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Ultraviolet B radiation-stimulated urocortin 1 is involved in tyrosinase-related protein 1 production in human melanoma HMV-II cells
Peptides 61 (2014) 93–97
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Short communication
Ultraviolet B radiation-stimulated urocortin 1 is involved in tyrosinase-related protein 1 production in human melanoma HMV-II cells Yutaka Watanuki a , Kazunori Kageyama a,b,∗ , Shinobu Takayasu a , Yasushi Matsuzaki c , Yasumasa Iwasaki d , Makoto Daimon a a
Department of Endocrinology and Metabolism, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan Department of Endocrinology, Metabolism, and Infectious Diseases, Hirosaki University School of Medicine & Hospital, 53 Hon-cho, Hirosaki, Aomori 036-8563, Japan c Department of Dermatology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan d Health Care Center, Kochi University, Kochi, Kochi 780-8520, Japan b
a r t i c l e
i n f o
Article history: Received 6 August 2014 Received in revised form 9 September 2014 Accepted 9 September 2014 Keywords: Corticotropin-releasing factor Urocortin 1 Nurr-1 Nur77 Tyrosinase-related protein 1
a b s t r a c t Ultraviolet B (UVB) radiation stimulates cutaneous melanin pigmentation. The melanosomal enzyme tyrosinase-related protein 1 (TRP1) is involved in the modulation of pigment production in response to this stressor. Recent molecular and biochemical analyses have revealed the presence of corticotropinreleasing factor (CRF) and urocortin 1 (Ucn1), together with their corresponding receptors, in mammalian skin. Although CRF and Ucn1 are thought to have potent effects on the skin system, their possible roles and regulations have yet to be determined fully. Our previous findings in human melanoma HMV-II cells suggest that both CRF and Ucn1 regulate TRP1 gene expression via Nurr-1/Nur77, transcription factors that constitute the nuclear receptor 4a subgroup of orphan nuclear receptors. HMV-II cells were found to express mainly Ucn1 mRNA. This study aimed to explore the effects of UVB on Ucn1 mRNA and TRP1 protein levels in HMV-II cells. UVB (30 mJ/cm2 ) increased Nurr-1, Nur77, and Ucn1 mRNA levels. UVB also increased TRP1 protein levels. Ucn1 knockdown inhibited the UVB-induced increases in TRP1 protein levels. These data suggest that UVB-stimulated Ucn1 contributes to TRP1 production via the transcription of both Nurr-1 and Nur77. Ucn1, produced in melanoma cells, acts on melanoma cells themselves in an autocrine manner. © 2014 Elsevier Inc. All rights reserved.
Introduction Corticotropin-releasing factor (CRF), which is synthesized in and secreted from the hypothalamic paraventricular nucleus in response to stress, stimulates adrenocorticotropic hormone (ACTH), which is encoded by the proopiomelanocortin (POMC) gene in pituitary corticotrophs [3,11]. Urocortin 1 (Ucn1), a 40-amino acid peptide originally cloned from the rat Edinger–Westphal nucleus, is a member of the CRF family of
∗ Corresponding author at: Department of Endocrinology and Metabolism, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan. Tel.: +81 172 39 5062; fax: +81 172 39 5063. E-mail address: [email protected] (K. Kageyama). http://dx.doi.org/10.1016/j.peptides.2014.09.009 0196-9781/© 2014 Elsevier Inc. All rights reserved.
peptides [26]. Ucn1 in the Edinger–Westphal nucleus may play a significant role in stress adaptation even in humans [12]. Both CRF and Ucn1 contribute to the stress response and cardiovascular and gonadal functions via G protein-coupled seven transmembrane CRF receptors [6,24,25]. CRF exhibits a high affinity for CRF receptor type 1 (CRF1 receptor; IC50 = 1.6 nM), but not for CRF receptor type 2b (CRF2b receptor; IC50 = 42 nM), while Ucn1 exhibits similar affinity for the CRF1 (IC50 = 0.16 nM) and CRF2b (IC50 = 0.86 nM) receptors [5]. Recent molecular and biochemical analyses have revealed the presence of CRF and Ucn1, together with their corresponding receptors, in mammalian skin [17,18]. The CRF or Ucn system also may exist in the skin [15,21]. CRF activates POMC gene transcription and ACTH release through CRF1 receptors in melanocytes or melanoma cells [9,13,23,30]. CRF also regulates cell viability, proliferation, and migration of skin cells [22,29]. In addition, CRF and Ucn1 stimulate the
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expression of tyrosinase-related enzymes in human hair follicle melanocytes [7]. Cutaneous melanin pigmentation occurs in response to external and internal stresses [4,19,20]. Ultraviolet B (UVB) radiation stimulates the production of ␣-melanocyte-stimulating hormone (␣-MSH) in human keratinocytes and melanocytes [1,14]. The melanosomal enzyme tyrosinase-related protein 1 (TRP1) is involved in the modulation of pigment production in response to the stressor. Both Nurr-1 and Nur77 are transcription factors that constitute the nuclear receptor 4a subgroup of orphan nuclear receptors [2]. They also have the characteristics of immediate early genes and are induced by a variety of extracellular signals. Both Nurr-1 and Nur77 are known to act via a nerve growth factorinducible factor-B response element (NBRE) in the TRP1 promoter. Indeed, putative NBREs have also been identified in the TRP1 promoter [27]. Our previous findings in human melanoma HMV-II cells suggest that both CRF and Ucn1 regulate TRP1 gene expression via Nurr-1/Nur77 [27]. HMV-II cells were found to express mainly Ucn1 mRNA. This study first aimed to explore the effects of UVB on Ucn1 gene expression and TRP1 protein levels using human melanoma HMV-II cells. To elucidate further the possible effects of endogenous Ucn1, TRP1 protein levels following UVB stimulation were examined further using small interfering RNA (siRNA) for Ucn1.
