Biochimica et Biophysica Acta 1851 (2015) 194–202
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CTGF/CCN2 exerts profibrotic action in myoblasts via the up-regulation of sphingosine kinase-1/S1P3 signaling axis: Implications in the action mechanism of TGFβ Gennaro Bruno a,1, Francesca Cencetti a,b,1, Irene Pertici a, Lukasz Japtok c, Caterina Bernacchioni a,b, Chiara Donati a,b, Paola Bruni a,b,⁎ a
Dipartimento di Scienze Biomediche Sperimentali e Cliniche “Mario Serio,” Università di Firenze, Viale G.B. Morgagni 50, Firenze 50134, Italy Istituto Interuniversitario di Miologia, Italy c Faculty of Mathematics and Natural Science, Institute of Nutritional Science, Department of Toxicology, University of Potsdam, Arthur-Scheunert Allee 114-116, Nuthetal, Potsdam 14558, Germany b
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Article history: Received 1 August 2014 Received in revised form 19 November 2014 Accepted 20 November 2014 Available online 29 November 2014 Keyword: Sphingosine kinase S1P3 receptor Connective tissue growth factor Myoblasts Transforming growth factor beta
a b s t r a c t The matricellular protein connective tissue growth factor (CTGF/CCN2) is recognized as key player in the onset of fibrosis in various tissues, including skeletal muscle. In many circumstances, CTGF has been shown to be induced by transforming growth factor beta (TGFβ) and accounting, at least in part, for its biological action. In this study it was verified that in cultured myoblasts CTGF/CCN2 causes their transdifferentiation into myofibroblasts by upregulating the expression of fibrosis marker proteins α-smooth muscle actin and transgelin. Interestingly, it was also found that the profibrotic effect exerted by CTGF/CCN2 was mediated by the sphingosine kinase (SK)-1/S1P3 signaling axis specifically induced by the treatment with the profibrotic cue. Following CTGF/CCN2-induced upregulation, S1P3 became the S1P receptor subtype expressed at the highest degree, at least at mRNA level, and was thus capable of readdressing the sphingosine 1-phosphate signaling towards fibrosis rather than myogenic differentiation. Another interesting finding is that CTGF/CCN2 silencing prevented the TGFβ-dependent up-regulation of SK1/S1P3 signaling axis and strongly reduced the profibrotic effect exerted by TGFβ, pointing at a crucial role of endogenous CTGF/CCN2 generated following TGFβ challenge in the transmission of at least part of its profibrotic effect. These results provide new insights into the molecular mechanism by which CTGF/CCN2 drives its biological action and strengthen the concept that SK1/S1P3 axis plays a critical role in the onset of fibrotic cell phenotype. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Inside-out sphingosine 1-phosphate (S1P) signaling represents a common molecular mechanism by which a wide array of extracellular agents, including hormones, cytokines, growth factors and neurotransmitters elicit, at least in part, their biological effects [1–3]. The molecular machinery necessary to activate in autocrine and/or in paracrine fashion one or more S1P receptor subtypes comprises sphingosine kinase(SK)-1, or less frequently SK2, both responsible for the intracellular formation of the bioactive sphingolipid S1P, and a membrane transporter that
Abbreviations: CTGF, connective tissue growth factor; TGFβ, transforming growth factor beta; SK, sphingosine kinase; S1P, sphingosine 1-phosphate; α-SMA, α-smooth muscle actin; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's modified phosphate-buffered saline; FCS, fetal calf serum; BSA, bovine serum albumin; siRNA, short interfering RNA; ECL, enhanced chemiluminescence; SDS–PAGE, sodium dodecyl sulfate– polyacrylamide gel electrophoresis; SCR, scrambled; PI, propidium iodide ⁎ Corresponding author at: Dipartimento di Scienze Biomediche Sperimentali e Cliniche “Mario Serio,” Viale G.B. Morgagni 50, 50134 Firenze, Italy. Tel.: +39 0552751204. E-mail address: paola.bruni@unifi.it (P. Bruni). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bbalip.2014.11.011 1388-1981/© 2014 Elsevier B.V. All rights reserved.
facilitates the release of generated S1P in the extracellular compartment [4]. Then activated S1P receptors, which are coupled to multiple heterotrimeric GTP binding proteins, coordinate the activation of numerous signaling pathways, thus accounting for the final biological response [5]. A wealth of experimental data support a key role of inside-out S1P signaling downstream of primary extracellular cues in the control of cell migration, proliferation and differentiation in a variety of cellular settings. In this regard, it has been firmly established that transforming growth factor beta (TGFβ) exploits S1P signaling axis to exert its profibrotic effect in fibroblasts. Indeed, SK1 is up-regulated by TGFβ in dermal fibroblasts and its expression is required for the induction of the tissue inhibitor of metalloproteases-1 that favors the deposition of extracellular matrix proteins by reducing their degradation [6]. Accordingly, it has been proved that SK1 and S1P2 are implicated in TGFβ-dependent biosynthesis of collagen in cardiac fibroblasts [7]. Moreover, TGFβ-induced transdifferentiation of lung fibroblasts into myofibroblasts, which plays a major role in the onset of pulmonary fibrosis, was found to rely on SK1 and both S1P2 and S1P3 [8]. Besides its major role in the transmission of the fibrotic effect of TGFβ in fibroblasts, S1P signaling has been also individuated as critical
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player in the onset of fibrotic phenotype in skeletal muscle precursor cells such as myoblasts. TGFβ was found to induce transdifferentiation of myoblasts into myofibroblasts, in turn responsible for extracellular matrix protein deposition, progressive replacement of the muscle fibers with connective tissue and impairment of skeletal muscle repair [9]. Notably, in cultured myoblasts S1P endogenously generated by SK1, via the ligation of S1P2, participates to the correct accomplishment of myogenic program that drives their differentiation into myotubes, multinucleated cells endowed with contractile properties that resemble skeletal myofibres [10]. In accordance, insulin-like growth factor-1, pivotal physiological regulator of myogenesis, exerts at least in part its biological action via SK1 activation and S1P2 engagement [11]. In contrast,
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TGFβ is capable of fully readdressing the inside-out signaling of S1P in myoblasts, by up-regulating SK1, that accounts for increased S1P production and enhancing the expression of S1P3 that, becoming the highest expressed receptor subtype, acquires a dominant signaling action [9]. Although the SK1/S1P3 axis has been identified as key player in the profibrotic action of TGFβ in myoblasts, at present is unknown whether it is implicated in the mechanism by which other profibrotic cues exert their biological action. Here, to address the overall relevance of the SK1/ S1P3 axis, we have examined whether this signaling pathway is downstream to another key inducer of skeletal muscle fibrosis such as connective tissue growth factor (CTGF), also known as CCN2, a member of the CCN family of secreted matricellular proteins. This protein, produced
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Fig. 1. CTGF exerts a profibrotic action in C2C12 myoblasts. (A) Dose–response effect of CTGF on the transdifferentiation of myoblasts into myofibrobalsts is evaluated after treatment with the indicated concentration of CTGF by analyzing the expression of the fibrosis marker Transgelin (Tagln) in 10 μg of protein in total cell lysate. Upper panel, Western blot analysis is performed by using specific anti-Tagln antibody. Lower panel, results are obtained by densitometric analysis, normalized to β-actin content in each specimen, and data are reported as mean ± SEM of three independent experiments, fold change compared to control, set as one. The effect of CTGF is statistically significant by one-way ANOVA, followed by Bonferroni's post hoc test, *P b 0.05. (B) Upper panel, time-dependent effect of CTGF in myoblasts is analyzed by measuring the expression of Tagln at the indicated time points. Lower panel, results are analyzed and reported as described in section A. The effect of CTGF at 16 h and 24 h is statistically significant by one-way ANOVA, followed by Bonferroni's post hoc test, *P b 0.05, **P b 0.01. (C) Immunofluorescence analysis of the fibrosis marker α-smooth muscle actin (α-SMA) is performed in myoblasts treated with CTGF for 24 h, using a specific mouse anti-α-SMA antibody, fluorescein-conjugated anti-mouse secondary antibody and propidium iodide (PI) staining (images are representative of three separate experiments).
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by myoblasts in response to TGFβ [12,13], has been shown to induce the expression of extracellular matrix constituents such as fibronectin and to cause also dedifferentiation of committed myoblasts [14]. Notably, in the present study, it is shown that the profibrotic effect exerted by CTGF requires the SK1/S1P3 axis, reinforcing the notion that SK1/S1P3 signaling axis is critical for transdifferentiation of myoblasts into myofibroblasts. Moreover, experimental evidence is presented for a key role of TGFβ-directed induction of CTGF expression in the switch to SK1/S1P3 signaling axis and the execution of the profibrotic response, providing a more detailed view onto the mechanisms that regulate the fibrotic response to TGFβ in myoblasts.
