional activity in vitro smooth muscle cell proliferation (black). (Bengal et al., 1992; Li et al., 1992). Using high throughput data, Because the MEF2A dataset was obtained from differentiating Blum et al. (2012) reported that c-Jun and MyoD coordinate muscle myoblasts, and given that several other AP-1 components apart from enhancers, indicating a more complex role for AP-1 in muscle than c-Jun and Fra-1 could be associated with MEF2 target genes, we previously anticipated (Blum et al., 2012). Additionally, the Fos determined the location of AP-1 consensus sequences containing the family member Fra-2 is thought to play a role in maintenance of the sequence TGAGTCA using cisGenome and allowing zero skeletal muscle satellite cell population (Alli et al., 2013). mismatches. From the mm9 genome, this search identified 264,537 Although MEF2 and AP-1 have individually been shown to AP-1 consensus sites. We focused on putative MEF2A and AP-1 function in the myogenic program, their potential interaction has not binding sites within ±10 kb of the transcription start site (TSS) of the been documented.