Biological and Pharmaceutical Bulletin
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Interferon-γ Attenuates 5-Hydroxytryptamine-Induced Melanogenesis in Primary Melanocyte
Jia ZhouJingjing LingFengfeng Ping
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2016 Volume 39 Issue 7 Pages 1091-1099

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Abstract

Interferon-γ (IFN-γ) is an important cytokine which can be secreted by keratinocytes or macrophages induced by UVB irradiation in skin. Mammalian skin cells have the capability to produce and metabolize 5-hydroxytryptamine (5-HT) whose cutaneous effects are mediated by the interactions with 5-HT receptors. Treatment with 5-HT resulted in a dose-dependent increase of tyrosinase (TYR) activity and melanin contents in normal human foreskin-derived epidermal melanocytes (NHEM), while with IFN-γ a decreased effect resulted. These regulatory results were due to changes of the expression levels of microphthalmia-associated transcription factor (MITF) and its downstream TYR, tyrosinase-related protein 1 (TRP-1) and dopachrome tautomerase (DCT). We proved here that 5-HTR1A/2A participated in the regulation of melanogenesis. IFN-γ could offset the pro-melanogenesis effect of 5-HT in NHEM and the intensity of this neutralization was unanticipated below the baseline level. IFN-γ neutralized the up-regulation effect of 5-HT on MITF and downstream TYR, TRP-1 and DCT. Though functioning as 5-HT1A/2A receptor during the melanogenesis process, IFN-γ played no role in 5-HT1A/2A receptor expressions. Our results also demonstrated that the inhibition of IFN-γ was reversible after its removal. Confusingly, the effect of cross-talk between 5-HT and IFN-γ on NHEM melanogenesis was irreversible. Whether treated with 5-HT for 5 d or 12 d, the pigmentation level neither recovered after displacing the IFN-γ-containing medium. In addition, IFN-γ was able to inhibit the inductive effect of 5-HT on NHEM migration. Taken together, the suppression of IFN-γ on 5-HT-induced melanogenesis further suggests the negative role of IFN-γ in inflammation-associated pigmentary changes.

Skin is the largest organ in human body, which plays a role as biologically active barrier to the external environment. It acts not only as a target for neuroendocrine signals but also a source of hormones and neurotransmitters, particularly the epidermis.1) Epidermis and dermis are the two main parts of skin. Epidermal melanocytes, derived from the neural crest, can produce melanin to absorb ultraviolet radiation and provide photoprotection.2) Melanin is synthesized in melanosomes, which are specialized lysosomerelated organelle (LRO) of pigment cells devoting to the biosynthesis, storage and transport of melanin.3) Melanosomes undergo 4 maturation stages, each characterized by unique ultrastructural morphology and melanin content.4,5) The principal role of the melanocyte is to produce the pigment within melanosomes and to transfer these organelles to upwards of 40 keratinocytes, which are neighboring melanocyte to constitute the ‘epidermal melanin unit.’ Melanocyte transport melanosomes from the formation site in the cell center to the principal site of transfer at dendritic tips. Accomplishing this feat, melanocyte needs to couple long-range, bidirectional microtubule-dependent transport of melanosomes along the length of dendrites with the capture and local movement of the organelles in actin-rich dendritic tips by the actin-based motor protein myosin Va.5,6) Studies have shown that there are four possible mechanisms described for melanosome transfer, including the cytophagocytosis model, membrane fusion model, shedding-phagocytosis model and exocytosis–endocytosis model.6) In addition, multifarious signaling pathways and transcription factors firmly manage melanocyte migration. These proteins and pathways provide and integrate signals to create the proper environment for its normal development and migration.2)

Melanin biosynthesis process is under a complex control mediated by multiple elements through different pathways. A series of pigment cell-specific melanosomal proteins are responsible for melanogenesis, such as tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1) and dopachrome tautomerase (DCT).79) Skin cells can produce hormones, neurotransmitters and neuropeptides and corresponding functional receptors, owing to which constitute the cutaneous equivalent of the hypothalamic–pituitary–adrenal (HPA) axis.10,11) It has recently uncovered that cutaneous expression of the biochemical machinery involved in the sequential transformation of L-tryptophan to serotonin and melatonin.12,13) Serotonin 5-hydroxytryptamine (5-HT) is a neurotransmitter whose actions are mediated via an interaction with receptors, including seven families (5-HT1–7 receptors) with at least 21 subtypes. Skin cells express mRNA encoding 5-HT receptors, 5-HT1A, -1B, -2A, -2B, -2C, and -7 receptor.14,15) 5-HT2A receptor was reported participating in the regulatory processes of 5-HT on melanogenesis.16) Our preliminary study also found that 5-HT1A receptor took a vital part in ‘brain–skin connection’ to regulate skin pigmentation.17)