Materials and methods Cell culture HMV-II cells were cultured as follows using a cell line provided by the RIKEN BRC through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. Human melanoma HMV-II cells were cultured in a T75 culture flask with Dulbecco’s modified Eagle’s medium/F-12 HAM supplemented with 10% fetal bovine serum, 50 g/mL streptomycin, and 50 U/mL penicillin at 37 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. Culture media were changed every 48 h, and the cells were subcultured once a week. For each experiment, the cells were plated in 6-well plates at 2.0 × 105 cells/well and the medium was changed every 48 h. At the end of each experiment, total cellular RNA or protein was collected and stored at −80 ◦ C until the relevant assay was performed. UVB irradiation UVB irradiation was performed as reported previously [10]. Cells in phosphate-buffered saline (PBS) were irradiated with UVB using two FL20S-E lamps (Toshiba, Tokyo, Japan) that emitted wavelengths of 280–320 nm with an emission peak at 312.5 nm and an intensity of 500 W/cm2 at the target area. The irradiation dose was 30 mJ/cm2 (25 cm distance for 70 s). After UVB irradiation, the cells were cultured in fresh growth medium. The irradiance of the UVB rays was determined using an UVR-3036/S2 radiometer and a UVB detector (Clinical Supply, Kakamigahara, Japan). RNA extraction The cells were incubated with medium for the indicated times following UVB stimulation. At the end of each experiment, total cellular RNA was extracted using an RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. cDNA was synthesized from total RNA (0.5 g) using random
hexamers as primers with the SuperScript First-Strand Synthesis System for Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer’s instructions.
Real-time RT-PCR Total cellular RNA extraction and cDNA synthesis were performed as described above. First strand cDNAs were then subjected to real-time PCR as follows. The expression levels of human Nurr-1, Nur77, and Ucn1 mRNA were evaluated using quantitative realtime PCR based on specific sets of primers and probes (TaqMan Gene Expression Assays; Applied Biosystems, Foster City, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to standardize the obtained values, because GAPDH mRNA levels did not change in any of the treatments. Each reaction consisted of 1× TaqMan Universal PCR Master Mix (Applied Biosystems), 1× TaqMan Gene Expression Assays Products (Hs00174941 m1 for human CRF, Hs01845155 s1 for human Ucn1, Hs00264218 s1 for human Ucn2, Hs01118813 m1 for human Nurr-1, Hs00374230 m1 for human Nur77, and Hs99999905 m1 for human GAPDH), and 1 L cDNA in a total volume of 25 L using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) as follows: 95 ◦ C for 10 min and then 40 cycles at 95 ◦ C for 15 s and 60 ◦ C for 1 min. The above assays involved specific sets of primers and a TaqMan probe spanning the exon–exon junction and should not, therefore, have been affected by DNA contamination. Data were collected and recorded with ABI PRISM 7000 SDS software (Applied Biosystems) and expressed as a function of the threshold cycle (CT ). The amplification efficacies for each gene of interest and the housekeeping gene amplimers were found to be identical when analyzed with diluted samples.
RNA interference experiments Ucn1 and control siRNAs were designed and purchased from QIAGEN. The cells were transfected with siRNA and HiPerFect transfection reagent (QIAGEN) according to the manufacturer’s protocol. For the measurement of Ucn1 mRNA levels, the cells, seeded into 6-well plates at a density of 2.0 × 105 cellswell, were incubated for 2 days in 1 mL culture medium containing siRNA for either control (siControl) or Ucn1 (siUcn1, Hs UCN 2), and then incubated with medium for 48 h after UVB stimulation. The expression levels of TRP1 and -actin proteins were examined by western blotting.
Western blotting HMV-II cells were plated in 6-well plates at approximately 70% confluence. At the end of the incubation period, the cells were washed twice with PBS and lysed with Laemmli sample buffer. Cell debris was pelleted by centrifugation, and the supernatant was recovered. Twenty micrograms of extract were boiled and used for electrophoresis on a gradient (4–20%) polyacrylamide gel. The proteins were then transferred to a polyvinylidene fluoride membrane (Daiichi Kagaku, Tokyo, Japan). After blocking with Detector Block® Buffer (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA), the membrane was incubated for 1 h, detected with anti-TRP1(1:5000 dilution) (sc-25543; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti--actin antibodies (1:10,000 dilution) (ab8227; Abcam, Cambridge, MA, USA), washed with PBS containing 0.05% Tween 20, and incubated
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Fig. 1. Time-dependent changes in the mRNA expression levels of Nurr-1, Nur77, and Ucn1 in HMV-II cells following UVB (30 mJ/cm2 ) stimulation. Control cells were treated with medium alone. The cells were treated in triplicate, with the average of 3 independent experiments shown (an average in triplicate was considered n = 1, n = 3). Statistical analysis was performed using one-way ANOVA, followed by a Fisher’s protected least-significant difference post hoc test. *P < 0.05 compared with control. The cells were incubated with medium following UVB (30 mJ/cm2 ) stimulation.
with horseradish peroxidase-labeled anti-rabbit immunoglobulin G (Daiichi Kagaku). The chemiluminescent substrate SuperSignalWest Pico (Pierce Chemical Co., Rockford, IL, USA) was used for detection and the membrane was exposed to BioMax film (Eastman Kodak Co., Rochester, NY, USA). -Actin expression was assessed as a loading control. Independent experiments were repeated three times and a representative blot shown. Statistical analysis Each in vitro experiment was performed at least three times. Samples were provided in triplicate for each group of experiments. Each value is expressed as the mean ± standard error of the mean. Statistical analysis was performed with analysis of variance (ANOVA), followed by a Fisher’s protected least-significant difference post hoc test. The level of statistical significance was set at P < 0.05. Results Time-dependent changes in the expression levels of Nurr-1, Nur77, and Ucn1 mRNA following UVB (30 mJ/cm2 ) stimulation in HMV-II cells We were unable to evaluate CRF and Ucn2 expression using quantitative real-time PCR due to their low mRNA levels. HMV-II
cells were then stimulated by 30 mJ/cm2 UVB to determine its effects on Nurr-1, Nur77, and Ucn1 mRNA levels. A time course study showed that UVB irradiation significantly increased both Nurr-1 and Nur77 mRNA levels at 6 h and 2 h, respectively, after stimulation (ANOVA, P < 0.05 and P < 0.005, respectively; Fig. 1A and B). UVB irradiation also significantly increased Ucn1 mRNA levels between 2 h and 6 h after stimulation (ANOVA, P < 0.01; Fig. 1C). The maximal effect of UVB was observed at 6 h, with an approximately 1.7-fold increase in Ucn1 mRNA levels compared to the basal level (Fig. 1C).
