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Krüppel-like factor 6 (KLF6) promotes cell proliferation in skeletal myoblasts in response to TGFβ/Smad3 signaling

Krüppel-like factor 6 (KLF6) promotes cell proliferation in skeletal myoblasts in response to... Background: 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. Methods: KLF6 protein expression levels and promoter activity were analyzed using standard cellular and molecular techniques in cell culture. Results: 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. Conclusions: 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. Keywords: Myoblasts, Krüppel-like factor 6, Transforming growth factor β, Cell proliferation Background glycoproteins in placental cells [3]. Since then, KLF6 has KLF6 is a member of the Krüppel-like Factors (KLF) been found to be expressed in most tissues including gene family which are a group of transcription factors neuronal, hindgut, heart and limb buds [4] and is local- that contain three highly conserved Cys -His type zinc ized in the nucleus [5]. Interestingly, homozygous null 2 2 fingers located in the C-terminus [1,2]. Subsequently, KLF6 mice result in failure in the development of the these proteins regulate a vast range of target genes by liver and yolk sac vasculature, resulting in early lethality preferentially binding to cognate GC-boxes or CACCC at (E)12.5 [4]. To date, the most well-established target elements. KLF6 was originally identified due to its ability gene of KLF6 is Transforming growth factor β (TGFβ) to regulate TATA-less gene promoters that can regulate and its receptors [6], and subsequent studies have shown a positive feedback loop by which TGFβ activation * Correspondence: jmcderm@yorku.ca enhances KLF6 transactivation properties through the for- Department of Biology, York University; York University, 4700 Keele St, mation of a Smad3-Sp1-KLF6 protein complex [7]. TGFβ Toronto, ON M3J 1P3, Canada and KLF6 cooperatively regulate a wide range of cellular Centre for Research in Mass Spectrometry, York University; York University, Toronto, ON M3J 1P3, Canada processes such as cell differentiation, proliferation and Full list of author information is available at the end of the article © 2013 Dionyssiou et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 2 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 epithelial-to-mesenchymal transitions (EMT) [8-13]. Re- KLF6 expression by TGFβ and cell proliferation but, im- cently KLF6 was identified as a myocyte enhancer factor 2 portantly did not re-activate the differentiation program (MEF2) target gene that is involved in neuronal cell sur- which is potently repressed by TGFβ signaling. Con- vival [14]. Since TGFβ and MEF2 are two key regulators versely, TGFβ treatment coupled with pharmacological of skeletal myogenesis and since KLF6 was identified in inhibition of MEK1/2, enhanced myotube formation but the myogenic transcriptome [15], we wanted to investigate had no effect on KLF6 expression and function. Loss of the role of KLF6 in skeletal muscle cells. function assays using siRNA targeting KLF6 revealed Regulation of skeletal myogenesis is a complex process. that KLF6 is required for cell proliferation. These experi- Initially paracrine factors instigate the migration of desig- ments tease apart two independent functions of TGFβ nated myotome progenitor cells to the dermomyotome re- signaling in myogenic cells. One is a repressive effect on gion of the somite. These proliferating cells grow and differentiation which is mediated by ERK activation, the divide until cell contact triggers differential gene expression other being an enhancement of proliferation, which is and activation of the MEF2 proteins and muscle regulatory dependent on Smad3 and KLF6. factors (MRFs). This cascade of events causes morpho- logical changes in the progenitor cells that allow them to Methods align and fuse to form multinucleated myotubes that can Plasmids eventually spontaneously contract as functional muscle fi- Expression plasmids for pcDNA3-MEF2D, pCMV β- bers. TGFβ antagonizes this process by preventing cells galactosidase [28,29], and reporter gene constructs for from exiting the cell cycle hence maintaining myoblasts in 3TP-lux [30], MCK-Luc [31], and MEF2-Luc [32] have a proliferative state. TGFβ ligands bind to atypeIIreceptor been previously described. KLF6 reporter constructs which becomes activated and autophosphorylated [16]. The pRMO6 and pROM6 ΔMEF2 were generously provided activated type II receptor can then phosphorylate and acti- by Dr. Nicolas P. Koritschoner (Faculty of Bioquimica y vate a type I receptor, which in turn phosphorylates Ciencias Biologicas, Universidad Nacional del Litoral, receptor-mediated Smads(2/3) enabling them to dimerize Santa Fe, Argentina). with Smad4 and translocate into the nucleus where they can bind to other transcription factors and DNA, to repress Antibodies essential muscle genes and the expression of their down- Anti-MEF2A rabbit polyclonal, anti-Myosin heavy chain stream targets [17,18]. In addition, TGFβ also regulates the mouse monoclonal and anti-Myogenin mouse monoclonal mitogen-activated protein kinase (MAPK) pathway, which antibodies were produced with the assistance of the York involves a cascade of protein kinases (MAPKKK, MAPKK, University (Toronto, Ontario, Canada) Animal Care Facility. MAPK) that become activated in sequence by G-proteins Anti-MEF2D (1:1000; BD Biosciences, Mississauga, in response to TGFβ binding its receptors [19-21]. Upon Ontario, Canada), Smad3, phospho-Smad3 and phospho- TGFβ activation, MEK1/2 (MAPKK) can phosphorylate ERK1/2 (1:1000; Cell Signaling, Toronto, Ontario, and activate Extracellular signal-regulated kinase (ERK)1/2 Canada), and KLF6, actin, and ERK1/2 (1:1000; Santa MAPK at conserved TEY sites, causing it to translocate Cruz, Santa Cruz, CA95060, US) were used for immuno- into the nucleus to regulate gene expression. These blotting experiments. Immunoglobulin G (IgG) was also two TGFβ-regulated pathways converge to inhibit the func- purchased from Santa Cruz Biotechnologies. tion of MEF2 and hence muscle-specific genes [22], and ul- timately result in cell proliferation [23,24]. Not surprisingly, Cell culture, transfections and drug treatments inhibition of either or both of these pathways, (either C2C12 cells were maintained in DMEM supplemented pharmacologically or through ectopically expressed Smad7, with 10% fetal bovine serum (HyClone, Rockford, IL61101, which can antagonize the canonical Smad-pathway), en- US), 1% L-glutamine and 1% penicillin-streptomycin. Cells hances myotube formation [25,26]. Crosstalk between these were maintained in a humidified, 37°C incubator with a 5% pathways is further supported by Smad7 antagonizing the CO atmosphere. For transfections, cells were seeded on repressive effects of MEK1 on MyoD [26,27]. pre-gelatin-coated plates 1 day prior to transfection and In this report, our goal was to assess the role of KLF6 were transfected according to the standard calcium phos- in myogenic cells based on its regulation by both phate method previously described by Perry et al., 2001. A MEF2D and TGFβ. We report that TGFβ upregulates mixture of 50 μl2.5 MCaCl per 25 μgDNA with an equal KLF6 specifically through a Smad3-dependent pathway, volume of 2× HeBS (2.8 M NaCl, 15 mM Na HPO ,50 2 4 which enhances proliferation in myoblasts. In addition, mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid we observed that 1) TGFβ enhanced KLF6 promoter ac- (HEPES), pH = 7.15) was used, and the cells were incubated tivation, and 2) that MEF2 is recruited to the KLF6 pro- overnight followed by washing and addition of fresh media. moter region but is not required for KLF6 activation by Drug treatments were used at the following concentrations: TGFβ. Pharmacological inhibition of Smad3 repressed 2ng/ml TGFβ,5 μM Sis3 and 10 μM U0126 as indicated. Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 3 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 siRNA gene silencing instructions. Precipitated proteins were separated by SDS siRNA targeting KLF6, MEF2D and non-specific scram- PAGE and immunoblotting of proteins was performed as ble RNA were purchased from Sigma. Transient trans- described above. fections were performed using TurboFect Transfection Reagent (R0531, Fermentas) according to the manufac- Chromatin immunoprecipitation (ChIP) turer’s instructions. Turbofect (Fermentas): a 1:2 mixture ChIP experiments followed the guidelines set by EZ ratio of DNA to turbofect reagent (including 4 ng/ml ChIP™ (Upsate) with minor modifications. Approxi- siRNA) in 200 μl serum-free DMEM was prepared for mately 1× 10 C2C12 cells were fixed with 1% formalde- 19-h incubation. hyde (Sigma) for 15 minutes at 37°C. Fixing was quenched by Glycine (Bioshop, Burlington, ON Canada) Immunocytochemistry at a final concentration of 0.125 M. Cells were collected C2C12 cells were treated as previously described by Salma in PBS containing phenylmethylsulfonyl fluoride (PMSF) and McDermott, 2012 [14], and incubated overnight (Sigma) and protease inhibitor cocktail (Roche, Laval, with at 4°C with primary MEF2D and KLF6 antibodies Quebec, Canada). Cells were collected at 5000 rpm for 5 (1:100) diluted in 1.5% goat serum. Cells were washed minutes at 4°C. Cells were lysed using Wash Buffer I (10 three times with PBS for 10 minutes and incubated with mM HEPES pH 6.5, 0.5 M ethylene glycol tetraacetic the appropriate tetramethyl rhodamine iso-thiocyanate acid (EGTA), 10 mM EDTA, 0.25% Triton X-100, prote- (TRITC)/fluorescein isothiocyanate (FITC)-conjugated ase inhibitor cocktail, PMSF) for 5 minutes on ice. Nu- secondary antibodies (1:500) in 1.5% goat serum (PBS) clei were collected and resuspended in Wash Buffer II for 2 h at room temperature (RT) following 4’,6- (10 mM HEPES pH 6.5, 0.5 mM EGTA, 1 mM EDTA, diaminidino-2-phenylindole (DAPI) staining for 15 mi- 200 mM NaCl, protease inhibitor cocktail, PMSF) for 10 nutes at RT. Cells were washed three times with PBS minutes on ice. Nuclei were again collected and then and cover slips were mounted with DAKO mounting treated with nuclear lysis buffer (50 mM Tris–HCl pH media (Dako) on glass slides. The fluorescence images 8.1, 10 mM EDTA, 1% SDS). Chromatin was sheared were captured using Fluoview 300 (Olympus). using a Misonix sonicator to produce 500 bp fragments. Crosslinked sheared chromatin was collected following Protein extractions, immunoblotting and reporter gene a 15-minute spin at maximum speed. Twenty percent assays of total chromatin was set aside as input. Sheared Cells were harvested using an NP-40 lysis buffer (0.5% crosslinked chromatin was diluted 1:10 with immuno- NP-40, 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10 mM precipitation (IP) dilution buffer (0.01% SDS, 1.1% sodium pyrophosphate, 1 mM ethylenediaminetetraacetic Triton-X 100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH acid (EDTA) (pH 8.0), 0.1 M NaF) containing 10 μg/ml 8.1, 167 mM NaCl) and incubated with antibody over- leupetin and aprotinin, 5 μg/ml pepstatin A, 0.2 mM night at 4°C with rocking. Protein G Dynabeads phenylmethylsulfonyl fluoride and 0.5 mM sodium (Invitrogen) were blocked with 20 μgsalmonsperm orthovanadate. Protein concentrations were determined DNA in IP dilution buffer (15 μl of beads + 135 μlIP using the Bradford method (Bio-Rad) with BSA as a dilution buffer + 20 μg salmon sperm DNA per IP) standard. We used 20 μg of total protein extracts for im- overnight at 4°C with rocking. We incubated 152 μlof munoblotting, diluted in sample buffer containing 5% β- pre-blocked beads with the IP reaction at 4°C for 1 h. mercaptoethanol, and boiled. Transcriptional assays were Dynabead-bound antibody-chromatin complexes were done using Luciferase reporter plasmids. The cells were washed using IP Wash Buffer I (20 mM Tris pH 8.1, 2 harvested for these assays using 20 mM Tris, (pH 7.4) and mM EDTA, 150 mM NaCl, 1% Triton-X 100, 0.1% 0.1% Triton-X 100, and the values obtained were normal- SDS) and II (20 mM Tris pH 8.1, 2 mM EDTA, 500 ized to β-galactosidase activity expressed from a constitu- mM NaCl, 1% Triton X-100, 0.1% SDS), each incu- tive SV40-driven expression vector and represented as bated for 10 minutes at 4°C, and followed with two relative light units (RLU), or in some cases, corrected Lu- washes in Tris-EDTA (TE) buffer at 4°C. Protein-DNA ciferase values for control, reporter alone transfections complexes were freed from Dynabeads through the were arbitrarily set to 1.0, and fold activation values were addition of elution buffer (0.1 M NaHCO3, 1% SDS) calculated. Bars represent the mean (n = 3) and error bars for 30 minutes at RT. To separate protein from DNA, represent the standard error of the mean (n = 3). samples were treated with 12 μlof5MNaCl (BioShop) at 65°C for 4 h or overnight. Protein was Co-immunoprecipitation assays further degraded by the addition of Proteinase K Protein extracts were prepared as described above. Immu- (Sigma), EDTA, Tris pH 6.5 for 1 h at 45°C. DNA noprecipitation was performed using the ExactaCruz samples were then purified using a PCR clean up kit kit (Santa Cruz Biotechnology), as per manufacturer’s (Qiagen, Mississauga, ON, Canada). Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 4 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 Hours in DM 0 24 48 72 96 120 KLF6 MEF2D Actin DAPI KLF6 DAPI KLF6 MEF2D Merged Merged MEF2D Myoblasts Myotubes MEF2 MEF2 binding site binding site Luc Luc - 507 pROM6 -507 pROM6 + 1 + 1 4.5 3.5 2.5 1.5 0.5 KLF6 M Δ EF2-Luc KLF6-Luc TGFβ (2 ng/ml) + + Figure 1 Western blot analysis reveals that Krüppel-like factor 6 (KLF6) and Myocyte enhancer factor 2 (MEF2D) are co-expressed in C2C12 myoblasts. (a) Myoblasts were cultured in growth medium (10% serum), followed by serum withdrawal (2%) for 144 h and harvested at 24-h time intervals. Cells were then lysed and equal amounts of protein (20 μg) were used for western blot analysis. The levels of the indicated proteins were assessed by a standard immunoblotting technique using specific primary antibodies for each. Actin was used as a loading control. (b) Immunocytochemistry reveals that KLF6 and MEF2D are co-localized in the nucleus at the myoblast stage but to a lesser extent in differentiated myotubes. C2C12 cells were treated as previously described by Salma and McDermott, 2012 [14]. We used 4’,6-diaminidino-2- phenylindole (DAPI) staining for nuclear staining; green and red were used for MEF2D and KLF6 respectively and were then merged. (c) Transforming growth factor β (TGFβ) treatment potentiates the KLF6 promoter region through MEF2. KLF6 promoter constructs (pROM6 Luc and pROM6 ΔMEF2 Luc) were used, and Luciferase activities were analyzed upon serum withdrawal, with and without 2 ng/ml TGFβ treatment as indicated. Fold Activity MB in GM Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 