It has been reported that interferon-γ (IFN-γ) is highly expressed in the lesions of vitiligo patients. Furthermore, UVB irradiation can induce the secretion of IFN-γ in keratinocytes or macrophages.18) IFN-γ signaling impedes maturation of the key organelle melanosome by concerted regulation of above mentioned pigmentation genes.19) The IFN-γ-mediated hypopigmentation cross-talk in delicately balancing skin pigmentation and its implications in disease pathophysiology have drawn researchers’ attention. Given the important functions of 5-HT and IFN-γ in melanogenesis, the objective of this study is to investigate the effect of the interaction between 5-HT and IFN-γ on melanocyte pigmentation.

MATERIALS AND METHODS

Reagents

5-HT, IFN-γ, dimethyl sulfoxide (DMSO), L-3,4-dihydroxyphenylalanine (L-DOPA), 3-isobutyl-1-methylxanthine (IBMX), phorbol esters (TPA), cholera toxin (CT), melanin standards, β-actin antibody and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Sigma-Aldrich (U.S.A.). 5-HT1A receptor agonist 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT), 5-HT2A receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI), 5-HT1A receptor antagonist WAY100635, and 5-HT2A receptor antagonist Ketanserin were from TOCRIS Bioscience (U.K.). TYR and TRP-1 antibodies were from Santa Cruz Biotechnology (U.S.A.). DCT, 5-HT1A receptor and 5-HT2A receptor antibodies were from Abcam Technology (U.K.). Primary antibody of p-Stat-1, p-cAMP response element binding protein (CREB) and t-CREB were from Cell Signaling Technology (U.S.A.). Enhanced bicinchoninic acid (BCA) protein assay kit, phenylmethylsulfonyl fluoride (PMSF) and cell lysis buffer for Western and IP were from Beyotime Institute of Biotechnology (China), total protein extraction kit was from Applygen Technologies Inc. (China). Other reagents were of the highest quality available.

Cell Culture

The studies on human material were approved by local ethic committee. Normal human foreskin-derived epidermal melanocytes (NHEM) were derived from young male adult foreskins (ethnic Han/aged 18 to 22 years) obtained at circumcision following standard protocols.20) Briefly, foreskins were cut into strips and digested with 0.25% trypsin at 4°C for 20 h. Epidermis was separated from dermis. The NHEM suspension was filtered and cells were washed twice at 1500 rpm for 5 min prior to resuspension in MCDB153 medium (Sigma), supplemented with insulin, IBMX, TPA, CT and 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO, U.S.A.). NHEM were grown in a humidified atmosphere with 5% CO2 at 37°C.

Tyrosinase Activity and Melanin Contents Assay

Tyrosinase activity, as the dopa oxidase here, was measured by the rate of L-DOPA oxidation as reported.21) NHEM were treated with drugs for 72 h, washed with ice-cold phosphate buffered saline (PBS), lysed by incubation in cell lysis buffer (1 mM PMSF) at 4°C for 20 min, and then lysates were centrifuged at 14000 rpm for 15 min to obtain the supernatant for activity assay and centrifugation for melanin contents assay. Protein concentrations were determined by BCA kit with bovine serum albumin (BSA) as a standard. 100 µL of supernatant containing the same 10 µg total proteins was added to each well in 96-well plate, and then mixed with 100 µL 0.1% L-DOPA in 0.1 M PBS (pH 6.8) (M/V). After incubation at 37°C for 0.5 h, the dopachrome was monitored by measuring the absorbance at 475 nm.

Total melanin in the cell pellet was dissolved in 100 µL of 1 N NaOH–10% DMSO for 1 h at 80°C, and solubilized melanin was measured at 405 nm. The melanin content was obtained by compared against standard curves made with synthetic melanin (Sigma). Then the relative value of melanin content was calculated as a percent of the control.