Changes in the expression levels of TRP1 protein following UVB (30 mJ/cm2 ) stimulation in HMV-II cells HMV-II cells were stimulated with UVB to determine its effects on the time-dependent changes in TRP1 protein levels. UVB irradiation (30 mJ/cm2 ) significantly increased TRP1 protein levels between 24 and 48 h after stimulation (Fig. 2A). We then examined the functional role of endogenous Ucn1 in HMV-II cells using siRNA for Ucn1. Ucn1 mRNA levels were reduced by 48% in cells transfected with siRNA against the Ucn1 gene. Ucn1 knockdown did not induce any significant changes in basal TRP1 protein levels compared with the control (Fig. 2B). UVB irradiation (30 mJ/cm2 ) increased TRP-1 protein levels at 48 h after stimulation (Fig. 2B).
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Fig. 2. Changes in the expression levels of TRP1 protein in HMV-II cells following UVB stimulation. Control cells were treated with medium alone. The cells were treated in triplicate, with the average of 3 independent experiments shown (an average in triplicate was considered n = 1, n = 3). Statistical analysis was performed using one-way ANOVA, followed by a Fisher’s protected least-significant difference post hoc test. *P < 0.05 compared with control. The cells were incubated with medium following UVB (30 mJ/cm2 ) stimulation. (A) Time-dependent changes in the expression levels of TRP1 protein following UVB (30 mJ/cm2 ) stimulation. (B) Effects of Ucn1 on TRP1 protein levels following UVB (30 mJ/cm2 ) stimulation. The cells, seeded into 6-well plates at a density of 2.0 × 105 cellswell, were incubated for 1 day in 1 mL culture medium containing siRNA for either control (siControl) or Ucn1 (siUcn1). One group of cells was then incubated with medium for 2 days after UVB (30 mJ/cm2 ) stimulation. + P< compared to siUcn1 (−)/UVB (+).
Ucn1 knockdown significantly inhibited the UVB-induced increases in TRP1 protein levels (Fig. 2B). Discussion In this study, UVB increased both Nurr-1 and Nur77 mRNA expression levels. Our previous data suggest that both CRF and Ucn1 stimulate Nurr-1/Nur77 mRNA levels and gene promoter activity in melanoma cells [27]. Both Nurr-1 and Nur77 are common mediators of the CRF/Ucn1-induced activation of the TRP1 gene promoter. In our previous study, TRP1 promoter activity was stimulated strongly by both Nurr-1 and Nur77 [27]. Taken together, these findings indicate that UVB-stimulated Ucn1 increases the transcription of both Nurr-1 and Nur77. In fact, Ucn1 mRNA expression levels were also stimulated following UVB irradiation in HMV-II cells. Ucn1 stimulated TRP1 gene transcription and protein expression levels in HMV-II cells [27]. Tyrosinase hydroxylates l-tyrosine to l-dihydroxyphenylalanine and is thought to be an early obligatory and rate-limiting step in melanogenesis, contributing to both eumelanogenic and pheomelanogenic pathways. TRP1 is involved in the terminal step of eumelanogenesis [8,28]. Thus, Ucn1-induced TRP1 may contribute to the maintenance of cutaneous melanin synthesis following UVB irradiation. HMV-II cells express the CRF1 receptor, but not the CRF2 receptor, suggesting that Ucn1 plays an endogenous role in HMV-II cells via the CRF1 receptor. A selective CRF1 receptor antagonist, but not a CRF2 receptor antagonist, had effects on the CRF/Ucn1-induced activation of TRP1 gene transcription as well as Nurr-1/Nur77 expression [27]. These results are consistent with those of previous studies showing the expression of the CRF1 receptor mainly in human skin cells such as melanocytes [17,18]. Taken together, the present findings suggest that the Ucn1-induced activation of TRP1
gene transcription, as well as Nurr-1/Nur77, is mediated mainly via the CRF1 receptor in an autocrine or paracrine manner in melanoma cells. It has been suggested that endogenous Ucn1 acts in an autocrine or paracrine manner in the skin. Protective skin pigmentation in response to stresses such as UVB occurs as a result of ␣-MSH production in keratinocytes and melanocytes. Ucn1 mRNA levels were increased in mouse skin following UVB irradiation ex vivo [16]. UVB-induced Ucn1 is produced in melanoma cells, and acts on melanoma cells themselves via the CRF1 receptor in an autocrine manner. UVB increased TRP1 protein levels, and an approximate 50% knockdown of Ucn1 mRNA levels partially inhibited the UVBinduced increases in TRP1 protein levels. Endogenous Ucn1 is at least partially involved in the regulation of the UVB-stimulated production of TRP1 in HMV-II cells. In conclusion, the present study demonstrated that UVB increased Nurr-1, Nur77, and Ucn1 mRNA levels. UVB also increased TRP1 protein levels. Ucn1 knockdown inhibited the UVBinduced increases of TRP1 protein levels. Thus, Ucn1 is produced in melanoma cells, and acts on melanoma cells themselves in an autocrine manner. Conflict of interest None of the authors have any potential conflicts of interest associated with this research. Acknowledgments We thank the Department of Pharmacology, Hirosaki University School of Medicine, Japan, for generously providing us with access to their ABI PRISM 7000 Sequence Detection System.