reagents, protease inhibitor cocktail, TRI Reagent® RNA Isolation Reagent, bovine serum albumin (BSA) and monoclonal anti-α-smooth muscle actin (α-SMA) antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), Dulbecco's modified phosphate-buffered saline (DPBS) and fetal calf serum (FCS) were obtained from Lonza (Basel, Switzerland). TGFβ1 and CTGF (lower molecular weight isoform of 11 kDa) were purchased from PeproTech (London, UK). The selective S1P1 antagonist, W146, and the specific S1P1/S1P3 antagonist, VPC23019, were from Avanti Polar Lipids (Alabaster, AL, USA). Short interfering RNA (siRNA) duplexes targeting specific gene of interest such as mouse CTGF (SASI_Mm01_00097842 and SASI_Mm01_00097843), mouse S1P3 (SASI_Mm01_00145233 and SASI_Mm01_00145234), mouse SK1 (SASI_Mm01_00033983 and SASI_Mm01_00033984), mouse SK2 (SASI_Mm01_00050883 and SASI_Mm01_00050884) and scrambled (SCR)-siRNA (Mission Universal Negative control no. 1) were from Sigma-Proligo (The Woodlands, TX, USA). Lipofectamine RNAiMAX® Transfection Reagent and all reagents and probes used to perform real-
2. Materials and methods 2.1. Materials Mouse skeletal muscle C2C12 cells were obtained from the European Collection of Cell Cultures (Salisbury, UK). Biochemicals, cell culture
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Fig. 2. CTGF induces SK1 expression and S1P production in murine myoblasts. (A) One microgram of total RNA extracted from C2C12 myoblasts, stimulated or not with 10 ng/ml CTGF for the indicated time intervals is analyzed by real-time RT-PCR. The quantification of SK1 and SK2 mRNA is based on the 2^(−ΔΔCt) method as described in the Materials and Methods section. The target gene of the unchallenged specimen is used as calibrator and the quantification of housekeeping gene 18S rRNA is performed in parallel. Data reported are means ± SEM of three independent experiments performed in triplicate. The effect of CTGF at 6 h and 18 h of treatment on SK1 mRNA levels is statistically significant by Student's t test (*P b 0.05). (B) Left panel, 10 μg of protein of total cell lysate is subjected to Western blot analysis by using specific anti-SK1 and anti-SK2 antibodies. Right panel, results are reported as described in Fig. 1A legend. The effect of CTGF on SK1 protein expression is statistically significant by Student's t test, *P b 0.05. (C) C2C12 myoblasts were treated with 10 ng/ml CTGF for the indicated time intervals; media were collected and subjected to S1P analysis. The effect of CTGF on S1P levels is statistically significant by Student's t test, *P b 0.05.
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time PCR were obtained from Life Technologies (Carlsbad, CA, USA). Goat anti-transgelin antibody was from Everest Biotech (Oxfordshire, OX, UK). Monoclonal antibody against β-actin was from Cytoskeleton Inc. (Denver, CO, USA). SK2 (N-terminal region) rabbit polyclonal and SK1 (central region) rabbit polyclonal antibodies were purchased from ECM Biosciences LLC (Versailles, KY USA). Secondary antibodies conjugated to horseradish peroxidase and rabbit polyclonal anti-CTGF antibody
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were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Fluorescein-conjugated anti-mouse secondary antibody and Vectashield® mounting medium were purchased from Vector Laboratories (Burlingame, CA, USA). Enhanced chemiluminescence (ECL) reagents were obtained from GE Healthcare Europe GmbH (Milan, Italy). Pharmacological inhibitors of SK (VPC96091 and VPC96047) were kindly provided by Prof. K. Lynch, University of Virginia, USA.
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Fig. 3. SK1 is involved in CTGF-mediated profibrotic effect. (A) C2C12 myoblasts are incubated in the presence of 1 μM VPC96047 or 1 μM VPC96091, 30 min before 10 ng/ml CTGF challenge for 24 h. Upper panel, the fibrosis marker protein Tagln is detected by Western Blotting as described in Fig. 1A legend. Lower panel, densitometric analysis is reported as described in Fig. 1A legend. The effect of VPC96047 and VPC96091 treatment on CTGF-induced profibrotic action is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, ## P b 0.01. (B) Left panel, quantitative analysis of SK1 and SK2 mRNA is performed by real-time RT-PCR in total RNA from C2C12 transfected with SCR-, SK1- or SK2- specific siRNA, as described in the Materials and Methods section. Results are expressed as fold change according to the 2^(−ΔΔCt) method, as described in Fig. 2A legend. The effect of siRNA transfection on SK1 and SK2 expression is statistically significant by Student's t test, **P b 0.01. Upper-right panel, the profibrotic response of CTGF is evaluated in SCR-, SK1- and SK2-siRNA-transfected C2C12 treated or not with 10 ng/ml CTGF by measuring Tagln expression performing Western blot analysis. Lower-right panel, data are reported as described in Fig. 1A legend. The effect of SK1 down-regulation on CTGF-induced Tagln expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, ##P b 0.01.
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10 μM W146, 1 μM VPC23019, 1 μM VPC96047 or 1 μM VPC96091, 30 min before agonist stimulation.
2.2. Cell culture and treatment with inhibitors
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The expression of the fibrosis marker, transgelin, was evaluated after 24 h of agonist challenge in total cell lysates obtained from confluent serum-starved C2C12 myoblasts. Ten micrograms of protein from total cell lysates were subjected to SDS–polyacrylamide gel electrophoresis
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Murine C2C12 myoblasts were routinely grown in DMEM containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in 5% CO2. For myofibroblasts transdifferentiation experiments, cells were seeded in p35 and when 90% confluent were shifted to DMEM without serum supplemented with 1 mg/ml BSA and incubated in the presence or absence of 5 ng/ml TGFβ1 or 10 ng/ml CTGF for the indicated times. In some of the experiments, cells were pre-treated with
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Fig. 4. Role of S1P3 in the profibrotic effect exerted by CTGF. (A) One microgram of total RNA extracted from C2C12 myoblasts, stimulated or not with 10 ng/ml CTGF for the indicated time intervals, is analyzed by real-time RT-PCR. S1P1–4 mRNA quantification is based on the 2^(−ΔΔCt) method, and the analysis is performed as described in Fig. 2A legend by using specific murine S1P1–4 gene expression assays. Upper panel, relative expression of S1P receptor subtypes is analyzed using as calibrator each receptor subtype in the control specimen. Lower panel, the expression profile of the S1P receptor subtypes is analyzed utilizing as calibrator S1P1. The effect of CTGF at 6 h and 18 h of treatment on S1P3 mRNA levels is statistically significant by Student's t test, **P b 0.01; ***P b 0.001. (B) C2C12 myoblasts are incubated in the presence of 10 μM W146 or 1 μM VPC23019, 30 min before 10 ng/ml CTGF challenge for 24 h. Upper panel, the fibrosis marker protein Tagln is detected as described in Fig. 1A legend. Lower panel, densitometric analysis is reported as described in Fig. 1A legend. The effect of VPC23019 treatment on CTGF-induced Tagln expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, ##P b 0.01. (C) Left panel, quantitative analysis of S1P3 mRNA is performed by real-time PCR in total RNA from C2C12 transfected with SCR- and specific S1P3-siRNA, as described in the Materials and Methods section. Results are expressed as fold change according to the 2^(−ΔΔCT) method, as described in Fig. 2A legend. The effect of siRNA transfection on S1P3 expression is statistically significant by Student's t test (*P b 0.05). Upper-right panel, the profibrotic response of CTGF is evaluated in SCR- and S1P3-siRNA-transfected C2C12 treated or not with 10 ng/ml CTGF by measuring Tagln expression using Western blot analysis. Lower-right panel, data are reported as described in Fig. 1A legend. The effect of S1P3 down-regulation on CTGF-induced Tagln expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test (##P b 0.01). The effect of CTGF treatment on Tagln expression in SCR-siRNA and S1P3-siRNA-transfected myoblasts is statistically significant by one-way ANOVA, followed by Bonferroni's post hoc test, **P b 0.01, *P b 0.05.
CTGF mRNA expression levels (2^-ΔΔ ΔΔ Ct)
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and fluorescein-conjugated anti-mouse secondary antibody for 1 h. For nuclear counter-staining, the specimen was incubated with propidium iodide (PI) solution (0.5 μM PI in 0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) for 15 min. Images were obtained using a Leica SP5 laser scanning confocal microscope with 63× objective.
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Fig. 5. Effect of TGFβ on CTGF mRNA expression. One microgram of total RNA extracted from C2C12 myoblasts, stimulated or not with 5 ng/ml TGFβ for the indicated time intervals, is analyzed by real-time RT-PCR to quantify CTGF mRNA expression according to the 2^(−ΔΔCt) method, as described in the Materials and Methods section. The quantification is performed as reported in Fig. 2A legend. The effect of TGFβ at all the indicated time intervals is statistically significant by one-way ANOVA, followed by Bonferroni's post hoc test, **P b 0.01; ***P b 0.001.
and Western blot analysis [15] and equally loaded protein was checked by expression of the nonmuscle-specific β isoform of actin [9]. 2.4. Quantitative real-time RT-PCR Total RNA (1 μg), extracted with TRI Reagent® from C2C12 myoblasts, was reverse transcribed using the High Capacity cDNA Reverse Transcription kit, according to the manufacturer's instructions. The quantification of SK1, SK2, CTGF and S1P receptor mRNA was performed by real-time PCR employing TaqMan gene expression assays. Each measurement was carried out in triplicate, using the automated ABI Prism 7700 Sequence Detector System essentially as previously described [16]. Simultaneous amplification of the target sequence (CTGF Mm01192932_g1, SK1 Mm00448841_g1, SK2 Mm00445021_m1, S1P1 Mm00514644_m1, S1P2 Mm01177794_m1, S1P3 Mm00515669_m1, S1P4 Mm00468695_s1) together with the housekeeping gene 18S rRNA was performed. Results were analyzed by ABI Prism Sequence Detection Systems software, version 1.7 (Applied Biosystems, Foster City, CA). The 2^(−ΔΔCt) method was applied as a comparative method of quantification [17], and data were normalized to ribosomal 18S RNA expression.
