Krüppel-like factor 6 (KLF6) promotes cell proliferation in skeletal myoblasts in response to TGFβ/Smad3 signaling
© Dionyssiou et al.; licensee BioMed Central Ltd. 2013
Received: 21 November 2012
Accepted: 15 February 2013
Published: 2 April 2013
Krüppel-like factor 6 (KLF6) has been recently identified as a MEF2D target gene involved in neuronal cell survival. In addition, KLF6 and TGFβ have been shown to regulate each other’s expression in non-myogenic cell types. Since MEF2D and TGFβ also fulfill crucial roles in skeletal myogenesis, we wanted to identify whether KLF6 functions in a myogenic context.
KLF6 protein expression levels and promoter activity were analyzed using standard cellular and molecular techniques in cell culture.
We found that KLF6 and MEF2D are co-localized in the nuclei of mononucleated but not multinucleated myogenic cells and, that the MEF2 cis element is a key component of the KLF6 promoter region. In addition, TGFβ potently enhanced KLF6 protein levels and this effect was repressed by pharmacological inhibition of Smad3. Interestingly, pharmacological inhibition of MEK/ERK (1/2) signaling resulted in re-activation of the differentiation program in myoblasts treated with TGFβ, which is ordinarily repressed by TGFβ treatment. Conversely, MEK/ERK (1/2) inhibition had no effect on TGFβ-induced KLF6 expression whereas Smad3 inhibition negated this effect, together supporting the existence of two separable arms of TGFβ signaling in myogenic cells. Loss of function analysis using siRNA-mediated KLF6 depletion resulted in enhanced myogenic differentiation whereas TGFβ stimulation of myoblast proliferation was reduced in KLF6 depleted cells.
Collectively these data implicate KLF6 in myoblast proliferation and survival in response to TGFβ with consequences for our understanding of muscle development and a variety of muscle pathologies.
KeywordsMyoblasts Krüppel-like factor 6 Transforming growth factor β Cell proliferation
Bovine serum albumin
Dulbecco’s modified Eagle’s serum
Ethylene glycol tetraacetic acid
Extracellular signal regulated kinase
Krüppel-like factor 6
Mitogen activated protein kinase
Muscle creatine kinase
Myocyte enhancer factor 2
MAP Kinase, ERK Kinase Kinase
Muscle regulatory factor
Myosin heavy chain
Quantitative polymerase chain reaction
Relative light units
Small interfering RNA
Transforming growth factor beta
Tetramethyl rhodamine iso-thiocyanate
KLF6 is a member of the Krüppel-like Factors (KLF) gene family which are a group of transcription factors that contain three highly conserved Cys2-His2 type zinc fingers located in the C-terminus [1, 2]. Subsequently, these proteins regulate a vast range of target genes by preferentially binding to cognate GC-boxes or CACCC elements. KLF6 was originally identified due to its ability to regulate TATA-less gene promoters that can regulate glycoproteins in placental cells . Since then, KLF6 has been found to be expressed in most tissues including neuronal, hindgut, heart and limb buds  and is localized in the nucleus . Interestingly, homozygous null KLF6 mice result in failure in the development of the liver and yolk sac vasculature, resulting in early lethality at (E)12.5 . To date, the most well-established target gene of KLF6 is Transforming growth factor β (TGFβ) and its receptors , and subsequent studies have shown a positive feedback loop by which TGFβ activation enhances KLF6 transactivation properties through the formation of a Smad3-Sp1-KLF6 protein complex . TGFβ and KLF6 cooperatively regulate a wide range of cellular processes such as cell differentiation, proliferation and epithelial-to-mesenchymal transitions (EMT) [8–13]. Recently KLF6 was identified as a myocyte enhancer factor 2 (MEF2) target gene that is involved in neuronal cell survival . Since TGFβ and MEF2 are two key regulators of skeletal myogenesis and since KLF6 was identified in the myogenic transcriptome , we wanted to investigate the role of KLF6 in skeletal muscle cells.
