Regulation of myotube formation by the actin-binding factor drebrin
© Mancini et al; licensee BioMed Central Ltd. 2011
Received: 19 August 2011
Accepted: 8 December 2011
Published: 8 December 2011
Myogenic differentiation involves cell-cycle arrest, activation of the muscle-specific transcriptome, and elongation, alignment and fusion of myoblasts into multinucleated myotubes. This process is controlled by promyogenic transcription factors and regulated by signaling pathways in response to extracellular cues. The p38 mitogen-activated protein kinase (p38 MAPK) pathway promotes the activity of several such transcription factors, including MyoD and MEF2, thereby controlling the muscle-specific transcription program. However, few p38-regulated genes that play a role in the regulation of myogenesis have been identified.
RNA interference (RNAi), chemical inhibition and immunofluorescence approaches were used to assess the role of drebrin in differentiation of primary mouse myoblasts and C2C12 cells.
In a search for p38-regulated genes that promote myogenic differentiation, we identified Dbn1, which encodes the actin-binding protein drebrin. Drebrin is an F-actin side-binding protein that remodels actin to facilitate the change of filopodia into dendritic spines during synaptogenesis in developing neurons. Dbn1 mRNA and protein are induced during differentiation of primary mouse and C2C12 myoblasts, and induction is substantially reduced by the p38 MAPK inhibitor SB203580. Primary myoblasts and C2C12 cells depleted of drebrin by RNAi display reduced levels of myogenin and myosin heavy chain and form multinucleated myotubes very inefficiently. Treatment of myoblasts with BTP2, a small-molecule inhibitor of drebrin, produces a phenotype similar to that produced by knockdown of drebrin, and the inhibitory effects of BTP2 are rescued by expression of a mutant form of drebrin that is unable to bind BTP2. Drebrin in myoblasts is enriched in cellular projections and cell cortices and at regions of cell-cell contact, all sites where F-actin, too, was concentrated.
Our findings reveal that Dbn1 expression is a target of p38 MAPK signaling during myogenesis and that drebrin promotes myoblast differentiation.
Keywordsmyoblast cell differentiation drebrin myotube actin
Myoblast differentiation is a multistep process that involves withdrawal from the cell cycle, acquisition of a cell type-specific transcriptional program and morphological changes that include elongation, alignment and fusion of myoblasts to form myofibers [1–4]. Whereas transcriptional regulation is at the core of myogenesis, the formation and growth of myofibers is also controlled by a variety of signaling ligands and their receptors, including insulin-like growth factor 1, fibroblast growth factors (FGFs), Wnts, transforming growth factor β superfamily members and others [1–3, 5]. Furthermore, the activity of MyoD and other promyogenic transcription factors is tightly controlled at the posttranslational level by signal transduction pathways, including phosphatidylinositol 3-kinase/Akt, integrin/focal adhesion kinase (FAK) and calcium/calcineurin [4, 6–9]. Among the signaling pathways that promote myogenesis, the p38α/β MAPK (p38α/β mitogen-activated protein kinase) pathway plays a prominent role. There is a persistent rise in p38α/β (hereafter simply "p38") activity during myoblast differentiation, and inhibition of p38 expression or activity blocks induction of select muscle-specific genes and myogenic differentiation [8, 10–13]. p38 phosphorylates substrates that drive muscle-specific gene expression at several levels, including MyoD dimerization with E proteins, Mef2 transcriptional activity, chromatin remodeling at muscle-specific genes and stability of myogenic mRNAs [11, 13–18]. Despite progress in understanding these proximal targets of p38's promyogenic actions, few p38-regulated genes that play a role in the regulation of myogenesis have been identified.
In addition to the acquisition of a muscle-specific transcriptional program, the changes in myoblast morphology that occur during differentiation indicate that dramatic alterations in the F-actin cytoskeleton are required for the formation of myofibers. Consistent with this notion, lamellipodia and filopodia, cellular structures that require actin remodeling, form dynamically during myoblast differentiation in vitro [19–21]. Furthermore, disruption of the F-actin cytoskeleton by chemical or other means inhibits various aspects of myoblast differentiation, including myoblast fusion [21–24]. At least some aspects of the cytoskeletal rearrangement and morphological changes that occur during differentiation are likely to be mediated by transcriptional induction of regulators of these processes, as expression of MyoD in fibroblasts induces not only expression of muscle-specific genes but also elongation and fusion into multinucleated myotubes .
