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Inhibitory effects of extracellular superoxide dismutase on ultraviolet B-induced melanogenesis in murine skin and melanocytes
Accepted Manuscript Inhibitory effects of extracellular superoxide dismutase on ultraviolet B-induced melanogenesis in murine skin and melanocytes
Hae-Young Kim, Shyam Kishor Sah, Sung S. Choi, Tae-Yoon Kim PII: DOI: Reference:
S0024-3205(18)30517-4 doi:10.1016/j.lfs.2018.08.056 LFS 15903
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
5 April 2018 13 August 2018 22 August 2018
Please cite this article as: Hae-Young Kim, Shyam Kishor Sah, Sung S. Choi, Tae-Yoon Kim , Inhibitory effects of extracellular superoxide dismutase on ultraviolet B-induced melanogenesis in murine skin and melanocytes. Lfs (2018), doi:10.1016/j.lfs.2018.08.056
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Inhibitory effects of extracellular superoxide dismutase on ultraviolet Binduced melanogenesis in murine skin and melanocytes
Hae-Young Kim1, Shyam Kishor Sah1, Sung S. Choi1, Tae-Yoon Kim1* Department of Dermatology, College of Medicine, The Catholic University of Korea, 505
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Banpo-dong, Seocho-gu, Seoul 06591, Republic of Korea.
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*Correspondence : Department of Dermatology, College of Medicine, The Catholic
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University of Korea, Seoul 06591, Republic of Korea, Tel.: 82 2 2258 6221; Fax : 82 2 3482
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8261; Email address: [email protected]
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Running title: Extracellular superoxide dismutase regulates melanogenesis
Abstract word: 223
Introduction word: 367 Discussion word: 1032 Conclusion: 38 Number of figures: 4 Number of supplementary table: 1
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Abstract Aims: Several anti-melanogenic molecules have been developed or identified, but their uses are limited due to either adverse effects or instability during the treatment. We aimed to evaluate the effects of extracellular superoxide dismutase (SOD3), a powerful antioxidant, as
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a candidate anti-melanogenic molecule. Main methods: UVB-induced reactive oxygen species (ROS) production and proliferation in
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melan-a cells was evaluated by 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate staining
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and bromodeoxyuridine incorporation assay, respectively. Quantitative real-time polymerase
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chain reaction and western blot were performed to detect the melanogenesis-related gene expression and downstream signaling. Anti-melanogenic effects of SOD3 were also
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evaluated using SOD3 transgenic mice under UVB exposure in-vivo condition. Key findings: SOD3 inhibited UVB-induced proliferation, ROS production and
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melanogenesis in melanocytes. Measurement of melanin content and tyrosinase activity
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assays showed that SOD3 significantly inhibited melanin synthesis. Moreover, these suppressive effects of SOD3 were dependent on the endothelin-1 (ET-1)/endothelin B
mitogen-activated
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receptor, protein kinase C, melanocortin 1 receptor/protein kinase A, Wnt7a/β-catenin, and protein
kinase
pathways,
with
concomitant
downregulation
of
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microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related proteins 1, dopachrome tautomerse. Interestingly, SOD3 was found to inhibit transforming growth factor-beta 1 (TGF-β1) to inactivate the ET-1 signaling pathway, and finally prevents the production of melanin. Significance: Our results provide novel insights into the role of SOD3 in melanocyte homeostasis and its uses as a potential biomedicine to treat hyperpigmentary conditions of the skin. Key words: Melanocytes, Melanogenesis, Extracellular superoxide dismutase, Endothelin-1 2
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1. Introduction Melanin is a pigment that is synthesized by melanocytes in the basal layer of the epidermis and is then transferred to epidermal keratinocytes via dendritic processes [1]. Melanin protects skin cells from ultraviolet (UV) light radiation. Repeated exposure to ultraviolet-B
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(UVB) is known to cause sunburn [2], and skin cancer via mutations in oncogenes and tumor suppressor genes [3-5]. Therefore, several studies attempt to understand the regulation of
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melanogenesis and focus on the treatment of hyperpigmentary skin disorders and the
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development of skin-whitening agents [2]. UVB modulates the secretion of melanocortin 1
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receptor (MC1R), stem cell factor (SCF), endothelin-1 (ET-1), and Wnt7a, and induces melanin production in melanocytes [6]. Similarly, cytokines such as interleukin-1 alpha (IL-
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1α), tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta 1 (TGF-β1), which are secreted by keratinocytes during UV exposure, regulate the production of strong
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mitogenic and melanogenic factors, such as ET-1 and alpha-melanocyte-stimulating hormone
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(α-MSH) [7-9]. ET-1, a 21 amino acid-peptide has also been shown to induce cell proliferation, melanin production, and melanocyte migration via activation of the protein
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kinase C (PKC) pathway, intracellular calcium mobilization, and the activation of nonreceptor tyrosine kinase activity [10-13].
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UV-induced reactive oxygen species (ROS) have been reported to accelerate skin aging and increase both melanocyte proliferation and melanin synthesis [14]. Although each cell has an antioxidant defense mechanism to alleviate ROS and protect skin from oxidative stress, excessive ROS production can override the antioxidant defense system and cause several skin diseases including hyperpigmentation of the skin [15,16]. Among the known several antioxidant enzymes, extracellular superoxide dismutase (SOD3), which are found in extracellular space plays a central role in ROS regulation. Previous reports have suggested that SOD3 could be an effective therapeutic reagent to treat several chronic inflammatory 3
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disorders, such as atopic dermatitis and psoriasis [17-20]. However, the effects of SOD3 and its role in melanogenesis are unclear. Thus, we hypothesized that SOD3 could play an important role in the maintenance of skin integrity and the regulation of melanogenesis. We further sought to determine the underlying mechanism of action of SOD3 in the regulation of
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melanogenesis in mouse and human melanocytes. Our results suggest a critical role for SOD3
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in maintaining skin homeostasis and alleviating skin hyperpigmentation.
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2. MATERIALS AND METHODS
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2.1. Cell culture and UVB irradiation
The murine melanocyte cell line, melan-a cells were purchased from Amore Pacific Company
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(Republic of Korea), and normal human epidermal melanocyte cells (NHEM) was obtained as a kind gift from Dr. Jae-Sung Hwang (Kyung Hee University, Republic of Korea). Melan-a
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cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (SH 30027.01,
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HyClone Laboratories, USA) supplemented with 10% fetal bovine serum (090450, Wisent, Quebec, Canada), 1% penicillin-streptomycin (LS202-02, Welgene, Republic of Korea) and
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200 nM 2-O-tetradecanoylphorbol-13-acetate (P8139, Sigma-Aldrich, USA), NHEM cells were cultured in Medium 254 (M-254-800, Gibco, USA) supplemented with Human
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Melanocyte Growth Supplement (HMGS, S-002-5, Gibco, USA) and 1% penicillinstreptomycin at 37ºC in a humidified 95% (v/v) mixture of air and 5% Carbon dioxide (CO2). UVB irradiations were determined using a previously described method [21].
2.2. Tyrosinase activity assay Melan-a cell were seeded at 5×105 cells per 4 mL in 60-mm dishes for 24 h, followed by pretreatment with 200 U/mL SOD3 for 5 h and UVB irradiation at 100 J/m². Cells were then further incubated for 72 h. Cells were subsequently harvested and lysed in 1% Triton X-100 4
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(9002-93-1, Amresco, Australia) containing phenylmethanesulfonyl fluoride (PMSF, 329-986, Sigma-Aldrich, USA) and protease inhibitor cocktail (P8340, Sigma-Aldrich, USA). The lysates were centrifuged at 13,200 rpm for 15 min to obtain the supernatant fraction; the protein concentration was then determined. Sample aliquots (each containing 20 µg protein)
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were mixed with 10 mM L-DOPA (59-92-7, Sigma-Aldrich, USA) and transferred to a 96-
absorbance of 475 nm after incubation at 37ºC for 30 min.