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References [1] Chakraborty AK, Funasaka Y, Slominski A, Ermak G, Hwang J, Pawelek JM, et al. Production and release of proopiomelanocortin (POMC) derived peptides by human melanocytes and keratinocytes in culture: regulation by ultraviolet B. Biochim Biophys Acta 1996;1313:130–8. [2] Davis IJ, Lau LF. Endocrine and neurogenic regulation of the orphan nuclear receptors Nur77 and Nurr-1 in the adrenal glands. Mol Cell Biol 1994;14:3469–83. [3] Gillies GE, Linton EA, Lowry PJ. Corticotropin releasing activity of the new CRF is potentiated several times by vasopressin. Nature 1982;299: 355–7. [4] Ito N, Ito T, Kromminga A, Bettermann A, Takigawa M, Kees F, et al. Human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal axis and synthesize cortisol. FASEB J 2005;19:1332–4. [5] Jahn O, Tezval H, van Werven L, Eckart K, Spiess J. Three-amino acid motifs of urocortin II and III determine their CRF receptor subtype selectivity. Neuropharmacology 2004;47:233–42. [6] Kageyama K, Bradbury MJ, Zhao L, Blount AL, Vale WW. Urocortin messenger ribonucleic acid: tissue distribution in the rat and regulation in thymus by lipopolysaccharide and glucocorticoids. Endocrinology 1999;140:5651–8. [7] Kauser S, Slominski A, Wei ET, Tobin DJ. Modulation of the human hair follicle pigmentary unit by corticotropin-releasing hormone and urocortin peptides. FASEB J 2006;20:882–95. [8] Kenny EE, Timpson NJ, Sikora M, Yee MC, Moreno-Estrada A, Eng C, et al. Melanesian blond hair is caused by an amino acid change in TYRP1. Science 2012;336:554. [9] Kim MH, Cho D, Kim HJ, Chong SJ, Lee KH, Yu DS, et al. Investigation of the corticotropin-releasing hormone-proopiomelanocortin axis in various skin tumours. Br J Dermatol 2006;155:910–5. [10] Kimura K, Matsuzaki Y, Nishikawa Y, Kitamura H, Akasaka E, Rokunohe D, et al. Characterization of retinoic acid-inducible gene-I (RIG-I) expression corresponding to viral infection and UVB in human keratinocytes. J Dermatol Sci 2012;66:64–70. [11] Mouri T, Itoi K, Takahashi K, Suda T, Murakami O, Yoshinaga K, et al. Colocalization of corticotropin-releasing factor and vasopressin in the paraventricular nucleus of the human hypothalamus. Neuroendocrinology 1993;57:34–9. [12] Pagida MA, Konstantinidou AE, Tsekoura E, Patsouris E, Panayotacopoulou MT. Immunohistochemical demonstration of urocortin 1 in Edinger–Westphal nucleus of the human neonate: colocalization with tyrosine hydroxylase under acute perinatal hypoxia. Neurosci Lett 2013;554:47–52. [13] Sato H, Nagashima Y, Chrousos GP, Ichihashi M, Funasak Y. The expression of corticotropin-releasing hormone in melanoma. Pigment Cell Res 2002;15:98–103. [14] Schauer E, Trautinger F, Kock A, Schwarz A, Bhardwaj R, Simon M, et al. Proopiomelanocortin derived peptides are synthesised and released by human keratinocytes. J Clin Invest 1994;93:2258–62.
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[15] Skobowiat C, Dowdy JC, Sayre RM, Tuckey RC, Slominski A. Cutaneous hypothalamic-pituitary-adrenal axis homolog: regulation by ultraviolet radiation. Am J Physiol Endocrinol Metab 2011;301:E484–543. [16] Skobowiat C, Nejati R, Lu L, Williams RW, Slominski AT. Genetic variation of the cutaneous HPA axis: an analysis of UVB-induced differential responses. Gene 2013;530:1. [17] Slominski AT, Botchkarev V, Choudhry M, Fazal N, Fechner K, Furkert J, et al. Cutaneous expression of CRH and CRH-R, Is there a “skin stress response system?”. Ann N Y Acad Sci 1999;885:287–311. [18] Slominski A, Pisarchik A, Tobin DJ, Mazurkiewicz JE, Wortsman J. Differential expression of a cutaneous corticotropin-releasing hormone system. Endocrinology 2004;145:941–50. [19] Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 2004;84:1155–228. [20] Slominski A, Wortsman J, Luger T, Paus R, Solomon S. Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress. Physiol Rev 2000;80:979–1020. [21] Slominski A, Wortsman J, Tuckey RC, Paus R. Differential expression of HPA axis homolog in the skin. Mol Cell Endocrinol 2007;265–256:143–9. [22] Slominski A, Zbytek B, Pisarchik A, Slominski RM, Zmijewski MA, Wortsman J. CRH functions as a growth factor/cytokine in the skin. J Cell Physiol 2006;206:780–91. [23] Slominski A, Zbytek B, Szczesniewski A, Semak I, Kaminski J, Sweatman T, et al. CRH stimulation of corticosteroids production in melanocytes is mediated by ACTH. Am J Physiol Endocrinol Metab 2005;288:E701–6. [24] Suda T, Kageyama K, Sakihara S, Nigawara T. Physiological roles of urocortins, human homologues of fish urotensin 1, and their receptors. Peptides 2004;25:1689–701. [25] Vale W, Spiess J, Rivier C, Rivier J. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and betaendorphin. Science 1981;213:1394–7. [26] Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, et al. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 1995;378:287–92. [27] Watanuki Y, Takayasu S, Kageyama K, Iwasaki Y, Sakihara S, Terui K, et al. Involvement of Nurr-1/Nur77 in corticotropin-releasing factor/urocortin1induced tyrosinase-related protein 1 gene transcription in human melanoma HMV-II cells. Mol Cell Endocrinol 2013;370:42–51. [28] Yamada M, Sakai K, Hayashi M, Hozumi Y, Abe Y, Kawaguchi M, et al. Oculocutaneous albinism type 3: a Japanese girl with novel mutations in TYRP1 gene. J Dermatol Sci 2011;64:217. [29] Yang Y, Park H, Yang Y, Kim TS, Bang SI, Cho D. Enhancement of cell migration by corticotropin-releasing hormone through ERK1/2 pathway in murine melanoma cell line, B16F10. Exp Dermatol 2007;16:22–7. [30] Zbytek B, Wortsman J, Slominski A. Characterization of a ultraviolet Binduced corticotropin-releasing hormone-proopiomelanocortin system in human melanocytes. Mol Endocrinol 2006;20:2539–47.
