- Email: [email protected]
3 pathways in dermal fibroblasts
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Transient receptor potential vanilloid-3 (TRPV3) channel induces dermal fibrosis via the TRPV3/TSLP/Smad2/3 pathways in dermal fibroblasts Ji-young Um, Seok Young Kang, Hyun Ji Kim, Bo Young Chung, Chun Wook Park, Hye One Kim* Department of Dermatology, College of Medicine, Hallym University, Kangnam Sacred Heart Hospital, Seoul, South Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 July 2019 Received in revised form 10 December 2019 Accepted 24 December 2019
Background: Excessive wound healing can lead to hypertrophic scars, which are not only a cosmetic issue but could also be itchy or painful. Previously, we reported that, in comparison with normal tissue, thymic stromal lymphopoietin (TSLP) expression was increased in skin burn scars when the activation of transient receptor potential vanilloid-3 (TRPV3) of keratinocytes was increased. However, the functional role of TRPV3 in dermal fibrosis remains unclear. Objective: We aimed to determine whether TRPV3 affects the collagen production of human primary dermal fibroblasts (HPDFs) and to investigate the mechanism involved. Method: Human primary dermal fibroblasts were cultured and transformed into myofibroblasts using TSLP and carvacrol. Expression levels of α-SMA, fibronectin, and COL1A1 were determined using qPCR, western blotting, and immunofluorescence staining. Ca2+ influx was measured using a calcium-sensitive fluorescent dye, Fura3-AM. Nuclear factor of activated T-cells (NFAT) and phosphorylated-Smad2/3 were determined by western blotting. Silencing of TRPV3 with TRPV3-specific small interference RNA was evaluated using qPCR and western blotting. Results: The expression levels of α-SMA, fibronectin, COL1A1, and TSLP were significantly increased in carvacrol-treated HPDFs. The expression levels of α-SMA, fibronectin, and COL1A1 were significantly increased by TSLP. The expression levels of TSLP and COL1A1 were significantly blocked by TRPV3 silencing in HPDFs. Regulation of Ca2+ influx and the expression levels of NFAT and p-Smad2/3 were significantly increased in carvacrol-treated HPDFs. ECM productions induced via the TRPV3/TSLP/Smad2/ 3 pathways. Conclusions: The activation of the TRPV3 channels regulates dermal fibrosis by reducing extracellular matrix production via the TRPV3/TSLP/Smad2/3 pathways in dermal fibroblasts. © 2019 Published by Elsevier B.V. on behalf of Japanese Society for Investigative Dermatology.
Keywords: Transient receptor potential vanilloid-3 Thymic stromal lymphopoietin Fibrosis
1. Introduction Excessive wound healing can lead to hypertrophic scars, which are not only cosmetic defects but could also be itchy or painful [1]. Scars affect a considerable number of people worldwide and the characteristic symptoms include local itching, pain, dysfunction, restricted movement, and other physical and psychosocial problems [2]. Although they pose no risk to life, these symptoms can
* Corresponding author at: Department of Dermatology, Kangnam Sacred Heart Hospital, College of Medicine, Hallym University, 948-1, Daerim-1-Dong, Yeongdeungpo-gu, Seoul 150-950, South Korea. E-mail addresses: [email protected] (J.-y. Um), [email protected] (S.Y. Kang), [email protected] (H.J. Kim), [email protected] (B.Y. Chung), [email protected] (C.W. Park), [email protected] (H.O. Kim).
cause a considerable reduction in the quality of life. Previously, we reported that the expression of transient receptor potential vanilloid-3 (TRPV3) and thymic stromal lymphopoietin (TSLP) was increased in the tissues of pruritic burn scars in comparison with that in normal skin tissues of the same patients [2]. Pruritic burn scars tend to be hypertrophic but the associated pathogenic mechanisms are still unclear [3]. Additionally, the functional role of TRPV3 and TSLP expression in dermal fibrosis also remains to be elucidated. Dermal fibrosis is known to be involved in extracellular matrix (ECM) accumulation, which has been proposed as an important factor in scar formation [4,10]. Increased fibroblasts and myofibroblasts have been observed in skin scars. Fibroblasts, which are found in the stroma and are the cellular source of ECM proteins, are involved in the process of scar growth [5]. Among the fibroblasts,
https://doi.org/10.1016/j.jdermsci.2019.12.011 0923-1811/ © 2019 Published by Elsevier B.V. on behalf of Japanese Society for Investigative Dermatology.
Please cite this article in press as: J.- Um, et al., Transient receptor potential vanilloid-3 (TRPV3) channel induces dermal fibrosis via the TRPV3/ TSLP/Smad2/3 pathways in dermal fibroblasts, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2019.12.011
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myofibroblasts are an activated cell phenotype with tissue contractile properties and a high capacity for ECM protein secretion [6]. A major process in the scar formation is the ECM accumulation caused by differentiation of fibroblasts into myofibroblasts. Collagens are the major structural components of the skin and represent a large family of extracellular proteins that impart specific physical properties to tissues. These proteins also have important functions during morphogenesis and growth [11]. As a member of the TRP family, TRPV3 channel has been reported to be expressed in many cell types and tissues [7]. Moreover, all TRP family proteins are permeable to Ca2+ [8,14–18]. In addition, it is known that TRPV3 could be directly or indirectly involved in cardiac hypertrophy [16]. However, little is known about the role of TRP3 in dermal fibrosis with regard to scar formation. Carvacrol [2-methyl-5-(1-methylethyl) phenol] is a monoterpenic phenol extracted from several natural herbs such as Origanum vulgare and Thymus vulgaris. It has been demonstrated that carvacrol possess a variety of biological and pharmacological activities, such as hepatoprotective, analgesic, antitumor, antioxidant, and anti-inflammatory properties [7]. Also, carvacrol is potent activator of the human ion channels TRPV3. Recent studies revealed that carvacrol might mediates action of TRPV3. It is strongly activated by carvacrol [8]. Recently, it was reported that carvacrol treatment suppresses autoimmune arthritis, indicating that carvacrol may be useful for treating chronic inflammatory diseases. Recent studies have revealed mechanisms underlying carvacrol-induced expression of type I collagen gene [9]. Although there are several reports on the effect of carvacrol on chronic inflammatory diseases and skin aging, its effect on dermal fibrosis has not yet been explored. The purpose of this study was to determine whether TRPV3 affects collagen production in human primary dermal fibroblasts and investigate the underlying mechanism of action of TRPV3 on human primary dermal fibroblasts (HPDFs). 2. Materials and methods 2.1. Reagents Carvacrol and recombinant TSLP were purchased from SigmaAldrich (St. Louis, MO). Carvacrol was dissolved in dimethyl sulfoxide, and TSLP was dissolved in 0.1 % BSA. The maximum final concentration of the dimethyl sulfoxide was less than 0.1 %. 2.2. Dermal tissue and fibroblast culture To obtain dermal tissue, 3 patients were recruited. Written informed consent was obtained from each patient before surgery, and the study was approved by the Bioethics Committee of Gangnam Sacred Heart Hospital of Hallym University (2018-01032). Dermal tissue samples were isolated from surgical tissues by enzymatic digestion with collagenase (500 U/ml, Sigma), hyaluronidase (30 U/ml, Sigma), and DNase (10 U/ml, Sigma). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10 % (v/v) heat-inactivated FBS (Invitrogen, Carlsbad, CA), 1 % (v/v) 1 104 units/mL penicillin, and 1 104 mg/mL streptomycin (Invitrogen). Purity of obtained dermal fibroblast was confirmed microscopically by characteristic spindle-shaped cell morphology. Approximately 95 % of the cells in cultured dermal fibroblast were positive for the fibroblast markers vimentin and Thy-1 (Santa Cruz Biotechnology, CA), and negative for the epithelial cell marker, E-cadherin (Santa Cruz Biotechnology). Experimental cells were obtained from the fourth cell passage.