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2.3. Measurement of melanin content
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well plate. Tyrosinase activity was evaluated by analyzing the oxidation of L-DOPA at an
Cellular melanin levels were determined using a previously described method [22]. Briefly,
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melan-a cell were seeded at 5×105 cells per 4 mL in 60-mm dishes for 24 h, followed by pre-
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treatment with 200 U/mL SOD3 for 5 h and UVB irradiation at 100 J/m². After 72 h, cells were washed with 1X PBS (pH 7.4) and homogenized in 1 mL of 1 M NaOH solution (1310-
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73-2, Sigma-Aldrich, USA). Cellular extracts (200 µL) were transferred into 96-well plates,
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and total melanin levels were determined at an absorbance of 405 nm using a SpectraMax Plus spectrophotometer (Biotek, USA).
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2.4. Western blot analysis
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Melan-a cell were harvested and lysed in radioimmunoprecipitation assay buffer (WH400028897, 3M, USA ) containing 2 mM EDTA, 137 mM NaCl (7647-14-5, Amresco, Australia ), 20 mM Tris–HCl (pH 8.0; 77-86-1, Amresco, Australia), 1 mM sodium orthovanadate, 10 mM NaF, 1 mM PMSF, 1% Triton X-100, 10% glycerol, and a protease inhibitor cocktail. Equal amounts of cell lysates were resolved by SDS-PAGE (EBA-1041, Elpis Biotech, Korea), and transferred to a polyvinylidene difluoride membrane (PVDF, IPVH00010, Sigma-Aldrich, USA). After blocking with 5% skim milk, the membranes were incubated with antibodies against tyrosinase, microphthalmia-associated transcription factor 5
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(MITF), ET-1, endothelin A receptor (EDNRA), endothelin B receptor (EDNRB), Wnt7a, SCF, PKCβ2, p-PKCβ2 (1:1,000) (Abcam, England), TGF-β1, p38, p-p38, p-JNK, JNK, cAMP response element binding (CREB), p-CREB, p-β-catenin (1:1,000; Cell Signaling Technology, USA), tyrosinase-related proteins 1 (TYRP1), dopachrome tautomerse (DCT),
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β-catenin, protein kinase A (PKA), p-PKA (Santa Cruz Biotechnology, USA), and MC1R (Bioss Antibodies, USA), followed by incubation with horseradish peroxidase-conjugated
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secondary antibody (Invitrogen Corporation, USA, and Life Technologies, USA). Proteins
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were detected by enhanced chemiluminescence (K-12045-D-50, Advansta, USA) and
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visualized using the LAS 3000 detection system (Fujifilm Corporation, Japan).
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2.5. cDNA synthesis and quantitative real-time PCR (qRT-PCR) Melan-a and NHEMs were seeded at 3×105 cells per 2 mL in 35-mm dishes for 24 h,
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followed by pre-treatment with 200 U/mL SOD3 for 5 h and UVB irradiation at 100 J/m².
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Cells were then incubated for an additional 24 h. Total RNA was extracted using the TRIzol reagent (15596018, Invitrogen, USA) following the manufacturer’s instructions. The cDNA was reverse-transcribed from 1 µg total RNA using the QuantiTech Reverse Transcription kit
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(Qiagen, Germany). PCR was performed using a Rotor-Gene 6000 (Corbett research, UK)
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and a QuantiTech SYBR Green PCR kit (Qiagen, Germany). The amplification program was one cycle for 10 min (95°C), followed by 35 cycles for 20 s at 95°C, 20 s at 55°C, and 20 s for 72°C. Previously designed primers for MITF, tyrosinase, TYRP1, DCT, ET-1, EDNRA, EDNRB, MC1R, Wnt7a, SCF, TGF-β1, and GAPDH are listed in Supplementary Table 1.
2.6. Detection of ROS For ROS analysis, melan-a cell were seeded at 3×105 cells per 2 mL in 6-well plates for 24 h, followed by pre-treatment with 200 U/mL SOD3 for 5 h and UVB irradiation at 100 J/m² for 6
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1 h. Cells were then washed three times with 1X PBS (pH 7.4), and suspended in Hank’s Balanced Salt Solution (10-508F, Lonza Walkersville, USA) containing 2 µM 6-carboxy2',7'-dichlorodihydrofluorescein diacetate (DCFDA, 21884, Sigma-Aldrich, USA) followed
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by a 30 min incubation at 37ºC. ROS levels were measured at 540 nm using a fluorimeter.
2.7. Bromodeoxyuridine (BrdU) incorporation assay
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BrdU incorporation assay was carried out as previously described [22]. Briefly, melan-a cell
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were seeded at 5×105 cells per 4 mL in 60-mm dishes for 24 h, followed by pre-treatment with 200 U/mL SOD3 for 5 h and UVB irradiation at 100 J/m². Cells were then treated with
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10 μM BrdU for 4 h prior to end point of experiment (at 24, 48 and 72h). The cells were
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washed with 1X PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were permeabilized with 0.5% triton X-100 and incubated with 2 N
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HCl/Triton×100 to denature the chromosomal DNA. The cells were washed to remove HCl,
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followed by incubation with 1% bovine serum albumin for blocking nonspecific binding. BrdU incorporation was determined by treating with APC-conjugated anti-BrdU
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mouse monoclonal antibody.
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2.8. In-vivo studies
Ten-week-old C57BL/6 (WT) mice and SOD3 transgenic C57BL/6 mice (TG) were used for in-vivo studies. SOD3 TG mice were generated as previously described [23]. Mice were treated according to the provision of the Catholic Ethics Committee in accordance with the Catholic University's National Institutes of Health Guidelines. C57BL/6 WT mice and SOD3 TG mice were exposed to UVB at 5 kJ/m2 twice on alternating days and sacrificed on day 5. Mouse back skin was collected for western blot analysis of melanogenesis-related proteins.
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2.9. Purification and activity assay of recombinant human SOD3 Recombinant SOD3 was prepared as previously described [24]. Briefly, HEK 293 cells were transiently transfected with the SOD3 construct for 48 h. The supernatant was collected, and purified using Ni Sepharose 6 fast flow bead (11-0012-38, GE Healthcare, UK) designed for
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purification of His-Tag proteins. The purified SOD3 was then dialyzed and activity was
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measured as per manufacturer’s instruction using a SOD assay kit (S311, Dojindo, Japan).
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2.10. Statistical analysis
Statistical significance was assessed by comparing mean ± S.D. values with one way
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ANOVA test, wherever appropriate. All experiments were performed in triplicate, and
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repeated at least three times unless otherwise stated. Numbers above the blots indicate the
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levels of band intensity normalized with respective β-actin.
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3. Results
3.1. SOD3 suppresses UVB-induced ROS production and proliferation of melan-a cells
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To understand the role of SOD3 in UVB-induced ROS production and proliferation of melana cells, we first pre-treatment melan-a cells with recombinant human SOD3 and examined the
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effects on UVB-induced ROS production and proliferation using fluorimetry and a BrdU incorporation assay, respectively. We found that SOD3 potently scavenges ROS in melan-a cells and suppresses UVB-induced cell proliferation (Fig. 1A). Notably, SOD3 alone suppressed the proliferation of melan-a cells and exerted more potent effects after 72 h (Fig. B).
3.2. SOD3 inhibits UVB-induced melanin production in melan-a cells
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Next, we examined melanin levels in melan-a cells after pre-treatment with SOD3 and exposure to UVB. We found that melanin content was significantly reduced in SOD3treatment cells compared with UVB alone-treated cells (Fig. 1C and D). To examine the mechanism behind the inhibitory effects of SOD3 on melanogenesis, intracellular tyrosinase
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activity in melan-a cells was measured. As shown in Fig. 1E, treatment with SOD3
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significantly inhibited tyrosinase activity by 60% compared with UVB alone-treated cells.
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3.3. SOD3 inhibits the expression of UVB-induced melanogenesis-related molecules in melan-a cells and NHEMs
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To elucidate the mechanism of melanogenesis, we examined the expression levels of MITF,
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tyrosinase, TYRP1, and DCT in melan-a and NHEM cells after SOD3 treatment for 24 h. SOD3 suppressed mRNA expressions levels of MITF, tyrosinase, TYRP1, and DCT in both
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cell types at 24 h. (Fig. 2A and B). Consistently, SOD3 also reduced tyrosinase, TYRP1,
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DCT and MITF expression at protein levels compared with untreated controls irradiated with UVB at 72 h. (Fig. 2C and D).
