Melanin production through novel processing of proopiomelanocortin in the extracellular compartment of the auricular skin of C57BL/6 mice after UV-irradiation

The production of melanin is regulated by α-melanocyte-stimulating hormone (α-MSH), which is produced from proopiomelanocortin (POMC). Keratinocytes release POMC along with lower levels of α-MSH and ACTH. To clarify the mechanism of melanogenesis after ultraviolet (UV)-irradiation, this study focused on the expression of POMC and POMC-derived peptides after UV-irradiation. Western blot analysis and immunoassays indicated that both POMC and α-MSH-like immunoreactivity (α-MSH-LI) increased after UV-irradiation. However, other POMC-derived products were very low. In hypophysectomized mice, α-MSH-LI increased to the same level as in control mice after UV-irradiation. Structural analysis revealed that the major α-MSH-LI product was ACTH(1–8). Furthermore, ACTH(1–8) competed with [125I]-α-MSH for receptor binding and increased melanin production via a melanocortin-1 receptor. These results suggested that melanin was produced through ACTH(1–8) after UV-irradiation. Trypsin-like enzymatic activity, which is responsible for POMC activation, increased after UV-irradiation and was identified as tryptase. In mast cell-deficient mice, which do not produce tryptase, α-MSH-LI levels were unchanged after UV-irradiation. The present study demonstrates the production of ACTH(1–8) from POMC by tryptase, which is a novel peptide-processing mechanism in the extracellular compartment of the skin.

circulation. It has been reported that these peptides, when originating from the pituitary, have an effect on skin pigmentation in human 12 . However, skin pigmentation is preferentially induced in areas irradiated with UV through an unknown induction mechanism. It is possible that skin pigmentation shows induced local responses to UV in irradiated areas.
The type 1 melanocortin receptor (MC-1R), expressed in melanocytes, contributes to melanin production 24 . α -MSH and ACTH are known agonists for MC1-R but the affinity of POMC for MC-1R is markedly low 23 . To clarify the mechanism of melanin production after UV-irradiation, we assessed the expression of POMC and POMC-derived peptides after UV-irradiation to the skin of the murine auricle. The present paper investigates melanin production through the novel processing of POMC in the skin's extracellular compartment after UV-irradiation.

Results
α-MSH-like immunoreactivity is detectable in UV-irradiated auricular tissue. To examine the mechanisms driving skin pigmentation following in vivo UV-irradiation, we exposed the right auricles of mice to UV light and kept left auricles as controls with no UV exposure. Skin melanin level increased significantly in UV-irradiated auricles [ Fig. 1A]. Semi-quantitative RT-PCR, Western blot analysis, and gel filtration chromatography revealed that the expression of POMC also increased in UV-irradiated auricles [ Fig. 1B-D]. Conversely, ACTH and pro-ACTH were detected at low levels in auricle extracts with or without UV-irradiation. In contrast, the pituitary abundantly contained pro-ACTH (13 kDa) and ACTH (4.5 kDa), in addition to POMC (32 kDa).
Structural analysis of peptides producing α-MSH-like immunoreactivity. We measured the molecular weights of peptides producing α -MSH-LI in the auricle using gel filtration chromatography. The gel filtration profile of α -MSH-LI in control auricles peaked at about 1.6 kDa, which we estimated as α -MSH. The α -MSH-LI in UV-irradiated auricles showed a main peak at about 1.1 kDa, while the 1.6 kDa peak was apparently undetected in UV-irradiated auricles [ Fig. 2A]. Pro-ACTH and ACTH, which are eluted in the void volume, were undetectable in auricles with or without UV-irradiation, in agreement with the Western blotting results. The 1.1 kDa α -MSH-LI peak was then analyzed by reverse-phase chromatography [ Fig. 2B]. The α -MSH-LI was eluted as one peak and identified as ACTH  Fig. 2D]. In contrast, acetylated α -MSH-related peptides were not detected in UV-irradiated auricles. These results indicated that ACTH (1)(2)(3)(4)(5)(6)(7)(8) was produced by cleaving the Arg and Lys residues of POMC, pro-ACTH or ACTH, reflecting the action of a trypsin-like protease. Expression of a trypsin-like protease in response to UV-irradiation. Trypsin-like protease activity was measured using a Boc-Phe-Ser-Arg-MCA cleavage assay. Trypsin-like protease activity was elevated in UV-irradiated auricles [ Fig. 4A]. The molecular weight of the trypsin-like protease was measured using gel filtration and Western blot analysis. The major fraction was a molecular mass of about 160 kDa after gel filtration [ Fig. 4B]. Western blot analysis revealed the presence of tryptase, a trypsin-like protease, with a molecular mass of about 30 kDa [ Fig 4C]. Tryptase forms a tetramer protein with a molecular mass of about 160 kDa, suggesting that tryptase is a major trypsin-like protease involved in POMC processing in UV-irradiated skin.  Response after UV-irradiation to the auricles in mast cell-deficient mice. To assess the role of tryptase in POMC processing, which is expressed in mast cells, we measured trypsin-like protease activity in auricles irradiated with UV. Trypsin-like protease activity increased markedly in UV-irradiated auricles of wild-type (WBB6F1-+ /+ ) mice, but not in UV-irradiated auricles of mast cell-deficient (WBB6F1-W/Wv) mice. The α -MSH-LI also increased in UV-irradiated auricles of wild-type mice, but not in mast cell-deficient mice [ Fig. 4E].