Drebrin is a ubiquitously expressed F-actin side-binding protein that is highly abundant in the brain [25, 26]. Drebrin contains an actin-depolymerizing factor II/cofilin-like domain, an actin-binding domain and two Homer-binding domains , and it remodels actin to facilitate the maturation of filopodia into dendritic spines during synaptogenesis in developing neurons (for reviews, see [27, 28]). It is localized in lamellipodia and filopodia, at sites of cell-cell contact and in adhesion plaques [27–32]. Furthermore, drebrin associates with several proteins that promote myoblast differentiation and/or fusion, including the microtubule plus-tip binding protein EB3 [33, 34]; the scaffold protein Homer [35, 36] and, via Homer, the small GTPase Cdc42 [35, 37, 38]; and the chemokine receptor CXCR4 [39, 40]. We report herein that drebrin expression is induced during differentiation of primary and C2C12 myoblasts in a p38 MAPK-dependent manner. Furthermore, depletion of drebrin by RNA interference (RNAi) or inhibition of its function with a small-molecule antagonist diminished expression of muscle-specific genes and myotube formation. Drebrin is therefore an actin-regulating factor induced during myogenic differentiation that promotes the differentiation process.
Primary myoblasts were isolated from wild-type mice at postnatal day 13 by the method of Rando and Blau  and maintained as previously described . Differentiation was induced by switching cultures from Ham's F-10 medium with 20% fetal bovine serum (FBS)/2.5 ng/ml basic FGF (R&D Systems, Minneapolis, MN, USA)/4% penicillin-streptomycin (growth medium (GM)) to DMEM with 5% horse serum (differentiation medium (DM)). C2C12 myoblasts were maintained in DMEM containing 15% FBS supplemented with 1% penicillin/streptomycin and L-glutamine (GM). Differentiation was induced by transferring to DMEM supplemented with 2% horse serum (DM). Where indicated, 5 μM SB203580 (Sigma-Aldrich, St Louis, MO, USA) or 5 μM BTP2 (Calbiochem, La Jolla, CA, USA) were added to cultures and replenished every 12 hours.
Expression of drebrin
Plasmids encoding murine GFP-tagged drebrin E were generated by subcloning cDNA encoding either wild-type murine Dbn1 E or the K270M, K271M mutant into the pEGFP-C1 vector (Clontech Laboratories, Inc, Mountain View, CA, USA). Plasmids were transfected into C2C12 cells using Lipofectamine 2000 reagent (Invitrogen/Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions.
For small hairpin RNA studies, four sequences against murine Dbn1 were initially chosen. The oligonucleotides were cloned into the pSUPER.puro vector (Oligoengine, Seattle, WA, USA) and transfected into C2C12 cells with Lipofectamine 2000 reagent. Puromycin-resistant cells were selected, pooled and examined by Western blot analysis for drebrin expression. The most effective sequences were chosen for further studies and corresponded to nucleotides 5' GCCACTTCGAGAACCAGAAAG 3' (siDbn1-1) and 5' AGGAAGAGCCATGTGCAAAGGT 3' (siDbn1-3) (NM_019813.3).
Twenty-four hours after selection, cultures were seeded onto growth factor-reduced Matrigel (R&D Systems)-coated dishes at a density of 150,000 cells/ml. Twenty-four hours later, cultures were switched to DM. At various time points later, cultures were fixed with 2% paraformaldehyde (PFA) and immunostained for myosin heavy chain (MHC) and drebrin expression. Sister cultures were lysed, and 30 μg of cleared cell extracts were examined by Western blot analysis as previously described . Drebrin expression was also knocked down by transfection of C2C12 cells or primary myoblasts with 200 nM drebrin-specific RNAi (4390771; Ambion/Life Technologies) or with 200 nM control nonsilencing RNAi Red (465318; Invitrogen/Life Technologies). RNAs were transfected with StemPro LipoMax reagent (Invitrogen/Life Technologies) according to the manufacturer's instructions. Thirty-six hours after transfection, cells were either harvested to assess drebrin expression by Western blot analysis or analyzed for differentiation potential tested by transfer to DM.
Western blot analyses were performed as described in Kang et al. . Briefly, cells were lysed in extraction buffer (50 mM Tris·HCl, pH 7.4, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM ethylenediaminetetraacetic acid, 1% Triton X-100) supplemented with protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Total proteins were resolved on SDS-polyacrylamide gels, transferred onto Immobilon polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) and probed with specific antibodies. Primary antibodies used were anti-drebrin (Abcam, Cambridge, MA, USA), anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-myogenin (F5D; Santa Cruz Biotechnology) and anti-MHC (MF20; Developmental Studies Hybridoma Bank, Department of Biology, University of Iowa, Iowa City, IA, USA). Membranes were reprobed with the appropriate horseradish peroxidase-conjugated secondary antibody (The Jackson Laboratory, Bar Harbor, ME, USA), and specific bands were visualized with an enhanced chemiluminescence detection system (Roche Applied Science, Indianapolis, IN, USA)
RNA was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). One microgram of total RNA was reverse-transcribed using the First-Strand cDNA Synthesis Kit (Invitrogen/Life Technologies). One-tenth of the cDNA was applied for real-time PCR using QuantiFast SYBR Green RT-PCR Kit (QIAGEN) and analyzed on a Bio-Rad Q5 cycler (Bio-Rad Laboratories, Hercules, CA, USA).