S1P was extracted and quantified as described [18]. Briefly, lipid extraction of conditioned cell media was performed using C17-S1P as internal standards. Sample analysis was carried out by rapid-resolution liquid chromatography-MS/MS using a Q-TOF 6530 mass spectrometer (Agilent Technologies, Waldbronn, Germany) operating in the positive ESI mode. The precursor ions of S1P (m/z 380.256) and C17-S1P (m/z 366.240) were cleaved into the fragment ions of m/z 264.270 and m/z 250.252, respectively. Quantification was performed with Mass Hunter Software (Agilent Technologies). 2.7. Cell transfection C2C12 myoblasts grown into tissue culture p35 plates (120.000 cells/well) were transfected with siRNA duplexes using Lipofectamine RNAiMAX®, according to the manufacturer's instructions, as previously described [9]. Briefly, Lipofectamine RNAiMAX® was incubated with specific siRNA duplexes in DMEM without serum and antibiotics at room temperature for 20 min, and afterward the lipid/RNA complexes were added with gentle agitation to cells to a final concentration of 50 nM in DMEM supplemented with serum. After 24 h, cells were shifted to DMEM without serum containing 1 mg/ml BSA and then used for the experiments at 48 h from the beginning of transfection. The specific gene knockdown was evaluated by real-time RT-PCR and occasionally confirmed by Western blot analysis. 2.8. Statistical analysis ImageJ software and analysis software Quantity One (Bio-Rad Laboratories, Hercules, CA) were used to perform densitometric analysis of the Western Blot bands. Graphical representations were obtained by GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Statistical analysis was performed using Student's t test, one-way ANOVA and two-way ANOVA followed by Bonferroni's post hoc test. Asterisks indicate statistical significance: *P b 0.05, **P b 0.01, ***P b 0.001, #P b 0.05, ##P b 0.01 and ###P b 0.001.
2.5. Immunostaining and fluorescence microscopy 3. Results C2C12 cells were seeded on microscope slides, pre-coated with 2% gelatin and then treated or not with 10 ng/ml CTGF. After 24 h, cells were fixed in 2% paraformaldehyde in PBS for 20 min and permeabilized in 0.1% Triton X-100–PBS for 30 min. Cells were then blocked with 3% BSA in DPBS for 1 h and incubated with anti-α-SMA antibody for 2 h
With the aim of investigating whether SK1/S1P3 signaling axis is mechanistically involved in the transmission of the profibrotic effect of CTGF in myoblasts, at first it was examined if in the adopted experimental conditions this protein was capable of inducing the expression
Fig. 6. CTGF expression is required for TGFβ-dependent profibrotic action and for TGFβ-induced SK1 and S1P3 expression in C2C12 myoblasts. (A) Upper left panel, quantitative analysis of CTGF mRNA is performed by real-time PCR in total RNA from C2C12 transfected with SCR- and specific murine CTGF-siRNA, as described in the Materials and Methods section. Results are expressed as fold change according to the 2^(−ΔΔCt) method, as described in Fig. 2A legend. The effect of siRNA transfection on CTGF mRNA expression is statistically significant by Student's t test, **P b 0.01. Lower left panel, CTGF expression is quantified by Western blot analysis in SCR- and CTGF-siRNA-transfected myoblasts, using a specific polyclonal anti-CTGF antibody. Densitometric analysis, normalized to β-actin content, is reported as fold change compared to control set as one, a representative experiment out of three with similar results. Upper-right panel, the profibrotic response of TGFβ is evaluated in SCR- and CTGF-siRNA-transfected cells treated or not with 5 ng/ml TGFβ for 18 h by measuring Tagln expression using Western blot analysis. Lower-right panel, data are reported as described in Fig. 1A legend. The effect of CTGF down-regulation on TGFβ-induced Tagln expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, #P b 0.05. (B) Left panel, quantitative mRNA measurement of SK1 is analyzed by real-time RT-PCR in total RNA from C2C12 transfected with SCR- and specific CTGF-siRNA, treated or not with 5 ng/ml TGFβ for 18 h. Results are showed as fold change according to the 2^(−ΔΔCt) method, as described in Fig. 2A legend. The effect of TGFβ on SK1 expression in SCR- and CTGF-siRNA-transfected myoblasts is statistically significant by one-way ANOVA followed by Bonferroni's post hoc test, ***P b 0.001 and *P b 0.05, respectively. The effect of CTGF-siRNA transfection on TGFβ-induced SK1 expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, #P b 0.05. Right panel, quantitative analysis of S1P3 is performed by real-time PCR in total RNA from C2C12 transfected with SCR- and specific CTGF-siRNA, treated or not with 5 ng/ml TGFβ for 18 h. Results are expressed as fold change according to the 2^(−ΔΔCt) method, as described in Fig. 2A legend. The effect of TGFβ on S1P3 expression in SCR-siRNAtransfected myoblasts is statistically significant by one-way ANOVA followed by Bonferroni's post hoc test, ***P b 0.001. The effect of CTGF-siRNA transfection on TGFβ-induced S1P3 expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, ##P b 0.01. Upper panel, SK1 protein expression is evaluated in SCR- and CTGF-siRNAtransfected C2C12 treated or not with 5 ng/ml TGFβ by Western blot analysis. Lower panel, data are reported as described in Fig. 1A legend. The effect of CTGF down-regulation on TGFβ-induced SK1 protein expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, ##P b 0.01.
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of transgelin recognized fibrosis marker protein previously observed to be significantly up-regulated by TGFβ in these cells [9]. As shown in Fig. 1A, CTGF dose-dependently enhanced at 24 h of incubation the expression of transgelin, measured by Western analysis, with the maximal
effect elicited at 10 ng/ml. This most efficacious concentration was then employed in all the subsequent experiments. The time course of the CTGF effect on transgelin expression was also investigated. The up-regulation of the fibrosis marker protein was already detectable at
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16 h and comparable to that observed at 24 h, while at 48 h it was no more appreciable (Fig. 1B). To further prove that CTGF was responsible for the transdifferentiation of myoblasts into myofibroblasts, confocal fluorescence analysis was performed. Results illustrated in Fig. 1C clearly show that α-SMA, key protein marker of fibrosis typically expressed in myofibroblasts, was greatly abundant at 24 h of CTGF treatment, whereas it was barely detectable in unchallenged myoblasts. Next, it was studied whether SK expression was regulated by CTGF. Data illustrated in Fig. 2A clearly show that already at 6 h, 10 ng/ml CTGF increased mRNA SK1, being the increase elicited at 18 h still robust; instead, SK2 mRNA content was not significantly affected. The up-regulation of SK1, but not that of SK2 following CTGF challenge, was also confirmed by the Western analysis of the protein content performed at 24 h (Fig. 2B). In agreement, 24 h treatment with 10 ng/ml CTGF provoked an appreciable increase of S1P content in the extracellular medium (Fig. 2C). Successive experiments were then addressed to understand whether the up-regulation of SK1 by CTGF was implicated in its profibrotic effect. Indeed, as illustrated in Fig. 3A, cell treatment with 1 μM VPC96047, specific pharmacological inhibitor of SK1 and SK2, or 1 μM VPC96091, selective inhibitor of the SK1 isoform, was capable of abrogating the enhancement of transgelin expression, supporting the hypothesis that at least SK1 is critically involved in the induction of the fibrotic response by CTGF. The involvement of SK1 or SK2 was further explored by transfecting myoblasts with selective siRNA that were efficacious in reducing the expression of SK1 or SK2 (Fig. 3B). In the same figure, it is shown that the protein content of transgelin was no more enhanced by CTGF challenge in cells where SK1 had been previously silenced, whereas SK2 downregulation did not affect the positive effect of CTGF on the levels of the fibrosis marker, confirming that SK1 is a critical player in the profibrotic response elicited by CTGF. The possible role of S1P receptors in the biological action of CTGF was then addressed. To this aim, it was examined whether treatment of myoblasts with CTGF could alter the expression of one or more S1P receptor subtypes. As depicted in Fig. 4A, S1P3 mRNA content was already enhanced by CTGF challenge at 6 h and still appreciably increased at 18 h, whereas the expression of S1P1, S1P2 or S1P4 was not affected. As a consequence, by analyzing S1P receptor expression profile, S1P1 was the most expressed in untreated cells whereas after 18 h of stimulation with CTGF S1P3 became the most abundant receptor subtype. Subsequently, the effect of pharmacological inhibition of S1P receptors on the profibrotic action of CTGF was studied. The blockade of S1P1 and S1P3 by myoblast treatment with 1 μM VPC23019 fully prevented the up-regulation of transgelin expression induced by CTGF, whereas the selective inhibition of S1P1 by cell treatment with 10 μM W146 did not alter the efficacy of the protein (Fig. 4B), suggesting the involvement of S1P3 in the transmission of CTGF action. The role of S1P3 was more deeply investigated in experiments where this receptor subtype was selectively silenced. Data illustrated in Fig. 4C show that cell transfection with specific siRNA efficaciously reduced the expression of S1P3 in control and CTGF-treated myoblasts. Interestingly, in this experimental condition, the enhancement of transgelin expression elicited by CTGF was significantly diminished further corroborating the critical involvement of S1P3 in conveying the biological effect elicited by CTGF. CTGF is reported to be autonomously produced by myoblasts in response to TGFβ challenge [12,13], thus playing a decisive role in the accomplishment of its final biological response. It was then regarded of interest to address whether S1P signaling axis could be exploited by endogenous CTGF for determining the fibrotic response directed by TGFβ, being this latter already known to depend on S1P signaling axis for inducing myoblast transdifferentiation into myofibroblasts [9]. For this purpose, at first it was examined whether, in the adopted experimental conditions, TGFβ effectively increased CTGF expression. As shown in Fig. 5, myoblast treatment with 5 ng/ml TGFβ resulted in an approximately thirty-fold increase in CTGF mRNA content as early as at 2 h of incubation, in full agreement with the notion that CTGF is encoded by