Discussion
Melanin plays an important role in protecting skin after UV-irradiation. α -MSH is a major catalyst for melanogenesis in the skin following UV-irradiation. α -MSH-LI and ACTH-LI may be present in culture media of normal keratinocytes, fibroblasts, melanocytes, and their cell extracts [13][14][15][16][17][18][19][20][21][22] . Although POMC and POMC-derived peptides such as ACTH and α -MSH are released from the intermedia of the pituitary, it is unlikely that pituitary secretions contribute to local pigmentation. In the present report, POMC-related peptides were detected in the UV-irradiated auricles of hypophysectomized mice. These results suggest that skin regulates its own pigmentation. Research by Slominski et al. supports this idea, revealing that keratinocytes and melanocytes released POMC-related peptides following stimulation by corticotrophin-releasing hormone (CRH) 25 . In addition, keratinocytes and melanocytes release large amounts of POMC compared to α -MSH and ACTH 23 . However, POMC only slightly influenced melanogenesis via MC-1R 23 . Recent studies showed small lung carcinoma cells can release neuropeptide progalanin that are activated in the extracellular compartment 26,27 and contribute to angiogenesis 28 . In the present study, we explored the idea that POMC released from keratinocytes is activated in the extracellular compartments of skin and involved in melanogenesis in the auricles after exposure to UV.
Western blot analysis, gel filtration chromatography and immunoassay indicated that both POMC production and α -MSH-LI increased in UV-irradiated auricles. However, other POMC-derived products, such as pro-ACTH and ACTH, were detected, but at very low levels, suggesting the processing mechanism of POMC varies between the pituitary and skin. α -MSH-LI can be present in blood and affect peripheral tissue 29 . Conversely, α -MSH-LI expression increases in UV-irradiated skin 30 , which The trypsin-like protease was identified using a Boc-Phe-Ser-Arg-MCA cleavage assay. Each bar and line represents the mean ± SEM (n = 4, *p < 0.05 vs. control). (B). After UV-irradiation the auricles were subjected to gel filtration chromatography to determine any trypsin-like protease activity. Arrows indicate molecular markers (160 K, aldolase: 30 K, carbonic anhydrase). Vo and Vt denote void volume (blue dextran) and bed volume (dibutyryl cAMP), respectively. (C). Western blotting analysis of tryptase and plasmin in the auricles after UV-irradiation. D and E. Changes in trypsin-like protease activity (D) and α -MSH-LI (E) in auricles of mast cell-deficient (WBB6F1-W/Wv) and wild-type mice (WBB6F1-+ /+ ) after UV-irradiation. Each bar and line represents the mean ± SEM (n = 4, *p < 0.05 vs. control).
C57BL/6 mice are well-studied models for exploring pigment mechanisms and expression of POMC and POMC-related peptides. ACTH, α -MSH, β -endorphin and β -MSH, all derived from POMC, as well as a corticotropin-releasing hormone receptor, were detected during a hair cycle [35][36][37][38] . Interestingly, the expressions of these peptides were regulated in space and time for each hair cycle stage. Additionally, corticotropin-releasing hormone regulated the expression of these POMC-related peptides in a similar manner to hypothalamus-pituitary regulation. In this study, UV-irradiation induced the expression of tryptase released from mast cells, resulting in ACTH(1-8) production from POMC and ACTH-related peptides. Melanin was produced in two forms, pheomelanin (yellow /red) and eumelanin (black). In the POMC-deficient C57BL/6 mice study, melanocytes continuously produced eumelanin and α -MSH caused a shift from producing pheomelanin to eumelanin 39 . These results show that α -MSH affected not only melanogenesis, but the level of eumelanin changed the color tone. In the present study, UV-irradiation produced black auricular skin in the C57BL/6 mice, supported by a previous report 39 . Trypsin-like enzymatic activity increases after UV-irradiation 40 . Tryptase is a major serine protease present in mast cell secretary vesicles. Previous studies report tryptase expression increases in normal human skin after UV-irradiation 40,41 . In the present study, Western blot analysis identified tryptase as the major trypsin-like enzyme responsible for processing POMC. In mast cell-deficient mice, which do not produce tryptase, trypsin-like protease activity and the α -MSH-LI were unchanged in the auricle after UV-irradiation, unlike the increases seen in wild-type mice. In UV-irradiated skin, tryptase contributes to the pigmentation process via protease-activated receptor-2 (PAR-2) activation. PAR-2 activation induces melanosome transfer by increasing the phagocytosis of melanosomes by keratinocytes 42 .