Dbn1, Myh3, Myog and Gapdh mRNA expression were quantified with the following primers. Dbn1 forward: 5' AGGCCAAGAAGGAGGAAGAG 3'; Dbn1 reverse: 5' TTCCTCCTGTGCTCCTCAAT 3'; Myh3 forward: 5' CAGAAATGGAGACACGGATCAGA 3'; Myh3 reverse: 5' AGAGGTGAAGTCACGGGTCTTTGCC 3'; Myog forward: 5'GGGCCCCTGGAAGAAAAG 3'; Myog reverse: 5'AGGAGGCGCTGTGGGAGT 3'; Gapdh forward: 5' TGCACCACCAACTGCTTA 3'; Gapdh reverse: 5' GATGCAGGGATGATGTTC 3'.
Immunostaining and microscopy
Cells were grown on either Matrigel- or collagen-coated dishes as indicated, fixed with 2% PFA for 10 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, blocked with PBS containing 5% goat serum for 1 hour and incubated overnight with the following primary antibodies: anti-MHC (clone MF-20; Developmental Studies Hybridoma Bank), anti-drebrin (rabbit; Abnova, Walnut, CA, USA) and Alexa Fluor 568 phalloidin (Invitrogen/Life Technologies). After being washed three times with PBS, cells were incubated with the appropriate secondary antibody conjugated with Alexa Fluor 488 or 568 (Invitrogen/Life Technologies) for 1 hour, counterstained with 4',6-diamidino-2-phenylindole (DAPI) and then visualized using a Nikon Eclipse TS100 inverted microscope (Nikon Instruments, Melville, NY, USA). Images were acquired using a ProgRes digital microscope camera system and software (Jenoptik AG, Jena, Germany).
Cell proliferation and cell death assays
Cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation as described by Kang et al. . Briefly, dividing cells were incubated in 20 mM BrdU for 2 hours. The medium was aspirated, and cells were immediately fixed for 30 minutes at -20°C with ice-cold 70% ethanol/30% glycine at pH 2.0. After being washed with PBS, cells were incubated with mouse anti-BrdU (Chemicon International/Millipore, Temecula, CA, USA) and anti-mouse Alexa Fluor 568 (Invitrogen/Life Technologies). Cells were counterstained with DAPI and visualized by the standard immunofluorescence protocol. Apoptosis was analyzed with the In Situ Cell Death Detection Kit (Roche Applied Science) according to the manufacturer's instructions.
Dbn1 is induced during myogenic differentiation
RNAi-mediated depletion of drebrin inhibits myoblast differentiation and myotube formation
The drebrin inhibitor BTP2 blocks myoblast differentiation and myotube formation
Myoblast differentiation involves expression of the muscle-specific transcriptional program and changes in cellular morphology, most strikingly among the latter, fusion into multinucleated myofibers. The p38 MAPK signaling pathway promotes myogenic differentiation, including myoblast fusion [6, 8]. The reported substrates of p38 that are involved in myogenesis are all involved with expression of muscle-specific genes [11–14, 16–18], yet few p38 target genes that regulate myogenesis have been identified. In this report, we identify Dbn1, encoding drebrin, as a gene that is induced during myoblast differentiation in an SB203580-sensitive manner and that promotes the differentiation process.
The inhibition of Dbn1 induction by SB203580 suggests that one or more p38-regulated transcription factors are involved, directly or indirectly, in the expression of Dbn1 during myoblast differentiation. MyoD and Mef2 family members are well-established as p38-regulated transcription factors that are central to muscle-specific gene expression and myogenesis, and they are therefore potential candidate regulators of Dbn1 expression . Investigators in a recent study identified MEK5/ERK5 as a signaling pathway in myoblasts that induces expression of the transcription factors Klf2 and Klf4. Klf2 and Klf4 in turn regulate a set of target genes involved in myoblast fusion, largely independently of MyoD and Mef2, and Dbn1 was among this set of ERK5 → Klf2/Klf4 target genes . Klf4 can be posttranslationally activated by p38 MAPK , providing another potential explanation for the sensitivity of Dbn1 induction to SB203580. The nonreceptor tyrosine kinase FAK is important for myoblast maturation and fusion, and Dbn1 induction failed to occur properly in primary myoblasts in which fusion was inhibited by forced expression of FAT (Focal Adhesion Targeting, a naturally occurring dominant-negative variant of FAK) (see Table S1 in ). Taken together, Dbn1 expression has been associated with myogenesis in several systems, but its regulation is likely to be complex, integrating input from multiple signaling pathways and, potentially, multiple transcription factors.
