Research Article 5329 Foxk1 promotes cell proliferation and represses myogenic differentiation by regulating Foxo4 and Mef2 Xiaozhong Shi1, Alicia M. Wallis1, Robert D. Gerard2, Kevin A. Voelker3, Robert W. Grange3, Ronald A. DePinho4, Mary G. Garry1 and Daniel J. Garry1,* 1Lillehei Heart Institute, University of Minnesota-Twin Cities, Minneapolis, MN 55455, USA 2Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA 3Department of Human Nutrition, Foods and Exercise, Virginia, USA Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA 4Belfer Institute for Applied Cancer Science, Departments of Medical Oncology, Medicine and Genetics, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA *Author for correspondence ([email protected]) Accepted 9 August 2012 Journal of Cell Science 125, 5329–5337 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.105239 Summary In response to severe injury, adult skeletal muscle exhibits a remarkable regenerative capacity due to a resident muscle stem/progenitor cell population. While a number of factors are expressed in the muscle progenitor cell (MPC) population, the molecular networks that govern this cell population remain an area of active investigation. In this study, utilizing knockdown techniques and overexpression of Foxk1 in the myogenic lineage, we observed dysregulation of Foxo and Mef2 downstream targets. Utilizing an array of technologies, we establish that Foxk1 represses the transcriptional activity of Foxo4 and Mef2 and physically interacts with Foxo4 and Mef2, thus promoting MPC proliferation and antagonizing the myogenic lineage differentiation program, respectively. Correspondingly, knockdown of Foxk1 in C2C12 myoblasts results in cell cycle arrest, and Foxk1 overexpression in C2C12CAR myoblasts retards muscle differentiation. Collectively, we have established that Foxk1 promotes MPC proliferation by repressing Foxo4 transcriptional activity and inhibits myogenic differentiation by repressing Mef2 activity. These studies enhance our understanding of the transcriptional networks that regulate the MPC population and muscle regeneration. Key words: Foxk1, Foxo4, Mef2, Cell proliferation, Cell differentiation Journal of Cell Science Introduction expression of the cyclin dependent kinase inhibitor, p21, and Adult skeletal muscle is a dynamic and highly regenerative tissue perturbed cell cycle kinetics of the muscle progenitor cell population due to a resident myogenic progenitor cell (MPC) population (Garry et al., 2000; Hawke et al., 2003a). Transgenic, molecular (Mauro, 1961). In response to a severe injury that involves more biological and biochemical studies have demonstrated that Sox15 is than 90% of the muscle, the MPC population is capable of a potent transcriptional activator of Foxk1 in the myogenic completely restoring the cellular architecture within a three-week progenitor cell population, although Foxk1’s downstream period. Recent studies using genetic mouse models and transcriptional program in this lineage has yet to be defined transcriptome analysis have identified molecular markers for (Meeson et al., 2007). Our recent studies have demonstrated that the MPC population that include Foxk1, CD29, C-met, integrin Foxk1 recruits Sin3/Sds3 repression complex and functions to alpha7, m-cadherin, Pax3, Pax7 and Syndecan3/4 (Biressi and activate the myogenic progenitor although the mechanisms are Rando, 2010; Shi and Garry, 2006). In addition, the C-met/Hgf, incompletely defined (Shi and Garry, 2012; Shi et al., 2012). Igf, Tgfb/Myostatin/Smad3/4, Notch/Numb signaling pathways Foxo proteins have been shown to have a broad functional role have also been shown to be essential for the MPC population in the regulation of catabolic pathways, cell cycle kinetics, cell (Buckingham and Vincent, 2009; Ten Broek et al., 2010). fate, aging and life span (Burgering, 2008; Ho et al., 2008; Despite these recent insights, the molecular networks that govern Partridge and Bru¨ning, 2008). Recent studies have demonstrated the MPC population remain an area of active investigation. that Foxo1 transgenic overexpression in skeletal muscle results in Forkhead/winged helix transcription factors are known to exert decreased body size, decreased muscle mass and increased important regulatory functions in developmental processes atrogin 1 (ubiquitin ligase) expression (Kamei et al., 2004). In including the determination of cell fate, cell cycle kinetics, cell addition, molecular biological and biochemical studies have differentiation and tissue morphogenesis (Hannenhalli and demonstrated that Foxo proteins directly interact with the Tgf- Kaestner, 2009; Myatt and Lam, 2007; Wijchers et al., 2006; beta downstream effectors, Smad3/4 