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a TGFβ-inducible immediate early gene that does not require de novo protein synthesis [19]. At more prolonged time points such as 18 and 24 h, the effect of TGFβ was further more potent. Then to examine the impact of TGFβ-induced CTGF expression onto the profibrotic effect of TGFβ, CTGF silencing was performed. Data illustrated in Fig. 6A show that myoblasts transfected with CTGFsiRNA exhibited a significant reduction of CTGF mRNA following 18 h treatment with 5 ng/ml TGFβ (upper left panel). In the same figure, it is shown that CTGF is appreciably down-regulated at protein level by specific silencing (lower left panel). In this experimental setting, the TGFβ-induced expression of transgelin was dramatically diminished when CTGF was specifically down-regulated, demonstrating that CTGF is a key transducer of TGFβ effect in myoblasts (Fig. 6A, right panel). Notably, results depicted in Fig. 6B show that the reduced expression of CTGF in response to TGFβ challenge was accompanied also by a diminished potency of TGFβ on SK1 mRNA induction and a full abrogation of SK1 protein up-regulation. Moreover, the TGFβ-dependent strong increase of S1P3 mRNA at 18 h of incubation was no more detectable when CTGF was silenced (Fig. 6C), supporting the view that SK1/S1P3 axis induced by CTGF is required for the TGFβ-dependent increase of fibrotic marker expression. 4. Discussion In this study, we have demonstrated that the increased expression of the profibrotic marker transgelin induced by CTGF in myoblasts requires SK1 and S1P3, which are both up-regulated by the treatment the profibrotic agent. This conclusion is drawn taking into consideration that the robustly enhanced expression of transgelin following cell treatment with CTGF was abolished when SK1 was blocked by a selective, isotype-specific, pharmacological inhibitor or specifically downregulated by gene silencing. Similarly, the up-regulation of transgelin by CTGF was no more detectable in myoblasts treated with the S1P1/ S1P3 antagonist VPC23019, but not with the S1P1 antagonist W146, while the profibrotic effect of CTGF was appreciably reduced when S1P3 was specifically silenced. These results point to a clear involvement of S1P3 in the biological action of CTGF and suggest that the partial inhibition observed when S1P3 was silenced could be attributable to the residual S1P3 expression following siRNA transfection. These experimental data are in accordance with a previous study of ours in which a strict dependence of the profibrotic effect of TGFβ on the induction of SK1/S1P3 axis in myoblasts was shown [9]. The present finding further supports the importance of SK1/S1P3 signaling axis in transmitting the fibrotic response in these cells. Moreover, it emphasizes the notion that extracellular cues can exploit S1P inside-out signaling to transmit specific biological responses not only by rapidly activating S1P receptors already present at plasma membrane but also by acting at more prolonged time points by increasing the expression of the biosynthetic molecular machinery required for S1P production and altering the pattern of S1P receptor expression, thus making S1P signaling axis furthermore versatile in the control of cell biology. CTGF is a protein involved in the extracellular matrix remodeling that occurs in a number of physiological processes but is also implicated in patho-physiological events. Importantly, its overproduction is associated with fibrosis of major organs [20]. A main reason of interest in CTGF depends on its induction by TGFβ and its role of mediator of at least some of the fibrogenic actions of TGFβ [21]. The involvement of CTGF in the fibrosis of skeletal muscle has been demonstrated in in vitro as well as in vivo studies. It has been reported several years ago that C2C12 myoblasts are able to express CTGF in response to TGFβ challenge and in the same study evidence was provided for the appreciable profibrotic action exerted by CTGF, via the induction of extracellular matrix protein expression, although the functional relationship between TGFβ and CTGF was not fully addressed [14]. Moreover, CTGF levels were found to be up-regulated in biopsies from muscular dystrophy patients [22], in accordance with the high degree of fibrosis that
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characterize these degenerative diseases, and reduced CTGF availability was recently reported capable of attenuating the severity of muscular dystrophy in mice [23]. The present study confirms that in myoblasts CTGF acts as profibrotic cue and provides clear-cut evidence that the TGFβ-induced expression of transgelin depends on the preceding upregulation of CTGF, thus functionally linking, at least partially, TGFβ fibrotic action in myoblasts to CTGF up-regulation. Differently from what observed for the most part of the extracellular agents, no canonical high affinity membrane receptor for CTGF has been identified thus far. In regard to its mechanism of action, CTGF is known to be able to interact with a variety of proteins present on the cell surface, including integrins and receptors such as TrkA, low-density lipoprotein receptor-related protein 1 and 6, as well as heparan sulfate proteoglycans and a number of growth factors [24]. The dissection of the signaling pathways triggered by CTGF to accomplish its biological action has been hampered by the occurrence of a wide variety of potential transducers and therefore not extensively investigated. Here, for the first time, it is reported that SK1/S1P3 signaling is downstream of CTGF in the array of molecular events leading to transgelin expression, thus providing new hints on the molecular mechanisms by which CTGF exerts its biological effect. Intriguingly, several line of evidence supports the view that S1P signaling system is involved in the regulation of CTGF expression. Indeed, S1P receptors via direct ligation of exogenous S1P, or via transactivation mediated by extracellular cues, were described to be coupled to the up-regulation of CTGF in different cell types [25–28], whereas the up-regulation of SK1 attenuated CTGF expression in podocytes [29]. The present identification of SK/S1P3 axis downstream of CTGF increases the knowledge on the complexity of functional interaction between CTGF and S1P signaling pathway. Presently, it remains to be established whether in a given cell type crossregulation takes place between S1P-induced CTGF expression and CTGF-directed S1P signaling, giving rise to a feed-forward, amplifying loop between S1P and CTGF. Remarkably, by performing the specific silencing of CTGF, it was demonstrated here that the up-regulation of SK1/S1P3 axis driven by TGFβ, necessary for causing the increase of transgelin expression, was actually mediated by its very rapid induction of CTGF expression. Therefore, CTGF generated following TGFβ appears to be the extracellular cue directly responsible for conveying the S1P inside-out signaling to act primarily via S1P3, that is coupled to fibrosis, rather than to S1P2, coupled instead to myogenesis [10,11]. Taking into account that SK/ S1P axis has been shown to be implicated in the transmission of the profibrotic effect of TGFβ in various types of fibroblasts [6–8], it will be of utmost interest to ascertain whether formation of CTGF in response to TGFβ is obligatorily required to stimulate S1P signaling axis. Gaining of further knowledge in the molecular mechanisms by which CTGF transmits its profibrotic effect appears to be highly desirable especially considering how CTGF represents an innovative therapeutic target in various types of tissue fibrosis. Acknowledgements This research was supported by grants from the University of Florence (ex 60%) to CD and the Fondazione Cassa di Risparmio di Lucca to PB (BRUNICRL12). The authors are indebted with Prof. K.L. Lynch and Dr. T.L. MacDonald, University of Virginia, Charlottesville, VA, USA, for the kind gift of VPC96047 and VPC96091. References [1] K. Takabe, S.W. Paugh, S. Milstien, S. Spiegel, “Inside-out” signaling of sphingosine1-phosphate: therapeutic targets, Pharmacol. Rev. 60 (2008) 181–195. [2] N.J. Pyne, S. Pyne, Sphingosine 1-phosphate, lysophosphatidic acid and growth factor signaling and termination, Biochim. Biophys. Acta 1781 (2008) 467–476.
[3] P. Xia, C. Wadham, Sphingosine 1-phosphate, a key mediator of the cytokine network: juxtacrine signaling, Cytokine Growth Factor Rev. 22 (2011) 45–53. [4] T. Nishi, N. Kobayashi, Y. Hisano, A. Kawahara, A. Yamaguchi, Molecular and physiological functions of sphingosine 1-phosphate transporters, Biochim. Biophys. Acta 1841 (2014) 759–765. [5] I. Ishii, N. Fukushima, X. Ye, J. Chun, Lysophospholipid receptors: signaling and biology, Annu. Rev. Biochem. 73 (2004) 321–354. [6] M. Yamanaka, D. Shegogue, H. Pei, S. Bu, A. Bielawska, J. Bielawski, et al., Sphingosine kinase 1 (SPHK1) is induced by transforming growth factor-beta and mediates TIMP-1 up-regulation, J. Biol. Chem. 279 (2004) 53994–54001. [7] N. Gellings Lowe, J.S. Swaney, K.M. Moreno, R.A. Sabbadini, Sphingosine-1-phosphate and sphingosine kinase are critical for transforming growth factor-betastimulated collagen production by cardiac fibroblasts, Cardiovasc. Res. 82 (2009) 303–312. [8] Y. Kono, T. Nishiuma, Y. Nishimura, Y. Kotani, T. Okada, S. Nakamura, et al., Sphingosine kinase 1 regulates differentiation of human and mouse lung fibroblasts mediated by TGF-beta1, Am. J. Respir. Cell Mol. Biol. 37 (2007) 395–404. [9] F. Cencetti, C. Bernacchioni, P. Nincheri, C. Donati, P. Bruni, Transforming growth factor-beta1 induces transdifferentiation of myoblasts into myofibroblasts via upregulation of sphingosine kinase-1/S1P3 axis, Mol. Biol. Cell 21 (2010) 1111–1124. [10] E. Meacci, F. Nuti, C. Donati, F. Cencetti, M. Farnararo, P. Bruni, Sphingosine kinase activity is required for myogenic differentiation of C2C12 myoblasts, J. Cell. Physiol. 214 (2008) 210–220. [11] C. Bernacchioni, F. Cencetti, S. Blescia, C. Donati, P. Bruni, Sphingosine kinase/sphingosine 1-phosphate axis: a new player for insulin-like growth factor-1-induced myoblast differentiation, Skelet. Muscle 2 (2012) 15. [12] J. Obreo, L. Diez-Marques, S. Lamas, A. Duwell, N. Eleno, C. Bernabeu, et al., Endoglin expression regulates basal and TGF-beta1-induced extracellular matrix synthesis in cultured L6E9 myoblasts, Cell. Physiol. Biochem. 14 (2004) 301–310. [13] N. Maeda, F. Kanda, S. Okuda, H. Ishihara, K. Chihara, Transforming growth factorbeta enhances connective tissue growth factor expression in L6 rat skeletal myotubes, Neuromuscul. Disord. 15 (2005) 790–793. [14] C. Vial, L.M. Zuniga, C. Cabello-Verrugio, P. Canon, R. Fadic, E. Brandan, Skeletal muscle cells express the profibrotic cytokine connective tissue growth factor (CTGF/ CCN2), which induces their dedifferentiation, J. Cell. Physiol. 215 (2008) 410–421. [15] P. Nincheri, C. Bernacchioni, F. Cencetti, C. Donati, P. Bruni, Sphingosine kinase-1/ S1P1 signalling axis negatively regulates mitogenic response elicited by PDGF in mouse myoblasts, Cell. Signal. 