2.3. Quantitative PCR HPDFs (5 106 cells/ml) were exposed to carvacrol and TSLP for 24 h. Total RNA was isolated per the manufacturer's instructions using TRIzol (Invitrogen). Two micrograms of RNA were reverse transcribed using PrimeScriptTM RT Master Mix (Takara Biotechnology, CO., LTD.) per the manufacturer’s protocol. Synthesized cDNA was used in the RT-qPCR assay. Moreover, SYBR GreenTM Premix Ex TaqTM II Kit (Takara Biotechnology) was used for performing RT-qPCR, using the following primers: α-SMA (sense sequence 50 -GGTGCTGTCTCTCTATGC CTCTGGA-30 , anti-sense sequence 50 -CCCATCAGGCAA CTCGATACTCTTC-30 ), GAPDH (sense sequence 50 -GTGGATATT GTTGCCATCA ATGACC -30 , anti-sense sequence 50 - GCCCCAGCCTTCTTCATGGTGGT-30 ). Fibronectin (sense sequence 50 -GGATGCTCCTG CTGTCAC-30 , anti-sense sequence 50 CTGTTTGAT CTGGACCTGCAG -30 ), TRPV3 (sense sequence 50 GCTGAAG AAGCGCATCTTTGCA-30 , anti-sense sequence 50 -TCATAGG CCTCCTCTGTGTACT-3). Measurements were conducted in triplicate and the fold change values were calculated using the DCt method. qPCR was performed using LightCycler96 instrument (Roche, Switzerland). Results were obtained from at least three independent experiments. 2.4. TSLP quantitation by enzyme-linked immunosorbent assay (ELISA) TSLP protein expression levels in HPDFs after treatment with carvacrol were evaluated by ELISA. HPDFs were exposed to various concentrations of carvacrol for 48 h and the supernatant from the cultured cells were collected. TSLP levels were measured using ELISA (Biolegend, San Diego, CA, USA). Briefly, each well was blocked with blocking buffer for 2 h and washed with buffer. Antibodies against TSLP were added to the media and incubated for 1 h. A substrate solution and stop solution were introduced sequentially and the optical density of each well was determined within 30 min using an automatic plate reader (BioTek Instruments Korea Ltd.). 2.5. Western blot analysis HPDFs were lysed using the RIPA Lysis solution (Thermo Scientific, USA) and centrifuged for 30 min at 10,000xg HPDF lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore Inc., Billerica, MA). Membranes were blocked with 5 % skim milk solution and incubated overnight at 4 C with the following primary antibodies: Fibronectin, GAPDH (Santa Cruz Biotechnology), α-SMA, Nuclear factor of activated T-cells (NFAT), p-Smad2/3 (Cell signaling technology Inc, USA), TRPV3, TSLP, Collagen I (Abcam, USA), TSLPR (Novus biological, USA). After incubation, the membranes were washed in Tris buffered saline0.1 % Tween-20 buffer and treated with peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G (IgG). The blots were visualized with HRP-conjugated secondary antibodies and ECL system (ATTO Corporation, JAPAN). 2.6. Immunofluorescence staining HPDFs were fixed with 4 % paraformaldehyde for 30 min. Cells were permeabilized with 0.2 % Triton X-100 in 1 % bovine serum albumin (BSA) for 10 min, blocked with 3 % BSA for one hour at room temperature and incubated overnight at 4 C with anti-α-SMA (Cell Signaling Technology, Boston, MA), anti-fibronectin (Santa Cruz Biotechnology), anti-TRPV3 (Abcam) and anti-TSLP (Novus Biologicals). Cells were then incubated in goat anti-Mouse HRP, goat anti-Rb HRP (Millipore) and the second antibody conjugated with
Please cite this article in press as: J.- Um, et al., Transient receptor potential vanilloid-3 (TRPV3) channel induces dermal fibrosis via the TRPV3/ TSLP/Smad2/3 pathways in dermal fibroblasts, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2019.12.011
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FITC (Abcam) at 1:200 for 1 h. Stained HPDFs were captured and visualized using a microscope (Leica Microsystems, Germany). For nuclear counterstaining, Vectashield mounting medium was used along with DAPI (Vector Laboratories, Burlingame, CA). 2.7. RNA interference HPDFs were plated on 6-well plates and grown to 60%–70% confluence. Control siRNAs or TRPV3 (50 nM) were synthesized by Santa Cruz Biotechnology. HPDFs were transiently transfected with siRNA using LipofectamineTM RNAiMax (Invitrogen) per the manufacturer’s instructions. These HPDFs were used for qPCR. 2.8. Measurement of [Ca2+]i [Ca2+]i was measured using a calcium-sensitive fluorescent dye, Fura3-AM. HPDFs were washed with PBS buffer containing 120 mM NaCl, 5.4 mM KCl, 1.5 mM CaCl2, 1 mM NaH2PO4 and
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10 mM HEPES (pH 7.4), and subsequently incubated with Fura3AM at 37 C for 1 h. Cells were then washed with Krebs—HEPES buffer and resuspended at 1 106 cells/ml in the same buffer. Three milliliters of the cell suspension were placed in a Fluorescence reader and fluorescence was monitored at 340 nm (lex1), 380 nm (lex2) and 510 nm (lem) every 5 s for 100 s. The changes in [Ca2+]i were represented as fluorescent intensity (FI) (excitation at 488 nm, emission at 530 nm). All [Ca2+]i analyses were processed in a single cell, and the results are expressed as relative fluorescence intensity. 2.9. Statistical analysis Results are shown as mean standard error of the mean. Statistical significance of differences between groups was assessed by one-way analysis of variance (ANOVA) for factorial comparisons and by the Tukey’s multiple comparison tests for multiple comparisons using Prism for Windows software (GraphPad
Fig. 1. Effect of Carvacrol on transient receptor potential vanilloid-3 (TRPV3) expression in human primary dermal fibroblasts. (a) mRNA expression levels of TRPV3 in HPDFs were examined by qPCR. (b) Protein expression levels of TRPV3 were determined by western blotting. (c) Localization of TRPV3 and TSLP was determined by immunofluorescence staining. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Values are means SEM. All experiments of at least three independent experiments. **p < 0.01 vs. control, ***p < 0.001 vs. control.
Please cite this article in press as: J.- Um, et al., Transient receptor potential vanilloid-3 (TRPV3) channel induces dermal fibrosis via the TRPV3/ TSLP/Smad2/3 pathways in dermal fibroblasts, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2019.12.011
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Software Inc., La Jolla, CA, USA). Results were obtained from at least three independent experiments.
activator carvacrol significantly increased TSLP and TSLPR mRNA and protein expression levels in HPDFs.