5 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 Quantitative (q)PCR MEF2A/D expression is not required for KLF6 protein ChIP-qPCR analysis of the KLF6 promoter was done expression in skeletal myoblasts using BioRad Sybr Green as per the user manual with a Since we had already observed that TGFβ regulates the final primer concentration of 0.5 μM. The antibody used KLF6 promoter through MEF2 we wanted to assess the in ChIP was 5 μg αMEF2 (sc-313X; Santa Cruz Biotech- effect of MEF2A/D knock down using RNA silencing nology, Inc.). The equivalent amount of rabbit IgG (12– (Figure 2a). Although siRNA2 for MEF2A appears to 370, Millipore) was used as a control in each ChIP. affect KLF6 expression slightly, this observation did Sequences of the primers flanking the ME2 site on the not indicate a strong and consistent effect. On the KLF6 promoter were: 5’-CTGCAACGTTGGGCTGTA-3’ other hand, siMEF2D appears to de-repress KLF6 ex- and 5’-TTGGAAAGACGTCTCACAGG-3’. Each sample pression. Since MEF2D is a potent Histone deacetylase 4 was run in triplicate and then analyzed using percent (HDAC4) co-factor, siMEF2D might be preventing the input or fold enrichment. recruitment of HDAC4 to the promoter and hence de- repressing KLF6. Contrary to our initial hypothesis, these data indicate that MEF2 is not necessarily required Results and discussion for KLF6 expression, or that its requirement is only at MEF2D and KLF6 expression and co-localization in the the myoblast stage when the cells are responsive to nucleus in skeletal myoblasts TGFβ signaling. To further analyze this observation, we Since KLF6 was identified in the skeletal muscle tran- assessed MEF2 recruitment on the KLF6 promoter with scriptome [15], and has also been shown to be an or without TGFβ treatment (Figure 2b). These data indi- MEF2D target gene that is involved in the cell survival cate that while MEF2 is indeed recruited to the KLF6 pathway in primary embryonal hippocampal neurons [14], and since MEF2D is also a crucial regulator of skeletal myogenesis, we wanted to investigate the role of KLF6 in skeletal myoblasts. We determined that KLF6 and MEF2D are indeed both co-expressed in C2C12 myoblasts, and are co-localized in the nucleus using western blot analysis and immunocytochemistry MEF2A respectively (Figures 1a and 1b). Endogenous expres- sion of KLF6 is detected in C2C12 myoblasts in growth MEF2D conditions and sustained upon serum withdrawal and throughout the course of myogenic differentiation up to 120 h. Interestingly, we observed that KLF6 protein KLF6 expression is downregulated at 48 h, upregulated at 72 h, downregulated at 96 h and upregulated again Actin at 120 h in a reproducible manner that is not easily explainable at this point (Figure 1a). Immunofluores- cence labeling was conducted to observe the cellular localization of KLF6 with respect to MEF2D in prolifer- MEF2 recruitment to 0.12 KLF6 Promoter ating myoblasts and then in differentiated myotubes. IgG 0.10 MEF2 The data indicated strong nuclear localization of both KLF6 (red) and MEF2D (green) in conjunction with nu- 0.08 clear (blue) DAPI staining in myoblasts, and less so in 0.06 differentiated myotubes (Figure 1b). Since TGFβ has 0.04 also been shown to regulate KLF6 expression, we tested the effect of TGFβ on previously characterized KLF6 0.02 reporter gene constructs (pROM6-Luc and pROM6- 0.00 Luc ΔMEF2). Serum was withdrawn 24 h after transfec- Control TGFβ tion and treatment with 2 ng/ml TGFβ for 24 h was Figure 2 Myocyte enhancer factor 2 (MEF2)A/D RNA silencing carried out as indicated in the figure. The data illus- reveals that MEF2A/D expression is not required for trates a 4-fold increase in transcriptional activity of endogenous Krüppel-like factor 6 (KLF6) protein expression (a). pROM6-Luc in response to TGFβ treatment, but no ef- In contrast siMEF2D appears to de-repress endogenous KLF6 protein levels. (b) Chromatin immunoprecipitation analysis of MEF2 fect on pROM6-Luc ΔMEF2, indicating that TGFβ reg- recruitment onto the KLF6 promoter revealed no change upon ulates the KLF6 promoter, which requires that the Transforming growth factor β (TGFβ) treatment. MEF2 cis element is intact (Figure 1c). % Input SCR siRNA1 siRNA2 SCR siRNA1 siRNA2 Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 6 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 promoter in C2C12 myoblasts, there is no change in an indicator of myogenic differentiation. Interestingly, MEF2 recruitment upon TGFβ treatment compared to pharmacological inhibition of Smad3 with 5 μMSis3 re- the control, implicating a different mechanism for TGFβ duced TGFβ-induced KLF6 protein expression but had no activation of KLF6. effect on myogenin. This indicates that TGFβ regulates KLF6 and myogenin through two distinct pathways. TGFβ regulates KLF6 through a Smad3-specific pathway Smad2/3 and phospho-Smad2/3 antibodies were used as and inhibits skeletal myogenesis through an MEK/ERK- positive controls for Sis3 treatment since Sis3 inhibits specific pathway Smad3 phosphorylation and hence its translocation into Since Smad3 is activated in proliferating myoblasts and the nucleus [33]. Since TGFβ also regulates the MEK is also regulated by TGFβ, we observed that Smad3, stands for MAP kinase, ERK kinase Kinase (MEK)/ERK along with MEF2 and KLF6, are co-expressed in skeletal (1/2) MAPK pathway we wanted to test the effect of myoblasts (Figure 3a). To further investigate the effect pharmacological inhibition of that pathway on KLF6 of TGFβ on KLF6 we used well-documented pharmaco- using 10 μM U0126. The data summarized in Figure 3c logical inhibitors of the Smad and ERK1/2 Mitogen acti- confirm that TGFβ induces KLF6 protein expression vated protein kinase (MAPK) pathways. We tested the while inhibiting myotube formation (using sarcomeric effect of TGFβ on KLF6 protein expression in C2C12 myosin heavy chain expression as an indicator). In this ex- myoblasts in the presence and absence of a Smad3 inhibi- periment Smad3 inhibition repressed TGFβ induction of tor, Sis3 (Figure 3b). The data in Figure 3b reveal that KLF6 but did not reverse the effects on Myosin heavy chain indeed, TGFβ treatment increases KLF6 protein levels (MyHC) (Figure 3c). Strikingly, pharmacological inhibition and this corresponded with a decrease in myogenin as of ERK1/2 had no effect on KLF6 levels but instead rescued DMSO + TGFβ ++ + Sis3 + U0126 + DMSO + TGFβ ++ ERK1/2 Sis3 + + pERK1/2 Smad2 Smad3 KLF6 pSmad2 pSmad3 Smad2/3 Myogenin pSmad3 KLF6 Actin Actin MyHC Figure 3 Western blot analysis revealed that Smad3 and Krüppel-like factor 6 (KLF6) are co-expressed in C2C12 myoblasts (a). Myogenin was used as a protein marker for differentiation and actin was used as a loading control. Pharmacological manipulation of the Transforming growth factor β (TGFβ) signaling pathway reveals that TGFβ regulates KLF6 protein expression through Smad3 but not MEK/ERK MAPK. (b) Western blot analysis indicates that 2 ng/ml TGFβ treatment elevates KLF6 protein expression and that this effect is abrogated in the presence of 5 μM of specific inhibitor of Smad3, Sis3. TGFβ treatment also inhibited the myogenic differentiation marker, myogenin protein expression level, and this effect was not abrogated by Sis3. (c) Western blot analysis revealed that TGFβ treatment enhances KLF6 expression through Smad3 but not ERK1/2 MAPK and that TGFβ treatment repressed myogenic differentiation through ERK1/2 MAPK but not Smad3; 10 μM U0126 was used as an inhibitor of the MEK/ERK MAPK pathway, 5 μM Sis3 was used for Smad3 inhibition and 2 ng/ml TGFβ were all used as indicated. Actin was used as a loading control. Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 7 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 myotube formation and MyHC expression, thus supporting silencing resulted in increased MyoD and myogenin the idea that TGFβ regulates KLF6 and myogenic differenti- protein expression (Figure 4b, upper panel) and this ation through Smad3 and ERK1/2 distinctively. corresponded with a 2.5-fold increase in muscle creatine kinase (MCK) promoter (Figure 4b, lower panel). Further- TGFβ induces cell proliferation in C2C12 myoblasts more, an MTT cell proliferation assay was performed, and through KLF6 the data showed that at 24 h, 2 ng/ml TGFβ treatment Since TGFβ represses skeletal myogenesis by retaining doubles the number of proliferating cells (Figure 4c). cells in a proliferative state, we wanted to test the effect of This effect is largely negated following KLF6 gene silen- KLF6 mRNA silencing using siRNA-mediated gene silen- cing, thus implicating KLF6 in the proliferative cing. siRNA3 was chosen as the most efficient in depleting response to TGFβ signaling. In support of this, siKLF6 KLF6 expression as shown in Figure 4a. Subsequent KLF6 on its own reduced the number of proliferating cells a b scr siKlf6(3) KLF6 MEF2A MyoD Myogenin Actin MTT Cell Proliferation 1 4 Assay MCK-Luc Assay 0.9 3.5 0.8 0.7 2.5 0.6 0.5 2 0.4 1.5 0.3 0.2 0.5 0.1 0 0 TGFβ (2 ng/ml) + + Scr siKLF6 (3) siKLF6 (3) + + TGFβ U0126 SiS3 Smad3 MEK1/2 KLF6 ERK1/2 Cell Proliferation Myogenin MyHC Skeletal Muscle Differentiation Figure 4 Krüppel-like factor 6 (KLF6) RNA silencing reveals that KLF6 protein expression was successfully repressed, particularly by siRNA3, which was used in subsequent experiments (a). (b) KLF6 RNA silencing resulted in (i) increased MyoD and myogenin protein levels, (ii) enhanced MCK Luciferase activity and, (iii) reduced transforming growth factor β (TGFβ) induced cell proliferation. (c) Cell proliferation was measured using the MTT cell proliferation assay kit. The number of proliferating cells is directly proportional to the absorbance at 570 nm. TGFβ treatment doubled the number of proliferating cells and this effect was repressed with KLF6 silencing. (d) A schematic summary of the data presented, in which TGFβ/Extracellular signal regulated kinase (ERK) signaling represses myogenic differentiation while TGFβ/Smad signaling regulates KLF6 gene expression and myoblast proliferation. Absorbance (570 nm) Fold activation Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 8 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 indicating a functional role in proliferation of skeletal 1P3, Canada. Centre for Research in Biomolecular Interactions, York University; York University, Toronto, ON M3J 1P3, Canada. myoblasts (Figure 4c). Received: 21 November 2012 Accepted: 15 February 2013 Published: 2 April 2013 Conclusions In this study we report a novel role for KLF6 in skeletal References myoblasts. Based on our data we propose that KLF6 is a 1. 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Kojima S, Hayashi S, Shimokado K, Suzuki Y, Shimada J, Crippa MP, Friedman KLF6 as a potential therapeutic target for such patho- SL: Transcriptional activation of urokinase by the Krüppel-like factor Zf9/ logical conditions, as well as for cancers, such as embry- COPEB activates latent TGF-beta1 in vascular endothelial cells. Blood 2000, 95:1309–16. onal rhabdomyosarcoma, where TGFβ promotes cell 7. Botella LM, Sanz-Rodriguez F, Komi Y, Fernandez A, Varela E, Garrido-Martin proliferation [35]. EM, Narla G, Friedman SL, Kojima S: TGF-β regulates the expression of transcription factor KLF6 and its splice variants and promotes co- Abbreviations operative transactivation of common target genes through Smad3-Sp1 Bp: Base pairs; BSA: Bovine serum albumin; ChIP: Chromatin -KLF6 interaction. Biochem J 2009, 419:485–495. immunoprecipitation; DAPI: 4’,6-diaminidino-2-phenylindole; 8. Haldar SM, Ibrahim OA, Jain MK: Krüppel-like Factors (KLFs) in muscle DM: Differentiation media; DMEM: Dulbecco’s modified Eagle’s serum; biology. J Mol Cell Cardiol 2000, 43:1–10. EDTA: Ethylenediaminetetraacetic acid; EGTA: Ethylene glycol tetraacetic acid; 9. Gehrau RC, D'Astolfo DS, Prieto C, Bocco JL, Koritschoner NP: Genomic EMT: Epithelial-to-mesenchymal transitions; ERK: Extracellular signal regulated organization and functional analysis of the gene encoding the Krüppel- kinase; FITC: Fluorescein isothiocyanate; GM: Growth media; HEPES: 4-(2- like transcription factor KLF6. Biochim Biophys Acta 2005, 1730:137–46. hydroxyethyl)-1-piperazineethanesulfonic acid; IgG: Immunoglobulin G; 10. Nandan MO, Yang VW: The role of Krüppel-like factors in the IP: Immunoprecipitation; KLF6: Krüppel-like factor 6; Luc: Luciferase; reprogramming of somatic cells to induced pluripotent stem cells. Histol MAPK: Mitogen activated protein kinase; MCK: Muscle creatine kinase; Histopathol 2009, 24:1343–1355. MEF2: Myocyte enhancer factor 2; MEK: MAP Kinase, ERK Kinase Kinase; 11. Jiang J, Chan YS, Loh YH, Cai J, Tong GQ, Lim CA, Robson P, Zhong S, Ng MRF: Muscle regulatory factor; MyHc: Myosin heavy chain; PBS: Phosphate- HH: A core Klf circuitry regulates self-renewal of embryonic stem cells. buffered saline; PMSF: Phenylmethylsulfonyl fluoride; qPCR: Quantitative Nat Cell Biol 2008, 10:353–360. polymerase chain reaction; RLU: Relative light units; RT: Room temperature; 12. Suske G, Bruford E, Philipsen S: Mammalian SP/KLF transcription factors: siRNA: Small interfering RNA; Sis3: Smad3 inhibitor; TE: Tris-EDTA; bring in the family. Genomics 2005, 85:551–556. TGFβ: Transforming growth factor beta; TRITC: Tetramethyl rhodamine 13. Holian J, Qi W, Kelly DJ, Zhang Y, Mreich E, Pollock CA, Chen XM: Role of iso-thiocyanate; U0126: MEK/ERK inhibitor. Krüppel-like factor 6 in transforming growth factor-β1-induced epithelial-mesenchymal transition of proximal tubule cells. Am J Physiol Renal Physiol 2008, 259:F1388–F1396. Competing interests 14. Salma J, McDermott JC: Suppression of a MEF2-KLF6 survival pathway by The authors declare that they have no competing interests. PKA signaling promotes apoptosis in embryonic hippocampal neurons. J Neurosci 2012, 32:2790–2803. Authors’ contributions 15. Blais A, Tsikitis M, Acosta-Alvear D, Sharan R, Kluger Y, Dynlacht BD: An MGD designed the experiments, performed drug treatments, siKLF6 initial blueprint for myogenic differentiation. Genes Dev 2005, 19:553–569. experiments, and KLF6 functional assays, and drafted the manuscript. JS 16. Luo K, Lodish HF: Positive and negative regulation of type II TGF-beta identified KLF6 as a MEF2D target gene and carried out co-localization and, receptor signal transduction by autophosphorylation on multiple serine immunofluorescence experiments. MB performed activity assays and western residues. EMBO J 1997, 16:1970–81. blotting. SW conducted ChIP analysis and siMEF2 experiments. LZ conducted 17. Liu D, Black BL, Derynck R: TGF-β inhibits muscle differentiation through western blots. JCM conceived of the study, and participated in its design functional repression of myogenic transcription factors by Smad3. Gen and coordination, and helped to draft the manuscript. All authors read and Dev 2001, 15:2950–2966. approved the final manuscript. 