22 (2010) 1688–1699. [16] C. Donati, F. Cencetti, P. Nincheri, C. Bernacchioni, S. Brunelli, E. Clementi, et al., Sphingosine 1-phosphate mediates proliferation and survival of mesoangioblasts, Stem Cells 25 (2007) 1713–1719. [17] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(− Delta Delta C(T)) method, Methods 25 (2001) 402–408. [18] L. Japtok, K. Schaper, W. Baumer, H.H. Radeke, S.K. Jeong, B. Kleuser, Sphingosine 1phosphate modulates antigen capture by murine Langerhans cells via the S1P2 receptor subtype, PLoS One 7 (2012) e49427. [19] A. Igarashi, H. Okochi, D.M. Bradham, G.R. Grotendorst, Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair, Mol. Biol. Cell 4 (1993) 637–645. [20] A. Leask, A. Holmes, D.J. Abraham, Connective tissue growth factor: a new and important player in the pathogenesis of fibrosis, Curr. Rheumatol. Rep. 4 (2002) 136–142. [21] G.R. Grotendorst, Connective tissue growth factor: a mediator of TGF-beta action on fibroblasts, Cytokine Growth Factor Rev. 8 (1997) 171–179. [22] G. Sun, K. Haginoya, Y. Wu, Y. Chiba, T. Nakanishi, A. Onuma, et al., Connective tissue growth factor is overexpressed in muscles of human muscular dystrophy, J. Neurol. Sci. 267 (2008) 48–56. [23] M.G. Morales, J. Gutierrez, C. Cabello-Verrugio, D. Cabrera, K.E. Lipson, R. Goldschmeding, et al., Reducing CTGF/CCN2 slows down mdx muscle dystrophy and improves cell therapy, Hum. Mol. Genet. 22 (2013) 4938–4951. [24] E. Brandan, J. Gutierrez, Role of proteoglycans in the regulation of the skeletal muscle fibrotic response, FEBS J. 280 (2013) 4109–4117. [25] S. Katsuma, Y. Ruike, T. Yano, M. Kimura, A. Hirasawa, G. Tsujimoto, Transcriptional regulation of connective tissue growth factor by sphingosine 1-phosphate in rat cultured mesangial cells, FEBS Lett. 579 (2005) 2576–2582. [26] M. Markiewicz, S.S. Nakerakanti, B. Kapanadze, A. Ghatnekar, M. Trojanowska, Connective tissue growth factor (CTGF/CCN2) mediates angiogenic effect of S1P in human dermal microvascular endothelial cells, Microcirculation 18 (2011) 1–11. [27] H.M. El-Shewy, M. Sohn, P. Wilson, M.H. Lee, S.M. Hammad, L.M. Luttrell, et al., Lowdensity lipoprotein induced expression of connective tissue growth factor via transactivation of sphingosine 1-phosphate receptors in mesangial cells, Mol. Endocrinol. 26 (2012) 833–845. [28] M.H. Li, T. Sanchez, A. Pappalardo, K.R. Lynch, T. Hla, F. Ferrer, Induction of antiproliferative connective tissue growth factor expression in Wilms' tumor cells by sphingosine-1-phosphate receptor 2, Mol. Cancer Res. 6 (2008) 1649–1656. [29] S. Ren, A. Babelova, K. Moreth, C. Xin, W. Eberhardt, A. Doller, et al., Transforming growth factor-beta2 upregulates sphingosine kinase-1 activity, which in turn attenuates the fibrotic response to TGF-beta2 by impeding CTGF expression, Kidney Int. 76 (2009) 857–867.
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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip
CTGF/CCN2 exerts profibrotic action in myoblasts via the up-regulation of sphingosine kinase-1/S1P3 signaling axis: Implications in the action mechanism of TGFβ Gennaro Bruno a,1, Francesca Cencetti a,b,1, Irene Pertici a, Lukasz Japtok c, Caterina Bernacchioni a,b, Chiara Donati a,b, Paola Bruni a,b,⁎ a
Dipartimento di Scienze Biomediche Sperimentali e Cliniche “Mario Serio,” Università di Firenze, Viale G.B. Morgagni 50, Firenze 50134, Italy Istituto Interuniversitario di Miologia, Italy c Faculty of Mathematics and Natural Science, Institute of Nutritional Science, Department of Toxicology, University of Potsdam, Arthur-Scheunert Allee 114-116, Nuthetal, Potsdam 14558, Germany b
a r t i c l e
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Article history: Received 1 August 2014 Received in revised form 19 November 2014 Accepted 20 November 2014 Available online 29 November 2014 Keyword: Sphingosine kinase S1P3 receptor Connective tissue growth factor Myoblasts Transforming growth factor beta
a b s t r a c t The matricellular protein connective tissue growth factor (CTGF/CCN2) is recognized as key player in the onset of fibrosis in various tissues, including skeletal muscle. In many circumstances, CTGF has been shown to be induced by transforming growth factor beta (TGFβ) and accounting, at least in part, for its biological action. In this study it was verified that in cultured myoblasts CTGF/CCN2 causes their transdifferentiation into myofibroblasts by upregulating the expression of fibrosis marker proteins α-smooth muscle actin and transgelin. Interestingly, it was also found that the profibrotic effect exerted by CTGF/CCN2 was mediated by the sphingosine kinase (SK)-1/S1P3 signaling axis specifically induced by the treatment with the profibrotic cue. Following CTGF/CCN2-induced upregulation, S1P3 became the S1P receptor subtype expressed at the highest degree, at least at mRNA level, and was thus capable of readdressing the sphingosine 1-phosphate signaling towards fibrosis rather than myogenic differentiation. Another interesting finding is that CTGF/CCN2 silencing prevented the TGFβ-dependent up-regulation of SK1/S1P3 signaling axis and strongly reduced the profibrotic effect exerted by TGFβ, pointing at a crucial role of endogenous CTGF/CCN2 generated following TGFβ challenge in the transmission of at least part of its profibrotic effect. These results provide new insights into the molecular mechanism by which CTGF/CCN2 drives its biological action and strengthen the concept that SK1/S1P3 axis plays a critical role in the onset of fibrotic cell phenotype. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Inside-out sphingosine 1-phosphate (S1P) signaling represents a common molecular mechanism by which a wide array of extracellular agents, including hormones, cytokines, growth factors and neurotransmitters elicit, at least in part, their biological effects [1–3]. The molecular machinery necessary to activate in autocrine and/or in paracrine fashion one or more S1P receptor subtypes comprises sphingosine kinase(SK)-1, or less frequently SK2, both responsible for the intracellular formation of the bioactive sphingolipid S1P, and a membrane transporter that
Abbreviations: CTGF, connective tissue growth factor; TGFβ, transforming growth factor beta; SK, sphingosine kinase; S1P, sphingosine 1-phosphate; α-SMA, α-smooth muscle actin; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's modified phosphate-buffered saline; FCS, fetal calf serum; BSA, bovine serum albumin; siRNA, short interfering RNA; ECL, enhanced chemiluminescence; SDS–PAGE, sodium dodecyl sulfate– polyacrylamide gel electrophoresis; SCR, scrambled; PI, propidium iodide ⁎ Corresponding author at: Dipartimento di Scienze Biomediche Sperimentali e Cliniche “Mario Serio,” Viale G.B. Morgagni 50, 50134 Firenze, Italy. Tel.: +39 0552751204. E-mail address: paola.bruni@unifi.it (P. Bruni). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bbalip.2014.11.011 1388-1981/© 2014 Elsevier B.V. All rights reserved.
facilitates the release of generated S1P in the extracellular compartment [4]. Then activated S1P receptors, which are coupled to multiple heterotrimeric GTP binding proteins, coordinate the activation of numerous signaling pathways, thus accounting for the final biological response [5]. A wealth of experimental data support a key role of inside-out S1P signaling downstream of primary extracellular cues in the control of cell migration, proliferation and differentiation in a variety of cellular settings. In this regard, it has been firmly established that transforming growth factor beta (TGFβ) exploits S1P signaling axis to exert its profibrotic effect in fibroblasts. Indeed, SK1 is up-regulated by TGFβ in dermal fibroblasts and its expression is required for the induction of the tissue inhibitor of metalloproteases-1 that favors the deposition of extracellular matrix proteins by reducing their degradation [6]. Accordingly, it has been proved that SK1 and S1P2 are implicated in TGFβ-dependent biosynthesis of collagen in cardiac fibroblasts [7]. Moreover, TGFβ-induced transdifferentiation of lung fibroblasts into myofibroblasts, which plays a major role in the onset of pulmonary fibrosis, was found to rely on SK1 and both S1P2 and S1P3 [8]. Besides its major role in the transmission of the fibrotic effect of TGFβ in fibroblasts, S1P signaling has been also individuated as critical
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player in the onset of fibrotic phenotype in skeletal muscle precursor cells such as myoblasts. TGFβ was found to induce transdifferentiation of myoblasts into myofibroblasts, in turn responsible for extracellular matrix protein deposition, progressive replacement of the muscle fibers with connective tissue and impairment of skeletal muscle repair [9]. Notably, in cultured myoblasts S1P endogenously generated by SK1, via the ligation of S1P2, participates to the correct accomplishment of myogenic program that drives their differentiation into myotubes, multinucleated cells endowed with contractile properties that resemble skeletal myofibres [10]. In accordance, insulin-like growth factor-1, pivotal physiological regulator of myogenesis, exerts at least in part its biological action via SK1 activation and S1P2 engagement [11]. In contrast,
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TGFβ is capable of fully readdressing the inside-out signaling of S1P in myoblasts, by up-regulating SK1, that accounts for increased S1P production and enhancing the expression of S1P3 that, becoming the highest expressed receptor subtype, acquires a dominant signaling action [9]. Although the SK1/S1P3 axis has been identified as key player in the profibrotic action of TGFβ in myoblasts, at present is unknown whether it is implicated in the mechanism by which other profibrotic cues exert their biological action. Here, to address the overall relevance of the SK1/ S1P3 axis, we have examined whether this signaling pathway is downstream to another key inducer of skeletal muscle fibrosis such as connective tissue growth factor (CTGF), also known as CCN2, a member of the CCN family of secreted matricellular proteins. This protein, produced
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Fig. 1. CTGF exerts a profibrotic action in C2C12 myoblasts. (A) Dose–response effect of CTGF on the transdifferentiation of myoblasts into myofibrobalsts is evaluated after treatment with the indicated concentration of CTGF by analyzing the expression of the fibrosis marker Transgelin (Tagln) in 10 μg of protein in total cell lysate. Upper panel, Western blot analysis is performed by using specific anti-Tagln antibody. Lower panel, results are obtained by densitometric analysis, normalized to β-actin content in each specimen, and data are reported as mean ± SEM of three independent experiments, fold change compared to control, set as one. The effect of CTGF is statistically significant by one-way ANOVA, followed by Bonferroni's post hoc test, *P b 0.05. (B) Upper panel, time-dependent effect of CTGF in myoblasts is analyzed by measuring the expression of Tagln at the indicated time points. Lower panel, results are analyzed and reported as described in section A. The effect of CTGF at 16 h and 24 h is statistically significant by one-way ANOVA, followed by Bonferroni's post hoc test, *P b 0.05, **P b 0.01. (C) Immunofluorescence analysis of the fibrosis marker α-smooth muscle actin (α-SMA) is performed in myoblasts treated with CTGF for 24 h, using a specific mouse anti-α-SMA antibody, fluorescein-conjugated anti-mouse secondary antibody and propidium iodide (PI) staining (images are representative of three separate experiments).