3. Results
3.3. Effect of carvacrol on extracellular matrix (ECM) production in HPDFs
3.1. Effect of Carvacrol on TRPV3 expression in HPDFs Carvacrol and TSLP were examined at various concentrations (data not shown). Carvacrol did not affect cell survival up to 400 mM concentration. Moreover, TSLP did not show any cytotoxic effects for 72 h at concentrations up to 200 ng/ml. To investigate the effect of carvacrol on TRPV3 and TSLP expression, Carvacrol was treated for 6 h and 24 h, respectively. mRNA and protein expression levels of TRPV3 were increased (Fig. 1a and b). Immunofluorescence staining analysis showed that expression of TRPV3 and TSLP was higher in carvacrol-treated HPDFs than in control and was localized and concentrated in the cytoplasm (Fig. 1c). These results indicated that carvacrol significantly induced TRPV3 activation compare to the control. 3.2. Effect of Carvacrol on TSLP and thymic stromal lymphopoietin receptor (TSLPR) expression in HPDFs To examine whether TRPV3 regulates TSLP and TSLPR expression in vitro, we performed qPCR (Fig. 2a), ELISA (Fig. 2b), and western blot (Fig. 2c). These results showed that the TRPV3
To investigate the effect of carvacrol on myofibroblast differentiation and ECM production, HPDFs were treated with carvacrol and a-SMA and collagen expression levels were determined. These results showed that carvacrol increased αSMA, fibronectin, and COL1A1 mRNA expression levels in HPDFs (Fig. 3a). The protein expression levels of both α-SMA, fibronectin and COL1A1 were also increased after treatment with carvacrol for 72 h (Fig. 3b and c). Because it is already known that myofibroblast differentiation and ECM production are induced by TGF-β1, we used it as a positive control. These results suggest that TRPV3 is involved in dermal fibrosis induced by carvacrol. 3.4. Effect of TSLP on ECM production in HPDFs To examine whether TSLP regulates myofibroblast differentiation and ECM production, we determined the expression of α-SMA and collagen. The results showed that TSLP significantly induced αSMA, fibronectin, and COL1A1 mRNA expression levels in HPDFs (Fig. 4a). Moreover, protein expression levels of α-SMA, fibronectin and COL1A1 increased (Fig. 4b and c). These results implied that
Fig. 2. Effect of Carvacrol on TSLP and TSLPR in human primary dermal fibroblasts. Expression levels of TSLP and TSLPR in HPDFs were examined by (a) qPCR, (b) ELISA, and (C) western blotting. GAPDH was used as a loading control. All experiments of at least three independent experiments. Values are means SEM. *p < 0.05 vs. control, ***p < 0.001 vs. control.
Please cite this article in press as: J.- Um, et al., Transient receptor potential vanilloid-3 (TRPV3) channel induces dermal fibrosis via the TRPV3/ TSLP/Smad2/3 pathways in dermal fibroblasts, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2019.12.011
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Fig. 3. Effect of Carvacrol on ECM production in human primary dermal fibroblasts. Expression levels of a-SMA, fibronectin, and COL1A1 mRNAs in HPDFs were examined by (a) qPCR, (b) immunofluorescence staining, and (c) western blotting. The cells were treated with TGF-β1 and Carvacrol. TGF-β1 was used as positive control. GAPDH was used as a loading control. All experiments of at least three independent experiments. Values are means SEM. *p < 0.05 vs. control, ***p < 0.001 vs. control.
Fig. 4. Effect of TSLP on extracellular matrix (ECM) in human primary dermal fibroblasts. mRNA expression levels of (a) a-SMA, (b) fibronectin, and (c) COL1A1 in HPDFs were examined by qPCR and (d) Protein expression levels of a-SMA and fibronectin were examined by western blotting. The cells were treated with TSLP. All experiments of at least three independent experiments. Values are means SEM. *p < 0.05 vs. control, **p < 0.01 vs. control, ***p < 0.001 vs. control.
TRPV3 activation promoted TSLP expression, and eventually resulted in dermal fibrosis. 3.5. Effect of TRPV3 silencing on TSLP and COL1A1 in HPDFs To further confirm the effect of TRPV3 on TSLP and COL1A1 expression, HPDFs were transfected with control siRNA or TRPV3-
siRNA. The results showed that TRPV3-siRNA significantly inhibited the carvacrol-induced expression of TSLP and COL1A1 compared with control siRNA (Fig. 5a–c). We also found that carvacrol significantly up-regulated TSLP and COL1A1 mRNA expression. Moreover, TRPV3-siRNA inhibited the protein expression of TRPV3, α-SMA, fibronectin and COL1A1 (Fig. 5d). These results suggest that COL1A1 and TSLP are regulated by carvacrol.
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Fig. 5. Effect of TRPV3 silencing on TSLP and ECM production in human primary dermal fibroblasts. Silencing TRPV3 with TRPV3-specific small interference RNA was determined by (a–c) qPCR and (d) western blotting. Cells were transfected with siControl or siTRPV3 for 24 h and stimulated with carvacrol for 3 h. All experiments of at least three independent experiments. Values are means SEM. ***p < 0.001 vs. siControl, †p < 0.05 vs. siControl+ Carvacrol.
3.6. Effect of carvacrol on Ca2+ influx, NFAT dephosphorylation, and Smad2/3 signaling in HPDFs
(Fig. 6d and f). Taken together, these results indicated that TRPV3 was involved in dermal fibrosis induced by p-Smad2/3.
It has been reported that TRPV3 activity is intimately associated with cytosolic Ca2+ influx. Intracellular Ca2+ ([Ca2+]i) could act as a secondary messenger involved in multiple cellular functions, including inflammation, molecular transportation, and gene transcription. Ca2+]i can induce NFAT. The dephosphorylation of NFAT is dependent on Ca2+ influx. Therefore, we examined whether carvacrol can induce Ca2+]i influx and dephosphorylation of NFAT in HPDFs. The results showed that carvacrol increased [Ca2+]i compared to control (Fig. 6a). Results of the effect of carvacrol on the dephosphorylation of NFAT are shown in Fig. 6b. We found that carvacrol-treated HPDFs showed significantly elevated Ca2+ level and dephosphorylated NFAT. These results indicated that TRPV3 is involved in NFAT signaling. To confirm if TRPV3 is involved in the pSmad2/3 signaling pathway, HPDFs were treated with carvacrol or sis3 (Smad 2/3 inhibitor), and phosphorylated Smad2/3 level was analyzed (Fig. 6c, and f). The results showed that carvacrol increased p-Smad2/3 level in HPDFs. To examine whether smad2/3 regulates carvacrol-induced myofibroblast differentiation and ECM production, we determined the expression of protein levels (Fig. 6e). We also found that sis3 (Smad 2/3 inhibitor) significantly inhibit α-SMA, fibronectin and COL1A1 protein expression. But TRPV3, TSLP not inhibit. TRPV3 and TSLP are up-regulator of Smad2/3. To check if TRPV3-TSLP is involved in the p-Smad2/3 signaling pathway, HPDFs were treated with TSLP, and p-Smad2/3 level was determined. Moreover, TSLP increased p-Smad2/3 level
4. Discussion In this report, we investigated whether TRPV3 affects myofibroblast differentiation and collagen production in HPDFs and identified the underlying mechanism of action of TRPV3 on HPDFs. We found that carvacrol activates TRPV3, which induces myofibroblast differentiation, collagen production, and TSLP expression, through activation of the TRPV3/Smad2/3 signaling pathway. TRPV3 is predominantly expressed in keratinocytes present in the skin, esophagus, distal colon, and cornea [1,2,24,28,30,33]; Its expression and function in dermal fibrosis are still largely unknown. Recent studies clarify that TRPV3 and TSLP are upregulated in the skin of patients with allergic dermatitis [26]. In our previous study, TRPV3 was demonstrated to facilitate TSLP release from keratinocytes derived from pruritic and hypertrophic burn scar tissues [2]. TRPV3 induced the expression of TSLP in HPDFs in the present study. TSLP, a protein belonging to the cytokine family, is known to be produced mainly by non-hematopoietic cells such as fibroblasts, epithelial cells, and different types of stromal or stromal-like cells [10–12,29]. Recently, it was reported that TSLP is involved in allergic diseases and fibrotic conditions such as asthma, allergic rhinitis, atopic dermatitis, systemic sclerosis, and scleroderma [12–15,27]. These diseases involve factors that promoted TSLP expression by increased intracellular Ca2+ levels. Also, epithelial
Please cite this article in press as: J.- Um, et al., Transient receptor potential vanilloid-3 (TRPV3) channel induces dermal fibrosis via the TRPV3/ TSLP/Smad2/3 pathways in dermal fibroblasts, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2019.12.011
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Fig. 6. Effect of Carvacrol on Ca2+ influx, NFAT, and p-Smad2/3 signal pathway in human primary dermal fibroblasts. (a) HPDFs were loaded with Fura-3 for 100 s and then washed with PBS buffer for [Ca2+]i measurement by Fluorescence plate Readers. (b, c) Expression levels of dephosphorylated NFAT and p-smad2/3 in HPDFs were examined by western blot. (d) Expression levels of P-smad2/3 in HPDFs were examined by western blotting. (e) HPDFs, pre-treated with sis3 (smad2/3 inhibitor) at 3 mM for 1 h, were treated with carvacrol or TSLP. (f) Hypothetical schema of the action of TRPV3 in HPDFs. All experiments of at least three independent experiments. The cells were treated with carvacrol. Values are means SEM. ***p < 0.001 vs. control.