Regulation of skeletal myogenesis is a complex process. Initially paracrine factors instigate the migration of designated myotome progenitor cells to the dermomyotome region of the somite. These proliferating cells grow and divide until cell contact triggers differential gene expression and activation of the MEF2 proteins and muscle regulatory factors (MRFs). This cascade of events causes morphological changes in the progenitor cells that allow them to align and fuse to form multinucleated myotubes that can eventually spontaneously contract as functional muscle fibers. TGFβ antagonizes this process by preventing cells from exiting the cell cycle hence maintaining myoblasts in a proliferative state. TGFβ ligands bind to a type II receptor which becomes activated and autophosphorylated . The activated type II receptor can then phosphorylate and activate a type I receptor, which in turn phosphorylates receptor-mediated Smads(2/3) enabling them to dimerize with Smad4 and translocate into the nucleus where they can bind to other transcription factors and DNA, to repress essential muscle genes and the expression of their downstream targets [17, 18]. In addition, TGFβ also regulates the mitogen-activated protein kinase (MAPK) pathway, which involves a cascade of protein kinases (MAPKKK, MAPKK, MAPK) that become activated in sequence by G-proteins in response to TGFβ binding its receptors [19–21]. Upon TGFβ activation, MEK1/2 (MAPKK) can phosphorylate and activate Extracellular signal-regulated kinase (ERK)1/2 MAPK at conserved TEY sites, causing it to translocate into the nucleus to regulate gene expression. These two TGFβ-regulated pathways converge to inhibit the function of MEF2 and hence muscle-specific genes , and ultimately result in cell proliferation [23, 24]. Not surprisingly, inhibition of either or both of these pathways, (either pharmacologically or through ectopically expressed Smad7, which can antagonize the canonical Smad-pathway), enhances myotube formation [25, 26]. Crosstalk between these pathways is further supported by Smad7 antagonizing the repressive effects of MEK1 on MyoD [26, 27].
In this report, our goal was to assess the role of KLF6 in myogenic cells based on its regulation by both MEF2D and TGFβ. We report that TGFβ upregulates KLF6 specifically through a Smad3-dependent pathway, which enhances proliferation in myoblasts. In addition, we observed that 1) TGFβ enhanced KLF6 promoter activation, and 2) that MEF2 is recruited to the KLF6 promoter region but is not required for KLF6 activation by TGFβ. Pharmacological inhibition of Smad3 repressed KLF6 expression by TGFβ and cell proliferation but, importantly did not re-activate the differentiation program which is potently repressed by TGFβ signaling. Conversely, TGFβ treatment coupled with pharmacological inhibition of MEK1/2, enhanced myotube formation but had no effect on KLF6 expression and function. Loss of function assays using siRNA targeting KLF6 revealed that KLF6 is required for cell proliferation. These experiments tease apart two independent functions of TGFβ signaling in myogenic cells. One is a repressive effect on differentiation which is mediated by ERK activation, the other being an enhancement of proliferation, which is dependent on Smad3 and KLF6.
Expression plasmids for pcDNA3-MEF2D, pCMV β-galactosidase [28, 29], and reporter gene constructs for 3TP-lux , MCK-Luc , and MEF2-Luc  have been previously described. KLF6 reporter constructs pRMO6 and pROM6 ΔMEF2 were generously provided by Dr. Nicolas P. Koritschoner (Faculty of Bioquimica y Ciencias Biologicas, Universidad Nacional del Litoral, Santa Fe, Argentina).
Anti-MEF2A rabbit polyclonal, anti-Myosin heavy chain mouse monoclonal and anti-Myogenin mouse monoclonal antibodies were produced with the assistance of the York University (Toronto, Ontario, Canada) Animal Care Facility. Anti-MEF2D (1:1000; BD Biosciences, Mississauga, Ontario, Canada), Smad3, phospho-Smad3 and phospho-ERK1/2 (1:1000; Cell Signaling, Toronto, Ontario, Canada), and KLF6, actin, and ERK1/2 (1:1000; Santa Cruz, Santa Cruz, CA95060, US) were used for immunoblotting experiments. Immunoglobulin G (IgG) was also purchased from Santa Cruz Biotechnologies.
