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Apocynin inhibits the upregulation of TGF-β1 expression and ROS production induced by TGF-β in skeletal muscle cells
Phytomedicine 22 (2015) 885–893
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Apocynin inhibits the upregulation of TGF-β 1 expression and ROS production induced by TGF-β in skeletal muscle cells Johanna Abrigo a,c, María Gabriela Morales a,c, Felipe Simon b,c, Daniel Cabrera d, Gabriella Di Capua a,c, Claudio Cabello-Verrugio a,c,∗ a Laboratorio de Biología y Fisiopatología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile b Laboratorio de Fisiopatología Integrativa, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile c Millennium Institute on Immunology and Immunotherapy, Santiago, Chile d Departamento de Ciencias Químicas y Biológicas, Universidad Bernardo O´Higgins, Santiago, Chile
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Article history: Received 2 March 2015 Revised 18 June 2015 Accepted 21 June 2015
Keywords: TGF-β 1 Smad ROS NOX Atrophy Skeletal muscle
a b s t r a c t Background: Pure apocynin, which can be traditionally isolated and purified from several plant species such as Picrorhiza kurroa Royle ex Benth (Scrophulariaceae), acts as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity inhibiting its production of reactive oxygen species (ROS). Transforming growth factor type beta 1 (TGF-β 1) is a growth factor that produces inhibition of myogenesis, diminution of regeneration and induction of atrophy in skeletal muscle. The typical signalling that is activated by TGF-β involves the Smad pathway. Purpose: To evaluate the effect of TGF-β and the effect of apocynin on TGF-β 1 expression in skeletal muscle cells. Study design: Controlled laboratory study. In vitro assays were performed with C2 C12 cells incubated with TGF-β 1 in presence or absence of apocynin (NOX inhibitor), SB525334 (TGF-β -receptor I inhibitor), or chelerythrine (PKC inhibitor). Methods: TGF-β 1 and atrogin-1 expression was evaluated by RT-qPCR and/or ELISA; Smad3 phosphorylation by western blot; Smad4 nuclear translocation by indirect immunofluorescence; and ROS levels by DCF probe fluorescent measurements. Results: We show that myoblasts respond to TGF-β 1 by increasing its own gene expression in a time- and dose-dependent fashion which was abolished by SB525334 and siRNA for Smad2/3. TGF-β 1 also induced ROS. Remarkably, apocynin inhibited the TGF-β 1 induced ROS as well as the autoinduction of TGF-β 1 gene expression. We also show that TGF-β -induced ROS production and TGF-β 1 expression require PKC activity as indicated by the inhibition using chelerythrine. Conclusion: These results strongly suggest that TGF-β induces its own expression through a TGF-β receptor/Smad-dependent mechanism and apocynin is able to inhibit this process, suggesting that requires NOX-induced ROS in skeletal muscle cells. © 2015 Elsevier GmbH. All rights reserved.
Introduction
Abbreviations: Ang-II, angiotensin II; NOX, NADPH oxidase; PKC, Ca2+ -dependent protein kinase C; ROS, reactive oxygen species; TGF-β 1, transforming growth factor type beta 1; Tβ R, TGF-β receptor. ∗ Corresponding author at Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile. Avenida República 239, Postal Code 8370146, Santiago, Chile. Tel.: 562 770 3665; fax: 562 698 0414. E-mail address: [email protected], [email protected] (C. Cabello-Verrugio). http://dx.doi.org/10.1016/j.phymed.2015.06.011 0944-7113/© 2015 Elsevier GmbH. All rights reserved.
Apocynin (4-hydroxy-3-methoxyacetophenone) was first reported to be isolated from the roots of Apocynum cannabinum L. (Apocynaceae), and its extracts were used for heart disease and edema. Later, apocynin was identified as an immunomodulatory constituent from the root of Picrorhiza kurroa Royle ex Benth (Scrophulariaceae), a native plant grown in the Himalayan region, well known in traditional Indian medicine for the treatment of various immunerelated diseases (Smit et al., 2000; Stefanska and Pawliczak, 2008). Current research has been focused on the hepatoprotective, antioxidant, and immune-modulating activity of its active constituents.
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Fig. 1. Chemical structure of apocynin.
Apocynin (Fig. 1) has been widely used as an efficient inhibitor of the complex nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity (Barbieri et al., 2004; Stuppner et al., 1995). Apocynin inhibits the assembly of NOX that is responsible for reactive oxygen species (ROS) production. Due to the selectivity of its inhibition, it has been used in the treatment of arthritis, bowel disease, asthma, atherosclerosis, and familial amyotrophic lateral sclerosis (Stefanska and Pawliczak, 2008). In addition, it has been reported that apocynin decreases the skeletal muscle wasting by decreasing the ROS levels. This finding arises to apocynin as a good candidate over another compounds derivative of the same natural herbal extract, such as picroside iii, pikuroside c, vanillic acid, picroside ii, kutkoside or picroside (Stuppner et al., 1995), to study the role of ROS in the biology and physiology of skeletal muscle. Transforming growth factor type beta 1 (TGF-β 1) has been demonstrated to be a main regulator of atrophy, fibrosis, myogenesis and regeneration within skeletal muscle (Burks and Cohn, 2011). These effects of TGF-β 1 are produced by the activation of the canonical or Smad-dependent pathway. Briefly, TGF-β signals through two transmembrane receptors, Tβ RI and Tβ RII, producing the phosphorylation of Smad2 and Smad3, and further allowing the association of these proteins with Smad4. This Smad complex translocates to the cell nucleus and mediates the transcriptional regulation of TGF-β target genes (Attisano and Wrana, 2000). Reactive oxygen species (ROS) have been involved in cellular responses that are dependent on TGF-β -signal transduction via the activation of redox-sensitive signalling pathways in several tissues (Simon et al., 2013). ROS has also been implicated in muscle homeostasis and functioning; furthermore, its disturbance has been involved in muscle diseases (Cabello-Verrugio et al., 2011a; Cozzoli et al., 2011). NOX, a multi-enzyme complex that converts molecular oxygen to ROS using NADPH as a substrate, is one of the main sources of ROS in several tissues including skeletal muscle (Cabello-Verrugio et al., 2011a). It has been described that the phosphorylation of the regulatory subunit p47phox by the Ca2+ -dependent protein kinase C (PKC) is the main activation mechanism of NOX (Babior, 2002), even in skeletal muscle cells that have been exposed to angiotensin II (Ang-II) (Cabello-Verrugio et al., 2011a; Morales et al., 2012). In muscle wasting, apocynin has protective effects on the diaphragm during prolonged mechanical ventilation (McClung et al., 2009) and in plantaris muscle after myocardial infarction (Bechara et al., 2014) by reducing ROS. Diapocynin, a dimer of apocynin, also prevented force loss of dystrophic muscle and reduced membrane damage (Ismail et al., 2014). These data suggest the importance of NOX-ROS axis in the pathogenic cascade leading to muscular diseases. In addition, it is relevant to evaluate the mechanisms by which apocynin exerts its beneficial activity in skeletal muscle. NOX modulates the response of skeletal muscle cells to myostatin and Ang-II, as well as regulates the increase of oxidative stress in Duchenne muscular dystrophy (Cabello-Verrugio et al., 2011a; Inoue et al., 2012). Whereas ROS and NOX have been implicated in the regulation of TGFβ response in several tissues, its involvement in TGF-β signalling or in the modulation of TGF-β 1 expression within skeletal muscle has not yet been elucidated.