Western Blot Analysis

The protein suspension was obtained as the method mentioned above. Western blot was performed as described previously.22) The primary antibodies used were TYR (C-19) (SC7833), TRP-1 (SC10443), DCT (ab74073), p-CREB (CST 9198), t-CREB (CST 9197), p-Stat-1(CST 9177), 5-HT1A receptor (ab121032), 5-HT2A receptor (ab66049) and β-actin (A1978). Proteins were visualized using an enhanced chemiluminescence detection system. Densitometric analysis was again performed by using the Quantity One (Bio-Rad) to scan the signals. Western blot assay results reported here are representative of at least 3 experiments.

Transwell Migration Assay

The bottom chambers of Transwell were filled with MCDB153 with 0.5% fetal bovine serum (FBS) supplemented with different concentrations of drugs, and the top chambers were seeded inactivated 5×104 cells per well NHEM in 200 µL MCDB153 (0.5% FBS). After 24 h of migration, the cells on the top surface of the membrane (nonmigrated cells) were scraped with a cotton swab and the cells spreading on the bottom sides of the membrane (migrated cells) were fixed with cold 4% paraformaldehyde for 30 min. After that, those migrated cells were stained with 0.1% hexamethylpararosaniline. Images were taken using Olympus inverted microscope and migrated cells were quantified by manual counting.

Statistical Analysis

All data were expressed as the mean±standard deviation (S.D.). Statistical analysis was performed with one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons tests. Significant differences were accepted when p<0.05.

RESULTS

Effect of 5-HT and IFN-γ on Tyrosinase Activity, Melanin Amount, and Enzymatic Cascade Expressions in NHEM

For identification of the primary melanocytes, the anti-Mitf and anti-Tyr antibodies were used as this cell type marker. As shown in Fig. 1A, about 99% of these attached cells were positive for the two markers and could be used for subsequent experiments.

Fig. 1. Effect of 5-HT and IFN-γ on Tyrosinase Activity, Melanin Amount, and Enzymatic Cascade Expressions in NHEM

(A) Identification of primary melanocytes. Cells were labeling with Mitf and Tyr antibodies. Nuclei were labeled with DAPI. (B) After incubation of with 5-HT (0, 10, 50 µM) and IFN-γ (0, 2.5, 5 ng/mL) for 72 h, cell viability was determined using a MTT assay. (C) Tyrosinase activity was determined by L-DOPA oxization as described in Materials and Methods by 5-HT (0, 10, 50 µM) and IFN-γ (0, 2.5, 5 ng/mL) for 72 h. (D) Melanin content was performed as described in Materials and Methods the same by 5-HT (0, 10, 50 µM) and IFN-γ (0, 2.5, 5 ng/mL) for 72 h. (E) The cells were treated with 50 µM of 5-HT and 5 ng/mL of IFN-γ for 72 h. Western blot assays were performed to examine TYR, TRP-1 and DCT expression levels, Total cellular proteins (20 µg/lane of NHEM) were subjected to 10% SDS-PAGE. Results are shown as the mean±S.D. and are representative of 3 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. * p<0.05, ** p<0.01, #p<0.05, ##p<0.01, compared with control.

To investigate the effect of 5-HT and IFN-γ on melanogenesis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed as the first step to examine whether 5-HT or IFN-γ was cytotoxic to NHEM. As shown in Fig. 1B, treatments with 5-HT (0, 10, 50 µM) and IFN-γ (0, 2.5, 5 ng/mL) were not cytotoxic to NHEM for 72 h. Moreover, 5-HT and IFN-γ both played little parts in cell proliferation. NHEM was then exposed to 5-HT and IFN-γ, their effects on TYR activity were measured by L-DOPA oxidation. As compared to treatment with medium only (untreated condition), treatment with 5-HT resulted in a dose-dependent increase of TYR activity in NHEM, while with IFN-γ obtained a decrease effect (Fig. 1C). In melanin content assay, to exclude the possibility that raise in melanin content may be induced by the cell proliferating effect, the absorbance of the same number of cells across 5-HT and IFN-γ concentrations were measured and results showed that melanin level was in a dose-dependent manner increased by 5-HT and decreased by IFN-γ in NHEM (Fig. 1D).