TSLP expression in pulmonary fibrosis and TSLP overexpression have been shown to contribute to excessive ECM synthesis [18,20]. Our data suggest that TSLP expression induces the production of αSMA (myofibroblast differentiation marker), fibronectin, and collagen in dermal fibroblasts and induces dermal fibrosis. We further examined the effects of carvacrol on dephosphorylated nuclear factor of activated T-cells (NFAT) by intracellular increased Ca2+ levels promote TSLP expression. Cordeiro et al. demonstrated that intracellular calcium levels serve as a modulator of epithelial wound healing [22,23,25]. Moreover, Ca2+ influx plays an essential role in wound healing of epithelial cells [20–22]. Our data showed that carvacrol increases intracellular Ca2+ levels and then triggers NFAT action to further increase TSLP expression. The activated T cell (NFAT) pathway plays a key role in electrical remodeling in pathological cardiac hypertrophy. Recent studies have clarified the effects of TRPV3 in pressure overload-induced cardiac hypertrophy and the relationship between TRPV3 activation and calcineurin/NFAT signaling. Intracellular calcium levels can regulate ECM remodeling [18–20].
The extracellular matrix (ECM), a three-dimensional network of extracellular macromolecules, tightly controls wound healing. Type I collagen (Col I), the most abundant collagen type in the human body, plays an essential role in cell motility control by interacting with the intracellular cytoskeleton [18]. It is expressed in the scar tissue as the end-product of the wound-healing process [21,31,33]. Recent study demonstrated that carvacrol plays an important role in regulating the cell migration [34]. These results suggest that regulation of the TRPV3 channel in excessive wound healing can lead to hypertrophic scars; this may represent a novel pathologic mechanism. Furthermore, because TRPV3 is also expressed in the epidermis and dermis, the regulation of this channel may reduce pruritus and scar formation [21]. Smad mediates collagen production by TGF-β1 in various cell types. It is already well-known that TGF-β1 is a potent stimulator and key mediator of fibrosis [4,5]. TGF-β1 has been widely recognized as a regulator of myofibroblast differentiation [5]; however, the role of the TRPV3-Smad pathway in dermal fibrosis
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remains unknown. Also, the best know pathway for TGF-β1 activity is the Smad-complex pathway, in which TGF- β1 binds to the Smadbinding elements of target genes [32]. This study aimed to investigate the possible collagen production-promoting activities of carvacrol and its mechanism of action in human dermal fibroblasts. Based on these results, we speculated that the activation of TRPV3 in dermal fibroblasts contributes to dermal fibrosis as that of epidermal keratinocytes associate with postburn pruritus in the hypertrophic burn scars. To our knowledge, this is the first study reporting the effect of carvacrol on HPDFs. In conclusion, this study showed that carvacrol induced myofibroblast differentiation and ECM production in HPDFs via the Smad2/3 pathway. The results of the study suggest that carvacrol induces ECM production through the Smad2/3 signaling pathway in skin dermal fibroblasts. Therefore, modulation of TRPV3 could prove to be a possible therapeutic target for dermal fibrosis. Funding This work was supported by the National Research Foundation of Korea (NRF) [grant number NRF-2017R1A2B4006252], the Korea Healthcare Technology R&D Project funded by the Ministry of Health & Welfare, Republic of Korea [grant number HI17C0597], and the Hallym University Research Fund [grant number HURF2017-83]. Declaration of Competing Interest The authors have no conflict of interest to declare Acknowledgements We thank for sharing reagents. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j. jdermsci.2019.12.011. References [1] L. Carswell, J. Borger, Hypertrophic Scarring Keloids, Treasure Island (FL): StatPearls Publishing, 2019 Jan-. [2] C.W. Park, H.J. Kim, Y.W. Choi, B.Y. Chung, S.Y. Woo, D.K. Song, et al., TRPV3 channel in keratinocytes in scars with post-burn pruritus, Int. J. Mol. Sci. 18 (November (11)) (2017) E2425. [3] Y.H. Choi, K.M. Kim, H.O. Kim, Y.C. Jang, I.S. Kwak, Clinical and histological correlation in post-burn hypertrophic scar for pain and itching sensation, Ann. Dermatol. 25 (November (4)) (2013) 428–433. [4] J.D. Thatcher, The TGF-beta signal transduction pathway, Sci. Signal. 3 (April (119)) (2010) tr4. [5] S. Bhattacharyya, A.K. Ghosh, J. Pannu, Y. Mori, S. Takagawa, G. Chen, et al., Fibroblast expression of the coactivator p300 governs the intensity of profibrotic response to transforming growth factor beta, Arthritis Rheum. 52 (April (4)) (2005) 1248–1258. [6] A. Al-Shami, R. Spolski, J. Kelly, A. Keane-Myers, W.J. Leonard, A role for TSLP in the development of inflammation in an asthma model, J. Exp. Med. 202 (September (6)) (2005) 829–839. [7] T.T. Cui, G.X. Wang, N.N. Wei, K. Wang, A pivotal role for the activation of TRPV3 channel in itch sensations induced by the natural skin sensitizer carvacrol, Acta Pharmacol. Sin. 39 (March (3)) (2018) 331–335. [8] J. Lee, E. Jung, H. Yu, Y. Kim, J. Ha, Y.S. Kim, et al., Mechanisms of carvacrolinduced expression of type I collagen gene, J. Dermatol. Sci. 52 (December (3)) (2008) 160–169. [9] T.T. Cui, G.X. Wang, N.N. Wei, K. Wang, A pivotal role for the activation of TRPV3 channel in itch sensations induced by the natural skin sensitizer carvacrol, Acta Pharmacol. Sin. 39 (March (3)) (2018) 331–335.