The purpose of this study is to assess the effects of TGF-β 1 on its own expression and the involvement of NOX-induced ROS in this process. Our results demonstrate that TGF-β 1 induces its own expression through Tβ RI and a Smad-dependent pathway. Moreover, TGF-β 1 also induces ROS that is dependent on NOX through Tβ RI. Interestingly, the Smad-dependent signalling is modulated by the NOXinduced ROS in response to TGF-β 1. This is translated into the modulation of transcriptional activity which is dependent on TGF-β 1, as evidenced by p3TP-lux activity and TGF-β 1 expression which were partially decreased by the use of the NOX inhibitor, apocynin. Concomitantly, we also show that TGF-β -induced ROS production and TGF-β 1 expression require PKC activity. Material and methods Cell cultures The skeletal muscle cell line C2 C12 (American Type Culture Collection, CRL-1772) was grown as described previously (Cabello-Verrugio et al., 2011b). The cells were serum-starved for 18 h and then subjected to the treatment with TGF-β 1 (Shenandoah Biotechnology Inc., Warwick, PA, USA) (Morales et al., 2012); for the inhibition of Tβ RI were pre-incubated for 1 h with the inhibitor SB525334 (5 μM, Tocris Bioscience, Bristol, UK); as antioxidant used was N-Acetyl Cysteine (NAC) (5 mM, Sigma, St Louis, Mo, USA); for the NOX inhibition were pre-incubated for 1 h with the inhibitor apocynin (1 mM, Sigma, St Louis, Mo, USA) or diphenylene iodonium (DPI) (1 μM, Sigma, St Louis, Mo, USA), and followed for the incubation with TGF-β 1 for the time indicated in each figure. Apocynin source Apocynin (4 -Hydroxy-3 -methoxyacetophenone) was obtained from Sigma (St Louis, Mo, USA) (Cat.# A10809). The reported purity from the compound is ≥98% by gas chromatography. Immunoblot analysis For the skeletal muscle extracts, the diaphragm muscles were homogenized in Tris-EDTA buffer with a cocktail of protease inhibitors and 1 mM PMSF. Proteins were subjected to SDS-PAGE, transferred onto PVDF membranes (Millipore, Billerica, MA, USA) and probed with rabbit anti-total Smad3 and rabbit anti-phospho Smad3 (Cell signalling, Danvers, MA, USA). All immunoreactions were visualised by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA). Images were acquired using Fotodyne FOTO/Analyst Luminary Workstations Systems (Fotodyne, Inc., Hartland, WI). Protein content was determined by densitometric scanning of immunoreactive bands and intensity values were obtained by densitometry of individual bands normalized against control. Immunofluorescence microscopy Intracellular location of Smad4 was analyzed by indirect immunofluorescence (Cabello-Verrugio and Brandan, 2007). Cells were grown on glass coverslips and then fixed in 4% paraformaldehyde, permeabilized with 0.05% Triton X-100 and incubated for 1 h with 1:100 mouse anti-Smad4 (Santa Cruz Biotechnology, Dallas, TX, USA) in buffer 50 mM Tris-HCl, pH 7.7, 0.1 M NaCl, and 2% bovine serum albumin. After antibody removal and several washes with the above buffer, bound antibodies were detected by incubating the cells for 30 min with 1:100 affinity-purified alexa fluor 488 dye-conjugated goat anti-mouse antibody (Life Technologies, Carlsbad, CA, USA). For nuclear staining, sections were incubated with 1 μg/ml Hoechst 33258 in PBS for 10 min. After rinsing, the sections were mounted with fluorescent mounting medium (Dako Corporation, Glostrup,
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Denmark) under glass cover slips, viewed and photographed on the Motic BA310 epifluorescence microscope (Motic, Hong Kong). Short interfering RNA (siRNA) transfection The following siRNAs were used: since TGF-β requires Smad2/3 intermediates to signal, specific siRNA for both Smad2/3 (Santa Cruz Biotechnology, Dallas, TX, USA) were used and control siRNA (Life Technologies, Carlsbad, CA, USA). Briefly, for transfection, myoblasts were seeded into six-well plates until they reached 60% confluence. Subsequently, cells were incubated during 6 h in 800 μl of Opti-MEM I containing siRNA for Smad2/3 or control siRNA plus 8 μl of lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). Following transfection, FBS was added to the medium, and the cells were cultured for a further 48 h. Cells were finally assayed for RNA isolation. RNA isolation, reverse transcription, and quantitative real-time PCR At the end of the treatment, total RNA was isolated from the cell cultures using Trizol (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. The total RNA (1 μg) was reversetranscribed to cDNA using random hexamers and superscript reverse transcriptase (Life Technologies, Carlsbad, CA, USA). Taqman quantitative real-time PCR, were performed using pre-designed primer sets for mouse TGF-β 1, atrogin-1 and housekeeping gene GAPDH (Taqman Assays-on-Demand, Applied Biosystems by Life Technologies, Carlsbad, CA, USA). All reactions were performed in triplicate on an Eco Real-Time PCR System (Illumina, San Diego, CA, USA). mRNA expression was quantified using the comparative Ct method (2 – CT), using GAPDH as the reference gene. The mRNA levels are expressed relative to the mean expression in the control group. The values correspond to the mean of the Ct value ± standard deviation (SD) of the three independent experiments (Morales et al., 2012). Enzyme-linked immunosorbent assay A TGF-β 1 ELISA assay was performed to determine the TGF-β 1 levels secreted to the medium in C2 C12 that was exposed to AngII under different conditions, following the manufacturer’s protocol (TGF-β 1 EIA kit, Enzo Life Science, USA). The results were normalised by protein amount and were expressed as fold of induction relative to control cells. Measurement of intracellular [Ca2+ ] by FACS Treated C2 C12 cells were harvested, resuspended, and loaded with Fura-3 (5 μM) for Ca2+ measurements (Life Technologies, Carlsbad, CA, USA) for 15–30 min at room temperature in the absence of light. They were then analyzed immediately by Fluorescence Activated Cell Sorting (FACS) using a flow cytometry system (FACSCanto, BD Biosciences, Bergen, NJ, USA). A minimum of 10,000 cells were analyzed per sample. The cellular intensity of the dyes was analyzed using FACSDiva software v4.1.1 (BD Biosciences, USA) (Cabello-Verrugio et al., 2011a). Measurement of the intracellular ROS levels The C2 C12 cells were treated and harvested with trypsin/EDTA. After two washes with ice-cold PBS, cells were resuspended, and loaded with the cell permeate dye dichlorodihydrofluorescein (H2-DCF-DA) (5 μM, Life Technologies, Carlsbad, CA, USA) for 15–30 min in the dark at room temperature. Then, cells were immediately analysed and the ROS production was measured by the increases in DCF fluorescence, as an indicator of ROS production. DCF fluorescence was measured using a microplate reader at wavelengths of 488 nm (excitation) and 510 nm (emission). The values are expressed as fold of
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induction normalized to control cells treated with vehicle. Alternatively, C2 C12 myoblasts were cultured on glass cover slips and further treated with TGF-β 1 (2.5 ng/ml) for 24 h. At the end of experiment, cells were washed with HBSS and incubated with H2-DCF-DA for 30 min at 37 °C. After two washes with HBSS and one wash with PBS, the cells were fixed with 4% paraformaldehyde for 10 min and washed with PBS. The cells were incubated with 1 μg/ml Hoechst 33258 in PBS for 10 min for nuclear staining. After rinsing, the cells were mounted with fluorescent mounting medium (Dako Corporation, Glostrup, Denmark), viewed and photographed on the Motic BA310 epifluorescence microscope (Motic, Hong Kong). Statistical analysis The statistical analysis was evaluated using one-way analysis of variance (ANOVA) with a post-hoc Bonferroni multiple-comparison test (Sigma Stat). A difference was considered statistically significant at a P value of <0.05. Results TGF-β induces its own expression via a canonical Smad-dependent pathway in skeletal muscle cells We evaluated the effects of TGF-β on its own expression in C2 C12 myoblasts. Fig. 2A shows that TGF-β increases its expression in a time- (Fig. 2A) and dose-dependent fashion (Fig. 2B), reaching the maximum effect with 2.5 ng/ml of TGF-β for 9 h. In addition, Fig. 2C shows that similar results were obtained for TGF-β 1 protein levels induced by TGF-β . Fig. 3A shows that the effect of TGF-β 1 on its own expression involves the kinase activity of Tβ RI since its incubation within a specific inhibitor of this receptor SB525334 completely abolishes the TGF-β -dependent increase of its expression. In order to test the involvement of the Smad pathway in the increase of TGF-β 1 induced by itself, we transfected C2 C12 cells with siRNA directed against Smad2/3 proteins as we have previously demonstrated (Painemal et al., 2013). Fig. 3B shows that TGF-β 1 expression is decreased by the knockdown of Smad2/3 proteins, suggesting its participation in the induction of TGF-β 1 expression. These results demonstrate that TGFβ is able to induce its own expression through a Smad-dependent mechanism in C2 C12 myoblasts. NADPH oxidase (NOX)-derived ROS production is increased by TGF-β in skeletal muscle cells In order to test the mechanisms that are involved in autoinduced TGF-β 1 expression, we evaluated whether skeletal muscle cells increase their intracellular ROS production in response to TGF-β . By means of fluorescent detection using a ROS-sensitive fluorescent probe DCF in C2 C12 cells (Fig. 4A), we determined that intracellular ROS levels were increased by TGF-β in a dose-dependent manner. The increment of ROS was also detected by the DCF probe through fluorescence microscopy (Fig. 4C). This increase in TGF-β -induced ROS was completely abolished in the presence of Tβ RI inhibitor SB525334 (Fig. 4B, C and D). These results suggest that TGF-β 1 induces an increase in ROS production through a Tβ RI-dependent mechanism. We then tested the participation of NOX in the production of ROS induced by TGF-β in skeletal muscle cells. Using the fluorescent detection of the DCF probe, we evaluated the effect of apocynin, an NOX inhibitor. In Fig. 4B, it is shown that apocynin at 1 mM completely prevented the increase in TGF-β 1-induced ROS production. The same results were obtained through DCF detection by fluorescence microscopy (Fig. 4C). The quantification of these results is shown in Fig. 4D. These results strongly suggest that TGF-β is able to induce NOXinduced ROS in skeletal muscle cells.