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Short communication
Ultraviolet B radiation-stimulated urocortin 1 is involved in tyrosinase-related protein 1 production in human melanoma HMV-II cells Yutaka Watanuki a , Kazunori Kageyama a,b,∗ , Shinobu Takayasu a , Yasushi Matsuzaki c , Yasumasa Iwasaki d , Makoto Daimon a a
Department of Endocrinology and Metabolism, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan Department of Endocrinology, Metabolism, and Infectious Diseases, Hirosaki University School of Medicine & Hospital, 53 Hon-cho, Hirosaki, Aomori 036-8563, Japan c Department of Dermatology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan d Health Care Center, Kochi University, Kochi, Kochi 780-8520, Japan b
a r t i c l e
i n f o
Article history: Received 6 August 2014 Received in revised form 9 September 2014 Accepted 9 September 2014 Keywords: Corticotropin-releasing factor Urocortin 1 Nurr-1 Nur77 Tyrosinase-related protein 1
a b s t r a c t Ultraviolet B (UVB) radiation stimulates cutaneous melanin pigmentation. The melanosomal enzyme tyrosinase-related protein 1 (TRP1) is involved in the modulation of pigment production in response to this stressor. Recent molecular and biochemical analyses have revealed the presence of corticotropinreleasing factor (CRF) and urocortin 1 (Ucn1), together with their corresponding receptors, in mammalian skin. Although CRF and Ucn1 are thought to have potent effects on the skin system, their possible roles and regulations have yet to be determined fully. Our previous findings in human melanoma HMV-II cells suggest that both CRF and Ucn1 regulate TRP1 gene expression via Nurr-1/Nur77, transcription factors that constitute the nuclear receptor 4a subgroup of orphan nuclear receptors. HMV-II cells were found to express mainly Ucn1 mRNA. This study aimed to explore the effects of UVB on Ucn1 mRNA and TRP1 protein levels in HMV-II cells. UVB (30 mJ/cm2 ) increased Nurr-1, Nur77, and Ucn1 mRNA levels. UVB also increased TRP1 protein levels. Ucn1 knockdown inhibited the UVB-induced increases in TRP1 protein levels. These data suggest that UVB-stimulated Ucn1 contributes to TRP1 production via the transcription of both Nurr-1 and Nur77. Ucn1, produced in melanoma cells, acts on melanoma cells themselves in an autocrine manner. © 2014 Elsevier Inc. All rights reserved.
Introduction Corticotropin-releasing factor (CRF), which is synthesized in and secreted from the hypothalamic paraventricular nucleus in response to stress, stimulates adrenocorticotropic hormone (ACTH), which is encoded by the proopiomelanocortin (POMC) gene in pituitary corticotrophs [3,11]. Urocortin 1 (Ucn1), a 40-amino acid peptide originally cloned from the rat Edinger–Westphal nucleus, is a member of the CRF family of
∗ Corresponding author at: Department of Endocrinology and Metabolism, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan. Tel.: +81 172 39 5062; fax: +81 172 39 5063. E-mail address: [email protected] (K. Kageyama). http://dx.doi.org/10.1016/j.peptides.2014.09.009 0196-9781/© 2014 Elsevier Inc. All rights reserved.
peptides [26]. Ucn1 in the Edinger–Westphal nucleus may play a significant role in stress adaptation even in humans [12]. Both CRF and Ucn1 contribute to the stress response and cardiovascular and gonadal functions via G protein-coupled seven transmembrane CRF receptors [6,24,25]. CRF exhibits a high affinity for CRF receptor type 1 (CRF1 receptor; IC50 = 1.6 nM), but not for CRF receptor type 2b (CRF2b receptor; IC50 = 42 nM), while Ucn1 exhibits similar affinity for the CRF1 (IC50 = 0.16 nM) and CRF2b (IC50 = 0.86 nM) receptors [5]. Recent molecular and biochemical analyses have revealed the presence of CRF and Ucn1, together with their corresponding receptors, in mammalian skin [17,18]. The CRF or Ucn system also may exist in the skin [15,21]. CRF activates POMC gene transcription and ACTH release through CRF1 receptors in melanocytes or melanoma cells [9,13,23,30]. CRF also regulates cell viability, proliferation, and migration of skin cells [22,29]. In addition, CRF and Ucn1 stimulate the
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expression of tyrosinase-related enzymes in human hair follicle melanocytes [7]. Cutaneous melanin pigmentation occurs in response to external and internal stresses [4,19,20]. Ultraviolet B (UVB) radiation stimulates the production of ␣-melanocyte-stimulating hormone (␣-MSH) in human keratinocytes and melanocytes [1,14]. The melanosomal enzyme tyrosinase-related protein 1 (TRP1) is involved in the modulation of pigment production in response to the stressor. Both Nurr-1 and Nur77 are transcription factors that constitute the nuclear receptor 4a subgroup of orphan nuclear receptors [2]. They also have the characteristics of immediate early genes and are induced by a variety of extracellular signals. Both Nurr-1 and Nur77 are known to act via a nerve growth factorinducible factor-B response element (NBRE) in the TRP1 promoter. Indeed, putative NBREs have also been identified in the TRP1 promoter [27]. Our previous findings in human melanoma HMV-II cells suggest that both CRF and Ucn1 regulate TRP1 gene expression via Nurr-1/Nur77 [27]. HMV-II cells were found to express mainly Ucn1 mRNA. This study first aimed to explore the effects of UVB on Ucn1 gene expression and TRP1 protein levels using human melanoma HMV-II cells. To elucidate further the possible effects of endogenous Ucn1, TRP1 protein levels following UVB stimulation were examined further using small interfering RNA (siRNA) for Ucn1.