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Transient receptor potential vanilloid-3 (TRPV3) channel induces dermal fibrosis via the TRPV3/TSLP/Smad2/3 pathways in dermal fibroblasts Ji-young Um, Seok Young Kang, Hyun Ji Kim, Bo Young Chung, Chun Wook Park, Hye One Kim* Department of Dermatology, College of Medicine, Hallym University, Kangnam Sacred Heart Hospital, Seoul, South Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 July 2019 Received in revised form 10 December 2019 Accepted 24 December 2019
Background: Excessive wound healing can lead to hypertrophic scars, which are not only a cosmetic issue but could also be itchy or painful. Previously, we reported that, in comparison with normal tissue, thymic stromal lymphopoietin (TSLP) expression was increased in skin burn scars when the activation of transient receptor potential vanilloid-3 (TRPV3) of keratinocytes was increased. However, the functional role of TRPV3 in dermal fibrosis remains unclear. Objective: We aimed to determine whether TRPV3 affects the collagen production of human primary dermal fibroblasts (HPDFs) and to investigate the mechanism involved. Method: Human primary dermal fibroblasts were cultured and transformed into myofibroblasts using TSLP and carvacrol. Expression levels of α-SMA, fibronectin, and COL1A1 were determined using qPCR, western blotting, and immunofluorescence staining. Ca2+ influx was measured using a calcium-sensitive fluorescent dye, Fura3-AM. Nuclear factor of activated T-cells (NFAT) and phosphorylated-Smad2/3 were determined by western blotting. Silencing of TRPV3 with TRPV3-specific small interference RNA was evaluated using qPCR and western blotting. Results: The expression levels of α-SMA, fibronectin, COL1A1, and TSLP were significantly increased in carvacrol-treated HPDFs. The expression levels of α-SMA, fibronectin, and COL1A1 were significantly increased by TSLP. The expression levels of TSLP and COL1A1 were significantly blocked by TRPV3 silencing in HPDFs. Regulation of Ca2+ influx and the expression levels of NFAT and p-Smad2/3 were significantly increased in carvacrol-treated HPDFs. ECM productions induced via the TRPV3/TSLP/Smad2/ 3 pathways. Conclusions: The activation of the TRPV3 channels regulates dermal fibrosis by reducing extracellular matrix production via the TRPV3/TSLP/Smad2/3 pathways in dermal fibroblasts. © 2019 Published by Elsevier B.V. on behalf of Japanese Society for Investigative Dermatology.
Keywords: Transient receptor potential vanilloid-3 Thymic stromal lymphopoietin Fibrosis
1. Introduction Excessive wound healing can lead to hypertrophic scars, which are not only cosmetic defects but could also be itchy or painful [1]. Scars affect a considerable number of people worldwide and the characteristic symptoms include local itching, pain, dysfunction, restricted movement, and other physical and psychosocial problems [2]. Although they pose no risk to life, these symptoms can
* Corresponding author at: Department of Dermatology, Kangnam Sacred Heart Hospital, College of Medicine, Hallym University, 948-1, Daerim-1-Dong, Yeongdeungpo-gu, Seoul 150-950, South Korea. E-mail addresses: [email protected] (J.-y. Um), [email protected] (S.Y. Kang), [email protected] (H.J. Kim), [email protected] (B.Y. Chung), [email protected] (C.W. Park), [email protected] (H.O. Kim).
cause a considerable reduction in the quality of life. Previously, we reported that the expression of transient receptor potential vanilloid-3 (TRPV3) and thymic stromal lymphopoietin (TSLP) was increased in the tissues of pruritic burn scars in comparison with that in normal skin tissues of the same patients [2]. Pruritic burn scars tend to be hypertrophic but the associated pathogenic mechanisms are still unclear [3]. Additionally, the functional role of TRPV3 and TSLP expression in dermal fibrosis also remains to be elucidated. Dermal fibrosis is known to be involved in extracellular matrix (ECM) accumulation, which has been proposed as an important factor in scar formation [4,10]. Increased fibroblasts and myofibroblasts have been observed in skin scars. Fibroblasts, which are found in the stroma and are the cellular source of ECM proteins, are involved in the process of scar growth [5]. Among the fibroblasts,
https://doi.org/10.1016/j.jdermsci.2019.12.011 0923-1811/ © 2019 Published by Elsevier B.V. on behalf of Japanese Society for Investigative Dermatology.
Please cite this article in press as: J.- Um, et al., Transient receptor potential vanilloid-3 (TRPV3) channel induces dermal fibrosis via the TRPV3/ TSLP/Smad2/3 pathways in dermal fibroblasts, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2019.12.011
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myofibroblasts are an activated cell phenotype with tissue contractile properties and a high capacity for ECM protein secretion [6]. A major process in the scar formation is the ECM accumulation caused by differentiation of fibroblasts into myofibroblasts. Collagens are the major structural components of the skin and represent a large family of extracellular proteins that impart specific physical properties to tissues. These proteins also have important functions during morphogenesis and growth [11]. As a member of the TRP family, TRPV3 channel has been reported to be expressed in many cell types and tissues [7]. Moreover, all TRP family proteins are permeable to Ca2+ [8,14–18]. In addition, it is known that TRPV3 could be directly or indirectly involved in cardiac hypertrophy [16]. However, little is known about the role of TRP3 in dermal fibrosis with regard to scar formation. Carvacrol [2-methyl-5-(1-methylethyl) phenol] is a monoterpenic phenol extracted from several natural herbs such as Origanum vulgare and Thymus vulgaris. It has been demonstrated that carvacrol possess a variety of biological and pharmacological activities, such as hepatoprotective, analgesic, antitumor, antioxidant, and anti-inflammatory properties [7]. Also, carvacrol is potent activator of the human ion channels TRPV3. Recent studies revealed that carvacrol might mediates action of TRPV3. It is strongly activated by carvacrol [8]. Recently, it was reported that carvacrol treatment suppresses autoimmune arthritis, indicating that carvacrol may be useful for treating chronic inflammatory diseases. Recent studies have revealed mechanisms underlying carvacrol-induced expression of type I collagen gene [9]. Although there are several reports on the effect of carvacrol on chronic inflammatory diseases and skin aging, its effect on dermal fibrosis has not yet been explored. The purpose of this study was to determine whether TRPV3 affects collagen production in human primary dermal fibroblasts and investigate the underlying mechanism of action of TRPV3 on human primary dermal fibroblasts (HPDFs). 2. Materials and methods 2.1. Reagents Carvacrol and recombinant TSLP were purchased from SigmaAldrich (St. Louis, MO). Carvacrol was dissolved in dimethyl sulfoxide, and TSLP was dissolved in 0.1 % BSA. The maximum final concentration of the dimethyl sulfoxide was less than 0.1 %. 2.2. Dermal tissue and fibroblast culture To obtain dermal tissue, 3 patients were recruited. Written informed consent was obtained from each patient before surgery, and the study was approved by the Bioethics Committee of Gangnam Sacred Heart Hospital of Hallym University (2018-01032). Dermal tissue samples were isolated from surgical tissues by enzymatic digestion with collagenase (500 U/ml, Sigma), hyaluronidase (30 U/ml, Sigma), and DNase (10 U/ml, Sigma). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10 % (v/v) heat-inactivated FBS (Invitrogen, Carlsbad, CA), 1 % (v/v) 1 104 units/mL penicillin, and 1 104 mg/mL streptomycin (Invitrogen). Purity of obtained dermal fibroblast was confirmed microscopically by characteristic spindle-shaped cell morphology. Approximately 95 % of the cells in cultured dermal fibroblast were positive for the fibroblast markers vimentin and Thy-1 (Santa Cruz Biotechnology, CA), and negative for the epithelial cell marker, E-cadherin (Santa Cruz Biotechnology). Experimental cells were obtained from the fourth cell passage.