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Fig. 2. TGF-β 1 induces its own expression in skeletal muscle cells. (A) C2 C12 myoblasts were incubated with TGF-β (2.5 ng/ml) and TGF-β 1 expression was determined by RT-qPCR in different times of treatment. The values correspond to the mean of Ct value ± standard deviation. (n = 3; ∗, P < 0.05 versus control). (B) C2 C12 cells were incubated with several concentrations of TGF-β 1 for 9 h. TGF-β 1 mRNA levels were measured as in A. (n = 3; ∗, P < 0.05 versus control). (C) Detection by ELISA of TGF-β 1 protein levels into the medium of C2 C12 exposed to TGF-β for 48 h. The values correspond to the mean ± standard deviation. (n = 3; ∗, P < 0.05 versus control).
Fig. 3. The induction of TGF-β 1 expression by itself is dependent on TGF-β receptor I and Smad2/3 proteins in skeletal muscle cells. (A) C2 C12 myoblasts were untreated (control) or treated with 2.5 ng/ml of TGF-β in the absence or presence of SB525334 (5 μM) for 9 h. TGF-β 1 expression was determined by RT-qPCR. The values correspond to the mean of Ct value ± standard deviation. (n = 3 ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β ). (B) C2 C12 cells, transfected with control siRNA or Smad2/3 siRNA, were untreated (control) or treated with 2.5 ng/ml of TGF-β for 9 h. TGF-β 1 expression was measured by RT-qPCR. The values correspond to the mean of Ct value ± standard deviation. (n = 3; ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β ).
Smad-dependent signalling requires NOX-derived ROS production induced by TGF-β in skeletal muscle cells
induced by TGF-β requires NOX-derived ROS in skeletal muscle cells.
We evaluated whether the increase of intracellular ROS production in response to TGF-β is required for the activation of Smad signalling by this growth factor. Fig. 5A and B show that phosphoSmad3 levels are decreased by 1 mM of apocynin (for 0.5 ng/ml of TGF-β from 3.48 ± 0.13 to 2.12 ± 0.21; for 1.0 ng/ml of TGFβ from 3.62 ± 0.13 to 2.01 ± 0.11), suggesting the participation of NOX-induced ROS. To directly assay the effect of ROS on Smad3 phosphorylation, we incubated cells with the antioxidant NAC. The Figs. S1A and 1B show that NAC at 5 mM decreases Smad3 phosphorylation induced by TGF-β 1 (from 3.23 ± 0.15 to 2.04 ± 0.15). In the same direction to the results showed in Fig. 5A and B, Fig. 5C and D show that apocynin at 1 mM decreases the nuclear translocation of Smad4 in response to TGF-β (from 68.2 ± 4.4 to 42.3 ± 7.2). Together, these results strongly suggest that Smad signalling
TGF-β 1 and atrogin-1 expression, and p3TP-lux activity induced by TGF-β are dependent on NOX-derived ROS production in skeletal muscle cells Since NOX-derived ROS production modulates the Smaddependent signalling that is induced by TGF-β , we evaluated the effects of apocynin on the TGF-β target genes such as the autoinduction of TGF-β 1 and p3TP-lux activity. Fig. 6A shows that the inhibition of NOX with 1 mM of apocynin prevented the increase of TGF-β 1 expression by 50%. Similarly, apocynin also reduced by 50% the p3TPlux transcriptional activity of p3TP-lux that was induced by TGF-β (Fig. 6B). In addition, the induction of atrogin-1 (Fig. 6C) by TGFβ 1 is also modulated by NOX-induced ROS. In all Fig. 6A, B and C, the total abolition of the TGF-β effect in the presence of SB525334
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Fig. 4. TGF-β -induced ROS are mediated by TGF-β receptor I and NAD(P)H oxidase in skeletal muscle cells. (A) C2 C12 myoblasts were incubated with different concentrations of TGF-β for 24 h. The ROS levels were determined by fluorescence detection of DCF probe. Values correspond to the mean ± standard deviation (n = 3; ∗, P < 0.05 versus without TGF-β ). For B and C: C2 C12 cells were pre-incubated with the vehicle, apocynin (1 mM) or SB525334 (5 μM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 24 h. Then, cells were loaded with DCF probe for ROS detection. (B) Detection of ROS by microplate reader. Values correspond to the mean ± standard deviation (n = 3; ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β ). (C) Detection of ROS by microscopy. The nuclei are detected by Hoechst stain. Bar: 100 μm. (D) Quantification of experiments showed in C. (n = 3; ∗, P < 0.05 versus vehicle; #, P < 0.05 versus TGF-β ).
is shown to corroborate Tβ RI’s participation. Together, our results suggest that TGF-β 1 expression and transcriptional activity of target gene induced by TGF-β dependent on NOX-derived ROS production in skeletal muscle cells. Protein kinase C is required for TGF-β -induced intracellular ROS production and TGF-β expression in skeletal muscle cells First, we show that TGF-β 1 increased levels of intracellular Ca2+ in C2 C12 cells. These levels were not affected by 1 mM of apocynin
(Fig. S2A) neither 5 μM of DPI, another NOX inhibitor (Fig. S2B). This result suggests that the NOX activity and ROS generation that are induced by TGF-β occur downstream of the Ca2+ increase. Fig. 7A illustrates that the inhibition of PKC activity by chelerythrine (10 μM) prevented TGF-β -induced ROS production in C2 C12 cells, as evaluated using a DCF probe. This diminution of ROS production by the inhibition of PKC has a functional effect on TGF-β activity. Thus, Fig. 7B shows that the inhibition of PKC reduced up to 60% of the induction of TGF-β 1 expression. A similar effect was observed when p3TPlux transcriptional activity (Fig. 7C), or atrogin-1 expression (Fig. 7D),
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Fig. 5. Apocynin attenuates the activation of Smad signalling induced by TGF-β in skeletal muscle cells. (A) C2 C12 cells were pre-incubated with the vehicle or apocynin (1 mM) for 1 h, and then incubated with TGF-β (1.0 or 2.5 ng/ml) for 30 min. The protein levels of the phosphorylated and total Smad3 were evaluated by Western blot. (B) Quantification of experiments showed in A. The values correspond to the mean ± standard deviation (n = 3; ∗, P < 0.05 versus control; #, P < 0.05 versus TGF-β ). (C) C2 C12 myoblasts were preincubated with the vehicle or apocynin (1 mM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 90 min. The location of Smad4 was evaluated by indirect immunofluorescence. Bar: 100 μm. (D) Quantification of experiments showed in C. (n = 3; ∗, P < 0.05 versus vehicle; #, P < 0.05 versus TGF-β ).
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Fig. 6. TGF-β 1 expression induced in response to TGF-β is attenuated by apocynin in skeletal muscle cells. (A) C2 C12 myoblasts were pre-incubated with the vehicle, apocynin (1 mM) or SB525334 (5 μM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 9 h. TGF-β 1 expression was determined by RT-qPCR. The values correspond to the mean of Ct value ± standard deviation. (n = 3; ∗, P < 0.05 versus untreated control cells; #, P < 0.05 versus TGF-β ). (B) C2 C12 cells were transiently transfected with p3TP-lux plasmid. After 24 h, cells were pre-incubated with the vehicle, apocynin (1 mM) or SB525334 (5 μM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 24 h. The luciferase activity and the values represent the mean ± standard deviation (n = 3; ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β ). (C) C2 C12 myoblasts were pre-incubated with the vehicle, apocynin (1 mM) or SB525334 (5 μM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 6 h. Atrogin-1 expression was determined by RT-qPCR. The values correspond to the mean of Ct value ± standard deviation. (n = 3; ∗, P < 0.05 versus untreated control cells; #, P < 0.05 versus TGF-β ).