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by myoblasts in response to TGFβ [12,13], has been shown to induce the expression of extracellular matrix constituents such as fibronectin and to cause also dedifferentiation of committed myoblasts [14]. Notably, in the present study, it is shown that the profibrotic effect exerted by CTGF requires the SK1/S1P3 axis, reinforcing the notion that SK1/S1P3 signaling axis is critical for transdifferentiation of myoblasts into myofibroblasts. Moreover, experimental evidence is presented for a key role of TGFβ-directed induction of CTGF expression in the switch to SK1/S1P3 signaling axis and the execution of the profibrotic response, providing a more detailed view onto the mechanisms that regulate the fibrotic response to TGFβ in myoblasts.
reagents, protease inhibitor cocktail, TRI Reagent® RNA Isolation Reagent, bovine serum albumin (BSA) and monoclonal anti-α-smooth muscle actin (α-SMA) antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), Dulbecco's modified phosphate-buffered saline (DPBS) and fetal calf serum (FCS) were obtained from Lonza (Basel, Switzerland). TGFβ1 and CTGF (lower molecular weight isoform of 11 kDa) were purchased from PeproTech (London, UK). The selective S1P1 antagonist, W146, and the specific S1P1/S1P3 antagonist, VPC23019, were from Avanti Polar Lipids (Alabaster, AL, USA). Short interfering RNA (siRNA) duplexes targeting specific gene of interest such as mouse CTGF (SASI_Mm01_00097842 and SASI_Mm01_00097843), mouse S1P3 (SASI_Mm01_00145233 and SASI_Mm01_00145234), mouse SK1 (SASI_Mm01_00033983 and SASI_Mm01_00033984), mouse SK2 (SASI_Mm01_00050883 and SASI_Mm01_00050884) and scrambled (SCR)-siRNA (Mission Universal Negative control no. 1) were from Sigma-Proligo (The Woodlands, TX, USA). Lipofectamine RNAiMAX® Transfection Reagent and all reagents and probes used to perform real-
2. Materials and methods 2.1. Materials Mouse skeletal muscle C2C12 cells were obtained from the European Collection of Cell Cultures (Salisbury, UK). Biochemicals, cell culture
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Fig. 2. CTGF induces SK1 expression and S1P production in murine myoblasts. (A) One microgram of total RNA extracted from C2C12 myoblasts, stimulated or not with 10 ng/ml CTGF for the indicated time intervals is analyzed by real-time RT-PCR. The quantification of SK1 and SK2 mRNA is based on the 2^(−ΔΔCt) method as described in the Materials and Methods section. The target gene of the unchallenged specimen is used as calibrator and the quantification of housekeeping gene 18S rRNA is performed in parallel. Data reported are means ± SEM of three independent experiments performed in triplicate. The effect of CTGF at 6 h and 18 h of treatment on SK1 mRNA levels is statistically significant by Student's t test (*P b 0.05). (B) Left panel, 10 μg of protein of total cell lysate is subjected to Western blot analysis by using specific anti-SK1 and anti-SK2 antibodies. Right panel, results are reported as described in Fig. 1A legend. The effect of CTGF on SK1 protein expression is statistically significant by Student's t test, *P b 0.05. (C) C2C12 myoblasts were treated with 10 ng/ml CTGF for the indicated time intervals; media were collected and subjected to S1P analysis. The effect of CTGF on S1P levels is statistically significant by Student's t test, *P b 0.05.
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time PCR were obtained from Life Technologies (Carlsbad, CA, USA). Goat anti-transgelin antibody was from Everest Biotech (Oxfordshire, OX, UK). Monoclonal antibody against β-actin was from Cytoskeleton Inc. (Denver, CO, USA). SK2 (N-terminal region) rabbit polyclonal and SK1 (central region) rabbit polyclonal antibodies were purchased from ECM Biosciences LLC (Versailles, KY USA). Secondary antibodies conjugated to horseradish peroxidase and rabbit polyclonal anti-CTGF antibody
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were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Fluorescein-conjugated anti-mouse secondary antibody and Vectashield® mounting medium were purchased from Vector Laboratories (Burlingame, CA, USA). Enhanced chemiluminescence (ECL) reagents were obtained from GE Healthcare Europe GmbH (Milan, Italy). Pharmacological inhibitors of SK (VPC96091 and VPC96047) were kindly provided by Prof. K. Lynch, University of Virginia, USA.
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Fig. 3. SK1 is involved in CTGF-mediated profibrotic effect. (A) C2C12 myoblasts are incubated in the presence of 1 μM VPC96047 or 1 μM VPC96091, 30 min before 10 ng/ml CTGF challenge for 24 h. Upper panel, the fibrosis marker protein Tagln is detected by Western Blotting as described in Fig. 1A legend. Lower panel, densitometric analysis is reported as described in Fig. 1A legend. The effect of VPC96047 and VPC96091 treatment on CTGF-induced profibrotic action is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, ## P b 0.01. (B) Left panel, quantitative analysis of SK1 and SK2 mRNA is performed by real-time RT-PCR in total RNA from C2C12 transfected with SCR-, SK1- or SK2- specific siRNA, as described in the Materials and Methods section. Results are expressed as fold change according to the 2^(−ΔΔCt) method, as described in Fig. 2A legend. The effect of siRNA transfection on SK1 and SK2 expression is statistically significant by Student's t test, **P b 0.01. Upper-right panel, the profibrotic response of CTGF is evaluated in SCR-, SK1- and SK2-siRNA-transfected C2C12 treated or not with 10 ng/ml CTGF by measuring Tagln expression performing Western blot analysis. Lower-right panel, data are reported as described in Fig. 1A legend. The effect of SK1 down-regulation on CTGF-induced Tagln expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, ##P b 0.01.
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10 μM W146, 1 μM VPC23019, 1 μM VPC96047 or 1 μM VPC96091, 30 min before agonist stimulation.
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Murine C2C12 myoblasts were routinely grown in DMEM containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in 5% CO2. For myofibroblasts transdifferentiation experiments, cells were seeded in p35 and when 90% confluent were shifted to DMEM without serum supplemented with 1 mg/ml BSA and incubated in the presence or absence of 5 ng/ml TGFβ1 or 10 ng/ml CTGF for the indicated times. In some of the experiments, cells were pre-treated with
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Fig. 4. Role of S1P3 in the profibrotic effect exerted by CTGF. (A) One microgram of total RNA extracted from C2C12 myoblasts, stimulated or not with 10 ng/ml CTGF for the indicated time intervals, is analyzed by real-time RT-PCR. S1P1–4 mRNA quantification is based on the 2^(−ΔΔCt) method, and the analysis is performed as described in Fig. 2A legend by using specific murine S1P1–4 gene expression assays. Upper panel, relative expression of S1P receptor subtypes is analyzed using as calibrator each receptor subtype in the control specimen. Lower panel, the expression profile of the S1P receptor subtypes is analyzed utilizing as calibrator S1P1. The effect of CTGF at 6 h and 18 h of treatment on S1P3 mRNA levels is statistically significant by Student's t test, **P b 0.01; ***P b 0.001. (B) C2C12 myoblasts are incubated in the presence of 10 μM W146 or 1 μM VPC23019, 30 min before 10 ng/ml CTGF challenge for 24 h. Upper panel, the fibrosis marker protein Tagln is detected as described in Fig. 1A legend. Lower panel, densitometric analysis is reported as described in Fig. 1A legend. The effect of VPC23019 treatment on CTGF-induced Tagln expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, ##P b 0.01. (C) Left panel, quantitative analysis of S1P3 mRNA is performed by real-time PCR in total RNA from C2C12 transfected with SCR- and specific S1P3-siRNA, as described in the Materials and Methods section. Results are expressed as fold change according to the 2^(−ΔΔCT) method, as described in Fig. 2A legend. The effect of siRNA transfection on S1P3 expression is statistically significant by Student's t test (*P b 0.05). Upper-right panel, the profibrotic response of CTGF is evaluated in SCR- and S1P3-siRNA-transfected C2C12 treated or not with 10 ng/ml CTGF by measuring Tagln expression using Western blot analysis. Lower-right panel, data are reported as described in Fig. 1A legend. The effect of S1P3 down-regulation on CTGF-induced Tagln expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test (##P b 0.01). The effect of CTGF treatment on Tagln expression in SCR-siRNA and S1P3-siRNA-transfected myoblasts is statistically significant by one-way ANOVA, followed by Bonferroni's post hoc test, **P b 0.01, *P b 0.05.