Cell culture, transfections and drug treatments
C2C12 cells were maintained in DMEM supplemented with 10% fetal bovine serum (HyClone, Rockford, IL61101, US), 1% L-glutamine and 1% penicillin-streptomycin. Cells were maintained in a humidified, 37°C incubator with a 5% CO2 atmosphere. For transfections, cells were seeded on pre-gelatin-coated plates 1 day prior to transfection and were transfected according to the standard calcium phosphate method previously described by Perry et al., 2001. A mixture of 50 μl 2.5 M CaCl2 per 25 μg DNA with an equal volume of 2× HeBS (2.8 M NaCl, 15 mM Na2HPO4, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH = 7.15) was used, and the cells were incubated overnight followed by washing and addition of fresh media. Drug treatments were used at the following concentrations: 2 ng/ml TGFβ, 5 μM Sis3 and 10 μM U0126 as indicated.
siRNA gene silencing
siRNA targeting KLF6, MEF2D and non-specific scramble RNA were purchased from Sigma. Transient transfections were performed using TurboFect Transfection Reagent (R0531, Fermentas) according to the manufacturer’s instructions. Turbofect (Fermentas): a 1:2 mixture ratio of DNA to turbofect reagent (including 4 ng/ml siRNA) in 200 μl serum-free DMEM was prepared for 19-h incubation.
C2C12 cells were treated as previously described by Salma and McDermott, 2012 , and incubated overnight with at 4°C with primary MEF2D and KLF6 antibodies (1:100) diluted in 1.5% goat serum. Cells were washed three times with PBS for 10 minutes and incubated with the appropriate tetramethyl rhodamine iso-thiocyanate (TRITC)/fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:500) in 1.5% goat serum (PBS) for 2 h at room temperature (RT) following 4’,6-diaminidino-2-phenylindole (DAPI) staining for 15 minutes at RT. Cells were washed three times with PBS and cover slips were mounted with DAKO mounting media (Dako) on glass slides. The fluorescence images were captured using Fluoview 300 (Olympus).
Protein extractions, immunoblotting and reporter gene assays
Cells were harvested using an NP-40 lysis buffer (0.5% NP-40, 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10 mM sodium pyrophosphate, 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), 0.1 M NaF) containing 10 μg/ml leupetin and aprotinin, 5 μg/ml pepstatin A, 0.2 mM phenylmethylsulfonyl fluoride and 0.5 mM sodium orthovanadate. Protein concentrations were determined using the Bradford method (Bio-Rad) with BSA as a standard. We used 20 μg of total protein extracts for immunoblotting, diluted in sample buffer containing 5% β-mercaptoethanol, and boiled. Transcriptional assays were done using Luciferase reporter plasmids. The cells were harvested for these assays using 20 mM Tris, (pH 7.4) and 0.1% Triton-X 100, and the values obtained were normalized to β-galactosidase activity expressed from a constitutive SV40-driven expression vector and represented as relative light units (RLU), or in some cases, corrected Luciferase values for control, reporter alone transfections were arbitrarily set to 1.0, and fold activation values were calculated. Bars represent the mean (n = 3) and error bars represent the standard error of the mean (n = 3).
Protein extracts were prepared as described above. Immunoprecipitation was performed using the ExactaCruz kit (Santa Cruz Biotechnology), as per manufacturer’s instructions. Precipitated proteins were separated by SDS PAGE and immunoblotting of proteins was performed as described above.