all of them induced by TGF-β were determined in C2 C12 myoblasts. These results suggest that PKC activity participates in TGF-β -induced ROS production and transcriptional activity in skeletal muscle cells. Together, these results suggest that PKC activity is partially required for TGF-β induced activity and required to activate NOX. Discussion This study demonstrates that TGF-β 1 induces its own expression in skeletal muscle cells through a mechanism that is dependent on Tβ RI, a Smad pathway and NOX-derived ROS. Interestingly, we demonstrated that pure apocynin decreased the increment of ROS and partially inhibited the auto-induction of TGF-β 1. In addition, NOX-induced ROS in response to TGF-β are able to modulate the canonical Smad signalling and another gene expression that is modulated by TGF-β such as p3TP-lux activity and atrogin-1 gene expression. Our results have relevance in several pathological conditions in skeletal muscle in which TGF-β levels and signalling has been shown to be increased, such as spinal muscular atrophy, Kennedy disease, Marfan syndrome, Duchenne muscular dystrophy and skeletal muscle atrophy by disuse or induced by cachexia (Burks and Cohn, 2011). In this context, our data show that the use of apocynin partially inhibits the effect and signalling dependent on TGF-β , decreasing the NOX-induced ROS production. These findings are in agreement with previous reports in which the use of apocynin has beneficial and protective effects on the diaphragm during prolonged mechanical ventilation (McClung et al., 2009), in plantaris muscle after myocardial infarction (Bechara et al., 2014) by reducing ROS, and also prevented force loss of dystrophic muscle and reduced membrane
damage (Ismail et al., 2014). Although the plant species from which apocynin has been isolated have not been used to evaluate their effect on muscle diseases, a previous report indicates that the treatment of diabetic rats, characterized by the loss of body weight and the increase of muscle wasting, with P. kurroa showed an increase on body weight compared to control animals, suggesting a protective role of this natural compound on muscle wasting (Husain et al., 2009). However in future experiments it will be important to evaluate the effect of the natural extract of P. kurroa on skeletal muscle function in order to develop natural therapies against muscle pathologies. One of the effects of TGF-β is to produce fibrosis in several tissues including skeletal muscle (MacDonald and Cohn, 2012). Several reports indicate that TGF-β requires ROS to induce fibrosis (Samarakoon et al., 2013). Thus, our data are in agreement with these reports since they indirectly suggest that the fibrotic effect mediated by TGF-β could require ROS in skeletal muscle. Interestingly, TGF-β also produces skeletal muscle atrophy, as has been recently reported (Mendias et al., 2012; Narola et al., 2013; Ohsawa et al., 2012; Zhang et al., 2014). However, the molecular mechanism and signalling pathways that are involved have not been elucidated yet. Our data show that the expression of atrogin-1, an E3 ligase involved in skeletal muscle atrophy, is induced by TGF-β 1 and also modulated by ROS in skeletal muscle cells. Thus, the participation of ROS seems to be a good candidate to evaluate, since its involvement in skeletal muscle atrophy induced by disuse or other causes has been suggested (Kondo et al., 1993; Semprun-Prieto et al., 2011). The critical role of the increase in the ROS levels that are mediated by NOX activation has been described in several pathological conditions (Simon et al., 2013). We, and others, have previously described that NOX induces ROS production in skeletal muscle cells
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Fig. 7. TGF-β 1 expression and ROS production in response to TGF-β require PKC activity in skeletal muscle cells. (A) C2 C12 cells were exposed to vehicle alone or chelerythrine (10 μM) in the absence (control) or presence of TGF-β (2.5 ng/ml), and DCF fluorescence from three independent experiments was measured. Value represent mean ± standard deviation (n = 3; ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β ). (B) C2 C12 myoblasts were pre-incubated with chelerythrine (10 μM) and then incubated with TGF-β (2.5 ng/ml). After 9 h, TGF-β 1 expression was determined by RT-qPCR. The values correspond to the mean of Ct value ± standard deviation. (n = 3; ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β 1). (C) C2 C12 cells transfected with p3TP-lux plasmid, were pre-incubated with the vehicle or chelerythrine (10 μM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 24 h. The luciferase activity and the values represent the mean ± standard deviation (n = 3; ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β ). (D) C2 C12 myoblasts were pre-incubated with the vehicle or chelerythrine (10 μM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 6 h. Atrogin-1 expression was determined by RT-qPCR. The values correspond to the mean of Ct value ± standard deviation. (n = 3; ∗, P < 0.05 versus untreated control cells; #, P < 0.05 versus TGF-β ).
(Cabello-Verrugio et al., 2011a; Espinosa et al., 2006). In this study, we show that TGF-β -induced ROS are generated by NOX activation. Several reports have shown that the expression and activity of NOX is relevant in skeletal muscle (Cabello-Verrugio et al., 2011a), including muscle diseases such as Duchenne muscular dystrophy or atrophy (Kim et al., 2013) in which TGF-β plays a critical role. The main mechanism to activate NOX is the Ca2+ -dependent PKCmediated serine phosphorylation of subunit p47phox (Babior, 2004). Our results demonstrate that an increase in intracellular Ca2+ levels induced by TGF-β would be a necessary event to activate NOX by PKC, since the inhibition of PKC activity produced a total abolition of ROS production, suggesting a regulation of NOX activation. This event is concomitant with the partial reduction of TGF-β -induced p3TPlux activity and TGF-β 1 expression in the presence of a PKC inhibitor to similar levels obtained when NOX inhibition was produced. Further experiments are needed to determine the direct role of PKC, and the possible isoform that is involved in the NOX-induced ROS dependent on TGF-β . Among the factors that regulate TGF-β expression is Ang-II, as we have recently demonstrated (Morales et al., 2014; Morales et al., 2012). Moreover, we also reported that the prolonged fibrotic effect induced by Ang-II in skeletal muscle is dependent on increased TGF-β expression (Morales et al., 2012). Interestingly, both Ang-II and TGFβ produce atrophy and fibrosis in skeletal muscle, two events that are observed in myopathies such as dystrophies (Acuna et al., 2014; Cabello-Verrugio et al., 2011a, 2012a; Cisternas et al., 2015; Mendias et al., 2012; Meneses et al., 2014; Morales et al., 2012; Narola et al., 2013). Thus, the stimulation of its own expression by TGF-β 1 seems
to be consistent with a mechanism to reinforce atrophy and fibrosis in which Ang-II can induce atrophy, TGF-β expression and fibrosis, and the further TGF-β 1 expression that is induced by itself perpetuates a fibrotic environment. Smad signalling is the main pathway through which TGF-β induces its biological activities. Thus, Smad signalling is critical for the TGF-β -induced inhibition of skeletal muscle differentiation (Massague et al., 1986). In addition, Smad signalling is key to modulating skeletal muscle fibrosis participating in the expression of extracellular matrix proteins such as collagens and fibronectin, or pro-fibrotic factors such as connective tissue growth factor (Acuna et al., 2014; Cabello-Verrugio et al., 2012b; Morales et al., 2011, 2013a, 2013b). In this context, our results are in agreement with these facts, since we show that the expression of TGF-β induced by itself requires Smad pathway activation, as demonstrated by Smad3 phosphorylation and the nuclear translocation of Smad4. However, we cannot disregard the participation of non-canonical or Smad-independent pathways in the modulation of TGF-β expression. In this line, we describe the modulation of p38MAPK on the TGF-β induction in response to Ang-II (Morales et al., 2012). Further experiments to study the effects of TGF-β -induced non-canonical signalling pathways on TGF-β 1 expression must be conducted.
Conclusions Our study is the first report that shows the autoinduction of TGF-β 1 through a TGF-β -receptor/Smad-dependent mechanism.
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Apocynin is able to inhibit this process, suggesting that requires NOXinduced ROS in skeletal muscle cells. It has direct relevance in the possible use of apocynin obtained from synthetic or natural sources as a potential molecule in the treatment of muscle diseases. Conflict of interest The authors confirm that there are no conflicts of interest. Acknowledgments This study was supported by research grants from AssociationFrancaise Contre Les Myopathies AFM 16670 (CCV); FONDECYT 1120380 (CCV), 3130593 (MGM), 3140396 (DC), 1121078 (FS); Millennium Institute on Immunology and Immunotherapy, P09-016-F (FS, CCV), UNAB DI-741-15/N. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2015.06.011. References Acuna, M.J., Pessina, P., Olguin, H., Cabrera, D., Vio, C.P., Bader, M., Munoz-Canoves, P., Santos, R.A., Cabello-Verrugio, C., Brandan, E., 2014. Restoration of muscle strength in dystrophic muscle by angiotensin-1-7 through inhibition of TGF-beta signalling. Hum. Mol. Genet. 23, 1237–1249. Attisano, L., Wrana, J.L., 2000. Smads as transcriptional co-modulators. Curr. Opin. Cell Biol. 12, 235–243. Babior, B.M., 2002. The activity of leukocyte NADPH oxidase: regulation by p47PHOX cysteine and serine residues. Antioxid. Redox. Signal 4, 35–38. Babior, B.M., 2004. NADPH oxidase. Curr. Opin. Immunol. 16, 42–47. Barbieri, S.S., Cavalca, V., Eligini, S., Brambilla, M., Caiani, A., Tremoli, E., Colli, S., 2004. Apocynin prevents cyclooxygenase 2 expression in human monocytes through NADPH oxidase and glutathione redox-dependent mechanisms. Free Radic. Biol. Med. 37, 156–165. Bechara, L.R., Moreira, J.B., Jannig, P.R., Voltarelli, V.A., Dourado, P.M., Vasconcelos, A.R., Scavone, C., Ramires, P.R., Brum, P.C., 2014. NADPH oxidase hyperactivity induces plantaris atrophy in heart failure rats. Int. J. Cardiol. 175, 499–507. Burks, T.N., Cohn, R.D., 2011. Role of TGF-beta signaling in inherited and acquired myopathies. Skelet. Muscle 1, 19. Cabello-Verrugio, C., Acuna, M.J., Morales, M.G., Becerra, A., Simon, F., Brandan, E., 2011a. Fibrotic response induced by angiotensin-II requires NAD(P)H oxidaseinduced reactive oxygen species (ROS) in skeletal muscle cells. Biochem. Biophys. Res. Commun. 410, 665–670. Cabello-Verrugio, C., Brandan, E., 2007. A novel modulatory mechanism of transforming growth factor-beta signaling through decorin and LRP-1. J. Biol. Chem. 282, 18842–18850. Cabello-Verrugio, C., Cordova, G., Salas, J.D., 2012a. Angiotensin II: role in skeletal muscle atrophy. Curr. Protein Pept. Sci. 13, 560–569. Cabello-Verrugio, C., Cordova, G., Vial, C., Zuniga, L.M., Brandan, E., 2011b. Connective tissue growth factor induction by lysophosphatidic acid requires transactivation of transforming growth factor type beta receptors and the JNK pathway. Cell Signal 23, 449–457. Cabello-Verrugio, C., Morales, M.G., Cabrera, D., Vio, C.P., Brandan, E., 2012b. Angiotensin II receptor type 1 blockade decreases CTGF/CCN2-mediated damage and fibrosis in normal and dystrophic skeletal muscles. J. Cell Mol. Med. 16, 752–764. Cisternas, F., Morales, M.G., Meneses, C., Simon, F., Brandan, E., Abrigo, J., Vazquez, Y., Cabello-Verrugio, C., 2015. Angiotensin-(1-7) decreases skeletal muscle atrophy induced by angiotensin II through a Mas receptor-dependent mechanism. Clin. Sci (Lond). 128, 307–319. Cozzoli, A., Nico, B., Sblendorio, V.T., Capogrosso, R.F., Dinardo, M.M., Longo, V., Gagliardi, S., Montagnani, M., De Luca, A., 2011. Enalapril treatment discloses an early role of angiotensin II in inflammation- and oxidative stress-related muscle damage in dystrophic mdx mice. Pharmacol. Res. 64, 482–492. Espinosa, A., Leiva, A., Pena, M., Muller, M., Debandi, A., Hidalgo, C., Carrasco, M.A., Jaimovich, E., 2006. Myotube depolarization generates reactive oxygen species through NAD(P)H oxidase; ROS-elicited Ca2+ stimulates ERK, CREB, early genes. J. Cell Physiol. 209, 379–388. Husain, G.M., Singh, P.N., Kumar, V., 2009. Antidiabetic activity of standardized extract of Picrorhiza kurroa in rat model of NIDDM. Drug Discov. Ther. 3, 88–92. Inoue, N., Kinugawa, S., Suga, T., Yokota, T., Hirabayashi, K., Kuroda, S., Okita, K., Tsutsui, H., 2012. Angiotensin II-induced reduction in exercise capacity is associated with increased oxidative stress in skeletal muscle. Am. J. Physiol. Heart Circ. Physiol. 302, H1202–H1210.