Materials and methods Cell culture HMV-II cells were cultured as follows using a cell line provided by the RIKEN BRC through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. Human melanoma HMV-II cells were cultured in a T75 culture flask with Dulbecco’s modified Eagle’s medium/F-12 HAM supplemented with 10% fetal bovine serum, 50 g/mL streptomycin, and 50 U/mL penicillin at 37 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. Culture media were changed every 48 h, and the cells were subcultured once a week. For each experiment, the cells were plated in 6-well plates at 2.0 × 105 cells/well and the medium was changed every 48 h. At the end of each experiment, total cellular RNA or protein was collected and stored at −80 ◦ C until the relevant assay was performed. UVB irradiation UVB irradiation was performed as reported previously [10]. Cells in phosphate-buffered saline (PBS) were irradiated with UVB using two FL20S-E lamps (Toshiba, Tokyo, Japan) that emitted wavelengths of 280–320 nm with an emission peak at 312.5 nm and an intensity of 500 W/cm2 at the target area. The irradiation dose was 30 mJ/cm2 (25 cm distance for 70 s). After UVB irradiation, the cells were cultured in fresh growth medium. The irradiance of the UVB rays was determined using an UVR-3036/S2 radiometer and a UVB detector (Clinical Supply, Kakamigahara, Japan). RNA extraction The cells were incubated with medium for the indicated times following UVB stimulation. At the end of each experiment, total cellular RNA was extracted using an RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. cDNA was synthesized from total RNA (0.5 g) using random
hexamers as primers with the SuperScript First-Strand Synthesis System for Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer’s instructions.
Real-time RT-PCR Total cellular RNA extraction and cDNA synthesis were performed as described above. First strand cDNAs were then subjected to real-time PCR as follows. The expression levels of human Nurr-1, Nur77, and Ucn1 mRNA were evaluated using quantitative realtime PCR based on specific sets of primers and probes (TaqMan Gene Expression Assays; Applied Biosystems, Foster City, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to standardize the obtained values, because GAPDH mRNA levels did not change in any of the treatments. Each reaction consisted of 1× TaqMan Universal PCR Master Mix (Applied Biosystems), 1× TaqMan Gene Expression Assays Products (Hs00174941 m1 for human CRF, Hs01845155 s1 for human Ucn1, Hs00264218 s1 for human Ucn2, Hs01118813 m1 for human Nurr-1, Hs00374230 m1 for human Nur77, and Hs99999905 m1 for human GAPDH), and 1 L cDNA in a total volume of 25 L using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) as follows: 95 ◦ C for 10 min and then 40 cycles at 95 ◦ C for 15 s and 60 ◦ C for 1 min. The above assays involved specific sets of primers and a TaqMan probe spanning the exon–exon junction and should not, therefore, have been affected by DNA contamination. Data were collected and recorded with ABI PRISM 7000 SDS software (Applied Biosystems) and expressed as a function of the threshold cycle (CT ). The amplification efficacies for each gene of interest and the housekeeping gene amplimers were found to be identical when analyzed with diluted samples.
RNA interference experiments Ucn1 and control siRNAs were designed and purchased from QIAGEN. The cells were transfected with siRNA and HiPerFect transfection reagent (QIAGEN) according to the manufacturer’s protocol. For the measurement of Ucn1 mRNA levels, the cells, seeded into 6-well plates at a density of 2.0 × 105 cellswell, were incubated for 2 days in 1 mL culture medium containing siRNA for either control (siControl) or Ucn1 (siUcn1, Hs UCN 2), and then incubated with medium for 48 h after UVB stimulation. The expression levels of TRP1 and -actin proteins were examined by western blotting.
Western blotting HMV-II cells were plated in 6-well plates at approximately 70% confluence. At the end of the incubation period, the cells were washed twice with PBS and lysed with Laemmli sample buffer. Cell debris was pelleted by centrifugation, and the supernatant was recovered. Twenty micrograms of extract were boiled and used for electrophoresis on a gradient (4–20%) polyacrylamide gel. The proteins were then transferred to a polyvinylidene fluoride membrane (Daiichi Kagaku, Tokyo, Japan). After blocking with Detector Block® Buffer (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA), the membrane was incubated for 1 h, detected with anti-TRP1(1:5000 dilution) (sc-25543; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti--actin antibodies (1:10,000 dilution) (ab8227; Abcam, Cambridge, MA, USA), washed with PBS containing 0.05% Tween 20, and incubated
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Fig. 1. Time-dependent changes in the mRNA expression levels of Nurr-1, Nur77, and Ucn1 in HMV-II cells following UVB (30 mJ/cm2 ) stimulation. Control cells were treated with medium alone. The cells were treated in triplicate, with the average of 3 independent experiments shown (an average in triplicate was considered n = 1, n = 3). Statistical analysis was performed using one-way ANOVA, followed by a Fisher’s protected least-significant difference post hoc test. *P < 0.05 compared with control. The cells were incubated with medium following UVB (30 mJ/cm2 ) stimulation.
with horseradish peroxidase-labeled anti-rabbit immunoglobulin G (Daiichi Kagaku). The chemiluminescent substrate SuperSignalWest Pico (Pierce Chemical Co., Rockford, IL, USA) was used for detection and the membrane was exposed to BioMax film (Eastman Kodak Co., Rochester, NY, USA). -Actin expression was assessed as a loading control. Independent experiments were repeated three times and a representative blot shown. Statistical analysis Each in vitro experiment was performed at least three times. Samples were provided in triplicate for each group of experiments. Each value is expressed as the mean ± standard error of the mean. Statistical analysis was performed with analysis of variance (ANOVA), followed by a Fisher’s protected least-significant difference post hoc test. The level of statistical significance was set at P < 0.05. Results Time-dependent changes in the expression levels of Nurr-1, Nur77, and Ucn1 mRNA following UVB (30 mJ/cm2 ) stimulation in HMV-II cells We were unable to evaluate CRF and Ucn2 expression using quantitative real-time PCR due to their low mRNA levels. HMV-II
cells were then stimulated by 30 mJ/cm2 UVB to determine its effects on Nurr-1, Nur77, and Ucn1 mRNA levels. A time course study showed that UVB irradiation significantly increased both Nurr-1 and Nur77 mRNA levels at 6 h and 2 h, respectively, after stimulation (ANOVA, P < 0.05 and P < 0.005, respectively; Fig. 1A and B). UVB irradiation also significantly increased Ucn1 mRNA levels between 2 h and 6 h after stimulation (ANOVA, P < 0.01; Fig. 1C). The maximal effect of UVB was observed at 6 h, with an approximately 1.7-fold increase in Ucn1 mRNA levels compared to the basal level (Fig. 1C).