2.3. Quantitative PCR HPDFs (5 106 cells/ml) were exposed to carvacrol and TSLP for 24 h. Total RNA was isolated per the manufacturer's instructions using TRIzol (Invitrogen). Two micrograms of RNA were reverse transcribed using PrimeScriptTM RT Master Mix (Takara Biotechnology, CO., LTD.) per the manufacturer’s protocol. Synthesized cDNA was used in the RT-qPCR assay. Moreover, SYBR GreenTM Premix Ex TaqTM II Kit (Takara Biotechnology) was used for performing RT-qPCR, using the following primers: α-SMA (sense sequence 50 -GGTGCTGTCTCTCTATGC CTCTGGA-30 , anti-sense sequence 50 -CCCATCAGGCAA CTCGATACTCTTC-30 ), GAPDH (sense sequence 50 -GTGGATATT GTTGCCATCA ATGACC -30 , anti-sense sequence 50 - GCCCCAGCCTTCTTCATGGTGGT-30 ). Fibronectin (sense sequence 50 -GGATGCTCCTG CTGTCAC-30 , anti-sense sequence 50 CTGTTTGAT CTGGACCTGCAG -30 ), TRPV3 (sense sequence 50 GCTGAAG AAGCGCATCTTTGCA-30 , anti-sense sequence 50 -TCATAGG CCTCCTCTGTGTACT-3). Measurements were conducted in triplicate and the fold change values were calculated using the DCt method. qPCR was performed using LightCycler96 instrument (Roche, Switzerland). Results were obtained from at least three independent experiments. 2.4. TSLP quantitation by enzyme-linked immunosorbent assay (ELISA) TSLP protein expression levels in HPDFs after treatment with carvacrol were evaluated by ELISA. HPDFs were exposed to various concentrations of carvacrol for 48 h and the supernatant from the cultured cells were collected. TSLP levels were measured using ELISA (Biolegend, San Diego, CA, USA). Briefly, each well was blocked with blocking buffer for 2 h and washed with buffer. Antibodies against TSLP were added to the media and incubated for 1 h. A substrate solution and stop solution were introduced sequentially and the optical density of each well was determined within 30 min using an automatic plate reader (BioTek Instruments Korea Ltd.). 2.5. Western blot analysis HPDFs were lysed using the RIPA Lysis solution (Thermo Scientific, USA) and centrifuged for 30 min at 10,000xg HPDF lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore Inc., Billerica, MA). Membranes were blocked with 5 % skim milk solution and incubated overnight at 4 C with the following primary antibodies: Fibronectin, GAPDH (Santa Cruz Biotechnology), α-SMA, Nuclear factor of activated T-cells (NFAT), p-Smad2/3 (Cell signaling technology Inc, USA), TRPV3, TSLP, Collagen I (Abcam, USA), TSLPR (Novus biological, USA). After incubation, the membranes were washed in Tris buffered saline0.1 % Tween-20 buffer and treated with peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G (IgG). The blots were visualized with HRP-conjugated secondary antibodies and ECL system (ATTO Corporation, JAPAN). 2.6. Immunofluorescence staining HPDFs were fixed with 4 % paraformaldehyde for 30 min. Cells were permeabilized with 0.2 % Triton X-100 in 1 % bovine serum albumin (BSA) for 10 min, blocked with 3 % BSA for one hour at room temperature and incubated overnight at 4 C with anti-α-SMA (Cell Signaling Technology, Boston, MA), anti-fibronectin (Santa Cruz Biotechnology), anti-TRPV3 (Abcam) and anti-TSLP (Novus Biologicals). Cells were then incubated in goat anti-Mouse HRP, goat anti-Rb HRP (Millipore) and the second antibody conjugated with
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FITC (Abcam) at 1:200 for 1 h. Stained HPDFs were captured and visualized using a microscope (Leica Microsystems, Germany). For nuclear counterstaining, Vectashield mounting medium was used along with DAPI (Vector Laboratories, Burlingame, CA). 2.7. RNA interference HPDFs were plated on 6-well plates and grown to 60%–70% confluence. Control siRNAs or TRPV3 (50 nM) were synthesized by Santa Cruz Biotechnology. HPDFs were transiently transfected with siRNA using LipofectamineTM RNAiMax (Invitrogen) per the manufacturer’s instructions. These HPDFs were used for qPCR. 2.8. Measurement of [Ca2+]i [Ca2+]i was measured using a calcium-sensitive fluorescent dye, Fura3-AM. HPDFs were washed with PBS buffer containing 120 mM NaCl, 5.4 mM KCl, 1.5 mM CaCl2, 1 mM NaH2PO4 and
3
10 mM HEPES (pH 7.4), and subsequently incubated with Fura3AM at 37 C for 1 h. Cells were then washed with Krebs—HEPES buffer and resuspended at 1 106 cells/ml in the same buffer. Three milliliters of the cell suspension were placed in a Fluorescence reader and fluorescence was monitored at 340 nm (lex1), 380 nm (lex2) and 510 nm (lem) every 5 s for 100 s. The changes in [Ca2+]i were represented as fluorescent intensity (FI) (excitation at 488 nm, emission at 530 nm). All [Ca2+]i analyses were processed in a single cell, and the results are expressed as relative fluorescence intensity. 2.9. Statistical analysis Results are shown as mean standard error of the mean. Statistical significance of differences between groups was assessed by one-way analysis of variance (ANOVA) for factorial comparisons and by the Tukey’s multiple comparison tests for multiple comparisons using Prism for Windows software (GraphPad
Fig. 1. Effect of Carvacrol on transient receptor potential vanilloid-3 (TRPV3) expression in human primary dermal fibroblasts. (a) mRNA expression levels of TRPV3 in HPDFs were examined by qPCR. (b) Protein expression levels of TRPV3 were determined by western blotting. (c) Localization of TRPV3 and TSLP was determined by immunofluorescence staining. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Values are means SEM. All experiments of at least three independent experiments. **p < 0.01 vs. control, ***p < 0.001 vs. control.
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Software Inc., La Jolla, CA, USA). Results were obtained from at least three independent experiments.
activator carvacrol significantly increased TSLP and TSLPR mRNA and protein expression levels in HPDFs.