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3.4. SOD3 abrogates UVB-stimulated expression of ET-1/EDNRA/EDNRB, MC1R, Wnt7a,
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and their downstream pathways To understand the underlying mechanism of the anti-melanogenic effect of SOD3, melan-a cells were pre-treatment with SOD3, and mRNA levels of ET-1, EDNRA, EDNRB, MC1R, Wnt7a, and SCF were evaluated by qRT-PCR. As shown in Fig. 3A, SOD3 significantly inhibited UVB-induced activation of ET-1, EDNRA, EDNRB, MC1R, and Wnt7a. In contrast, SCF was unaffected by SOD3 treatment compared to UVB-treated melan-a cells. Consistent with these data a protein expression of ET-1, EDNRA, EDNRB, MC1R, and Wnt7 were also downregulated, as shown by western blot analysis. Next, we evaluated the effects 9
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of SOD3 on UVB-induced phosphorylation of downstream molecules such as PKCβ2, PKA, CREB, β-catenin, and mitogen-activated protein kinase (MAPK) by western blot analysis. Our data showed that SOD3 markedly suppressed the phosphorylation of PKCβ2, PKA,
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CREB, β-catenin, p38, and JNK (Fig. 3B-E).
3.5. Effects of SOD3 on the ET-1/ EDNRA/EDNRB axis are mediated by TGF-β1
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TGF-β1 is a potent regulator of ET-1 expression and function [25]. To examine the possible
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role of TGF-β1 on melanogenesis and its regulation by SOD3, we performed qRT-PCR and western blot analysis to evaluate the expression of TGF-β1 during UVB exposure over time.
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UVB induced a robust upregulation of TGF-β1 at 24 h, which was downregulated after
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treatment with SOD3 (Fig. 4A and B). Furthermore, recombinant TGF-β treatment of melana cells induced a significant upregulation of ET-1, EDNRA, and EDNRB mRNA levels (Fig.
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4C). However, the increase in mRNA expression levels by UVB was markedly suppressed
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after cells were treated with the TGF-β1 inhibitor LY-364947 (Fig. 4D), suggesting that the role of SOD3 in the suppression of melanogenesis occurs via the downregulation of ET-1
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expression, which is possibly mediated by TGF-β1.
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3.6. Overexpression of SOD3 in mice inhibits UVB-induced skin pigmentation and melanogenesis-related proteins expression Next, to confirm the effect of SOD3 on UVB-induced skin melanogenesis in vivo, we used transgenic (TG) mice overexpressing human SOD3 (SOD3 TG mice) that were subjected to UVB exposure at 5 kJ/m2 twice every other day and sacrificed on day 5 (Fig. 4E). Although there was no apparent overall phenotype in the mice due to UVB, western blot analysis of back skin showed that the expression levels of the master regulators of melanogenesis such as MITF, TYRP1, DCT, and other pigmentation inducing ET-1/EDNRA/EDNRB/TGF-β1 axis 10
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were increased and activated, respectively in WT mice skin compared to with SOD3 TG mouse skin after UVB exposure (Fig. 4F and G).
4. DISCUSSION
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Melanin is synthesized by melanin-producing cells of the skin and plays an important role in protecting the skin by absorbing UV rays, toxins and chemicals. However, skin exposed to
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excessive UV radiation can cause erythema, freckles, and even skin cancer. UV radiation has
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been shown to induce DNA damage in epidermal cells and is one of the most important
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causes of skin cancer [26]. In addition, skin exposed to UV radiation can produce ROS and melanin, and ROS levels can cause cellular damage, leading to immune dysregulation,
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photoaging and various skin disorders [27]. Oxidative stress caused by excessive production of intracellular oxidizing species and decreased cellular antioxidant capacity can lead to
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mutagenesis or cell death from damage to nucleic acids, lipids and proteins [28]. There is
of melanomas [29].
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increasing evidence that oxidative stress plays an important role in the onset and progression
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Several compounds such as arbutin, kojic acid, licorice extract, ellagic acid, oxyresveratrol, chlorophorin, norartocarpanone, and ascorbic acid have been developed for use in skin
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whitening or the treatment of skin pigmentation disorders [30-32]. Most of these compounds decreased melanin production by inhibition of tyrosinase but are poorly known to be used in clinical settings due to efficacy and safety issues. For example, hydroquinone is effective for whitening, but it affects melanosome degradation and membrane structure of melanocytes, leading to apoptosis, which is followed by vitiligo side effects [33]. Therefore, Long-term safety and efficacy of SOD3 treatment should also be evaluated before using it in clinical settings. In general, drugs having an antioxidant activity are believed to exhibit whitening, anti-aging 11
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and anti-inflammatory effects [34]. Among known antioxidant enzymes, SOD plays a central role in protecting cells from the effects of superoxide radicals that either directly modify proteins or produce derivatives such as H2O2 and ONOO- [35]. SOD deficient mice have been shown to exhibit increased sensitivity to organ impairment, oxygen toxicity, and
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inflammation. Exogenous SOD has been shown to mitigate inflammatory responses and prevent inflammatory diseases involving oxidative stress [36]. Several reports have suggested
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that SOD3 could be an effective therapeutic to treat the inflammatory skin disorders such as
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atopic dermatitis, psoriasis, and asthma [17-20].
Unlike SOD1 and SOD2, SOD3 is an extracellular antioxidant enzyme that functions without
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infiltrating cells. SOD3 is suggested to suppress inflammatory response at least by part
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reducing the ROS and through non-enzymatic manner [23]. TG mice overexpressing SOD3 were found to be resistant to inflammation [37], whereas SOD1 TG mice showed neuronal
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abnormalities and are not resistant to inflammation [38]. Interestingly, SOD3 is shown to
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have a half-life around 20 h whereas SOD1 has a very short half-life (~ 20 min) in blood [39]. These encouraging results led us to investigate the use of SOD3 as a potential treatment for hyperpigmentary skin disorders.
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Here, we demonstrated the effect of SOD3 on UVB-induced melanin production and also
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showed that SOD3 suppresses UVB-induced proliferative response in melanocytes. SOD3 treatment at 200 U/mL inhibits tyrosinase activity, suppress the expression of MITF, tyroninase, TYRP1, and DCT in melanocytes both at mRNA and protein levels. Moreover, qRT-PCR analysis revealed that the key upstream regulators of MITF, tyrosinase, TYRP1, DCT, and melanin production such as ET-1, EDNRA, EDNRB, MC1R, and Wnt7a were downregulated after SOD3 treatment. Downstream signaling networks of those upstream regulators such as PKCβ2, PKA, CREB, β-catenin, and MAPKs were also inhibited after treatment with SOD3. In contrast, SCF mRNA and protein levels were unaffected by SOD3. 12
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We also investigated the effects of SOD3 on UVB-induced melanogenesis in SOD3 TG mice. Here, we found the robust downregulation of melanogenesis-related markers in SOD3 TG mouse skin without any side effects, suggesting a protective role for SOD3 in hyperpigmentary skin disorders.
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Endothelin is a cyclic family group that includes three isoforms of ET-1, ET-2 and ET-3 in mammals [40]. ET-1 is the most abundant isoform found in the skin, whereas ET-2 is found
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in kidney and intestine, and ET-3 is found in the central nervous system. ET-1 is a well-
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known autocrine factor for melanocyte growth, tyrosinase activation, and keratinocyte regeneration, including vasoconstriction, cell proliferation, and moisture and sodium
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excretion control [41,42]. One of the key regulators of ET-1 is TGF-β, a regulatory cytokine
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that induces ET-1 gene in vascular endothelial cells [43,44]. Here, we demonstrated the regulatory pathway involved in melanogenesis between TGF-β and ET-1. Reports have
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shown that the inhibition of TGF-β signaling reduces ET-1 expression, thereby reducing the
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effects of hypoxia on neonatal lungs [45-47]. In perfused isolated adult rat lungs, TGF-β increases ET-1 synthesis, whereas in isolated alveolar epithelial cells, ET-1 increases TGF-β1 synthesis and signaling via EDNRA and induces the epithelial-mesenchymal transition [48].