Changes in the expression levels of TRP1 protein following UVB (30 mJ/cm2 ) stimulation in HMV-II cells HMV-II cells were stimulated with UVB to determine its effects on the time-dependent changes in TRP1 protein levels. UVB irradiation (30 mJ/cm2 ) significantly increased TRP1 protein levels between 24 and 48 h after stimulation (Fig. 2A). We then examined the functional role of endogenous Ucn1 in HMV-II cells using siRNA for Ucn1. Ucn1 mRNA levels were reduced by 48% in cells transfected with siRNA against the Ucn1 gene. Ucn1 knockdown did not induce any significant changes in basal TRP1 protein levels compared with the control (Fig. 2B). UVB irradiation (30 mJ/cm2 ) increased TRP-1 protein levels at 48 h after stimulation (Fig. 2B).
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Fig. 2. Changes in the expression levels of TRP1 protein in HMV-II cells following UVB stimulation. Control cells were treated with medium alone. The cells were treated in triplicate, with the average of 3 independent experiments shown (an average in triplicate was considered n = 1, n = 3). Statistical analysis was performed using one-way ANOVA, followed by a Fisher’s protected least-significant difference post hoc test. *P < 0.05 compared with control. The cells were incubated with medium following UVB (30 mJ/cm2 ) stimulation. (A) Time-dependent changes in the expression levels of TRP1 protein following UVB (30 mJ/cm2 ) stimulation. (B) Effects of Ucn1 on TRP1 protein levels following UVB (30 mJ/cm2 ) stimulation. The cells, seeded into 6-well plates at a density of 2.0 × 105 cellswell, were incubated for 1 day in 1 mL culture medium containing siRNA for either control (siControl) or Ucn1 (siUcn1). One group of cells was then incubated with medium for 2 days after UVB (30 mJ/cm2 ) stimulation. + P< compared to siUcn1 (−)/UVB (+).
Ucn1 knockdown significantly inhibited the UVB-induced increases in TRP1 protein levels (Fig. 2B). Discussion In this study, UVB increased both Nurr-1 and Nur77 mRNA expression levels. Our previous data suggest that both CRF and Ucn1 stimulate Nurr-1/Nur77 mRNA levels and gene promoter activity in melanoma cells [27]. Both Nurr-1 and Nur77 are common mediators of the CRF/Ucn1-induced activation of the TRP1 gene promoter. In our previous study, TRP1 promoter activity was stimulated strongly by both Nurr-1 and Nur77 [27]. Taken together, these findings indicate that UVB-stimulated Ucn1 increases the transcription of both Nurr-1 and Nur77. In fact, Ucn1 mRNA expression levels were also stimulated following UVB irradiation in HMV-II cells. Ucn1 stimulated TRP1 gene transcription and protein expression levels in HMV-II cells [27]. Tyrosinase hydroxylates l-tyrosine to l-dihydroxyphenylalanine and is thought to be an early obligatory and rate-limiting step in melanogenesis, contributing to both eumelanogenic and pheomelanogenic pathways. TRP1 is involved in the terminal step of eumelanogenesis [8,28]. Thus, Ucn1-induced TRP1 may contribute to the maintenance of cutaneous melanin synthesis following UVB irradiation. HMV-II cells express the CRF1 receptor, but not the CRF2 receptor, suggesting that Ucn1 plays an endogenous role in HMV-II cells via the CRF1 receptor. A selective CRF1 receptor antagonist, but not a CRF2 receptor antagonist, had effects on the CRF/Ucn1-induced activation of TRP1 gene transcription as well as Nurr-1/Nur77 expression [27]. These results are consistent with those of previous studies showing the expression of the CRF1 receptor mainly in human skin cells such as melanocytes [17,18]. Taken together, the present findings suggest that the Ucn1-induced activation of TRP1
gene transcription, as well as Nurr-1/Nur77, is mediated mainly via the CRF1 receptor in an autocrine or paracrine manner in melanoma cells. It has been suggested that endogenous Ucn1 acts in an autocrine or paracrine manner in the skin. Protective skin pigmentation in response to stresses such as UVB occurs as a result of ␣-MSH production in keratinocytes and melanocytes. Ucn1 mRNA levels were increased in mouse skin following UVB irradiation ex vivo [16]. UVB-induced Ucn1 is produced in melanoma cells, and acts on melanoma cells themselves via the CRF1 receptor in an autocrine manner. UVB increased TRP1 protein levels, and an approximate 50% knockdown of Ucn1 mRNA levels partially inhibited the UVBinduced increases in TRP1 protein levels. Endogenous Ucn1 is at least partially involved in the regulation of the UVB-stimulated production of TRP1 in HMV-II cells. In conclusion, the present study demonstrated that UVB increased Nurr-1, Nur77, and Ucn1 mRNA levels. UVB also increased TRP1 protein levels. Ucn1 knockdown inhibited the UVBinduced increases of TRP1 protein levels. Thus, Ucn1 is produced in melanoma cells, and acts on melanoma cells themselves in an autocrine manner. Conflict of interest None of the authors have any potential conflicts of interest associated with this research. Acknowledgments We thank the Department of Pharmacology, Hirosaki University School of Medicine, Japan, for generously providing us with access to their ABI PRISM 7000 Sequence Detection System.