3. Results
3.3. Effect of carvacrol on extracellular matrix (ECM) production in HPDFs
3.1. Effect of Carvacrol on TRPV3 expression in HPDFs Carvacrol and TSLP were examined at various concentrations (data not shown). Carvacrol did not affect cell survival up to 400 mM concentration. Moreover, TSLP did not show any cytotoxic effects for 72 h at concentrations up to 200 ng/ml. To investigate the effect of carvacrol on TRPV3 and TSLP expression, Carvacrol was treated for 6 h and 24 h, respectively. mRNA and protein expression levels of TRPV3 were increased (Fig. 1a and b). Immunofluorescence staining analysis showed that expression of TRPV3 and TSLP was higher in carvacrol-treated HPDFs than in control and was localized and concentrated in the cytoplasm (Fig. 1c). These results indicated that carvacrol significantly induced TRPV3 activation compare to the control. 3.2. Effect of Carvacrol on TSLP and thymic stromal lymphopoietin receptor (TSLPR) expression in HPDFs To examine whether TRPV3 regulates TSLP and TSLPR expression in vitro, we performed qPCR (Fig. 2a), ELISA (Fig. 2b), and western blot (Fig. 2c). These results showed that the TRPV3
To investigate the effect of carvacrol on myofibroblast differentiation and ECM production, HPDFs were treated with carvacrol and a-SMA and collagen expression levels were determined. These results showed that carvacrol increased αSMA, fibronectin, and COL1A1 mRNA expression levels in HPDFs (Fig. 3a). The protein expression levels of both α-SMA, fibronectin and COL1A1 were also increased after treatment with carvacrol for 72 h (Fig. 3b and c). Because it is already known that myofibroblast differentiation and ECM production are induced by TGF-β1, we used it as a positive control. These results suggest that TRPV3 is involved in dermal fibrosis induced by carvacrol. 3.4. Effect of TSLP on ECM production in HPDFs To examine whether TSLP regulates myofibroblast differentiation and ECM production, we determined the expression of α-SMA and collagen. The results showed that TSLP significantly induced αSMA, fibronectin, and COL1A1 mRNA expression levels in HPDFs (Fig. 4a). Moreover, protein expression levels of α-SMA, fibronectin and COL1A1 increased (Fig. 4b and c). These results implied that
Fig. 2. Effect of Carvacrol on TSLP and TSLPR in human primary dermal fibroblasts. Expression levels of TSLP and TSLPR in HPDFs were examined by (a) qPCR, (b) ELISA, and (C) western blotting. GAPDH was used as a loading control. All experiments of at least three independent experiments. Values are means SEM. *p < 0.05 vs. control, ***p < 0.001 vs. control.
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Fig. 3. Effect of Carvacrol on ECM production in human primary dermal fibroblasts. Expression levels of a-SMA, fibronectin, and COL1A1 mRNAs in HPDFs were examined by (a) qPCR, (b) immunofluorescence staining, and (c) western blotting. The cells were treated with TGF-β1 and Carvacrol. TGF-β1 was used as positive control. GAPDH was used as a loading control. All experiments of at least three independent experiments. Values are means SEM. *p < 0.05 vs. control, ***p < 0.001 vs. control.
Fig. 4. Effect of TSLP on extracellular matrix (ECM) in human primary dermal fibroblasts. mRNA expression levels of (a) a-SMA, (b) fibronectin, and (c) COL1A1 in HPDFs were examined by qPCR and (d) Protein expression levels of a-SMA and fibronectin were examined by western blotting. The cells were treated with TSLP. All experiments of at least three independent experiments. Values are means SEM. *p < 0.05 vs. control, **p < 0.01 vs. control, ***p < 0.001 vs. control.
TRPV3 activation promoted TSLP expression, and eventually resulted in dermal fibrosis. 3.5. Effect of TRPV3 silencing on TSLP and COL1A1 in HPDFs To further confirm the effect of TRPV3 on TSLP and COL1A1 expression, HPDFs were transfected with control siRNA or TRPV3-
siRNA. The results showed that TRPV3-siRNA significantly inhibited the carvacrol-induced expression of TSLP and COL1A1 compared with control siRNA (Fig. 5a–c). We also found that carvacrol significantly up-regulated TSLP and COL1A1 mRNA expression. Moreover, TRPV3-siRNA inhibited the protein expression of TRPV3, α-SMA, fibronectin and COL1A1 (Fig. 5d). These results suggest that COL1A1 and TSLP are regulated by carvacrol.
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Fig. 5. Effect of TRPV3 silencing on TSLP and ECM production in human primary dermal fibroblasts. Silencing TRPV3 with TRPV3-specific small interference RNA was determined by (a–c) qPCR and (d) western blotting. Cells were transfected with siControl or siTRPV3 for 24 h and stimulated with carvacrol for 3 h. All experiments of at least three independent experiments. Values are means SEM. ***p < 0.001 vs. siControl, †p < 0.05 vs. siControl+ Carvacrol.
3.6. Effect of carvacrol on Ca2+ influx, NFAT dephosphorylation, and Smad2/3 signaling in HPDFs
(Fig. 6d and f). Taken together, these results indicated that TRPV3 was involved in dermal fibrosis induced by p-Smad2/3.
It has been reported that TRPV3 activity is intimately associated with cytosolic Ca2+ influx. Intracellular Ca2+ ([Ca2+]i) could act as a secondary messenger involved in multiple cellular functions, including inflammation, molecular transportation, and gene transcription. Ca2+]i can induce NFAT. The dephosphorylation of NFAT is dependent on Ca2+ influx. Therefore, we examined whether carvacrol can induce Ca2+]i influx and dephosphorylation of NFAT in HPDFs. The results showed that carvacrol increased [Ca2+]i compared to control (Fig. 6a). Results of the effect of carvacrol on the dephosphorylation of NFAT are shown in Fig. 6b. We found that carvacrol-treated HPDFs showed significantly elevated Ca2+ level and dephosphorylated NFAT. These results indicated that TRPV3 is involved in NFAT signaling. To confirm if TRPV3 is involved in the pSmad2/3 signaling pathway, HPDFs were treated with carvacrol or sis3 (Smad 2/3 inhibitor), and phosphorylated Smad2/3 level was analyzed (Fig. 6c, and f). The results showed that carvacrol increased p-Smad2/3 level in HPDFs. To examine whether smad2/3 regulates carvacrol-induced myofibroblast differentiation and ECM production, we determined the expression of protein levels (Fig. 6e). We also found that sis3 (Smad 2/3 inhibitor) significantly inhibit α-SMA, fibronectin and COL1A1 protein expression. But TRPV3, TSLP not inhibit. TRPV3 and TSLP are up-regulator of Smad2/3. To check if TRPV3-TSLP is involved in the p-Smad2/3 signaling pathway, HPDFs were treated with TSLP, and p-Smad2/3 level was determined. Moreover, TSLP increased p-Smad2/3 level
4. Discussion In this report, we investigated whether TRPV3 affects myofibroblast differentiation and collagen production in HPDFs and identified the underlying mechanism of action of TRPV3 on HPDFs. We found that carvacrol activates TRPV3, which induces myofibroblast differentiation, collagen production, and TSLP expression, through activation of the TRPV3/Smad2/3 signaling pathway. TRPV3 is predominantly expressed in keratinocytes present in the skin, esophagus, distal colon, and cornea [1,2,24,28,30,33]; Its expression and function in dermal fibrosis are still largely unknown. Recent studies clarify that TRPV3 and TSLP are upregulated in the skin of patients with allergic dermatitis [26]. In our previous study, TRPV3 was demonstrated to facilitate TSLP release from keratinocytes derived from pruritic and hypertrophic burn scar tissues [2]. TRPV3 induced the expression of TSLP in HPDFs in the present study. TSLP, a protein belonging to the cytokine family, is known to be produced mainly by non-hematopoietic cells such as fibroblasts, epithelial cells, and different types of stromal or stromal-like cells [10–12,29]. Recently, it was reported that TSLP is involved in allergic diseases and fibrotic conditions such as asthma, allergic rhinitis, atopic dermatitis, systemic sclerosis, and scleroderma [12–15,27]. These diseases involve factors that promoted TSLP expression by increased intracellular Ca2+ levels. Also, epithelial
Please cite this article in press as: J.- Um, et al., Transient receptor potential vanilloid-3 (TRPV3) channel induces dermal fibrosis via the TRPV3/ TSLP/Smad2/3 pathways in dermal fibroblasts, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2019.12.011
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Fig. 6. Effect of Carvacrol on Ca2+ influx, NFAT, and p-Smad2/3 signal pathway in human primary dermal fibroblasts. (a) HPDFs were loaded with Fura-3 for 100 s and then washed with PBS buffer for [Ca2+]i measurement by Fluorescence plate Readers. (b, c) Expression levels of dephosphorylated NFAT and p-smad2/3 in HPDFs were examined by western blot. (d) Expression levels of P-smad2/3 in HPDFs were examined by western blotting. (e) HPDFs, pre-treated with sis3 (smad2/3 inhibitor) at 3 mM for 1 h, were treated with carvacrol or TSLP. (f) Hypothetical schema of the action of TRPV3 in HPDFs. All experiments of at least three independent experiments. The cells were treated with carvacrol. Values are means SEM. ***p < 0.001 vs. control.