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Apocynin inhibits the upregulation of TGF-β 1 expression and ROS production induced by TGF-β in skeletal muscle cells Johanna Abrigo a,c, María Gabriela Morales a,c, Felipe Simon b,c, Daniel Cabrera d, Gabriella Di Capua a,c, Claudio Cabello-Verrugio a,c,∗ a Laboratorio de Biología y Fisiopatología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile b Laboratorio de Fisiopatología Integrativa, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile c Millennium Institute on Immunology and Immunotherapy, Santiago, Chile d Departamento de Ciencias Químicas y Biológicas, Universidad Bernardo O´Higgins, Santiago, Chile
a r t i c l e
i n f o
Article history: Received 2 March 2015 Revised 18 June 2015 Accepted 21 June 2015
Keywords: TGF-β 1 Smad ROS NOX Atrophy Skeletal muscle
a b s t r a c t Background: Pure apocynin, which can be traditionally isolated and purified from several plant species such as Picrorhiza kurroa Royle ex Benth (Scrophulariaceae), acts as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity inhibiting its production of reactive oxygen species (ROS). Transforming growth factor type beta 1 (TGF-β 1) is a growth factor that produces inhibition of myogenesis, diminution of regeneration and induction of atrophy in skeletal muscle. The typical signalling that is activated by TGF-β involves the Smad pathway. Purpose: To evaluate the effect of TGF-β and the effect of apocynin on TGF-β 1 expression in skeletal muscle cells. Study design: Controlled laboratory study. In vitro assays were performed with C2 C12 cells incubated with TGF-β 1 in presence or absence of apocynin (NOX inhibitor), SB525334 (TGF-β -receptor I inhibitor), or chelerythrine (PKC inhibitor). Methods: TGF-β 1 and atrogin-1 expression was evaluated by RT-qPCR and/or ELISA; Smad3 phosphorylation by western blot; Smad4 nuclear translocation by indirect immunofluorescence; and ROS levels by DCF probe fluorescent measurements. Results: We show that myoblasts respond to TGF-β 1 by increasing its own gene expression in a time- and dose-dependent fashion which was abolished by SB525334 and siRNA for Smad2/3. TGF-β 1 also induced ROS. Remarkably, apocynin inhibited the TGF-β 1 induced ROS as well as the autoinduction of TGF-β 1 gene expression. We also show that TGF-β -induced ROS production and TGF-β 1 expression require PKC activity as indicated by the inhibition using chelerythrine. Conclusion: These results strongly suggest that TGF-β induces its own expression through a TGF-β receptor/Smad-dependent mechanism and apocynin is able to inhibit this process, suggesting that requires NOX-induced ROS in skeletal muscle cells. © 2015 Elsevier GmbH. All rights reserved.
Introduction
Abbreviations: Ang-II, angiotensin II; NOX, NADPH oxidase; PKC, Ca2+ -dependent protein kinase C; ROS, reactive oxygen species; TGF-β 1, transforming growth factor type beta 1; Tβ R, TGF-β receptor. ∗ Corresponding author at Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile. Avenida República 239, Postal Code 8370146, Santiago, Chile. Tel.: 562 770 3665; fax: 562 698 0414. E-mail address: [email protected], [email protected] (C. Cabello-Verrugio). http://dx.doi.org/10.1016/j.phymed.2015.06.011 0944-7113/© 2015 Elsevier GmbH. All rights reserved.
Apocynin (4-hydroxy-3-methoxyacetophenone) was first reported to be isolated from the roots of Apocynum cannabinum L. (Apocynaceae), and its extracts were used for heart disease and edema. Later, apocynin was identified as an immunomodulatory constituent from the root of Picrorhiza kurroa Royle ex Benth (Scrophulariaceae), a native plant grown in the Himalayan region, well known in traditional Indian medicine for the treatment of various immunerelated diseases (Smit et al., 2000; Stefanska and Pawliczak, 2008). Current research has been focused on the hepatoprotective, antioxidant, and immune-modulating activity of its active constituents.
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Fig. 1. Chemical structure of apocynin.
Apocynin (Fig. 1) has been widely used as an efficient inhibitor of the complex nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity (Barbieri et al., 2004; Stuppner et al., 1995). Apocynin inhibits the assembly of NOX that is responsible for reactive oxygen species (ROS) production. Due to the selectivity of its inhibition, it has been used in the treatment of arthritis, bowel disease, asthma, atherosclerosis, and familial amyotrophic lateral sclerosis (Stefanska and Pawliczak, 2008). In addition, it has been reported that apocynin decreases the skeletal muscle wasting by decreasing the ROS levels. This finding arises to apocynin as a good candidate over another compounds derivative of the same natural herbal extract, such as picroside iii, pikuroside c, vanillic acid, picroside ii, kutkoside or picroside (Stuppner et al., 1995), to study the role of ROS in the biology and physiology of skeletal muscle. Transforming growth factor type beta 1 (TGF-β 1) has been demonstrated to be a main regulator of atrophy, fibrosis, myogenesis and regeneration within skeletal muscle (Burks and Cohn, 2011). These effects of TGF-β 1 are produced by the activation of the canonical or Smad-dependent pathway. Briefly, TGF-β signals through two transmembrane receptors, Tβ RI and Tβ RII, producing the phosphorylation of Smad2 and Smad3, and further allowing the association of these proteins with Smad4. This Smad complex translocates to the cell nucleus and mediates the transcriptional regulation of TGF-β target genes (Attisano and Wrana, 2000). Reactive oxygen species (ROS) have been involved in cellular responses that are dependent on TGF-β -signal transduction via the activation of redox-sensitive signalling pathways in several tissues (Simon et al., 2013). ROS has also been implicated in muscle homeostasis and functioning; furthermore, its disturbance has been involved in muscle diseases (Cabello-Verrugio et al., 2011a; Cozzoli et al., 2011). NOX, a multi-enzyme complex that converts molecular oxygen to ROS using NADPH as a substrate, is one of the main sources of ROS in several tissues including skeletal muscle (Cabello-Verrugio et al., 2011a). It has been described that the phosphorylation of the regulatory subunit p47phox by the Ca2+ -dependent protein kinase C (PKC) is the main activation mechanism of NOX (Babior, 2002), even in skeletal muscle cells that have been exposed to angiotensin II (Ang-II) (Cabello-Verrugio et al., 2011a; Morales et al., 2012). In muscle wasting, apocynin has protective effects on the diaphragm during prolonged mechanical ventilation (McClung et al., 2009) and in plantaris muscle after myocardial infarction (Bechara et al., 2014) by reducing ROS. Diapocynin, a dimer of apocynin, also prevented force loss of dystrophic muscle and reduced membrane damage (Ismail et al., 2014). These data suggest the importance of NOX-ROS axis in the pathogenic cascade leading to muscular diseases. In addition, it is relevant to evaluate the mechanisms by which apocynin exerts its beneficial activity in skeletal muscle. NOX modulates the response of skeletal muscle cells to myostatin and Ang-II, as well as regulates the increase of oxidative stress in Duchenne muscular dystrophy (Cabello-Verrugio et al., 2011a; Inoue et al., 2012). Whereas ROS and NOX have been implicated in the regulation of TGFβ response in several tissues, its involvement in TGF-β signalling or in the modulation of TGF-β 1 expression within skeletal muscle has not yet been elucidated.