CTGF mRNA expression levels (2^-ΔΔ ΔΔ Ct)
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and fluorescein-conjugated anti-mouse secondary antibody for 1 h. For nuclear counter-staining, the specimen was incubated with propidium iodide (PI) solution (0.5 μM PI in 0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) for 15 min. Images were obtained using a Leica SP5 laser scanning confocal microscope with 63× objective.
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Fig. 5. Effect of TGFβ on CTGF mRNA expression. One microgram of total RNA extracted from C2C12 myoblasts, stimulated or not with 5 ng/ml TGFβ for the indicated time intervals, is analyzed by real-time RT-PCR to quantify CTGF mRNA expression according to the 2^(−ΔΔCt) method, as described in the Materials and Methods section. The quantification is performed as reported in Fig. 2A legend. The effect of TGFβ at all the indicated time intervals is statistically significant by one-way ANOVA, followed by Bonferroni's post hoc test, **P b 0.01; ***P b 0.001.
and Western blot analysis [15] and equally loaded protein was checked by expression of the nonmuscle-specific β isoform of actin [9]. 2.4. Quantitative real-time RT-PCR Total RNA (1 μg), extracted with TRI Reagent® from C2C12 myoblasts, was reverse transcribed using the High Capacity cDNA Reverse Transcription kit, according to the manufacturer's instructions. The quantification of SK1, SK2, CTGF and S1P receptor mRNA was performed by real-time PCR employing TaqMan gene expression assays. Each measurement was carried out in triplicate, using the automated ABI Prism 7700 Sequence Detector System essentially as previously described [16]. Simultaneous amplification of the target sequence (CTGF Mm01192932_g1, SK1 Mm00448841_g1, SK2 Mm00445021_m1, S1P1 Mm00514644_m1, S1P2 Mm01177794_m1, S1P3 Mm00515669_m1, S1P4 Mm00468695_s1) together with the housekeeping gene 18S rRNA was performed. Results were analyzed by ABI Prism Sequence Detection Systems software, version 1.7 (Applied Biosystems, Foster City, CA). The 2^(−ΔΔCt) method was applied as a comparative method of quantification [17], and data were normalized to ribosomal 18S RNA expression.
S1P was extracted and quantified as described [18]. Briefly, lipid extraction of conditioned cell media was performed using C17-S1P as internal standards. Sample analysis was carried out by rapid-resolution liquid chromatography-MS/MS using a Q-TOF 6530 mass spectrometer (Agilent Technologies, Waldbronn, Germany) operating in the positive ESI mode. The precursor ions of S1P (m/z 380.256) and C17-S1P (m/z 366.240) were cleaved into the fragment ions of m/z 264.270 and m/z 250.252, respectively. Quantification was performed with Mass Hunter Software (Agilent Technologies). 2.7. Cell transfection C2C12 myoblasts grown into tissue culture p35 plates (120.000 cells/well) were transfected with siRNA duplexes using Lipofectamine RNAiMAX®, according to the manufacturer's instructions, as previously described [9]. Briefly, Lipofectamine RNAiMAX® was incubated with specific siRNA duplexes in DMEM without serum and antibiotics at room temperature for 20 min, and afterward the lipid/RNA complexes were added with gentle agitation to cells to a final concentration of 50 nM in DMEM supplemented with serum. After 24 h, cells were shifted to DMEM without serum containing 1 mg/ml BSA and then used for the experiments at 48 h from the beginning of transfection. The specific gene knockdown was evaluated by real-time RT-PCR and occasionally confirmed by Western blot analysis. 2.8. Statistical analysis ImageJ software and analysis software Quantity One (Bio-Rad Laboratories, Hercules, CA) were used to perform densitometric analysis of the Western Blot bands. Graphical representations were obtained by GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Statistical analysis was performed using Student's t test, one-way ANOVA and two-way ANOVA followed by Bonferroni's post hoc test. Asterisks indicate statistical significance: *P b 0.05, **P b 0.01, ***P b 0.001, #P b 0.05, ##P b 0.01 and ###P b 0.001.
2.5. Immunostaining and fluorescence microscopy 3. Results C2C12 cells were seeded on microscope slides, pre-coated with 2% gelatin and then treated or not with 10 ng/ml CTGF. After 24 h, cells were fixed in 2% paraformaldehyde in PBS for 20 min and permeabilized in 0.1% Triton X-100–PBS for 30 min. Cells were then blocked with 3% BSA in DPBS for 1 h and incubated with anti-α-SMA antibody for 2 h
With the aim of investigating whether SK1/S1P3 signaling axis is mechanistically involved in the transmission of the profibrotic effect of CTGF in myoblasts, at first it was examined if in the adopted experimental conditions this protein was capable of inducing the expression
Fig. 6. CTGF expression is required for TGFβ-dependent profibrotic action and for TGFβ-induced SK1 and S1P3 expression in C2C12 myoblasts. (A) Upper left panel, quantitative analysis of CTGF mRNA is performed by real-time PCR in total RNA from C2C12 transfected with SCR- and specific murine CTGF-siRNA, as described in the Materials and Methods section. Results are expressed as fold change according to the 2^(−ΔΔCt) method, as described in Fig. 2A legend. The effect of siRNA transfection on CTGF mRNA expression is statistically significant by Student's t test, **P b 0.01. Lower left panel, CTGF expression is quantified by Western blot analysis in SCR- and CTGF-siRNA-transfected myoblasts, using a specific polyclonal anti-CTGF antibody. Densitometric analysis, normalized to β-actin content, is reported as fold change compared to control set as one, a representative experiment out of three with similar results. Upper-right panel, the profibrotic response of TGFβ is evaluated in SCR- and CTGF-siRNA-transfected cells treated or not with 5 ng/ml TGFβ for 18 h by measuring Tagln expression using Western blot analysis. Lower-right panel, data are reported as described in Fig. 1A legend. The effect of CTGF down-regulation on TGFβ-induced Tagln expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, #P b 0.05. (B) Left panel, quantitative mRNA measurement of SK1 is analyzed by real-time RT-PCR in total RNA from C2C12 transfected with SCR- and specific CTGF-siRNA, treated or not with 5 ng/ml TGFβ for 18 h. Results are showed as fold change according to the 2^(−ΔΔCt) method, as described in Fig. 2A legend. The effect of TGFβ on SK1 expression in SCR- and CTGF-siRNA-transfected myoblasts is statistically significant by one-way ANOVA followed by Bonferroni's post hoc test, ***P b 0.001 and *P b 0.05, respectively. The effect of CTGF-siRNA transfection on TGFβ-induced SK1 expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, #P b 0.05. Right panel, quantitative analysis of S1P3 is performed by real-time PCR in total RNA from C2C12 transfected with SCR- and specific CTGF-siRNA, treated or not with 5 ng/ml TGFβ for 18 h. Results are expressed as fold change according to the 2^(−ΔΔCt) method, as described in Fig. 2A legend. The effect of TGFβ on S1P3 expression in SCR-siRNAtransfected myoblasts is statistically significant by one-way ANOVA followed by Bonferroni's post hoc test, ***P b 0.001. The effect of CTGF-siRNA transfection on TGFβ-induced S1P3 expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, ##P b 0.01. Upper panel, SK1 protein expression is evaluated in SCR- and CTGF-siRNAtransfected C2C12 treated or not with 5 ng/ml TGFβ by Western blot analysis. Lower panel, data are reported as described in Fig. 1A legend. The effect of CTGF down-regulation on TGFβ-induced SK1 protein expression is statistically significant by two-way ANOVA, followed by Bonferroni's post hoc test, ##P b 0.01.