18. Kollias HD, McDermott JC: Transforming growth factor – β and myostatin signaling in skeletal muscle. J Appl Physiol 2008, 104:579–587. Acknowlegement 19. Yue J, Mulder KM: Activation of the mitogen-activated protein kinase We would like to thank Canadian Institutes for Health Research (CIHR) for pathway by transforming growth factor-β. Methods Mol Biol 2000, funding this work. 142:125–131. 20. Pelicci G, Lanfrancone L, Grignani F, McGlade J, Cavallo F, Forni G, Nicoletti I, Author details Grignani F, Pawson T, Pelicci PG: A novel transforming protein (SHC) with Department of Biology, York University; York University, 4700 Keele St, an SH2 domain is implicated in mitogenic signal transduction. Cell 1992, Toronto, ON M3J 1P3, Canada. Centre for Research in Mass Spectrometry, 70:93–104. York University; York University, Toronto, ON M3J 1P3, Canada. Muscle 21. Derynck R, Zhang YE: Smad-dependent and Smad-independent pathways Health Research Centre, York University; York University, Toronto, ON M3J in TGF-β family signalling. Nature 2003, 425:577–584. Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 9 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 22. Liu D, Kang JS, Derynck R: TGF-β-activated Smad3 represses MEF2- depedent transcription in myogenic differentiation. EMBO 2004, 23:1557–1566. 23. Liu D, Black BL, Derynck R: TGF-β inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3. Genes Dev 2001, 15:2950–2966. 24. Jungert K, Buck A, Buchholz M, Wagner M, Adler G, Gres TM, Ellenrieder V: Smad-Sp1 complexes mediated TGFβ-induced early transcription of oncogenic Smad7 in pancreatic cancer cells. Carcinogenesis 2006, 27:2392–2401. 25. Kollias HD, Perry RL, Miyake T, Aziz A, McDermott JC: Smad7 promotes and enhances skeletal muscle differentiation. Mol Cell Biol 2006, 26:6248–6260. 26. Miyake T, Alli NS, McDermott JC: Nuclear function of Smad7 promotes myogenesis. Mol Cell Biol 2010, 30:722–735. 27. Perry RL, Parker MH, Rudnicki MA: Activated MEK1 binds the nuclear MyoD transcriptional complex to repress transactivation. Mol Cell 2001, 8:291–301. 28. Du M, Perry RL, Nowacki NB, Gordon JW, Salma J, Zhao J, Aziz A, Chan J, Siu KW, McDermott JC: Protein Kinase A represses skeletal myogenesis by targeting myocyte enhancer-binding factor 2D. Mol Cell Biol 2008, 28:2952–2970. 29. Perry RL, Yang C, Soora N, Salma J, Marback M, Naghibi L, Ilyas H, Chan J, Gordon JW, McDermott JC: Direct interaction between myocyte enhancer factor 2 (MEF2) and protein phosphatase 1 alpha represses MEF2- dependent gene expression. Mol Cell Biol 2009, 29:3355–3366. 30. Wrana JL, Attisano L, Carcamo J, Zentella A, Doody J, Laiho M, Wang XF, Massague J: TGF beta signals through heteromeric protein kinase receptor complex. Cell 1992, 71:1003–1014. 31. Donoviel DB, Shield MA, Buskin JN, Haugen HS, Clegg CH, Hauschka SD: Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice. Mol Cell Biol 1996, 16:1649–1658. 32. Quinn ZA, Yang CC, Wrana JL, McDermott JC: Smad proteins function as co-modulators for MEF2 transcriptional regulatory proteins. Nucleic Acids Res 2001, 29:732–742. 33. Jinnin M, Ihn H, Tamaki K: Characterization of SIS3, a novel and specific inhibitor of Smad3, and its effect on transforming growth factor-beta1 -induced extracellular matrix expression. Mol Pharmacol 2006, 69:597–607. 34. Burks TN, Cohn RD: Role of TGF-β signaling in inherited and acquired myopathies. Skeletal Muscle 2011, 1:19. 35. Bouché M, Canipari R, Melchionna R, Willems D, Senni MI, Molinaro M: TGF- beta autocrine loop regulates cell growth and myogenic differentiation in human rhabdomyosarcoma cells. FASEB J 2000, 14:1147–58. doi:10.1186/2044-5040-3-7 Cite this article as: Dionyssiou et al.: Krüppel-like factor 6 (KLF6) promotes cell proliferation in skeletal myoblasts in response to TGFβ/ Smad3 signaling. Skeletal Muscle 2013 3:7. 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Krüppel-like factor 6 (KLF6) promotes cell proliferation in skeletal myoblasts in response to TGFβ/Smad3 signaling

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Copyright © 2013 by Dionyssiou et al.; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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2044-5040
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10.1186/2044-5040-3-7
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23547561
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Abstract

Background: 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. Methods: KLF6 protein expression levels and promoter activity were analyzed using standard cellular and molecular techniques in cell culture. Results: 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. Conclusions: 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. Keywords: Myoblasts, Krüppel-like factor 6, Transforming growth factor β, Cell proliferation Background glycoproteins in placental cells [3]. Since then, KLF6 has KLF6 is a member of the Krüppel-like Factors (KLF) been found to be expressed in most tissues including gene family which are a group of transcription factors neuronal, hindgut, heart and limb buds [4] and is local- that contain three highly conserved Cys -His type zinc ized in the nucleus [5]. Interestingly, homozygous null 2 2 fingers located in the C-terminus [1,2]. Subsequently, KLF6 mice result in failure in the development of the these proteins regulate a vast range of target genes by liver and yolk sac vasculature, resulting in early lethality preferentially binding to cognate GC-boxes or CACCC at (E)12.5 [4]. To date, the most well-established target elements. KLF6 was originally identified due to its ability gene of KLF6 is Transforming growth factor β (TGFβ) to regulate TATA-less gene promoters that can regulate and its receptors [6], and subsequent studies have shown a positive feedback loop by which TGFβ activation * Correspondence: jmcderm@yorku.ca enhances KLF6 transactivation properties through the for- Department of Biology, York University; York University, 4700 Keele St, mation of a Smad3-Sp1-KLF6 protein complex [7]. TGFβ Toronto, ON M3J 1P3, Canada and KLF6 cooperatively regulate a wide range of cellular Centre for Research in Mass Spectrometry, York University; York University, Toronto, ON M3J 1P3, Canada processes such as cell differentiation, proliferation and Full list of author information is available at the end of the article © 2013 Dionyssiou et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 2 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 epithelial-to-mesenchymal transitions (EMT) [8-13]. Re- KLF6 expression by TGFβ and cell proliferation but, im- cently KLF6 was identified as a myocyte enhancer factor 2 portantly did not re-activate the differentiation program (MEF2) target gene that is involved in neuronal cell sur- which is potently repressed by TGFβ signaling. Con- vival [14]. Since TGFβ and MEF2 are two key regulators versely, TGFβ treatment coupled with pharmacological of skeletal myogenesis and since KLF6 was identified in inhibition of MEK1/2, enhanced myotube formation but the myogenic transcriptome [15], we wanted to investigate had no effect on KLF6 expression and function. Loss of the role of KLF6 in skeletal muscle cells. function assays using siRNA targeting KLF6 revealed Regulation of skeletal myogenesis is a complex process. that KLF6 is required for cell proliferation. These experi- Initially paracrine factors instigate the migration of desig- ments tease apart two independent