Since 5-HT and IFN-γ regulated TYR activity and melanin synthesis, we further explored whether they affected the expressions of enzymatic cascade in NHEM. Levels of TYR, TRP-1 and DCT protein expression were up-regulated by 5-HT, while partly or sharply down-regulated by IFN-γ (Fig. 1E). Furthermore, PKA signaling pathway was also examined. The activation of CREB was detected by Western blot analysis. As the data showed in Fig. 1F, 5-HT treatment for 120 min induced CREB phosphorylation.

5-HT1A/2A Receptor Participated in Melanogenesis Process of NHEM

The participation and adjustment function of 5-HTR1A/2A were proved by the usages of 5-HT1A receptor agonist 8-OH-DPAT, 5-HT2A receptor agonist DOI, 5-HT1A receptor antagonist WAY100635, and 5-HT2A receptor antagonist Ketanserin. As shown in Fig. 2A, both 8-OH-DPAT and DOI increased tyrosinase activity in NHEM. The effect of 5-HT on tyrosinase activity were inhibited by 5-HT1A receptor antagonist WAY100635, and 5-HT2A receptor antagonist Ketanserin. The same, melanin biosynthesis in NHEM was increased by 8-OH-DPAT and DOI (Fig. 2B). WAY100635/Ketanserin inhibited the impact of 5-HT on melanogenesis. These data indicated the involvements of 5-HT1A/2A receptor in mediating the pro-melanogenesis effect of 5-HT in NHEM.

Fig. 2. 5-HT1A/2A Receptor Participated in Melanogenesis Process of NHEM

Here, 5-HT1A receptor agonist 8-OH-DPAT and antagonist WAY100635, 5-HT2A receptor agonist DOI and antagonist Ketanserin were used. NHEM was pretreated with 10 µM of WAY100635/Ketanserin for 1 h before 5-HT was applied for 72 h at 50 µM. Ten micro molar of 8-OH-DPAT/DOI and 50 µM of 5-HT were applied for 72 h. (A) Tyrosinase activity was determined by L-DOPA oxization as described in Materials and Methods. (B) Melanin content was performed as described in Materials and Methods. Results are shown as the mean±S.D. and are representative of 3 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. * p<0.05, ** p<0.01, compared with control; #p<0.05, ##p<0.01, compared with 5-HT group.

Effect of Cross-Talk between 5-HT and IFN-γ on NHEM

Consistent with our prediction, when treatment with 50 µM of 5-HT and 5 ng/mL of IFN-γ together, IFN-γ could offset the pro-melanogenesis effect of 5-HT on NHEM. However, the intense of this neutralization was unanticipated below the baseline level, shown in Figs. 3A and B. Along with this influence of cross-talk on NHEM, the enzymatic cascade expressions, including TYR, TRP-1 and DCT, were changed correspondingly (Fig. 3C). Since IFN-γ could regulate melanosome maturation, the underlying mechanisms seized our attention.

Fig. 3. Effect of Cross-Talk between 5-HT and IFN-γ on NHEM

Cells were divided into 4 groups, control group and medicated groups incubation with 5-HT, IFN-γ and (5-HT+IFN-γ). After treatments with 50 µM of 5-HT, 5 ng/mL of IFN-γ, 50 µM of 5-HT+5 ng/mL of IFN-γ for 72 h, (A) tyrosinase activity was determined by L-DOPA oxization as described in Materials and Methods. (B) Melanin content was performed as described in Materials and Methods. 5-HT increased tyrosinase activity and melanin synthesis, while IFN-γ played an opposite role. IFN-γ could offset the pro-melanogenesis effect of 5-HT in NHEM. (C) Enzymatic cascade expressions, including TYR, TRP-1 and DCT were inhibited treated by (5-HT+IFN-γ) together, correspondingly. Results are shown as the mean±S.D. and are representative of 3 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ** p<0.01, ##p<0.01, compared with control; &p<0.05, &&p< 0.01, compared with 5-HT group.

IFN-γ Activated Stat-1 and Played No Role in 5-HT1A/2A Receptor Expression

Given the implication of 5-HT1A/2A receptor in the regulation of melanogenesis, the next step examining the impact of IFN-γ on 5-HT1A/2A receptor expression and function was operated. Working together with IFN-γ, pro-melanogenesis effect of both 8-OH-DPAT and DOI was reversed to be below control. WAY100635 and Ketanserin were without affecting NHEM function themselves. Furthermore, either WAY100635 or Ketanserin did not exert an effect on IFN-γ (Figs. 4A, B). Western blot assay demonstrated that IFN-γ activated Stat-1 (Fig. 4C), but had no effect on the expression level of 5-HT1A/2A receptor (Fig. 4D).