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of transgelin recognized fibrosis marker protein previously observed to be significantly up-regulated by TGFβ in these cells [9]. As shown in Fig. 1A, CTGF dose-dependently enhanced at 24 h of incubation the expression of transgelin, measured by Western analysis, with the maximal
effect elicited at 10 ng/ml. This most efficacious concentration was then employed in all the subsequent experiments. The time course of the CTGF effect on transgelin expression was also investigated. The up-regulation of the fibrosis marker protein was already detectable at
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16 h and comparable to that observed at 24 h, while at 48 h it was no more appreciable (Fig. 1B). To further prove that CTGF was responsible for the transdifferentiation of myoblasts into myofibroblasts, confocal fluorescence analysis was performed. Results illustrated in Fig. 1C clearly show that α-SMA, key protein marker of fibrosis typically expressed in myofibroblasts, was greatly abundant at 24 h of CTGF treatment, whereas it was barely detectable in unchallenged myoblasts. Next, it was studied whether SK expression was regulated by CTGF. Data illustrated in Fig. 2A clearly show that already at 6 h, 10 ng/ml CTGF increased mRNA SK1, being the increase elicited at 18 h still robust; instead, SK2 mRNA content was not significantly affected. The up-regulation of SK1, but not that of SK2 following CTGF challenge, was also confirmed by the Western analysis of the protein content performed at 24 h (Fig. 2B). In agreement, 24 h treatment with 10 ng/ml CTGF provoked an appreciable increase of S1P content in the extracellular medium (Fig. 2C). Successive experiments were then addressed to understand whether the up-regulation of SK1 by CTGF was implicated in its profibrotic effect. Indeed, as illustrated in Fig. 3A, cell treatment with 1 μM VPC96047, specific pharmacological inhibitor of SK1 and SK2, or 1 μM VPC96091, selective inhibitor of the SK1 isoform, was capable of abrogating the enhancement of transgelin expression, supporting the hypothesis that at least SK1 is critically involved in the induction of the fibrotic response by CTGF. The involvement of SK1 or SK2 was further explored by transfecting myoblasts with selective siRNA that were efficacious in reducing the expression of SK1 or SK2 (Fig. 3B). In the same figure, it is shown that the protein content of transgelin was no more enhanced by CTGF challenge in cells where SK1 had been previously silenced, whereas SK2 downregulation did not affect the positive effect of CTGF on the levels of the fibrosis marker, confirming that SK1 is a critical player in the profibrotic response elicited by CTGF. The possible role of S1P receptors in the biological action of CTGF was then addressed. To this aim, it was examined whether treatment of myoblasts with CTGF could alter the expression of one or more S1P receptor subtypes. As depicted in Fig. 4A, S1P3 mRNA content was already enhanced by CTGF challenge at 6 h and still appreciably increased at 18 h, whereas the expression of S1P1, S1P2 or S1P4 was not affected. As a consequence, by analyzing S1P receptor expression profile, S1P1 was the most expressed in untreated cells whereas after 18 h of stimulation with CTGF S1P3 became the most abundant receptor subtype. Subsequently, the effect of pharmacological inhibition of S1P receptors on the profibrotic action of CTGF was studied. The blockade of S1P1 and S1P3 by myoblast treatment with 1 μM VPC23019 fully prevented the up-regulation of transgelin expression induced by CTGF, whereas the selective inhibition of S1P1 by cell treatment with 10 μM W146 did not alter the efficacy of the protein (Fig. 4B), suggesting the involvement of S1P3 in the transmission of CTGF action. The role of S1P3 was more deeply investigated in experiments where this receptor subtype was selectively silenced. Data illustrated in Fig. 4C show that cell transfection with specific siRNA efficaciously reduced the expression of S1P3 in control and CTGF-treated myoblasts. Interestingly, in this experimental condition, the enhancement of transgelin expression elicited by CTGF was significantly diminished further corroborating the critical involvement of S1P3 in conveying the biological effect elicited by CTGF. CTGF is reported to be autonomously produced by myoblasts in response to TGFβ challenge [12,13], thus playing a decisive role in the accomplishment of its final biological response. It was then regarded of interest to address whether S1P signaling axis could be exploited by endogenous CTGF for determining the fibrotic response directed by TGFβ, being this latter already known to depend on S1P signaling axis for inducing myoblast transdifferentiation into myofibroblasts [9]. For this purpose, at first it was examined whether, in the adopted experimental conditions, TGFβ effectively increased CTGF expression. As shown in Fig. 5, myoblast treatment with 5 ng/ml TGFβ resulted in an approximately thirty-fold increase in CTGF mRNA content as early as at 2 h of incubation, in full agreement with the notion that CTGF is encoded by
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a TGFβ-inducible immediate early gene that does not require de novo protein synthesis [19]. At more prolonged time points such as 18 and 24 h, the effect of TGFβ was further more potent. Then to examine the impact of TGFβ-induced CTGF expression onto the profibrotic effect of TGFβ, CTGF silencing was performed. Data illustrated in Fig. 6A show that myoblasts transfected with CTGFsiRNA exhibited a significant reduction of CTGF mRNA following 18 h treatment with 5 ng/ml TGFβ (upper left panel). In the same figure, it is shown that CTGF is appreciably down-regulated at protein level by specific silencing (lower left panel). In this experimental setting, the TGFβ-induced expression of transgelin was dramatically diminished when CTGF was specifically down-regulated, demonstrating that CTGF is a key transducer of TGFβ effect in myoblasts (Fig. 6A, right panel). Notably, results depicted in Fig. 6B show that the reduced expression of CTGF in response to TGFβ challenge was accompanied also by a diminished potency of TGFβ on SK1 mRNA induction and a full abrogation of SK1 protein up-regulation. Moreover, the TGFβ-dependent strong increase of S1P3 mRNA at 18 h of incubation was no more detectable when CTGF was silenced (Fig. 6C), supporting the view that SK1/S1P3 axis induced by CTGF is required for the TGFβ-dependent increase of fibrotic marker expression. 4. Discussion In this study, we have demonstrated that the increased expression of the profibrotic marker transgelin induced by CTGF in myoblasts requires SK1 and S1P3, which are both up-regulated by the treatment the profibrotic agent. This conclusion is drawn taking into consideration that the robustly enhanced expression of transgelin following cell treatment with CTGF was abolished when SK1 was blocked by a selective, isotype-specific, pharmacological inhibitor or specifically downregulated by gene silencing. Similarly, the up-regulation of transgelin by CTGF was no more detectable in myoblasts treated with the S1P1/ S1P3 antagonist VPC23019, but not with the S1P1 antagonist W146, while the profibrotic effect of CTGF was appreciably reduced when S1P3 was specifically silenced. These results point to a clear involvement of S1P3 in the biological action of CTGF and suggest that the partial inhibition observed when S1P3 was silenced could be attributable to the residual S1P3 expression following siRNA transfection. These experimental data are in accordance with a previous study of ours in which a strict dependence of the profibrotic effect of TGFβ on the induction of SK1/S1P3 axis in myoblasts was shown [9]. The present finding further supports the importance of SK1/S1P3 signaling axis in transmitting the fibrotic response in these cells. Moreover, it emphasizes the notion that extracellular cues can exploit S1P inside-out signaling to transmit specific biological responses not only by rapidly activating S1P receptors already present at plasma membrane but also by acting at more prolonged time points by increasing the expression of the biosynthetic molecular machinery required for S1P production and altering the pattern of S1P receptor expression, thus making S1P signaling axis furthermore versatile in the control of cell biology. CTGF is a protein involved in the extracellular matrix remodeling that occurs in a number of physiological processes but is also implicated in patho-physiological events. Importantly, its overproduction is associated with fibrosis of major organs [20]. A main reason of interest in CTGF depends on its induction by TGFβ and its role of mediator of at least some of the fibrogenic actions of TGFβ [21]. The involvement of CTGF in the fibrosis of skeletal muscle has been demonstrated in in vitro as well as in vivo studies. It has been reported several years ago that C2C12 myoblasts are able to express CTGF in response to TGFβ challenge and in the same study evidence was provided for the appreciable profibrotic action exerted by CTGF, via the induction of extracellular matrix protein expression, although the functional relationship between TGFβ and CTGF was not fully addressed [14]. Moreover, CTGF levels were found to be up-regulated in biopsies from muscular dystrophy patients [22], in accordance with the high degree of fibrosis that
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characterize these degenerative diseases, and reduced CTGF availability was recently reported capable of attenuating the severity of muscular dystrophy in mice [23]. The present study confirms that in myoblasts CTGF acts as profibrotic cue and provides clear-cut evidence that the TGFβ-induced expression of transgelin depends on the preceding upregulation of CTGF, thus functionally linking, at least partially, TGFβ fibrotic action in myoblasts to CTGF up-regulation. Differently from what observed for the most part of the extracellular agents, no canonical high affinity membrane receptor for CTGF has been identified thus far. In regard to its mechanism of action, CTGF is known to be able to interact with a variety of proteins present on the cell surface, including integrins and receptors such as TrkA, low-density lipoprotein receptor-related protein 1 and 6, as well as heparan sulfate proteoglycans and a number of growth factors [24]. The dissection of the signaling pathways triggered by CTGF to accomplish its biological action has been hampered by the occurrence of a wide variety of potential transducers and therefore not extensively investigated. Here, for the first time, it is reported that SK1/S1P3 signaling is downstream of CTGF in the array of molecular events leading to transgelin expression, thus providing new hints on the molecular mechanisms by which CTGF exerts its biological effect. Intriguingly, several line of evidence supports the view that S1P signaling system is involved in the regulation of CTGF expression. Indeed, S1P receptors via direct ligation of exogenous S1P, or via transactivation mediated by extracellular cues, were described to be coupled to the up-regulation of CTGF in different cell types [25–28], whereas the up-regulation of SK1 attenuated CTGF expression in podocytes [29]. The present identification of SK/S1P3 axis downstream of CTGF increases the knowledge on the complexity of functional interaction between CTGF and S1P signaling pathway. Presently, it remains to be established whether in a given cell type crossregulation takes place between S1P-induced CTGF expression and CTGF-directed S1P signaling, giving rise to a feed-forward, amplifying loop between S1P and CTGF. Remarkably, by performing the specific silencing of CTGF, it was demonstrated here that the up-regulation of SK1/S1P3 axis driven by TGFβ, necessary for causing the increase of transgelin expression, was actually mediated by its very rapid induction of CTGF expression. Therefore, CTGF generated following TGFβ appears to be the extracellular cue directly responsible for conveying the S1P inside-out signaling to act primarily via S1P3, that is coupled to fibrosis, rather than to S1P2, coupled instead to myogenesis [10,11]. Taking into account that SK/ S1P axis has been shown to be implicated in the transmission of the profibrotic effect of TGFβ in various types of fibroblasts [6–8], it will be of utmost interest to ascertain whether formation of CTGF in response to TGFβ is obligatorily required to stimulate S1P signaling axis. Gaining of further knowledge in the molecular mechanisms by which CTGF transmits its profibrotic effect appears to be highly desirable especially considering how CTGF represents an innovative therapeutic target in various types of tissue fibrosis. Acknowledgements This research was supported by grants from the University of Florence (ex 60%) to CD and the Fondazione Cassa di Risparmio di Lucca to PB (BRUNICRL12). The authors are indebted with Prof. K.L. Lynch and Dr. T.L. MacDonald, University of Virginia, Charlottesville, VA, USA, for the kind gift of VPC96047 and VPC96091. References [1] K. Takabe, S.W. Paugh, S. Milstien, S. Spiegel, “Inside-out” signaling of sphingosine1-phosphate: therapeutic targets, Pharmacol. Rev. 60 (2008) 181–195. [2] N.J. Pyne, S. Pyne, Sphingosine 1-phosphate, lysophosphatidic acid and growth factor signaling and termination, Biochim. Biophys. Acta 1781 (2008) 467–476.
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