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References [1] Chakraborty AK, Funasaka Y, Slominski A, Ermak G, Hwang J, Pawelek JM, et al. Production and release of proopiomelanocortin (POMC) derived peptides by human melanocytes and keratinocytes in culture: regulation by ultraviolet B. Biochim Biophys Acta 1996;1313:130–8. [2] Davis IJ, Lau LF. Endocrine and neurogenic regulation of the orphan nuclear receptors Nur77 and Nurr-1 in the adrenal glands. Mol Cell Biol 1994;14:3469–83. [3] Gillies GE, Linton EA, Lowry PJ. Corticotropin releasing activity of the new CRF is potentiated several times by vasopressin. Nature 1982;299: 355–7. [4] Ito N, Ito T, Kromminga A, Bettermann A, Takigawa M, Kees F, et al. Human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal axis and synthesize cortisol. FASEB J 2005;19:1332–4. [5] Jahn O, Tezval H, van Werven L, Eckart K, Spiess J. Three-amino acid motifs of urocortin II and III determine their CRF receptor subtype selectivity. Neuropharmacology 2004;47:233–42. [6] Kageyama K, Bradbury MJ, Zhao L, Blount AL, Vale WW. Urocortin messenger ribonucleic acid: tissue distribution in the rat and regulation in thymus by lipopolysaccharide and glucocorticoids. Endocrinology 1999;140:5651–8. [7] Kauser S, Slominski A, Wei ET, Tobin DJ. Modulation of the human hair follicle pigmentary unit by corticotropin-releasing hormone and urocortin peptides. FASEB J 2006;20:882–95. [8] Kenny EE, Timpson NJ, Sikora M, Yee MC, Moreno-Estrada A, Eng C, et al. Melanesian blond hair is caused by an amino acid change in TYRP1. Science 2012;336:554. [9] Kim MH, Cho D, Kim HJ, Chong SJ, Lee KH, Yu DS, et al. Investigation of the corticotropin-releasing hormone-proopiomelanocortin axis in various skin tumours. Br J Dermatol 2006;155:910–5. [10] Kimura K, Matsuzaki Y, Nishikawa Y, Kitamura H, Akasaka E, Rokunohe D, et al. Characterization of retinoic acid-inducible gene-I (RIG-I) expression corresponding to viral infection and UVB in human keratinocytes. J Dermatol Sci 2012;66:64–70. [11] Mouri T, Itoi K, Takahashi K, Suda T, Murakami O, Yoshinaga K, et al. Colocalization of corticotropin-releasing factor and vasopressin in the paraventricular nucleus of the human hypothalamus. Neuroendocrinology 1993;57:34–9. [12] Pagida MA, Konstantinidou AE, Tsekoura E, Patsouris E, Panayotacopoulou MT. Immunohistochemical demonstration of urocortin 1 in Edinger–Westphal nucleus of the human neonate: colocalization with tyrosine hydroxylase under acute perinatal hypoxia. Neurosci Lett 2013;554:47–52. [13] Sato H, Nagashima Y, Chrousos GP, Ichihashi M, Funasak Y. The expression of corticotropin-releasing hormone in melanoma. Pigment Cell Res 2002;15:98–103. [14] Schauer E, Trautinger F, Kock A, Schwarz A, Bhardwaj R, Simon M, et al. Proopiomelanocortin derived peptides are synthesised and released by human keratinocytes. J Clin Invest 1994;93:2258–62.
97
[15] Skobowiat C, Dowdy JC, Sayre RM, Tuckey RC, Slominski A. Cutaneous hypothalamic-pituitary-adrenal axis homolog: regulation by ultraviolet radiation. Am J Physiol Endocrinol Metab 2011;301:E484–543. [16] Skobowiat C, Nejati R, Lu L, Williams RW, Slominski AT. Genetic variation of the cutaneous HPA axis: an analysis of UVB-induced differential responses. Gene 2013;530:1. [17] Slominski AT, Botchkarev V, Choudhry M, Fazal N, Fechner K, Furkert J, et al. Cutaneous expression of CRH and CRH-R, Is there a “skin stress response system?”. Ann N Y Acad Sci 1999;885:287–311. [18] Slominski A, Pisarchik A, Tobin DJ, Mazurkiewicz JE, Wortsman J. Differential expression of a cutaneous corticotropin-releasing hormone system. Endocrinology 2004;145:941–50. [19] Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev 2004;84:1155–228. [20] Slominski A, Wortsman J, Luger T, Paus R, Solomon S. Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress. Physiol Rev 2000;80:979–1020. [21] Slominski A, Wortsman J, Tuckey RC, Paus R. Differential expression of HPA axis homolog in the skin. Mol Cell Endocrinol 2007;265–256:143–9. [22] Slominski A, Zbytek B, Pisarchik A, Slominski RM, Zmijewski MA, Wortsman J. CRH functions as a growth factor/cytokine in the skin. J Cell Physiol 2006;206:780–91. [23] Slominski A, Zbytek B, Szczesniewski A, Semak I, Kaminski J, Sweatman T, et al. CRH stimulation of corticosteroids production in melanocytes is mediated by ACTH. Am J Physiol Endocrinol Metab 2005;288:E701–6. [24] Suda T, Kageyama K, Sakihara S, Nigawara T. Physiological roles of urocortins, human homologues of fish urotensin 1, and their receptors. Peptides 2004;25:1689–701. [25] Vale W, Spiess J, Rivier C, Rivier J. Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and betaendorphin. Science 1981;213:1394–7. [26] Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, et al. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 1995;378:287–92. [27] Watanuki Y, Takayasu S, Kageyama K, Iwasaki Y, Sakihara S, Terui K, et al. Involvement of Nurr-1/Nur77 in corticotropin-releasing factor/urocortin1induced tyrosinase-related protein 1 gene transcription in human melanoma HMV-II cells. Mol Cell Endocrinol 2013;370:42–51. [28] Yamada M, Sakai K, Hayashi M, Hozumi Y, Abe Y, Kawaguchi M, et al. Oculocutaneous albinism type 3: a Japanese girl with novel mutations in TYRP1 gene. J Dermatol Sci 2011;64:217. [29] Yang Y, Park H, Yang Y, Kim TS, Bang SI, Cho D. Enhancement of cell migration by corticotropin-releasing hormone through ERK1/2 pathway in murine melanoma cell line, B16F10. Exp Dermatol 2007;16:22–7. [30] Zbytek B, Wortsman J, Slominski A. Characterization of a ultraviolet Binduced corticotropin-releasing hormone-proopiomelanocortin system in human melanocytes. Mol Endocrinol 2006;20:2539–47.