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In melanocyte stem cells, TGF-β1 has been shown to not only induce cell cycle arrest, but to
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inhibit melanogenesis by downregulating MITF, the main transcriptional regulator of melanocyte differentiation, and its downstream melanin-producing gene [49]. TGF-β1 is a multifunctional cytokine that plays an important role in the induction of apoptosis, cell growth inhibition and other physiological and pathological responses. TGF-β1 can acts as either a tumor suppressor in early stages of carcinogenesis by promoting either cell cycle arrest or apoptosis of normal epithelial cells or a tumor promoter in later stages of tumorigenesis, leading to tumor cell invasion and metastasis [50,51]. Thus, the functions and effects of TGF-β1 are different depending on cell type and microenvironment. Our data 13
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revealed that TGF-β1 is crucial in ET-1 mediated melanogenesis in melan-a cells. The contrasting results may be due to pleiotropic nature of TGF-β1, which can elicit different functions depending on the cell type, as well as on the type and intensity of stimuli involved. Delayed skin pigmentation can occur for a number of reasons: an increase in the number of
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melanin-producing cells in response to UVB or UVA, the synthesis of new melanin bodies, an increase in enzyme hysteresis, and the progression of metastasis to keratinocytes, which
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typically occurs between 48 and 72 h after UVB exposure [52]. Recent studies have primarily
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focused on compounds that inhibit tyrosinase and, hence, melanin production [53]. However, efficient therapeutics with broad effects is desirable. We believe that the antioxidant SOD3
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has a broad spectrum effect and is expected to be safer and less toxic to humans than
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conventional treatments.
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5. Conclusion
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In conclusion, SOD3 effectively inhibits UVB-induced ROS and cell proliferation, and significantly inhibits melanogenesis. SOD3 may be useful as a potential therapeutic for
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cosmetic use.
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hyperpigmentary skin disorders and could also be developed as a whitening agent for
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Conflict of interest The authors declare that no conflict of interests exists.
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Acknowledgment This research was supported by Korea Health Technology R&D Project through the Korea
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Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare,
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Republic of Korea (grant number: HI17C0616) and Bio & Medical Technology Development
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Program of the National Research Foundation (NRF), funded by the Ministry of Science, ICT
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& Future Planning (NRF-2013M3A9A9050567).
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Figure legends Fig.1. SOD3 reduces UVB-induced ROS levels and suppresses proliferations and melanin production in melan-a cells. (A) Cells were pre-treatment with 200 U/mL SOD3 for 5 h, exposed to UVB at 100 J/m² and
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incubated for further 1 h. ROS level was evaluated by staining the cells with DCFDA at 2 µM. ROS levels were measured at 540 nm using a fluorimeter. (B) BrdU incorporation assay
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was performed to evaluate the effects of SOD3 on UVB-induced melan-a cell proliferation at
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24, 48, and 72 h and analyzed by flow cytometry. Three independent experiments were
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performed in triplicate. (C) Melan-a cells were pre-treatment with 200 U/mL SOD3, exposed to UVB at 100 J/m² and incubated for 3 days cells were harvested and images were taken. (D)
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Melan-a cells were pre-treatment with 200 U/mL SOD3 for 5 h, exposed to UVB at 100 J/m², and incubated for 3 days. Cells were lysed with 1 M NaOH and absorbance at 450 nm was
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measured to evaluate total melanin content. (E) Melan-a cells were pre-treatment with 200
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U/mL SOD3 for 5 h, exposed to UVB at 100 J/m² and incubated for 3 days. Effects of SOD3 on tyrosinase activity were measured at 475 nm by spectrophotometry. Each bar represents
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the mean ± S.D. of three independent experiments. ##P < 0.01, ###P < 0.001 (control vs. UVB-exposed cells), *P < 0.05; ***P < 0.001 (UVB-exposed cells vs. SOD3 and UVB-
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treated cells).
Fig.2. Effects of SOD3 on melanogenesis related genes. Melan-a cells and NHEMs were pre-treatment with 200 U/mL SOD3 for 5 h, exposed to UVB at 100 J/m², and further incubated for 24 h for mRNA expression analysis and 3 days for western blot analysis. (A and B) Cells were harvested for mRNA isolation and expression of MITF, tyrosinase, TYRP1, and DCT by qRT-PCR. Data are represented as the mean ± S.D. of three independent experiments. ##P < 0.01, ###P < 0.001 (control vs. UVB-exposed 22
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cells); *P < 0.05, **P < 0.01 (UVB-exposed cells vs. SOD3 and UVB-treated cells). (C and D) Both cells were pre-treated with 200 U/mL SOD3 for 5 h exposed to UVB at 100 J/m², and incubated the cells for 3 days. Cells were harvested and the expression of MITF, tyrosinase, TYRP1, and DCT was evaluated by western blot analysis. Numerical values on
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the blots represent densitometry units (DU). The control was set to 1 DU. All data represent
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three independent experiments.
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Fig.3. Evaluation of receptors/genes involved in melanogenesis.
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(A) Melan-a cells were pre-treatment with 200 U/mL SOD3 for 5 h, exposed to UVB at 100 J/m², and further incubated for 24 h. Cells were harvested for mRNA isolation and expression
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of ET-1, EDNRA, EDNRB, MC1R, Wnt7a, and SCF by qRT-PCR. Data represent the mean ± S.D. ##P < 0.01, ###P < 0.001 (control vs. UVB-exposed cells); *P < 0.05, **P < 0.01,
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***P < 0.001 (UVB-exposed cells vs. SOD3 and UVB-treated cells). (B-E) Protein
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expression levels of ET-1, EDNRA, EDNRB, MC1R, SCF, Wnt7a and β-actin were harvested 24 h after UVB 100J/m² exposed. Exposed to UVB at 100 J/m² and incubated the cells for 15
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min, cells were harvested and the expression the phosphorylation of PKCβ2, PKA, CREB, βcatenin, p38, JNK and β-actin was evaluated by western blot analysis. Numerical values on
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the blots represent DU. The control was normalized to 1 DU. All data represent three independent experiments.
Fig.4. SOD3 inhibits TGF-β1, a critical component of the ET-1 activation pathway and effects of UVB-induced melanogenesis on SOD3 TG mouse skin. Melan-a cells were exposed to UVB and incubated for 3, 6, 10, or 24 h. (A) mRNA and protein expression levels of TGF-β1 were evaluated by qRT-PCR and western blot, respectively. Data represent three independent experiments as the mean ± S.D. ***P < 0.001 23
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(control vs. UVB-exposed cells). (B) Melan-a cells were pre-treatment with 200 U/mL SOD3 for 5 h, exposed to UVB at 100 J/m² and further incubated the cells for 24 h. Cells were harvested for mRNA expression analysis by qRT-PCR and protein levels were evaluated by western blot. Data represent the mean ± S.D. ## P < 0.01, ###P < 0.001 (control vs. UVB-
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exposed cells); $$P < 0.01 (control vs. SOD3-treated cells.); **P < 0.01 (UVB-exposed cells vs. SOD3 and UVB-treated cells). (C) Melan-a cells were treated with 10 ng/mL TGF-β and
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incubated for 3, 6, 10, or 24 h. mRNA expression levels of ET-1, EDNRA and EDNRB were
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evaluated by qRT-PCR. Data represent the means ± S.D. *P < 0.05 (control vs. TGF-βtreated cells.), **P < 0.01 (control vs. TGF-β-treated cells.) ***P < 0.001 (control vs. TGF-β-
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treated cells.). (D) Melan-a cells were pre-treated with the TGF-β inhibitor LY-364947 at 10
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μM/mL for 1 h, exposed to UVB and incubated for 3, 6, 10, or 24 h. Cells were harvested for ET-1 mRNA expression analysis by qRT-PCR, and ET-1 protein levels were evaluated by
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western blot. Numerical values on the blots represent DU. The control was set to 1 DU. Data
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represent the mean ± S.D. ##P < 0.01 (control vs. UVB-exposed cells); *P < 0.05, **P < 0.01, ***P < 0.001 (UVB-exposed cells vs. UVB-exposed cells and LY364947-treated cells). (E) An experimental scheme for UVB-induced melanogenesis on C57BL/6 mouse. Mice were
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irradiated with UVB (5 kJ/m2) twice and sacrificed on day 5. (F and G) Melanogenesis
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related protein levels in mouse skin such as MITF, Tyrosinase, TYRP1, DCT, SOD3, TGFβ1, ET-1, EDNRA, EDNRB, and β-actin were evaluated by western blotting. Numerical values on the blots represent DU. The control was set to 1 DU. All data represent three independent experiments.