functions of TGFβ nated myotome progenitor cells to the dermomyotome re- signaling in myogenic cells. One is a repressive effect on gion of the somite. These proliferating cells grow and differentiation which is mediated by ERK activation, the divide until cell contact triggers differential gene expression other being an enhancement of proliferation, which is and activation of the MEF2 proteins and muscle regulatory dependent on Smad3 and KLF6. factors (MRFs). This cascade of events causes morpho- logical changes in the progenitor cells that allow them to Methods align and fuse to form multinucleated myotubes that can Plasmids eventually spontaneously contract as functional muscle fi- Expression plasmids for pcDNA3-MEF2D, pCMV β- bers. TGFβ antagonizes this process by preventing cells galactosidase [28,29], and reporter gene constructs for from exiting the cell cycle hence maintaining myoblasts in 3TP-lux [30], MCK-Luc [31], and MEF2-Luc [32] have a proliferative state. TGFβ ligands bind to atypeIIreceptor been previously described. KLF6 reporter constructs which becomes activated and autophosphorylated [16]. The pRMO6 and pROM6 ΔMEF2 were generously provided activated type II receptor can then phosphorylate and acti- by Dr. Nicolas P. Koritschoner (Faculty of Bioquimica y vate a type I receptor, which in turn phosphorylates Ciencias Biologicas, Universidad Nacional del Litoral, receptor-mediated Smads(2/3) enabling them to dimerize Santa Fe, Argentina). with Smad4 and translocate into the nucleus where they can bind to other transcription factors and DNA, to repress Antibodies essential muscle genes and the expression of their down- Anti-MEF2A rabbit polyclonal, anti-Myosin heavy chain stream targets [17,18]. In addition, TGFβ also regulates the mouse monoclonal and anti-Myogenin mouse monoclonal mitogen-activated protein kinase (MAPK) pathway, which antibodies were produced with the assistance of the York involves a cascade of protein kinases (MAPKKK, MAPKK, University (Toronto, Ontario, Canada) Animal Care Facility. MAPK) that become activated in sequence by G-proteins Anti-MEF2D (1:1000; BD Biosciences, Mississauga, in response to TGFβ binding its receptors [19-21]. Upon Ontario, Canada), Smad3, phospho-Smad3 and phospho- TGFβ activation, MEK1/2 (MAPKK) can phosphorylate ERK1/2 (1:1000; Cell Signaling, Toronto, Ontario, and activate Extracellular signal-regulated kinase (ERK)1/2 Canada), and KLF6, actin, and ERK1/2 (1:1000; Santa MAPK at conserved TEY sites, causing it to translocate Cruz, Santa Cruz, CA95060, US) were used for immuno- into the nucleus to regulate gene expression. These blotting experiments. Immunoglobulin G (IgG) was also two TGFβ-regulated pathways converge to inhibit the func- purchased from Santa Cruz Biotechnologies. tion of MEF2 and hence muscle-specific genes [22], and ul- timately result in cell proliferation [23,24]. Not surprisingly, Cell culture, transfections and drug treatments inhibition of either or both of these pathways, (either C2C12 cells were maintained in DMEM supplemented pharmacologically or through ectopically expressed Smad7, with 10% fetal bovine serum (HyClone, Rockford, IL61101, which can antagonize the canonical Smad-pathway), en- US), 1% L-glutamine and 1% penicillin-streptomycin. Cells hances myotube formation [25,26]. Crosstalk between these were maintained in a humidified, 37°C incubator with a 5% pathways is further supported by Smad7 antagonizing the CO atmosphere. For transfections, cells were seeded on repressive effects of MEK1 on MyoD [26,27]. pre-gelatin-coated plates 1 day prior to transfection and In this report, our goal was to assess the role of KLF6 were transfected according to the standard calcium phos- in myogenic cells based on its regulation by both phate method previously described by Perry et al., 2001. A MEF2D and TGFβ. We report that TGFβ upregulates mixture of 50 μl2.5 MCaCl per 25 μgDNA with an equal KLF6 specifically through a Smad3-dependent pathway, volume of 2× HeBS (2.8 M NaCl, 15 mM Na HPO ,50 2 4 which enhances proliferation in myoblasts. In addition, mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid we observed that 1) TGFβ enhanced KLF6 promoter ac- (HEPES), pH = 7.15) was used, and the cells were incubated tivation, and 2) that MEF2 is recruited to the KLF6 pro- overnight followed by washing and addition of fresh media. moter region but is not required for KLF6 activation by Drug treatments were used at the following concentrations: TGFβ. Pharmacological inhibition of Smad3 repressed 2ng/ml TGFβ,5 μM Sis3 and 10 μM U0126 as indicated. Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 3 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 siRNA gene silencing instructions. Precipitated proteins were separated by SDS siRNA targeting KLF6, MEF2D and non-specific scram- PAGE and immunoblotting of proteins was performed as ble RNA were purchased from Sigma. Transient trans- described above. fections were performed using TurboFect Transfection Reagent (R0531, Fermentas) according to the manufac- Chromatin immunoprecipitation (ChIP) turer’s instructions. Turbofect (Fermentas): a 1:2 mixture ChIP experiments followed the guidelines set by EZ ratio of DNA to turbofect reagent (including 4 ng/ml ChIP™ (Upsate) with minor modifications. Approxi- siRNA) in 200 μl serum-free DMEM was prepared for mately 1× 10 C2C12 cells were fixed with 1% formalde- 19-h incubation. hyde (Sigma) for 15 minutes at 37°C. Fixing was quenched by Glycine (Bioshop, Burlington, ON Canada) Immunocytochemistry at a final concentration of 0.125 M. Cells were collected C2C12 cells were treated as previously described by Salma in PBS containing phenylmethylsulfonyl fluoride (PMSF) and McDermott, 2012 [14], and incubated overnight (Sigma) and protease inhibitor cocktail (Roche, Laval, with at 4°C with primary MEF2D and KLF6 antibodies Quebec, Canada). Cells were collected at 5000 rpm for 5 (1:100) diluted in 1.5% goat serum. Cells were washed minutes at 4°C. Cells were lysed using Wash Buffer I (10 three times with PBS for 10 minutes and incubated with mM HEPES pH 6.5, 0.5 M ethylene glycol tetraacetic the appropriate tetramethyl rhodamine iso-thiocyanate acid (EGTA), 10 mM EDTA, 0.25% Triton X-100, prote- (TRITC)/fluorescein isothiocyanate (FITC)-conjugated ase inhibitor cocktail, PMSF) for 5 minutes on ice. Nu- secondary antibodies (1:500) in 1.5% goat serum (PBS) clei were collected and resuspended in Wash Buffer II for 2 h at room temperature (RT) following 4’,6- (10 mM HEPES pH 6.5, 0.5 mM EGTA, 1 mM EDTA, diaminidino-2-phenylindole (DAPI) staining for 15 mi- 200 mM NaCl, protease inhibitor cocktail, PMSF) for 10 nutes at RT. Cells were washed three times with PBS minutes on ice. Nuclei were again collected and then and cover slips were mounted with DAKO mounting treated with nuclear lysis buffer (50 mM Tris–HCl pH media (Dako) on glass slides. The fluorescence images 8.1, 10 mM EDTA, 1% SDS). Chromatin was sheared were captured using Fluoview 300 (Olympus). using a Misonix sonicator to produce 500 bp fragments. Crosslinked sheared chromatin was collected following Protein extractions, immunoblotting and reporter gene a 15-minute spin at maximum speed. Twenty percent assays of total chromatin was set aside as input. Sheared Cells were harvested using an NP-40 lysis buffer (0.5% crosslinked chromatin was diluted 1:10 with immuno- NP-40, 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10 mM