Chromatin immunoprecipitation (ChIP)
ChIP experiments followed the guidelines set by EZ ChIP™ (Upsate) with minor modifications. Approximately 1× 107 C2C12 cells were fixed with 1% formaldehyde (Sigma) for 15 minutes at 37°C. Fixing was quenched by Glycine (Bioshop, Burlington, ON Canada) at a final concentration of 0.125 M. Cells were collected in PBS containing phenylmethylsulfonyl fluoride (PMSF) (Sigma) and protease inhibitor cocktail (Roche, Laval, Quebec, Canada). Cells were collected at 5000 rpm for 5 minutes at 4°C. Cells were lysed using Wash Buffer I (10 mM HEPES pH 6.5, 0.5 M ethylene glycol tetraacetic acid (EGTA), 10 mM EDTA, 0.25% Triton X-100, protease inhibitor cocktail, PMSF) for 5 minutes on ice. Nuclei were collected and resuspended in Wash Buffer II (10 mM HEPES pH 6.5, 0.5 mM EGTA, 1 mM EDTA, 200 mM NaCl, protease inhibitor cocktail, PMSF) for 10 minutes on ice. Nuclei were again collected and then treated with nuclear lysis buffer (50 mM Tris–HCl pH 8.1, 10 mM EDTA, 1% SDS). Chromatin was sheared using a Misonix sonicator to produce 500 bp fragments. Crosslinked sheared chromatin was collected following a 15-minute spin at maximum speed. Twenty percent of total chromatin was set aside as input. Sheared crosslinked chromatin was diluted 1:10 with immunoprecipitation (IP) dilution buffer (0.01% SDS, 1.1% Triton-X 100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8.1, 167 mM NaCl) and incubated with antibody overnight at 4°C with rocking. Protein G Dynabeads (Invitrogen) were blocked with 20 μg salmon sperm DNA in IP dilution buffer (15 μl of beads + 135 μl IP dilution buffer + 20 μg salmon sperm DNA per IP) overnight at 4°C with rocking. We incubated 152 μl of pre-blocked beads with the IP reaction at 4°C for 1 h. Dynabead-bound antibody-chromatin complexes were washed using IP Wash Buffer I (20 mM Tris pH 8.1, 2 mM EDTA, 150 mM NaCl, 1% Triton-X 100, 0.1% SDS) and II (20 mM Tris pH 8.1, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), each incubated for 10 minutes at 4°C, and followed with two washes in Tris-EDTA (TE) buffer at 4°C. Protein-DNA complexes were freed from Dynabeads through the addition of elution buffer (0.1 M NaHCO3, 1% SDS) for 30 minutes at RT. To separate protein from DNA, samples were treated with 12 μl of 5 M NaCl (BioShop) at 65°C for 4 h or overnight. Protein was further degraded by the addition of Proteinase K (Sigma), EDTA, Tris pH 6.5 for 1 h at 45°C. DNA samples were then purified using a PCR clean up kit (Qiagen, Mississauga, ON, Canada).
ChIP-qPCR analysis of the KLF6 promoter was done using BioRad Sybr Green as per the user manual with a final primer concentration of 0.5 μM. The antibody used in ChIP was 5 μg αMEF2 (sc-313X; Santa Cruz Biotechnology, Inc.). The equivalent amount of rabbit IgG (12–370, Millipore) was used as a control in each ChIP. Sequences of the primers flanking the ME2 site on the KLF6 promoter were: 5’-CTGCAACGTTGGGCTGTA-3’ and 5’-TTGGAAAGACGTCTCACAGG-3’. Each sample was run in triplicate and then analyzed using percent input or fold enrichment.
Results and discussion
MEF2D and KLF6 expression and co-localization in the nucleus in skeletal myoblasts
MEF2A/D expression is not required for KLF6 protein expression in skeletal myoblasts
TGFβ regulates KLF6 through a Smad3-specific pathway and inhibits skeletal myogenesis through an MEK/ERK-specific pathway
TGFβ induces cell proliferation in C2C12 myoblasts through KLF6
In this study we report a novel role for KLF6 in skeletal myoblasts. Based on our data we propose that KLF6 is a downstream effector of the TGFβ/Smad3 pathway that regulates cell proliferation in skeletal myoblasts. We identify Smad3 as a key regulator of KLF6 expression, through TGFβ. In addition we were able to functionally distinguish between the TGFβ/Smad and TGFβ/MAPK pathways in that TGFβ inhibits skeletal myogenesis through the MEK/ERK (1/2) MAPK pathway and concomitantly enhances cell proliferation through Smad3-mediated induction of KLF6 expression. Our findings are summarized in Figure 4d. Many myopathies and muscle loss disorders have been linked with increased TGFβ signaling  and hence, our findings identify KLF6 as a potential therapeutic target for such pathological conditions, as well as for cancers, such as embryonal rhabdomyosarcoma, where TGFβ promotes cell proliferation .
We would like to thank Canadian Institutes for Health Research (CIHR) for funding this work.
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