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Figure 1
Figure 2
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Hae-Young Kim, Shyam Kishor Sah, Sung S. Choi, Tae-Yoon Kim PII: DOI: Reference:
S0024-3205(18)30517-4 doi:10.1016/j.lfs.2018.08.056 LFS 15903
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
5 April 2018 13 August 2018 22 August 2018
Please cite this article as: Hae-Young Kim, Shyam Kishor Sah, Sung S. Choi, Tae-Yoon Kim , Inhibitory effects of extracellular superoxide dismutase on ultraviolet B-induced melanogenesis in murine skin and melanocytes. Lfs (2018), doi:10.1016/j.lfs.2018.08.056
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Inhibitory effects of extracellular superoxide dismutase on ultraviolet Binduced melanogenesis in murine skin and melanocytes
Hae-Young Kim1, Shyam Kishor Sah1, Sung S. Choi1, Tae-Yoon Kim1* Department of Dermatology, College of Medicine, The Catholic University of Korea, 505
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1
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Banpo-dong, Seocho-gu, Seoul 06591, Republic of Korea.
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*Correspondence : Department of Dermatology, College of Medicine, The Catholic
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University of Korea, Seoul 06591, Republic of Korea, Tel.: 82 2 2258 6221; Fax : 82 2 3482
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8261; Email address: [email protected]
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Running title: Extracellular superoxide dismutase regulates melanogenesis
Abstract word: 223
Introduction word: 367 Discussion word: 1032 Conclusion: 38 Number of figures: 4 Number of supplementary table: 1
1
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Abstract Aims: Several anti-melanogenic molecules have been developed or identified, but their uses are limited due to either adverse effects or instability during the treatment. We aimed to evaluate the effects of extracellular superoxide dismutase (SOD3), a powerful antioxidant, as
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a candidate anti-melanogenic molecule. Main methods: UVB-induced reactive oxygen species (ROS) production and proliferation in
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melan-a cells was evaluated by 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate staining
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and bromodeoxyuridine incorporation assay, respectively. Quantitative real-time polymerase
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chain reaction and western blot were performed to detect the melanogenesis-related gene expression and downstream signaling. Anti-melanogenic effects of SOD3 were also
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evaluated using SOD3 transgenic mice under UVB exposure in-vivo condition. Key findings: SOD3 inhibited UVB-induced proliferation, ROS production and
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melanogenesis in melanocytes. Measurement of melanin content and tyrosinase activity
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assays showed that SOD3 significantly inhibited melanin synthesis. Moreover, these suppressive effects of SOD3 were dependent on the endothelin-1 (ET-1)/endothelin B
mitogen-activated
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receptor, protein kinase C, melanocortin 1 receptor/protein kinase A, Wnt7a/β-catenin, and protein
kinase
pathways,
with
concomitant
downregulation
of
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microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related proteins 1, dopachrome tautomerse. Interestingly, SOD3 was found to inhibit transforming growth factor-beta 1 (TGF-β1) to inactivate the ET-1 signaling pathway, and finally prevents the production of melanin. Significance: Our results provide novel insights into the role of SOD3 in melanocyte homeostasis and its uses as a potential biomedicine to treat hyperpigmentary conditions of the skin. Key words: Melanocytes, Melanogenesis, Extracellular superoxide dismutase, Endothelin-1 2
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1. Introduction Melanin is a pigment that is synthesized by melanocytes in the basal layer of the epidermis and is then transferred to epidermal keratinocytes via dendritic processes [1]. Melanin protects skin cells from ultraviolet (UV) light radiation. Repeated exposure to ultraviolet-B
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(UVB) is known to cause sunburn [2], and skin cancer via mutations in oncogenes and tumor suppressor genes [3-5]. Therefore, several studies attempt to understand the regulation of
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melanogenesis and focus on the treatment of hyperpigmentary skin disorders and the
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development of skin-whitening agents [2]. UVB modulates the secretion of melanocortin 1
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receptor (MC1R), stem cell factor (SCF), endothelin-1 (ET-1), and Wnt7a, and induces melanin production in melanocytes [6]. Similarly, cytokines such as interleukin-1 alpha (IL-
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1α), tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta 1 (TGF-β1), which are secreted by keratinocytes during UV exposure, regulate the production of strong
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mitogenic and melanogenic factors, such as ET-1 and alpha-melanocyte-stimulating hormone
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(α-MSH) [7-9]. ET-1, a 21 amino acid-peptide has also been shown to induce cell proliferation, melanin production, and melanocyte migration via activation of the protein
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kinase C (PKC) pathway, intracellular calcium mobilization, and the activation of nonreceptor tyrosine kinase activity [10-13].
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UV-induced reactive oxygen species (ROS) have been reported to accelerate skin aging and increase both melanocyte proliferation and melanin synthesis [14]. Although each cell has an antioxidant defense mechanism to alleviate ROS and protect skin from oxidative stress, excessive ROS production can override the antioxidant defense system and cause several skin diseases including hyperpigmentation of the skin [15,16]. Among the known several antioxidant enzymes, extracellular superoxide dismutase (SOD3), which are found in extracellular space plays a central role in ROS regulation. Previous reports have suggested that SOD3 could be an effective therapeutic reagent to treat several chronic inflammatory 3
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disorders, such as atopic dermatitis and psoriasis [17-20]. However, the effects of SOD3 and its role in melanogenesis are unclear. Thus, we hypothesized that SOD3 could play an important role in the maintenance of skin integrity and the regulation of melanogenesis. We further sought to determine the underlying mechanism of action of SOD3 in the regulation of
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melanogenesis in mouse and human melanocytes. Our results suggest a critical role for SOD3
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in maintaining skin homeostasis and alleviating skin hyperpigmentation.
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2. MATERIALS AND METHODS
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2.1. Cell culture and UVB irradiation
The murine melanocyte cell line, melan-a cells were purchased from Amore Pacific Company
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(Republic of Korea), and normal human epidermal melanocyte cells (NHEM) was obtained as a kind gift from Dr. Jae-Sung Hwang (Kyung Hee University, Republic of Korea). Melan-a
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cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (SH 30027.01,
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HyClone Laboratories, USA) supplemented with 10% fetal bovine serum (090450, Wisent, Quebec, Canada), 1% penicillin-streptomycin (LS202-02, Welgene, Republic of Korea) and
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200 nM 2-O-tetradecanoylphorbol-13-acetate (P8139, Sigma-Aldrich, USA), NHEM cells were cultured in Medium 254 (M-254-800, Gibco, USA) supplemented with Human
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Melanocyte Growth Supplement (HMGS, S-002-5, Gibco, USA) and 1% penicillinstreptomycin at 37ºC in a humidified 95% (v/v) mixture of air and 5% Carbon dioxide (CO2). UVB irradiations were determined using a previously described method [21].
2.2. Tyrosinase activity assay Melan-a cell were seeded at 5×105 cells per 4 mL in 60-mm dishes for 24 h, followed by pretreatment with 200 U/mL SOD3 for 5 h and UVB irradiation at 100 J/m². Cells were then further incubated for 72 h. Cells were subsequently harvested and lysed in 1% Triton X-100 4
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(9002-93-1, Amresco, Australia) containing phenylmethanesulfonyl fluoride (PMSF, 329-986, Sigma-Aldrich, USA) and protease inhibitor cocktail (P8340, Sigma-Aldrich, USA). The lysates were centrifuged at 13,200 rpm for 15 min to obtain the supernatant fraction; the protein concentration was then determined. Sample aliquots (each containing 20 µg protein)
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were mixed with 10 mM L-DOPA (59-92-7, Sigma-Aldrich, USA) and transferred to a 96-
absorbance of 475 nm after incubation at 37ºC for 30 min.
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2.3. Measurement of melanin content
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well plate. Tyrosinase activity was evaluated by analyzing the oxidation of L-DOPA at an
Cellular melanin levels were determined using a previously described method [22]. Briefly,
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melan-a cell were seeded at 5×105 cells per 4 mL in 60-mm dishes for 24 h, followed by pre-
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treatment with 200 U/mL SOD3 for 5 h and UVB irradiation at 100 J/m². After 72 h, cells were washed with 1X PBS (pH 7.4) and homogenized in 1 mL of 1 M NaOH solution (1310-
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73-2, Sigma-Aldrich, USA). Cellular extracts (200 µL) were transferred into 96-well plates,
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and total melanin levels were determined at an absorbance of 405 nm using a SpectraMax Plus spectrophotometer (Biotek, USA).