precipitation (IP) dilution buffer (0.01% SDS, 1.1% sodium pyrophosphate, 1 mM ethylenediaminetetraacetic Triton-X 100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH acid (EDTA) (pH 8.0), 0.1 M NaF) containing 10 μg/ml 8.1, 167 mM NaCl) and incubated with antibody over- leupetin and aprotinin, 5 μg/ml pepstatin A, 0.2 mM night at 4°C with rocking. Protein G Dynabeads phenylmethylsulfonyl fluoride and 0.5 mM sodium (Invitrogen) were blocked with 20 μgsalmonsperm orthovanadate. Protein concentrations were determined DNA in IP dilution buffer (15 μl of beads + 135 μlIP using the Bradford method (Bio-Rad) with BSA as a dilution buffer + 20 μg salmon sperm DNA per IP) standard. We used 20 μg of total protein extracts for im- overnight at 4°C with rocking. We incubated 152 μlof munoblotting, diluted in sample buffer containing 5% β- pre-blocked beads with the IP reaction at 4°C for 1 h. mercaptoethanol, and boiled. Transcriptional assays were Dynabead-bound antibody-chromatin complexes were done using Luciferase reporter plasmids. The cells were washed using IP Wash Buffer I (20 mM Tris pH 8.1, 2 harvested for these assays using 20 mM Tris, (pH 7.4) and mM EDTA, 150 mM NaCl, 1% Triton-X 100, 0.1% 0.1% Triton-X 100, and the values obtained were normal- SDS) and II (20 mM Tris pH 8.1, 2 mM EDTA, 500 ized to β-galactosidase activity expressed from a constitu- mM NaCl, 1% Triton X-100, 0.1% SDS), each incu- tive SV40-driven expression vector and represented as bated for 10 minutes at 4°C, and followed with two relative light units (RLU), or in some cases, corrected Lu- washes in Tris-EDTA (TE) buffer at 4°C. Protein-DNA ciferase values for control, reporter alone transfections complexes were freed from Dynabeads through the were arbitrarily set to 1.0, and fold activation values were addition of elution buffer (0.1 M NaHCO3, 1% SDS) calculated. Bars represent the mean (n = 3) and error bars for 30 minutes at RT. To separate protein from DNA, represent the standard error of the mean (n = 3). samples were treated with 12 μlof5MNaCl (BioShop) at 65°C for 4 h or overnight. Protein was Co-immunoprecipitation assays further degraded by the addition of Proteinase K Protein extracts were prepared as described above. Immu- (Sigma), EDTA, Tris pH 6.5 for 1 h at 45°C. DNA noprecipitation was performed using the ExactaCruz samples were then purified using a PCR clean up kit kit (Santa Cruz Biotechnology), as per manufacturer’s (Qiagen, Mississauga, ON, Canada). Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 4 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 Hours in DM 0 24 48 72 96 120 KLF6 MEF2D Actin DAPI KLF6 DAPI KLF6 MEF2D Merged Merged MEF2D Myoblasts Myotubes MEF2 MEF2 binding site binding site Luc Luc - 507 pROM6 -507 pROM6 + 1 + 1 4.5 3.5 2.5 1.5 0.5 KLF6 M Δ EF2-Luc KLF6-Luc TGFβ (2 ng/ml) + + Figure 1 Western blot analysis reveals that Krüppel-like factor 6 (KLF6) and Myocyte enhancer factor 2 (MEF2D) are co-expressed in C2C12 myoblasts. (a) Myoblasts were cultured in growth medium (10% serum), followed by serum withdrawal (2%) for 144 h and harvested at 24-h time intervals. Cells were then lysed and equal amounts of protein (20 μg) were used for western blot analysis. The levels of the indicated proteins were assessed by a standard immunoblotting technique using specific primary antibodies for each. Actin was used as a loading control. (b) Immunocytochemistry reveals that KLF6 and MEF2D are co-localized in the nucleus at the myoblast stage but to a lesser extent in differentiated myotubes. C2C12 cells were treated as previously described by Salma and McDermott, 2012 [14]. We used 4’,6-diaminidino-2- phenylindole (DAPI) staining for nuclear staining; green and red were used for MEF2D and KLF6 respectively and were then merged. (c) Transforming growth factor β (TGFβ) treatment potentiates the KLF6 promoter region through MEF2. KLF6 promoter constructs (pROM6 Luc and pROM6 ΔMEF2 Luc) were used, and Luciferase activities were analyzed upon serum withdrawal, with and without 2 ng/ml TGFβ treatment as indicated. Fold Activity MB in GM Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 5 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 Quantitative (q)PCR MEF2A/D expression is not required for KLF6 protein ChIP-qPCR analysis of the KLF6 promoter was done expression in skeletal myoblasts using BioRad Sybr Green as per the user manual with a Since we had already observed that TGFβ regulates the final primer concentration of 0.5 μM. The antibody used KLF6 promoter through MEF2 we wanted to assess the in ChIP was 5 μg αMEF2 (sc-313X; Santa Cruz Biotech- effect of MEF2A/D knock down using RNA silencing nology, Inc.). The equivalent amount of rabbit IgG (12– (Figure 2a). Although siRNA2 for MEF2A appears to 370, Millipore) was used as a control in each ChIP. affect KLF6 expression slightly, this observation did Sequences of the primers flanking the ME2 site on the not indicate a strong and consistent effect. On the KLF6 promoter were: 5’-CTGCAACGTTGGGCTGTA-3’ other hand, siMEF2D appears to de-repress KLF6 ex- and 5’-TTGGAAAGACGTCTCACAGG-3’. Each sample pression. Since MEF2D is a potent Histone deacetylase 4 was run in triplicate and then analyzed using percent (HDAC4) co-factor, siMEF2D might be preventing the input or fold enrichment. recruitment of HDAC4 to the promoter and hence de- repressing KLF6. Contrary to our initial hypothesis, these data indicate that MEF2 is not necessarily required Results and discussion for KLF6 expression, or that its requirement is only at MEF2D and KLF6 expression and co-localization in the the myoblast stage when the cells are responsive to nucleus in skeletal myoblasts TGFβ signaling. To further analyze this observation, we Since KLF6 was identified in the skeletal muscle tran- assessed MEF2 recruitment on the KLF6 promoter with scriptome [15], and has also been shown to be an or without TGFβ treatment (Figure 2b). These data indi- MEF2D target gene that is involved in the cell survival cate that while MEF2 is indeed recruited to the KLF6 pathway in primary embryonal hippocampal neurons [14], and since MEF2D is also a crucial regulator of skeletal myogenesis, we wanted to investigate the role of KLF6 in skeletal myoblasts. We determined that KLF6 and MEF2D are indeed both co-expressed in C2C12 myoblasts, and are co-localized in the nucleus using western blot analysis and immunocytochemistry MEF2A respectively (Figures 1a and 1b). Endogenous expres- sion of KLF6 is detected in C2C12 myoblasts in growth MEF2D conditions and sustained upon serum withdrawal and throughout the course of myogenic differentiation up to 120 h. Interestingly, we observed that KLF6 protein KLF6 expression is downregulated at 48 h, upregulated at 72 h, downregulated at 96 h and upregulated again Actin at 120 h in a reproducible manner that is not easily explainable at this point (Figure 1a). Immunofluores- cence labeling was conducted to observe the cellular localization of KLF6 with respect to MEF2D in prolifer- MEF2 recruitment to 0.12 KLF6 Promoter ating myoblasts and then in differentiated myotubes. IgG 0.10 MEF2 The data indicated strong nuclear localization of both KLF6 (red) and MEF2D (green) in conjunction with nu- 0.08 clear (blue) DAPI staining in myoblasts, and less so in 0.06 differentiated myotubes (Figure 1b). Since TGFβ has 0.04 also been shown to regulate KLF6 expression, we tested the effect of TGFβ on previously characterized KLF6 0.02 