RNAi-mediated depletion of drebrin or inhibition of drebrin function with the chemical inhibitor BTP2 led to decreases in expression of myogenin and MHC and inhibition of myotube formation. Drebrin encodes an F-actin side-binding protein and is best known for its role in remodeling actin to promote maturation of filopodia into dendritic spines during synaptogenesis in developing neurons [27, 28]. Production and remodeling of F-actin-enriched structures is a key event in myoblast fusion , and it is logical to posit that drebrin may participate in such events. However, the fact that depletion or chemical inhibition of drebrin reduced the levels of myogenin, which is itself required for differentiation, raises the possibility that the diminished fusion into myotubes seen upon loss of drebrin function may occur as a consequence of a more generalized block in differentiation due to suboptimal myogenin induction. The decrease in MHC expression seen in drebrin-inhibited cells and the fact that, based on time courses of BTP2 treatment, drebrin function was not required after 60 hours in DM, supports the idea that regulation of myogenin expression is likely involved with at least some portion of drebrin's actions in myogenesis. Consistent with this notion, there is evidence to suggest that proper regulation of cytoskeletal structures may be important in muscle-specific gene expression. For example, muscle-specific gene expression is hindered when myoblasts are treated with the F-actin remodeling inhibitor latrunculin B or depleted of the microtubule-binding protein EB1 [21, 24]. Additionally, Gussoni and colleagues  reported that C6ORF32, a cytoplasmic protein induced during myoblast differentiation that promotes filopodia formation, is important for expression of myogenin and MHC. Furthermore, depletion in cultured mammalian myoblasts of factors specific for myoblast fusion in Drosophila (for example, Dock1, Brag2) not only blocks formation of multinucleated cells but also decreases the percentage of cells that express muscle-specific markers [21, 52]. It seems logical that at least some aspects of muscle-specific transcription and myoblast fusion would be coordinately regulated via cytoskeletal dynamics, and, if so, drebrin would be well-placed to participate in such coordinated regulation. Consistent with this likelihood, our findings indicate that drebrin in myoblasts is enriched in cellular projections, cell cortices and regions of cell-cell contact, all sites where we found F-actin was also concentrated.
Drebrin interacts with several other proteins that have been implicated in myoblast differentiation and fusion. For example, drebrin binds the microtubule plus-tip binding protein EB3 in axonal growth cones. This interaction is important for growth cone formation and axonal growth and provides a link between F-actin and dynamic microtubules [30, 33]. EB3 is important for myoblast fusion, and its expression is induced during myoblast differentiation with kinetics similar to Dbn1 induction . Drebrin also binds to the scaffold protein Homer . Homer2b promotes signaling by the calcineurin/Nuclear Factor of Activated T Cells (NFAT) pathway and enhances myoblast differentiation and myotube formation . NFAT family members regulate several aspects of myogenesis . Interestingly, the drebrin inhibitor BTP2 was originally identified in a screen for small-molecule antagonists of NFAT activity and was subsequently shown to bind directly to drebrin . In addition to their ability to regulate calcium signaling, Homer proteins associate with Cdc42 , a small GTPase involved in myoblast differentiation and fusion [37, 38]. Finally, drebrin binds to the cytoplasmic tail of the chemokine receptor CXCR4 . CXCR4 promotes myoblast and myocyte migration . Given the plethora of drebrin interactions of potential relevance, extensive structure-function studies are likely necessary to identify the most important ones for drebrin's actions in myogenesis.
In this study, we identified Dbn1 as a gene induced during myoblast differentiation in a p38 MAPK-dependent manner. Dbn1 encodes drebrin, an actin-binding protein that localizes to cellular extensions, cell cortices and sites of cell-cell contact. Inhibition of drebrin function, either by RNAi-mediated depletion or by the small-molecule inhibitor BTP2, reveals a role for drebrin in myogenic differentiation.
Dulbecco's modified Eagle's medium
green fluorescent protein
myosin heavy chain
reverse transcriptase polymerase chain reaction
small interfering RNA.
We are grateful to Min Lu and Mingi Hong for critiquing the manuscript. This work was supported by a grant from the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (AR46207; to RSK) and Grant-in-Aid for Scientific Research (A) from MEXT of Japan (19200029; to TS).
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