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2.4. Western blot analysis
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Melan-a cell were harvested and lysed in radioimmunoprecipitation assay buffer (WH400028897, 3M, USA ) containing 2 mM EDTA, 137 mM NaCl (7647-14-5, Amresco, Australia ), 20 mM Tris–HCl (pH 8.0; 77-86-1, Amresco, Australia), 1 mM sodium orthovanadate, 10 mM NaF, 1 mM PMSF, 1% Triton X-100, 10% glycerol, and a protease inhibitor cocktail. Equal amounts of cell lysates were resolved by SDS-PAGE (EBA-1041, Elpis Biotech, Korea), and transferred to a polyvinylidene difluoride membrane (PVDF, IPVH00010, Sigma-Aldrich, USA). After blocking with 5% skim milk, the membranes were incubated with antibodies against tyrosinase, microphthalmia-associated transcription factor 5
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(MITF), ET-1, endothelin A receptor (EDNRA), endothelin B receptor (EDNRB), Wnt7a, SCF, PKCβ2, p-PKCβ2 (1:1,000) (Abcam, England), TGF-β1, p38, p-p38, p-JNK, JNK, cAMP response element binding (CREB), p-CREB, p-β-catenin (1:1,000; Cell Signaling Technology, USA), tyrosinase-related proteins 1 (TYRP1), dopachrome tautomerse (DCT),
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β-catenin, protein kinase A (PKA), p-PKA (Santa Cruz Biotechnology, USA), and MC1R (Bioss Antibodies, USA), followed by incubation with horseradish peroxidase-conjugated
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secondary antibody (Invitrogen Corporation, USA, and Life Technologies, USA). Proteins
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were detected by enhanced chemiluminescence (K-12045-D-50, Advansta, USA) and
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visualized using the LAS 3000 detection system (Fujifilm Corporation, Japan).
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2.5. cDNA synthesis and quantitative real-time PCR (qRT-PCR) Melan-a and NHEMs were seeded at 3×105 cells per 2 mL in 35-mm dishes for 24 h,
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followed by pre-treatment with 200 U/mL SOD3 for 5 h and UVB irradiation at 100 J/m².
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Cells were then incubated for an additional 24 h. Total RNA was extracted using the TRIzol reagent (15596018, Invitrogen, USA) following the manufacturer’s instructions. The cDNA was reverse-transcribed from 1 µg total RNA using the QuantiTech Reverse Transcription kit
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(Qiagen, Germany). PCR was performed using a Rotor-Gene 6000 (Corbett research, UK)
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and a QuantiTech SYBR Green PCR kit (Qiagen, Germany). The amplification program was one cycle for 10 min (95°C), followed by 35 cycles for 20 s at 95°C, 20 s at 55°C, and 20 s for 72°C. Previously designed primers for MITF, tyrosinase, TYRP1, DCT, ET-1, EDNRA, EDNRB, MC1R, Wnt7a, SCF, TGF-β1, and GAPDH are listed in Supplementary Table 1.
2.6. Detection of ROS For ROS analysis, melan-a cell were seeded at 3×105 cells per 2 mL in 6-well plates for 24 h, followed by pre-treatment with 200 U/mL SOD3 for 5 h and UVB irradiation at 100 J/m² for 6
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1 h. Cells were then washed three times with 1X PBS (pH 7.4), and suspended in Hank’s Balanced Salt Solution (10-508F, Lonza Walkersville, USA) containing 2 µM 6-carboxy2',7'-dichlorodihydrofluorescein diacetate (DCFDA, 21884, Sigma-Aldrich, USA) followed
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by a 30 min incubation at 37ºC. ROS levels were measured at 540 nm using a fluorimeter.
2.7. Bromodeoxyuridine (BrdU) incorporation assay
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BrdU incorporation assay was carried out as previously described [22]. Briefly, melan-a cell
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were seeded at 5×105 cells per 4 mL in 60-mm dishes for 24 h, followed by pre-treatment with 200 U/mL SOD3 for 5 h and UVB irradiation at 100 J/m². Cells were then treated with
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10 μM BrdU for 4 h prior to end point of experiment (at 24, 48 and 72h). The cells were
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washed with 1X PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were permeabilized with 0.5% triton X-100 and incubated with 2 N
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HCl/Triton×100 to denature the chromosomal DNA. The cells were washed to remove HCl,
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followed by incubation with 1% bovine serum albumin for blocking nonspecific binding. BrdU incorporation was determined by treating with APC-conjugated anti-BrdU
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mouse monoclonal antibody.
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2.8. In-vivo studies
Ten-week-old C57BL/6 (WT) mice and SOD3 transgenic C57BL/6 mice (TG) were used for in-vivo studies. SOD3 TG mice were generated as previously described [23]. Mice were treated according to the provision of the Catholic Ethics Committee in accordance with the Catholic University's National Institutes of Health Guidelines. C57BL/6 WT mice and SOD3 TG mice were exposed to UVB at 5 kJ/m2 twice on alternating days and sacrificed on day 5. Mouse back skin was collected for western blot analysis of melanogenesis-related proteins.
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2.9. Purification and activity assay of recombinant human SOD3 Recombinant SOD3 was prepared as previously described [24]. Briefly, HEK 293 cells were transiently transfected with the SOD3 construct for 48 h. The supernatant was collected, and purified using Ni Sepharose 6 fast flow bead (11-0012-38, GE Healthcare, UK) designed for
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purification of His-Tag proteins. The purified SOD3 was then dialyzed and activity was
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measured as per manufacturer’s instruction using a SOD assay kit (S311, Dojindo, Japan).
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2.10. Statistical analysis
Statistical significance was assessed by comparing mean ± S.D. values with one way
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ANOVA test, wherever appropriate. All experiments were performed in triplicate, and
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repeated at least three times unless otherwise stated. Numbers above the blots indicate the
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levels of band intensity normalized with respective β-actin.
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3. Results
3.1. SOD3 suppresses UVB-induced ROS production and proliferation of melan-a cells
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To understand the role of SOD3 in UVB-induced ROS production and proliferation of melana cells, we first pre-treatment melan-a cells with recombinant human SOD3 and examined the
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effects on UVB-induced ROS production and proliferation using fluorimetry and a BrdU incorporation assay, respectively. We found that SOD3 potently scavenges ROS in melan-a cells and suppresses UVB-induced cell proliferation (Fig. 1A). Notably, SOD3 alone suppressed the proliferation of melan-a cells and exerted more potent effects after 72 h (Fig. B).
3.2. SOD3 inhibits UVB-induced melanin production in melan-a cells
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Next, we examined melanin levels in melan-a cells after pre-treatment with SOD3 and exposure to UVB. We found that melanin content was significantly reduced in SOD3treatment cells compared with UVB alone-treated cells (Fig. 1C and D). To examine the mechanism behind the inhibitory effects of SOD3 on melanogenesis, intracellular tyrosinase
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activity in melan-a cells was measured. As shown in Fig. 1E, treatment with SOD3
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significantly inhibited tyrosinase activity by 60% compared with UVB alone-treated cells.
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3.3. SOD3 inhibits the expression of UVB-induced melanogenesis-related molecules in melan-a cells and NHEMs
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To elucidate the mechanism of melanogenesis, we examined the expression levels of MITF,
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tyrosinase, TYRP1, and DCT in melan-a and NHEM cells after SOD3 treatment for 24 h. SOD3 suppressed mRNA expressions levels of MITF, tyrosinase, TYRP1, and DCT in both
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cell types at 24 h. (Fig. 2A and B). Consistently, SOD3 also reduced tyrosinase, TYRP1,
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DCT and MITF expression at protein levels compared with untreated controls irradiated with UVB at 72 h. (Fig. 2C and D).