TSLP expression in pulmonary fibrosis and TSLP overexpression have been shown to contribute to excessive ECM synthesis [18,20]. Our data suggest that TSLP expression induces the production of αSMA (myofibroblast differentiation marker), fibronectin, and collagen in dermal fibroblasts and induces dermal fibrosis. We further examined the effects of carvacrol on dephosphorylated nuclear factor of activated T-cells (NFAT) by intracellular increased Ca2+ levels promote TSLP expression. Cordeiro et al. demonstrated that intracellular calcium levels serve as a modulator of epithelial wound healing [22,23,25]. Moreover, Ca2+ influx plays an essential role in wound healing of epithelial cells [20–22]. Our data showed that carvacrol increases intracellular Ca2+ levels and then triggers NFAT action to further increase TSLP expression. The activated T cell (NFAT) pathway plays a key role in electrical remodeling in pathological cardiac hypertrophy. Recent studies have clarified the effects of TRPV3 in pressure overload-induced cardiac hypertrophy and the relationship between TRPV3 activation and calcineurin/NFAT signaling. Intracellular calcium levels can regulate ECM remodeling [18–20].
The extracellular matrix (ECM), a three-dimensional network of extracellular macromolecules, tightly controls wound healing. Type I collagen (Col I), the most abundant collagen type in the human body, plays an essential role in cell motility control by interacting with the intracellular cytoskeleton [18]. It is expressed in the scar tissue as the end-product of the wound-healing process [21,31,33]. Recent study demonstrated that carvacrol plays an important role in regulating the cell migration [34]. These results suggest that regulation of the TRPV3 channel in excessive wound healing can lead to hypertrophic scars; this may represent a novel pathologic mechanism. Furthermore, because TRPV3 is also expressed in the epidermis and dermis, the regulation of this channel may reduce pruritus and scar formation [21]. Smad mediates collagen production by TGF-β1 in various cell types. It is already well-known that TGF-β1 is a potent stimulator and key mediator of fibrosis [4,5]. TGF-β1 has been widely recognized as a regulator of myofibroblast differentiation [5]; however, the role of the TRPV3-Smad pathway in dermal fibrosis
Please cite this article in press as: J.- Um, et al., Transient receptor potential vanilloid-3 (TRPV3) channel induces dermal fibrosis via the TRPV3/ TSLP/Smad2/3 pathways in dermal fibroblasts, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2019.12.011
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remains unknown. Also, the best know pathway for TGF-β1 activity is the Smad-complex pathway, in which TGF- β1 binds to the Smadbinding elements of target genes [32]. This study aimed to investigate the possible collagen production-promoting activities of carvacrol and its mechanism of action in human dermal fibroblasts. Based on these results, we speculated that the activation of TRPV3 in dermal fibroblasts contributes to dermal fibrosis as that of epidermal keratinocytes associate with postburn pruritus in the hypertrophic burn scars. To our knowledge, this is the first study reporting the effect of carvacrol on HPDFs. In conclusion, this study showed that carvacrol induced myofibroblast differentiation and ECM production in HPDFs via the Smad2/3 pathway. The results of the study suggest that carvacrol induces ECM production through the Smad2/3 signaling pathway in skin dermal fibroblasts. Therefore, modulation of TRPV3 could prove to be a possible therapeutic target for dermal fibrosis. Funding This work was supported by the National Research Foundation of Korea (NRF) [grant number NRF-2017R1A2B4006252], the Korea Healthcare Technology R&D Project funded by the Ministry of Health & Welfare, Republic of Korea [grant number HI17C0597], and the Hallym University Research Fund [grant number HURF2017-83]. Declaration of Competing Interest The authors have no conflict of interest to declare Acknowledgements We thank for sharing reagents. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j. jdermsci.2019.12.011. References [1] L. Carswell, J. Borger, Hypertrophic Scarring Keloids, Treasure Island (FL): StatPearls Publishing, 2019 Jan-. [2] C.W. Park, H.J. Kim, Y.W. Choi, B.Y. Chung, S.Y. Woo, D.K. Song, et al., TRPV3 channel in keratinocytes in scars with post-burn pruritus, Int. J. Mol. Sci. 18 (November (11)) (2017) E2425. [3] Y.H. Choi, K.M. Kim, H.O. Kim, Y.C. Jang, I.S. Kwak, Clinical and histological correlation in post-burn hypertrophic scar for pain and itching sensation, Ann. Dermatol. 25 (November (4)) (2013) 428–433. [4] J.D. Thatcher, The TGF-beta signal transduction pathway, Sci. Signal. 3 (April (119)) (2010) tr4. [5] S. Bhattacharyya, A.K. Ghosh, J. Pannu, Y. Mori, S. Takagawa, G. Chen, et al., Fibroblast expression of the coactivator p300 governs the intensity of profibrotic response to transforming growth factor beta, Arthritis Rheum. 52 (April (4)) (2005) 1248–1258. [6] A. Al-Shami, R. Spolski, J. Kelly, A. Keane-Myers, W.J. Leonard, A role for TSLP in the development of inflammation in an asthma model, J. Exp. Med. 202 (September (6)) (2005) 829–839. [7] T.T. Cui, G.X. Wang, N.N. Wei, K. Wang, A pivotal role for the activation of TRPV3 channel in itch sensations induced by the natural skin sensitizer carvacrol, Acta Pharmacol. Sin. 39 (March (3)) (2018) 331–335. [8] J. Lee, E. Jung, H. Yu, Y. Kim, J. Ha, Y.S. Kim, et al., Mechanisms of carvacrolinduced expression of type I collagen gene, J. Dermatol. Sci. 52 (December (3)) (2008) 160–169. [9] T.T. Cui, G.X. Wang, N.N. Wei, K. Wang, A pivotal role for the activation of TRPV3 channel in itch sensations induced by the natural skin sensitizer carvacrol, Acta Pharmacol. Sin. 39 (March (3)) (2018) 331–335.
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Please cite this article in press as: J.- Um, et al., Transient receptor potential vanilloid-3 (TRPV3) channel induces dermal fibrosis via the TRPV3/ TSLP/Smad2/3 pathways in dermal fibroblasts, J Dermatol Sci (2020), https://doi.org/10.1016/j.jdermsci.2019.12.011