The purpose of this study is to assess the effects of TGF-β 1 on its own expression and the involvement of NOX-induced ROS in this process. Our results demonstrate that TGF-β 1 induces its own expression through Tβ RI and a Smad-dependent pathway. Moreover, TGF-β 1 also induces ROS that is dependent on NOX through Tβ RI. Interestingly, the Smad-dependent signalling is modulated by the NOXinduced ROS in response to TGF-β 1. This is translated into the modulation of transcriptional activity which is dependent on TGF-β 1, as evidenced by p3TP-lux activity and TGF-β 1 expression which were partially decreased by the use of the NOX inhibitor, apocynin. Concomitantly, we also show that TGF-β -induced ROS production and TGF-β 1 expression require PKC activity. Material and methods Cell cultures The skeletal muscle cell line C2 C12 (American Type Culture Collection, CRL-1772) was grown as described previously (Cabello-Verrugio et al., 2011b). The cells were serum-starved for 18 h and then subjected to the treatment with TGF-β 1 (Shenandoah Biotechnology Inc., Warwick, PA, USA) (Morales et al., 2012); for the inhibition of Tβ RI were pre-incubated for 1 h with the inhibitor SB525334 (5 μM, Tocris Bioscience, Bristol, UK); as antioxidant used was N-Acetyl Cysteine (NAC) (5 mM, Sigma, St Louis, Mo, USA); for the NOX inhibition were pre-incubated for 1 h with the inhibitor apocynin (1 mM, Sigma, St Louis, Mo, USA) or diphenylene iodonium (DPI) (1 μM, Sigma, St Louis, Mo, USA), and followed for the incubation with TGF-β 1 for the time indicated in each figure. Apocynin source Apocynin (4 -Hydroxy-3 -methoxyacetophenone) was obtained from Sigma (St Louis, Mo, USA) (Cat.# A10809). The reported purity from the compound is ≥98% by gas chromatography. Immunoblot analysis For the skeletal muscle extracts, the diaphragm muscles were homogenized in Tris-EDTA buffer with a cocktail of protease inhibitors and 1 mM PMSF. Proteins were subjected to SDS-PAGE, transferred onto PVDF membranes (Millipore, Billerica, MA, USA) and probed with rabbit anti-total Smad3 and rabbit anti-phospho Smad3 (Cell signalling, Danvers, MA, USA). All immunoreactions were visualised by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA). Images were acquired using Fotodyne FOTO/Analyst Luminary Workstations Systems (Fotodyne, Inc., Hartland, WI). Protein content was determined by densitometric scanning of immunoreactive bands and intensity values were obtained by densitometry of individual bands normalized against control. Immunofluorescence microscopy Intracellular location of Smad4 was analyzed by indirect immunofluorescence (Cabello-Verrugio and Brandan, 2007). Cells were grown on glass coverslips and then fixed in 4% paraformaldehyde, permeabilized with 0.05% Triton X-100 and incubated for 1 h with 1:100 mouse anti-Smad4 (Santa Cruz Biotechnology, Dallas, TX, USA) in buffer 50 mM Tris-HCl, pH 7.7, 0.1 M NaCl, and 2% bovine serum albumin. After antibody removal and several washes with the above buffer, bound antibodies were detected by incubating the cells for 30 min with 1:100 affinity-purified alexa fluor 488 dye-conjugated goat anti-mouse antibody (Life Technologies, Carlsbad, CA, USA). For nuclear staining, sections were incubated with 1 μg/ml Hoechst 33258 in PBS for 10 min. After rinsing, the sections were mounted with fluorescent mounting medium (Dako Corporation, Glostrup,
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Denmark) under glass cover slips, viewed and photographed on the Motic BA310 epifluorescence microscope (Motic, Hong Kong). Short interfering RNA (siRNA) transfection The following siRNAs were used: since TGF-β requires Smad2/3 intermediates to signal, specific siRNA for both Smad2/3 (Santa Cruz Biotechnology, Dallas, TX, USA) were used and control siRNA (Life Technologies, Carlsbad, CA, USA). Briefly, for transfection, myoblasts were seeded into six-well plates until they reached 60% confluence. Subsequently, cells were incubated during 6 h in 800 μl of Opti-MEM I containing siRNA for Smad2/3 or control siRNA plus 8 μl of lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). Following transfection, FBS was added to the medium, and the cells were cultured for a further 48 h. Cells were finally assayed for RNA isolation. RNA isolation, reverse transcription, and quantitative real-time PCR At the end of the treatment, total RNA was isolated from the cell cultures using Trizol (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. The total RNA (1 μg) was reversetranscribed to cDNA using random hexamers and superscript reverse transcriptase (Life Technologies, Carlsbad, CA, USA). Taqman quantitative real-time PCR, were performed using pre-designed primer sets for mouse TGF-β 1, atrogin-1 and housekeeping gene GAPDH (Taqman Assays-on-Demand, Applied Biosystems by Life Technologies, Carlsbad, CA, USA). All reactions were performed in triplicate on an Eco Real-Time PCR System (Illumina, San Diego, CA, USA). mRNA expression was quantified using the comparative Ct method (2 – CT), using GAPDH as the reference gene. The mRNA levels are expressed relative to the mean expression in the control group. The values correspond to the mean of the Ct value ± standard deviation (SD) of the three independent experiments (Morales et al., 2012). Enzyme-linked immunosorbent assay A TGF-β 1 ELISA assay was performed to determine the TGF-β 1 levels secreted to the medium in C2 C12 that was exposed to AngII under different conditions, following the manufacturer’s protocol (TGF-β 1 EIA kit, Enzo Life Science, USA). The results were normalised by protein amount and were expressed as fold of induction relative to control cells. Measurement of intracellular [Ca2+ ] by FACS Treated C2 C12 cells were harvested, resuspended, and loaded with Fura-3 (5 μM) for Ca2+ measurements (Life Technologies, Carlsbad, CA, USA) for 15–30 min at room temperature in the absence of light. They were then analyzed immediately by Fluorescence Activated Cell Sorting (FACS) using a flow cytometry system (FACSCanto, BD Biosciences, Bergen, NJ, USA). A minimum of 10,000 cells were analyzed per sample. The cellular intensity of the dyes was analyzed using FACSDiva software v4.1.1 (BD Biosciences, USA) (Cabello-Verrugio et al., 2011a). Measurement of the intracellular ROS levels The C2 C12 cells were treated and harvested with trypsin/EDTA. After two washes with ice-cold PBS, cells were resuspended, and loaded with the cell permeate dye dichlorodihydrofluorescein (H2-DCF-DA) (5 μM, Life Technologies, Carlsbad, CA, USA) for 15–30 min in the dark at room temperature. Then, cells were immediately analysed and the ROS production was measured by the increases in DCF fluorescence, as an indicator of ROS production. DCF fluorescence was measured using a microplate reader at wavelengths of 488 nm (excitation) and 510 nm (emission). The values are expressed as fold of
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induction normalized to control cells treated with vehicle. Alternatively, C2 C12 myoblasts were cultured on glass cover slips and further treated with TGF-β 1 (2.5 ng/ml) for 24 h. At the end of experiment, cells were washed with HBSS and incubated with H2-DCF-DA for 30 min at 37 °C. After two washes with HBSS and one wash with PBS, the cells were fixed with 4% paraformaldehyde for 10 min and washed with PBS. The cells were incubated with 1 μg/ml Hoechst 33258 in PBS for 10 min for nuclear staining. After rinsing, the cells were mounted with fluorescent mounting medium (Dako Corporation, Glostrup, Denmark), viewed and photographed on the Motic BA310 epifluorescence microscope (Motic, Hong Kong). Statistical analysis The statistical analysis was evaluated using one-way analysis of variance (ANOVA) with a post-hoc Bonferroni multiple-comparison test (Sigma Stat). A difference was considered statistically significant at a P value of <0.05. Results TGF-β induces its own expression via a canonical Smad-dependent pathway in skeletal muscle cells We evaluated the effects of TGF-β on its own expression in C2 C12 myoblasts. Fig. 2A shows that TGF-β increases its expression in a time- (Fig. 2A) and dose-dependent fashion (Fig. 2B), reaching the maximum effect with 2.5 ng/ml of TGF-β for 9 h. In addition, Fig. 2C shows that similar results were obtained for TGF-β 1 protein levels induced by TGF-β . Fig. 3A shows that the effect of TGF-β 1 on its own expression involves the kinase activity of Tβ RI since its incubation within a specific inhibitor of this receptor SB525334 completely abolishes the TGF-β -dependent increase of its expression. In order to test the involvement of the Smad pathway in the increase of TGF-β 1 induced by itself, we transfected C2 C12 cells with siRNA directed against Smad2/3 proteins as we have previously demonstrated (Painemal et al., 2013). Fig. 3B shows that TGF-β 1 expression is decreased by the knockdown of Smad2/3 proteins, suggesting its participation in the induction of TGF-β 1 expression. These results demonstrate that TGFβ is able to induce its own expression through a Smad-dependent mechanism in C2 C12 myoblasts. NADPH oxidase (NOX)-derived ROS production is increased by TGF-β in skeletal muscle cells In order to test the mechanisms that are involved in autoinduced TGF-β 1 expression, we evaluated whether skeletal muscle cells increase their intracellular ROS production in response to TGF-β . By means of fluorescent detection using a ROS-sensitive fluorescent probe DCF in C2 C12 cells (Fig. 4A), we determined that intracellular ROS levels were increased by TGF-β in a dose-dependent manner. The increment of ROS was also detected by the DCF probe through fluorescence microscopy (Fig. 4C). This increase in TGF-β -induced ROS was completely abolished in the presence of Tβ RI inhibitor SB525334 (Fig. 4B, C and D). These results suggest that TGF-β 1 induces an increase in ROS production through a Tβ RI-dependent mechanism. We then tested the participation of NOX in the production of ROS induced by TGF-β in skeletal muscle cells. Using the fluorescent detection of the DCF probe, we evaluated the effect of apocynin, an NOX inhibitor. In Fig. 4B, it is shown that apocynin at 1 mM completely prevented the increase in TGF-β 1-induced ROS production. The same results were obtained through DCF detection by fluorescence microscopy (Fig. 4C). The quantification of these results is shown in Fig. 4D. These results strongly suggest that TGF-β is able to induce NOXinduced ROS in skeletal muscle cells.