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3.4. SOD3 abrogates UVB-stimulated expression of ET-1/EDNRA/EDNRB, MC1R, Wnt7a,
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and their downstream pathways To understand the underlying mechanism of the anti-melanogenic effect of SOD3, melan-a cells were pre-treatment with SOD3, and mRNA levels of ET-1, EDNRA, EDNRB, MC1R, Wnt7a, and SCF were evaluated by qRT-PCR. As shown in Fig. 3A, SOD3 significantly inhibited UVB-induced activation of ET-1, EDNRA, EDNRB, MC1R, and Wnt7a. In contrast, SCF was unaffected by SOD3 treatment compared to UVB-treated melan-a cells. Consistent with these data a protein expression of ET-1, EDNRA, EDNRB, MC1R, and Wnt7 were also downregulated, as shown by western blot analysis. Next, we evaluated the effects 9
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of SOD3 on UVB-induced phosphorylation of downstream molecules such as PKCβ2, PKA, CREB, β-catenin, and mitogen-activated protein kinase (MAPK) by western blot analysis. Our data showed that SOD3 markedly suppressed the phosphorylation of PKCβ2, PKA,
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CREB, β-catenin, p38, and JNK (Fig. 3B-E).
3.5. Effects of SOD3 on the ET-1/ EDNRA/EDNRB axis are mediated by TGF-β1
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TGF-β1 is a potent regulator of ET-1 expression and function [25]. To examine the possible
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role of TGF-β1 on melanogenesis and its regulation by SOD3, we performed qRT-PCR and western blot analysis to evaluate the expression of TGF-β1 during UVB exposure over time.
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UVB induced a robust upregulation of TGF-β1 at 24 h, which was downregulated after
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treatment with SOD3 (Fig. 4A and B). Furthermore, recombinant TGF-β treatment of melana cells induced a significant upregulation of ET-1, EDNRA, and EDNRB mRNA levels (Fig.
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4C). However, the increase in mRNA expression levels by UVB was markedly suppressed
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after cells were treated with the TGF-β1 inhibitor LY-364947 (Fig. 4D), suggesting that the role of SOD3 in the suppression of melanogenesis occurs via the downregulation of ET-1
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expression, which is possibly mediated by TGF-β1.
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3.6. Overexpression of SOD3 in mice inhibits UVB-induced skin pigmentation and melanogenesis-related proteins expression Next, to confirm the effect of SOD3 on UVB-induced skin melanogenesis in vivo, we used transgenic (TG) mice overexpressing human SOD3 (SOD3 TG mice) that were subjected to UVB exposure at 5 kJ/m2 twice every other day and sacrificed on day 5 (Fig. 4E). Although there was no apparent overall phenotype in the mice due to UVB, western blot analysis of back skin showed that the expression levels of the master regulators of melanogenesis such as MITF, TYRP1, DCT, and other pigmentation inducing ET-1/EDNRA/EDNRB/TGF-β1 axis 10
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were increased and activated, respectively in WT mice skin compared to with SOD3 TG mouse skin after UVB exposure (Fig. 4F and G).
4. DISCUSSION
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Melanin is synthesized by melanin-producing cells of the skin and plays an important role in protecting the skin by absorbing UV rays, toxins and chemicals. However, skin exposed to
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excessive UV radiation can cause erythema, freckles, and even skin cancer. UV radiation has
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been shown to induce DNA damage in epidermal cells and is one of the most important
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causes of skin cancer [26]. In addition, skin exposed to UV radiation can produce ROS and melanin, and ROS levels can cause cellular damage, leading to immune dysregulation,
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photoaging and various skin disorders [27]. Oxidative stress caused by excessive production of intracellular oxidizing species and decreased cellular antioxidant capacity can lead to
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mutagenesis or cell death from damage to nucleic acids, lipids and proteins [28]. There is
of melanomas [29].
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increasing evidence that oxidative stress plays an important role in the onset and progression
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Several compounds such as arbutin, kojic acid, licorice extract, ellagic acid, oxyresveratrol, chlorophorin, norartocarpanone, and ascorbic acid have been developed for use in skin
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whitening or the treatment of skin pigmentation disorders [30-32]. Most of these compounds decreased melanin production by inhibition of tyrosinase but are poorly known to be used in clinical settings due to efficacy and safety issues. For example, hydroquinone is effective for whitening, but it affects melanosome degradation and membrane structure of melanocytes, leading to apoptosis, which is followed by vitiligo side effects [33]. Therefore, Long-term safety and efficacy of SOD3 treatment should also be evaluated before using it in clinical settings. In general, drugs having an antioxidant activity are believed to exhibit whitening, anti-aging 11
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and anti-inflammatory effects [34]. Among known antioxidant enzymes, SOD plays a central role in protecting cells from the effects of superoxide radicals that either directly modify proteins or produce derivatives such as H2O2 and ONOO- [35]. SOD deficient mice have been shown to exhibit increased sensitivity to organ impairment, oxygen toxicity, and
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inflammation. Exogenous SOD has been shown to mitigate inflammatory responses and prevent inflammatory diseases involving oxidative stress [36]. Several reports have suggested
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that SOD3 could be an effective therapeutic to treat the inflammatory skin disorders such as
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atopic dermatitis, psoriasis, and asthma [17-20].
Unlike SOD1 and SOD2, SOD3 is an extracellular antioxidant enzyme that functions without
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infiltrating cells. SOD3 is suggested to suppress inflammatory response at least by part
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reducing the ROS and through non-enzymatic manner [23]. TG mice overexpressing SOD3 were found to be resistant to inflammation [37], whereas SOD1 TG mice showed neuronal
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abnormalities and are not resistant to inflammation [38]. Interestingly, SOD3 is shown to
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have a half-life around 20 h whereas SOD1 has a very short half-life (~ 20 min) in blood [39]. These encouraging results led us to investigate the use of SOD3 as a potential treatment for hyperpigmentary skin disorders.
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Here, we demonstrated the effect of SOD3 on UVB-induced melanin production and also
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showed that SOD3 suppresses UVB-induced proliferative response in melanocytes. SOD3 treatment at 200 U/mL inhibits tyrosinase activity, suppress the expression of MITF, tyroninase, TYRP1, and DCT in melanocytes both at mRNA and protein levels. Moreover, qRT-PCR analysis revealed that the key upstream regulators of MITF, tyrosinase, TYRP1, DCT, and melanin production such as ET-1, EDNRA, EDNRB, MC1R, and Wnt7a were downregulated after SOD3 treatment. Downstream signaling networks of those upstream regulators such as PKCβ2, PKA, CREB, β-catenin, and MAPKs were also inhibited after treatment with SOD3. In contrast, SCF mRNA and protein levels were unaffected by SOD3. 12
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We also investigated the effects of SOD3 on UVB-induced melanogenesis in SOD3 TG mice. Here, we found the robust downregulation of melanogenesis-related markers in SOD3 TG mouse skin without any side effects, suggesting a protective role for SOD3 in hyperpigmentary skin disorders.
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Endothelin is a cyclic family group that includes three isoforms of ET-1, ET-2 and ET-3 in mammals [40]. ET-1 is the most abundant isoform found in the skin, whereas ET-2 is found
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in kidney and intestine, and ET-3 is found in the central nervous system. ET-1 is a well-
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known autocrine factor for melanocyte growth, tyrosinase activation, and keratinocyte regeneration, including vasoconstriction, cell proliferation, and moisture and sodium
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excretion control [41,42]. One of the key regulators of ET-1 is TGF-β, a regulatory cytokine
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that induces ET-1 gene in vascular endothelial cells [43,44]. Here, we demonstrated the regulatory pathway involved in melanogenesis between TGF-β and ET-1. Reports have
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shown that the inhibition of TGF-β signaling reduces ET-1 expression, thereby reducing the
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effects of hypoxia on neonatal lungs [45-47]. In perfused isolated adult rat lungs, TGF-β increases ET-1 synthesis, whereas in isolated alveolar epithelial cells, ET-1 increases TGF-β1 synthesis and signaling via EDNRA and induces the epithelial-mesenchymal transition [48].