reporter gene constructs (pROM6-Luc and pROM6- 0.00 Luc ΔMEF2). Serum was withdrawn 24 h after transfec- Control TGFβ tion and treatment with 2 ng/ml TGFβ for 24 h was Figure 2 Myocyte enhancer factor 2 (MEF2)A/D RNA silencing carried out as indicated in the figure. The data illus- reveals that MEF2A/D expression is not required for trates a 4-fold increase in transcriptional activity of endogenous Krüppel-like factor 6 (KLF6) protein expression (a). pROM6-Luc in response to TGFβ treatment, but no ef- In contrast siMEF2D appears to de-repress endogenous KLF6 protein levels. (b) Chromatin immunoprecipitation analysis of MEF2 fect on pROM6-Luc ΔMEF2, indicating that TGFβ reg- recruitment onto the KLF6 promoter revealed no change upon ulates the KLF6 promoter, which requires that the Transforming growth factor β (TGFβ) treatment. MEF2 cis element is intact (Figure 1c). % Input SCR siRNA1 siRNA2 SCR siRNA1 siRNA2 Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 6 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 promoter in C2C12 myoblasts, there is no change in an indicator of myogenic differentiation. Interestingly, MEF2 recruitment upon TGFβ treatment compared to pharmacological inhibition of Smad3 with 5 μMSis3 re- the control, implicating a different mechanism for TGFβ duced TGFβ-induced KLF6 protein expression but had no activation of KLF6. effect on myogenin. This indicates that TGFβ regulates KLF6 and myogenin through two distinct pathways. TGFβ regulates KLF6 through a Smad3-specific pathway Smad2/3 and phospho-Smad2/3 antibodies were used as and inhibits skeletal myogenesis through an MEK/ERK- positive controls for Sis3 treatment since Sis3 inhibits specific pathway Smad3 phosphorylation and hence its translocation into Since Smad3 is activated in proliferating myoblasts and the nucleus [33]. Since TGFβ also regulates the MEK is also regulated by TGFβ, we observed that Smad3, stands for MAP kinase, ERK kinase Kinase (MEK)/ERK along with MEF2 and KLF6, are co-expressed in skeletal (1/2) MAPK pathway we wanted to test the effect of myoblasts (Figure 3a). To further investigate the effect pharmacological inhibition of that pathway on KLF6 of TGFβ on KLF6 we used well-documented pharmaco- using 10 μM U0126. The data summarized in Figure 3c logical inhibitors of the Smad and ERK1/2 Mitogen acti- confirm that TGFβ induces KLF6 protein expression vated protein kinase (MAPK) pathways. We tested the while inhibiting myotube formation (using sarcomeric effect of TGFβ on KLF6 protein expression in C2C12 myosin heavy chain expression as an indicator). In this ex- myoblasts in the presence and absence of a Smad3 inhibi- periment Smad3 inhibition repressed TGFβ induction of tor, Sis3 (Figure 3b). The data in Figure 3b reveal that KLF6 but did not reverse the effects on Myosin heavy chain indeed, TGFβ treatment increases KLF6 protein levels (MyHC) (Figure 3c). Strikingly, pharmacological inhibition and this corresponded with a decrease in myogenin as of ERK1/2 had no effect on KLF6 levels but instead rescued DMSO + TGFβ ++ + Sis3 + U0126 + DMSO + TGFβ ++ ERK1/2 Sis3 + + pERK1/2 Smad2 Smad3 KLF6 pSmad2 pSmad3 Smad2/3 Myogenin pSmad3 KLF6 Actin Actin MyHC Figure 3 Western blot analysis revealed that Smad3 and Krüppel-like factor 6 (KLF6) are co-expressed in C2C12 myoblasts (a). Myogenin was used as a protein marker for differentiation and actin was used as a loading control. Pharmacological manipulation of the Transforming growth factor β (TGFβ) signaling pathway reveals that TGFβ regulates KLF6 protein expression through Smad3 but not MEK/ERK MAPK. (b) Western blot analysis indicates that 2 ng/ml TGFβ treatment elevates KLF6 protein expression and that this effect is abrogated in the presence of 5 μM of specific inhibitor of Smad3, Sis3. TGFβ treatment also inhibited the myogenic differentiation marker, myogenin protein expression level, and this effect was not abrogated by Sis3. (c) Western blot analysis revealed that TGFβ treatment enhances KLF6 expression through Smad3 but not ERK1/2 MAPK and that TGFβ treatment repressed myogenic differentiation through ERK1/2 MAPK but not Smad3; 10 μM U0126 was used as an inhibitor of the MEK/ERK MAPK pathway, 5 μM Sis3 was used for Smad3 inhibition and 2 ng/ml TGFβ were all used as indicated. Actin was used as a loading control. Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 7 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 myotube formation and MyHC expression, thus supporting silencing resulted in increased MyoD and myogenin the idea that TGFβ regulates KLF6 and myogenic differenti- protein expression (Figure 4b, upper panel) and this ation through Smad3 and ERK1/2 distinctively. corresponded with a 2.5-fold increase in muscle creatine kinase (MCK) promoter (Figure 4b, lower panel). Further- TGFβ induces cell proliferation in C2C12 myoblasts more, an MTT cell proliferation assay was performed, and through KLF6 the data showed that at 24 h, 2 ng/ml TGFβ treatment Since TGFβ represses skeletal myogenesis by retaining doubles the number of proliferating cells (Figure 4c). cells in a proliferative state, we wanted to test the effect of This effect is largely negated following KLF6 gene silen- KLF6 mRNA silencing using siRNA-mediated gene silen- cing, thus implicating KLF6 in the proliferative cing. siRNA3 was chosen as the most efficient in depleting response to TGFβ signaling. In support of this, siKLF6 KLF6 expression as shown in Figure 4a. Subsequent KLF6 on its own reduced the number of proliferating cells a b scr siKlf6(3) KLF6 MEF2A MyoD Myogenin Actin MTT Cell Proliferation 1 4 Assay MCK-Luc Assay 0.9 3.5 0.8 0.7 2.5 0.6 0.5 2 0.4 1.5 0.3 0.2 0.5 0.1 0 0 TGFβ (2 ng/ml) + + Scr siKLF6 (3) siKLF6 (3) + + TGFβ U0126 SiS3 Smad3 MEK1/2 KLF6 ERK1/2 Cell Proliferation Myogenin MyHC Skeletal Muscle Differentiation Figure 4 Krüppel-like factor 6 (KLF6) RNA silencing reveals that KLF6 protein expression was successfully repressed, particularly by siRNA3, which was used in subsequent experiments (a). (b) KLF6 RNA silencing resulted in (i) increased MyoD and myogenin protein levels, (ii) enhanced MCK Luciferase activity and, (iii) reduced transforming growth factor β (TGFβ) induced cell proliferation. (c) Cell proliferation was measured using the MTT cell proliferation assay kit. The number of proliferating cells is directly proportional to the absorbance at 570 nm. TGFβ treatment doubled the number of proliferating cells and this effect was repressed with KLF6 silencing. (d) A schematic summary of the data presented, in which TGFβ/Extracellular signal regulated kinase (ERK) signaling represses myogenic differentiation while TGFβ/Smad signaling regulates KLF6 gene expression and myoblast proliferation. Absorbance (570 nm) Fold activation Dionyssiou et al. Skeletal Muscle 2013, 3:7 Page 8 of 9 http://www.skeletalmusclejournal.com/content/3/1/7 indicating a functional role in proliferation of skeletal 1P3, Canada. Centre for Research in Biomolecular Interactions, York University; York University, Toronto, ON M3J 1P3, Canada. myoblasts (Figure 4c). Received: 21 November 2012 Accepted: 15 February 2013 Published: 2 April 2013 Conclusions In this study we report a novel role for KLF6 in skeletal References myoblasts. Based on our data we propose that KLF6 is a 1. 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Skeletal MuscleSpringer Journals

Published: Apr 2, 2013

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