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Fig. 2. TGF-β 1 induces its own expression in skeletal muscle cells. (A) C2 C12 myoblasts were incubated with TGF-β (2.5 ng/ml) and TGF-β 1 expression was determined by RT-qPCR in different times of treatment. The values correspond to the mean of Ct value ± standard deviation. (n = 3; ∗, P < 0.05 versus control). (B) C2 C12 cells were incubated with several concentrations of TGF-β 1 for 9 h. TGF-β 1 mRNA levels were measured as in A. (n = 3; ∗, P < 0.05 versus control). (C) Detection by ELISA of TGF-β 1 protein levels into the medium of C2 C12 exposed to TGF-β for 48 h. The values correspond to the mean ± standard deviation. (n = 3; ∗, P < 0.05 versus control).
Fig. 3. The induction of TGF-β 1 expression by itself is dependent on TGF-β receptor I and Smad2/3 proteins in skeletal muscle cells. (A) C2 C12 myoblasts were untreated (control) or treated with 2.5 ng/ml of TGF-β in the absence or presence of SB525334 (5 μM) for 9 h. TGF-β 1 expression was determined by RT-qPCR. The values correspond to the mean of Ct value ± standard deviation. (n = 3 ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β ). (B) C2 C12 cells, transfected with control siRNA or Smad2/3 siRNA, were untreated (control) or treated with 2.5 ng/ml of TGF-β for 9 h. TGF-β 1 expression was measured by RT-qPCR. The values correspond to the mean of Ct value ± standard deviation. (n = 3; ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β ).
Smad-dependent signalling requires NOX-derived ROS production induced by TGF-β in skeletal muscle cells
induced by TGF-β requires NOX-derived ROS in skeletal muscle cells.
We evaluated whether the increase of intracellular ROS production in response to TGF-β is required for the activation of Smad signalling by this growth factor. Fig. 5A and B show that phosphoSmad3 levels are decreased by 1 mM of apocynin (for 0.5 ng/ml of TGF-β from 3.48 ± 0.13 to 2.12 ± 0.21; for 1.0 ng/ml of TGFβ from 3.62 ± 0.13 to 2.01 ± 0.11), suggesting the participation of NOX-induced ROS. To directly assay the effect of ROS on Smad3 phosphorylation, we incubated cells with the antioxidant NAC. The Figs. S1A and 1B show that NAC at 5 mM decreases Smad3 phosphorylation induced by TGF-β 1 (from 3.23 ± 0.15 to 2.04 ± 0.15). In the same direction to the results showed in Fig. 5A and B, Fig. 5C and D show that apocynin at 1 mM decreases the nuclear translocation of Smad4 in response to TGF-β (from 68.2 ± 4.4 to 42.3 ± 7.2). Together, these results strongly suggest that Smad signalling
TGF-β 1 and atrogin-1 expression, and p3TP-lux activity induced by TGF-β are dependent on NOX-derived ROS production in skeletal muscle cells Since NOX-derived ROS production modulates the Smaddependent signalling that is induced by TGF-β , we evaluated the effects of apocynin on the TGF-β target genes such as the autoinduction of TGF-β 1 and p3TP-lux activity. Fig. 6A shows that the inhibition of NOX with 1 mM of apocynin prevented the increase of TGF-β 1 expression by 50%. Similarly, apocynin also reduced by 50% the p3TPlux transcriptional activity of p3TP-lux that was induced by TGF-β (Fig. 6B). In addition, the induction of atrogin-1 (Fig. 6C) by TGFβ 1 is also modulated by NOX-induced ROS. In all Fig. 6A, B and C, the total abolition of the TGF-β effect in the presence of SB525334
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Fig. 4. TGF-β -induced ROS are mediated by TGF-β receptor I and NAD(P)H oxidase in skeletal muscle cells. (A) C2 C12 myoblasts were incubated with different concentrations of TGF-β for 24 h. The ROS levels were determined by fluorescence detection of DCF probe. Values correspond to the mean ± standard deviation (n = 3; ∗, P < 0.05 versus without TGF-β ). For B and C: C2 C12 cells were pre-incubated with the vehicle, apocynin (1 mM) or SB525334 (5 μM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 24 h. Then, cells were loaded with DCF probe for ROS detection. (B) Detection of ROS by microplate reader. Values correspond to the mean ± standard deviation (n = 3; ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β ). (C) Detection of ROS by microscopy. The nuclei are detected by Hoechst stain. Bar: 100 μm. (D) Quantification of experiments showed in C. (n = 3; ∗, P < 0.05 versus vehicle; #, P < 0.05 versus TGF-β ).
is shown to corroborate Tβ RI’s participation. Together, our results suggest that TGF-β 1 expression and transcriptional activity of target gene induced by TGF-β dependent on NOX-derived ROS production in skeletal muscle cells. Protein kinase C is required for TGF-β -induced intracellular ROS production and TGF-β expression in skeletal muscle cells First, we show that TGF-β 1 increased levels of intracellular Ca2+ in C2 C12 cells. These levels were not affected by 1 mM of apocynin
(Fig. S2A) neither 5 μM of DPI, another NOX inhibitor (Fig. S2B). This result suggests that the NOX activity and ROS generation that are induced by TGF-β occur downstream of the Ca2+ increase. Fig. 7A illustrates that the inhibition of PKC activity by chelerythrine (10 μM) prevented TGF-β -induced ROS production in C2 C12 cells, as evaluated using a DCF probe. This diminution of ROS production by the inhibition of PKC has a functional effect on TGF-β activity. Thus, Fig. 7B shows that the inhibition of PKC reduced up to 60% of the induction of TGF-β 1 expression. A similar effect was observed when p3TPlux transcriptional activity (Fig. 7C), or atrogin-1 expression (Fig. 7D),
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Fig. 5. Apocynin attenuates the activation of Smad signalling induced by TGF-β in skeletal muscle cells. (A) C2 C12 cells were pre-incubated with the vehicle or apocynin (1 mM) for 1 h, and then incubated with TGF-β (1.0 or 2.5 ng/ml) for 30 min. The protein levels of the phosphorylated and total Smad3 were evaluated by Western blot. (B) Quantification of experiments showed in A. The values correspond to the mean ± standard deviation (n = 3; ∗, P < 0.05 versus control; #, P < 0.05 versus TGF-β ). (C) C2 C12 myoblasts were preincubated with the vehicle or apocynin (1 mM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 90 min. The location of Smad4 was evaluated by indirect immunofluorescence. Bar: 100 μm. (D) Quantification of experiments showed in C. (n = 3; ∗, P < 0.05 versus vehicle; #, P < 0.05 versus TGF-β ).