and transcriptionally co- Yang et al., 2009). We have previously established that Foxk1 is activate the cyclin dependent kinase inhibitor, p21CIP and restricted to the MSC/MPC population in adult skeletal muscle maintain the hematopoietic stem cell population in a quiescent (Garry et al., 1997). Foxk1 deficient mice have severely impaired state (Seoane et al., 2004; Tothova et al., 2007). skeletal muscle regeneration, decreased number of muscle In the present study, we have utilized an array of techniques to progenitor cells, impaired progenitor cell activation, increased uncover the functional role of Foxk1 in the MPC population. We 5330 Journal of Cell Science 125 (22) have knocked down Foxk1 using siRNA techniques and Foxk1 represses transcription through a DNA-binding overexpressed Foxk1 using a transgenic technique in the independent mechanism myogenic lineage. Our analysis of Foxk1 knockdown and The interaction between the winged helix domain (WHD) of overexpression revealed dysregulation of Foxo and Mef2 Foxk1/Foxk2 and the consensus motif has been characterized downstream target genes, respectively. We demonstrate that using NMR spectroscopy and crystallography techniques Foxk1 directly interacts with Foxo4 and represses Foxo4 (Chuang et al., 2002; Liu et al., 2002; Tsai et al., 2006). Using transcriptional activity, and that the repression of Foxo4 results these techniques, a number of conserved amino acid residues in decreased p21 expression and increased cellular proliferation within the WHD have been shown to contact with the DNA (Liu of the MPC population. We further demonstrate that Foxk1 binds et al., 2002; Tsai et al., 2006). To further investigate the to and represses Mef2c thereby restraining myogenic transcriptional repression of Foxk1, we constructed two Foxk1 differentiation. Collectively, our current data concerning Foxk1 WHD (winged helix domain) mutants: K333A and R340A as provide direct evidence for a specific role for members of this these conserved amino acids were important in DNA-binding Forkhead gene family in the regulation of progenitor/stem cell (Liu et al., 2002; Tsai et al., 2006) (Fig. 2A). These mutations did function. not affect the protein stability in vitro (Fig. 2B). We observed that the DNA binding ability is attenuated in the K333A mutant Results and abolished in the R340A mutant (Fig. 2C). Transcriptional Foxk1 is required for the cell cycle progression assays revealed that both mutants did not affect the Foxk1 Our previous studies have defined the expression of Foxk1 in repression activity in two distinct promoter-reporter constructs myogenic progenitor cells. Further, we have reported that loss of (Fig. 2D,E). Collectively, these studies suggested that Foxk1 Foxk1 resulted in the perturbation of skeletal muscle regeneration represses transcription through a DNA-binding independent due to impaired cell cycle regulation of the myogenic progenitor mechanism. cell population. To further define the underlying mechanisms for Foxk1, we knocked down Foxk1 in C2C12 cells using siRNA Foxk1 represses and interacts with Foxo4 oligonucleotides. From the four candidates, we identified two Our above studies support the hypothesis that Foxk1 regulates siRNA olgionucleotides, which efficiently knocked down Foxk1 gene expression via Foxo proteins. To test our hypothesis, we in C2C12 cells (Fig. 1A). Using these reagents, we analyzed the used conventional transcriptional assays to evaluate the role of effect of Foxk1 knockdown on cell cycle kinetics. As shown in Foxk1 on Foxo transcriptional activity. We transfected a Fig. 1B, the knockdown of Foxk1 resulted in cell cycle arrest multimerized Foxo binding element (86FBE) fused to the using FACS analysis, which was further quantified in Fig. 1C. luciferase reporter to evaluate Foxo transcriptional activity in The gene expression studies revealed the upregulation of Foxo the presence and absence of Foxo factors and Foxk1 in C2C12 target genes (Fig. 1D). In addition, we observed decreased myoblasts. As expected, we observed that Foxo4 was a potent cellular proliferation with Foxk1 siRNA treatment (Fig. 1E). transcriptional activator of gene expression (Fig. 3A) (Shi et al., Taken together, these data support the notion that Foxk1 has an 2010). In a dose-dependent manner, Foxk1 repressed Foxo4 important functional role in the proliferation of the myogenic transcriptional activity (Fig. 3A; supplementary material Fig. Journal of Cell Science progenitor cell population. S1A). Knockdown of Foxk1 enhanced the transcriptional activity Fig. 1. Foxk1 promotes cellular proliferation. (A) Selection of Foxk1 siRNA oligonucleotides using qPCR analysis. All four siRNA oligonucleotides knocked down