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In melanocyte stem cells, TGF-β1 has been shown to not only induce cell cycle arrest, but to
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inhibit melanogenesis by downregulating MITF, the main transcriptional regulator of melanocyte differentiation, and its downstream melanin-producing gene [49]. TGF-β1 is a multifunctional cytokine that plays an important role in the induction of apoptosis, cell growth inhibition and other physiological and pathological responses. TGF-β1 can acts as either a tumor suppressor in early stages of carcinogenesis by promoting either cell cycle arrest or apoptosis of normal epithelial cells or a tumor promoter in later stages of tumorigenesis, leading to tumor cell invasion and metastasis [50,51]. Thus, the functions and effects of TGF-β1 are different depending on cell type and microenvironment. Our data 13
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revealed that TGF-β1 is crucial in ET-1 mediated melanogenesis in melan-a cells. The contrasting results may be due to pleiotropic nature of TGF-β1, which can elicit different functions depending on the cell type, as well as on the type and intensity of stimuli involved. Delayed skin pigmentation can occur for a number of reasons: an increase in the number of
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melanin-producing cells in response to UVB or UVA, the synthesis of new melanin bodies, an increase in enzyme hysteresis, and the progression of metastasis to keratinocytes, which
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typically occurs between 48 and 72 h after UVB exposure [52]. Recent studies have primarily
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focused on compounds that inhibit tyrosinase and, hence, melanin production [53]. However, efficient therapeutics with broad effects is desirable. We believe that the antioxidant SOD3
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has a broad spectrum effect and is expected to be safer and less toxic to humans than
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conventional treatments.
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5. Conclusion
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In conclusion, SOD3 effectively inhibits UVB-induced ROS and cell proliferation, and significantly inhibits melanogenesis. SOD3 may be useful as a potential therapeutic for
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cosmetic use.
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hyperpigmentary skin disorders and could also be developed as a whitening agent for
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Conflict of interest The authors declare that no conflict of interests exists.
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Acknowledgment This research was supported by Korea Health Technology R&D Project through the Korea
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Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare,
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Republic of Korea (grant number: HI17C0616) and Bio & Medical Technology Development
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Program of the National Research Foundation (NRF), funded by the Ministry of Science, ICT
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& Future Planning (NRF-2013M3A9A9050567).
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Figure legends Fig.1. SOD3 reduces UVB-induced ROS levels and suppresses proliferations and melanin production in melan-a cells. (A) Cells were pre-treatment with 200 U/mL SOD3 for 5 h, exposed to UVB at 100 J/m² and
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incubated for further 1 h. ROS level was evaluated by staining the cells with DCFDA at 2 µM. ROS levels were measured at 540 nm using a fluorimeter. (B) BrdU incorporation assay
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was performed to evaluate the effects of SOD3 on UVB-induced melan-a cell proliferation at
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24, 48, and 72 h and analyzed by flow cytometry. Three independent experiments were
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performed in triplicate. (C) Melan-a cells were pre-treatment with 200 U/mL SOD3, exposed to UVB at 100 J/m² and incubated for 3 days cells were harvested and images were taken. (D)
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Melan-a cells were pre-treatment with 200 U/mL SOD3 for 5 h, exposed to UVB at 100 J/m², and incubated for 3 days. Cells were lysed with 1 M NaOH and absorbance at 450 nm was
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measured to evaluate total melanin content. (E) Melan-a cells were pre-treatment with 200
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U/mL SOD3 for 5 h, exposed to UVB at 100 J/m² and incubated for 3 days. Effects of SOD3 on tyrosinase activity were measured at 475 nm by spectrophotometry. Each bar represents
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the mean ± S.D. of three independent experiments. ##P < 0.01, ###P < 0.001 (control vs. UVB-exposed cells), *P < 0.05; ***P < 0.001 (UVB-exposed cells vs. SOD3 and UVB-
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treated cells).
Fig.2. Effects of SOD3 on melanogenesis related genes. Melan-a cells and NHEMs were pre-treatment with 200 U/mL SOD3 for 5 h, exposed to UVB at 100 J/m², and further incubated for 24 h for mRNA expression analysis and 3 days for western blot analysis. (A and B) Cells were harvested for mRNA isolation and expression of MITF, tyrosinase, TYRP1, and DCT by qRT-PCR. Data are represented as the mean ± S.D. of three independent experiments. ##P < 0.01, ###P < 0.001 (control vs. UVB-exposed 22
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cells); *P < 0.05, **P < 0.01 (UVB-exposed cells vs. SOD3 and UVB-treated cells). (C and D) Both cells were pre-treated with 200 U/mL SOD3 for 5 h exposed to UVB at 100 J/m², and incubated the cells for 3 days. Cells were harvested and the expression of MITF, tyrosinase, TYRP1, and DCT was evaluated by western blot analysis. Numerical values on
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the blots represent densitometry units (DU). The control was set to 1 DU. All data represent
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three independent experiments.
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Fig.3. Evaluation of receptors/genes involved in melanogenesis.
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(A) Melan-a cells were pre-treatment with 200 U/mL SOD3 for 5 h, exposed to UVB at 100 J/m², and further incubated for 24 h. Cells were harvested for mRNA isolation and expression
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of ET-1, EDNRA, EDNRB, MC1R, Wnt7a, and SCF by qRT-PCR. Data represent the mean ± S.D. ##P < 0.01, ###P < 0.001 (control vs. UVB-exposed cells); *P < 0.05, **P < 0.01,
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***P < 0.001 (UVB-exposed cells vs. SOD3 and UVB-treated cells). (B-E) Protein
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expression levels of ET-1, EDNRA, EDNRB, MC1R, SCF, Wnt7a and β-actin were harvested 24 h after UVB 100J/m² exposed. Exposed to UVB at 100 J/m² and incubated the cells for 15
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min, cells were harvested and the expression the phosphorylation of PKCβ2, PKA, CREB, βcatenin, p38, JNK and β-actin was evaluated by western blot analysis. Numerical values on
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the blots represent DU. The control was normalized to 1 DU. All data represent three independent experiments.
Fig.4. SOD3 inhibits TGF-β1, a critical component of the ET-1 activation pathway and effects of UVB-induced melanogenesis on SOD3 TG mouse skin. Melan-a cells were exposed to UVB and incubated for 3, 6, 10, or 24 h. (A) mRNA and protein expression levels of TGF-β1 were evaluated by qRT-PCR and western blot, respectively. Data represent three independent experiments as the mean ± S.D. ***P < 0.001 23
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(control vs. UVB-exposed cells). (B) Melan-a cells were pre-treatment with 200 U/mL SOD3 for 5 h, exposed to UVB at 100 J/m² and further incubated the cells for 24 h. Cells were harvested for mRNA expression analysis by qRT-PCR and protein levels were evaluated by western blot. Data represent the mean ± S.D. ## P < 0.01, ###P < 0.001 (control vs. UVB-
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exposed cells); $$P < 0.01 (control vs. SOD3-treated cells.); **P < 0.01 (UVB-exposed cells vs. SOD3 and UVB-treated cells). (C) Melan-a cells were treated with 10 ng/mL TGF-β and
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incubated for 3, 6, 10, or 24 h. mRNA expression levels of ET-1, EDNRA and EDNRB were
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evaluated by qRT-PCR. Data represent the means ± S.D. *P < 0.05 (control vs. TGF-βtreated cells.), **P < 0.01 (control vs. TGF-β-treated cells.) ***P < 0.001 (control vs. TGF-β-
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treated cells.). (D) Melan-a cells were pre-treated with the TGF-β inhibitor LY-364947 at 10
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μM/mL for 1 h, exposed to UVB and incubated for 3, 6, 10, or 24 h. Cells were harvested for ET-1 mRNA expression analysis by qRT-PCR, and ET-1 protein levels were evaluated by
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western blot. Numerical values on the blots represent DU. The control was set to 1 DU. Data
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represent the mean ± S.D. ##P < 0.01 (control vs. UVB-exposed cells); *P < 0.05, **P < 0.01, ***P < 0.001 (UVB-exposed cells vs. UVB-exposed cells and LY364947-treated cells). (E) An experimental scheme for UVB-induced melanogenesis on C57BL/6 mouse. Mice were
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irradiated with UVB (5 kJ/m2) twice and sacrificed on day 5. (F and G) Melanogenesis
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related protein levels in mouse skin such as MITF, Tyrosinase, TYRP1, DCT, SOD3, TGFβ1, ET-1, EDNRA, EDNRB, and β-actin were evaluated by western blotting. Numerical values on the blots represent DU. The control was set to 1 DU. All data represent three independent experiments.
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