Conclusions
The present study demonstrates the local production of ACTH(1-8) from POMC and POMC-related peptides by tryptase, which is a novel peptide-processing mechanism in the skin's extracellular compartment. This process may play an important role in the pigmentation of skin following UV irradiation.

Experimental procedures
Experimental animals. Male C57BL/6 mice, mast cell-deficient male WBB6F1-W/Wv mice (8 weeks old), and non-deficient male WBB6F1-+ /+ mice (8 weeks old) were purchased from Japan SLC, Inc. Prior to experiments, mice were housed under standard laboratory conditions (23 ± 1 °C, 55 ± 5% humidity) and had access to tap water and food ad libitum. Lights were automatically turned on at 8:00 and off at 20:00. C57BL/6 mice were hypophysectomized through the external ear canal. Successful removal of the pituitary gland was confirmed by post-experimental craniotomy.
Sample preparation and measurement of melanin content. The right auricle was exposed to UV-irradiation at 500 mJ cm −2 using a 11FL-40SE lamp (Toshiba, Tokyo, Japan). Twenty-four hours after UV-irradiation, the auricle was removed and immediately boiled in 5 ml of 0.1 M acetic acid for 10 minutes. The auricles were cooled in an ice bath, homogenized in 0.1 M acetic acid, and centrifuged at 10,000 rpm for 30 minutes. The supernatants were stored at − 80 °C for RIA or Western blot analysis. The pellets were dissolved in 1 M NaOH at 60 °C, and melanin content was measured by a spectrophotometer at 475 nm. The pituitary extracts for Western blotting were prepared according to the same method as the auricle samples.
Gel filtration chromatography. Gel filtration was carried out on a Sephadex G-25 fine column Analytical reverse-phase HPLC. Analytical reverse-phase HPLC separations were achieved using a SLC-6B high-performance liquid chromatograph system (Shimazu, Kyoto, Japan) equipped with a SPD-7A detector. The α -MSH-like immunoreactivity (α -MSH-LI) fraction obtained by gel filtration was loaded onto an ODS column (4.6 × 150 mm, Myghtysil RP-18 GP 5 μ m, Kanto chemical Co. inc., Tokyo, Japan). The column was eluted with a linear gradient of CH 3 CN in 0.01 N HCl from 0% to 60% for 30 minutes at a flow rate of 1 ml/min. The fractionated samples were lyophilized, and then dissolved in the standard diluent for the radioimmunoassay.
Mass spectrometric analysis. The α -MSH-LI fraction obtained by reverse-phase HPLC was applied to the target and mixed with saturated α -cyano-4-hydroxycinnamic acid (CHCA) solution. Mass spectrometry experiments were carried out on a Bruker Ultraflex TOF/TOF (Bruker Daltonics, Bremen, Germany). All searches were carried out using Flex Analysis software (Bruker Daltonics, Bremen, Germany) and post source decay (PSD) fragment ion spectra identified using the Mascot protein sequence database (Matrix science Inc. UK).