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Fig. 6. TGF-β 1 expression induced in response to TGF-β is attenuated by apocynin in skeletal muscle cells. (A) C2 C12 myoblasts were pre-incubated with the vehicle, apocynin (1 mM) or SB525334 (5 μM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 9 h. TGF-β 1 expression was determined by RT-qPCR. The values correspond to the mean of Ct value ± standard deviation. (n = 3; ∗, P < 0.05 versus untreated control cells; #, P < 0.05 versus TGF-β ). (B) C2 C12 cells were transiently transfected with p3TP-lux plasmid. After 24 h, cells were pre-incubated with the vehicle, apocynin (1 mM) or SB525334 (5 μM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 24 h. The luciferase activity and the values represent the mean ± standard deviation (n = 3; ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β ). (C) C2 C12 myoblasts were pre-incubated with the vehicle, apocynin (1 mM) or SB525334 (5 μM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 6 h. Atrogin-1 expression was determined by RT-qPCR. The values correspond to the mean of Ct value ± standard deviation. (n = 3; ∗, P < 0.05 versus untreated control cells; #, P < 0.05 versus TGF-β ).
all of them induced by TGF-β were determined in C2 C12 myoblasts. These results suggest that PKC activity participates in TGF-β -induced ROS production and transcriptional activity in skeletal muscle cells. Together, these results suggest that PKC activity is partially required for TGF-β induced activity and required to activate NOX. Discussion This study demonstrates that TGF-β 1 induces its own expression in skeletal muscle cells through a mechanism that is dependent on Tβ RI, a Smad pathway and NOX-derived ROS. Interestingly, we demonstrated that pure apocynin decreased the increment of ROS and partially inhibited the auto-induction of TGF-β 1. In addition, NOX-induced ROS in response to TGF-β are able to modulate the canonical Smad signalling and another gene expression that is modulated by TGF-β such as p3TP-lux activity and atrogin-1 gene expression. Our results have relevance in several pathological conditions in skeletal muscle in which TGF-β levels and signalling has been shown to be increased, such as spinal muscular atrophy, Kennedy disease, Marfan syndrome, Duchenne muscular dystrophy and skeletal muscle atrophy by disuse or induced by cachexia (Burks and Cohn, 2011). In this context, our data show that the use of apocynin partially inhibits the effect and signalling dependent on TGF-β , decreasing the NOX-induced ROS production. These findings are in agreement with previous reports in which the use of apocynin has beneficial and protective effects on the diaphragm during prolonged mechanical ventilation (McClung et al., 2009), in plantaris muscle after myocardial infarction (Bechara et al., 2014) by reducing ROS, and also prevented force loss of dystrophic muscle and reduced membrane
damage (Ismail et al., 2014). Although the plant species from which apocynin has been isolated have not been used to evaluate their effect on muscle diseases, a previous report indicates that the treatment of diabetic rats, characterized by the loss of body weight and the increase of muscle wasting, with P. kurroa showed an increase on body weight compared to control animals, suggesting a protective role of this natural compound on muscle wasting (Husain et al., 2009). However in future experiments it will be important to evaluate the effect of the natural extract of P. kurroa on skeletal muscle function in order to develop natural therapies against muscle pathologies. One of the effects of TGF-β is to produce fibrosis in several tissues including skeletal muscle (MacDonald and Cohn, 2012). Several reports indicate that TGF-β requires ROS to induce fibrosis (Samarakoon et al., 2013). Thus, our data are in agreement with these reports since they indirectly suggest that the fibrotic effect mediated by TGF-β could require ROS in skeletal muscle. Interestingly, TGF-β also produces skeletal muscle atrophy, as has been recently reported (Mendias et al., 2012; Narola et al., 2013; Ohsawa et al., 2012; Zhang et al., 2014). However, the molecular mechanism and signalling pathways that are involved have not been elucidated yet. Our data show that the expression of atrogin-1, an E3 ligase involved in skeletal muscle atrophy, is induced by TGF-β 1 and also modulated by ROS in skeletal muscle cells. Thus, the participation of ROS seems to be a good candidate to evaluate, since its involvement in skeletal muscle atrophy induced by disuse or other causes has been suggested (Kondo et al., 1993; Semprun-Prieto et al., 2011). The critical role of the increase in the ROS levels that are mediated by NOX activation has been described in several pathological conditions (Simon et al., 2013). We, and others, have previously described that NOX induces ROS production in skeletal muscle cells
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Fig. 7. TGF-β 1 expression and ROS production in response to TGF-β require PKC activity in skeletal muscle cells. (A) C2 C12 cells were exposed to vehicle alone or chelerythrine (10 μM) in the absence (control) or presence of TGF-β (2.5 ng/ml), and DCF fluorescence from three independent experiments was measured. Value represent mean ± standard deviation (n = 3; ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β ). (B) C2 C12 myoblasts were pre-incubated with chelerythrine (10 μM) and then incubated with TGF-β (2.5 ng/ml). After 9 h, TGF-β 1 expression was determined by RT-qPCR. The values correspond to the mean of Ct value ± standard deviation. (n = 3; ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β 1). (C) C2 C12 cells transfected with p3TP-lux plasmid, were pre-incubated with the vehicle or chelerythrine (10 μM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 24 h. The luciferase activity and the values represent the mean ± standard deviation (n = 3; ∗, P < 0.05 versus untreated control; #, P < 0.05 versus TGF-β ). (D) C2 C12 myoblasts were pre-incubated with the vehicle or chelerythrine (10 μM) for 1 h, and then incubated with TGF-β (2.5 ng/ml) for 6 h. Atrogin-1 expression was determined by RT-qPCR. The values correspond to the mean of Ct value ± standard deviation. (n = 3; ∗, P < 0.05 versus untreated control cells; #, P < 0.05 versus TGF-β ).
(Cabello-Verrugio et al., 2011a; Espinosa et al., 2006). In this study, we show that TGF-β -induced ROS are generated by NOX activation. Several reports have shown that the expression and activity of NOX is relevant in skeletal muscle (Cabello-Verrugio et al., 2011a), including muscle diseases such as Duchenne muscular dystrophy or atrophy (Kim et al., 2013) in which TGF-β plays a critical role. The main mechanism to activate NOX is the Ca2+ -dependent PKCmediated serine phosphorylation of subunit p47phox (Babior, 2004). Our results demonstrate that an increase in intracellular Ca2+ levels induced by TGF-β would be a necessary event to activate NOX by PKC, since the inhibition of PKC activity produced a total abolition of ROS production, suggesting a regulation of NOX activation. This event is concomitant with the partial reduction of TGF-β -induced p3TPlux activity and TGF-β 1 expression in the presence of a PKC inhibitor to similar levels obtained when NOX inhibition was produced. Further experiments are needed to determine the direct role of PKC, and the possible isoform that is involved in the NOX-induced ROS dependent on TGF-β . Among the factors that regulate TGF-β expression is Ang-II, as we have recently demonstrated (Morales et al., 2014; Morales et al., 2012). Moreover, we also reported that the prolonged fibrotic effect induced by Ang-II in skeletal muscle is dependent on increased TGF-β expression (Morales et al., 2012). Interestingly, both Ang-II and TGFβ produce atrophy and fibrosis in skeletal muscle, two events that are observed in myopathies such as dystrophies (Acuna et al., 2014; Cabello-Verrugio et al., 2011a, 2012a; Cisternas et al., 2015; Mendias et al., 2012; Meneses et al., 2014; Morales et al., 2012; Narola et al., 2013). Thus, the stimulation of its own expression by TGF-β 1 seems
to be consistent with a mechanism to reinforce atrophy and fibrosis in which Ang-II can induce atrophy, TGF-β expression and fibrosis, and the further TGF-β 1 expression that is induced by itself perpetuates a fibrotic environment. Smad signalling is the main pathway through which TGF-β induces its biological activities. Thus, Smad signalling is critical for the TGF-β -induced inhibition of skeletal muscle differentiation (Massague et al., 1986). In addition, Smad signalling is key to modulating skeletal muscle fibrosis participating in the expression of extracellular matrix proteins such as collagens and fibronectin, or pro-fibrotic factors such as connective tissue growth factor (Acuna et al., 2014; Cabello-Verrugio et al., 2012b; Morales et al., 2011, 2013a, 2013b). In this context, our results are in agreement with these facts, since we show that the expression of TGF-β induced by itself requires Smad pathway activation, as demonstrated by Smad3 phosphorylation and the nuclear translocation of Smad4. However, we cannot disregard the participation of non-canonical or Smad-independent pathways in the modulation of TGF-β expression. In this line, we describe the modulation of p38MAPK on the TGF-β induction in response to Ang-II (Morales et al., 2012). Further experiments to study the effects of TGF-β -induced non-canonical signalling pathways on TGF-β 1 expression must be conducted.
Conclusions Our study is the first report that shows the autoinduction of TGF-β 1 through a TGF-β -receptor/Smad-dependent mechanism.
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