Fig. 4. IFN-γ Activated Stat-1 but Played No Role in 5-HT1A/2A Receptor Expression

(A) Cells were divided into 6 groups, control group and medicated groups incubation with 5 ng/mL of IFN-γ, (10 µM of 8-OH-DPAT+5 ng/mL of IFN-γ), (10 µM of DOI+5 ng/mL of IFN-γ), (10 µM of WAY100635+5 ng/mL of IFN-γ) and (10 µM of Ketanserin+5 ng/mL of IFN-γ), treated for 72 h. NHEM was serum-starved for 48 h, and then pretreated with 10 µM of WAY100635/ Ketanserin for 1 h before IFN-γ was applied. Tyrosinase activity was determined by L-DOPA oxization as described in Materials and Methods. (B) Melanin content was performed as described in Materials and Methods. (C) Cells were treated with 5-HT and IFN-γ respectively for 30 min, the activation of Stat-1 was examined by Western blot assay. (D) Western blot assay demonstrated that IFN-γ was without affecting any of the expression level of 5-HTR1A/2A. Results are shown as the mean±S.D. and are representative of 3 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ** p<0.01, compared with control; ^p<0.05, ^^p<0.01, compared with IFN-γ group; ##p<0.01, compared with 8-OH-DPAT group; $$p<0.01, compared with DOI group; %%p<0.01, compared with WAY100635 group; &&p<0.01, compared with Ketanserin group.

Effect of Cross-Talk between 5-HT and IFN-γ on NHEM Was Irreversible

It has been reported that hypopigmentation mediated by IFN-γ was reversible.19) Primary human melanocytes were treated with IFN-γ for 7 d and then allowed to recover for 5 d (day 12) by fresh medium without IFN-γ, the pigmentation process reinitiated. In accordance with those reported in the previous papers, our results also demonstrated that the inhibition of IFN-γ was reversible after the removal of IFN-γ, as shown in the second columns of Figs. 5A and B. Treatment with 5-HT for additional 5 d could not increase the pigmentation anymore (comparing the fourth column with the fifth in Figs. 5A, B). Though the reversibility of the influence of IFN-γ on melanogenesis was proved, the effect of cross-talk between 5-HT and IFN-γ on NHEM was irreversible. Treated with 5-HT for 7 d or 12 d soever, the pigmentation level of primary melanocytes did not recover after displacing the IFN-γ-containing medium (the sixth and seventh column in Figs. 5A, B).

Fig. 5. Effect of Cross-Talk between 5-HT and IFN-γ on NHEM Was Irreversible

Cells were divided into 6 groups, control group treated for 12 d, medicated groups incubation with 5 ng/mL of IFN-γ for 7 d and medium only recovered for 5 d, 50 µM of 5-HT for 7 d and medium only for 5 d, 50 µM of 5-HT for 12 d, (50 µM of 5-HT+5 ng/mL of IFN-γ) for 7 d and medium only recovered for 5 d, (50 µM of 5-HT+5 ng/mL of IFN-γ) for 7 d and 50 µM of 5-HT for other 5 d. (A) Tyrosinase activity was determined by L-DOPA oxization as described in Materials and Methods. (B) Melanin content was performed as described in Materials and Methods. Results are shown as the mean±S.D. and are representative of 3 independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. ** p<0.01, compared with control; ##p<0.01, compared with 5-HT treated for 7 d; &&p<0.01, compared with 5-HT treated for 12 d.

IFN-γ Inhibited 5-HT to Induce NHEM Migration

There exist an important functional association between melanocytes and keratinocytes. Many of the currently identified cytokines can be produced by keratinocytes and secreted to melanocytes to regulate their proliferation, differentiation and migration. Whatever the shedding spheroid granules mode or the filopodia delivery mode, the mobility of melanocytes is crucial. Melanosomes need melanocytes to provide proper transferring environment for pigmentation. The effects of 5-HT and IFN-γ on NHEM migration were observed. As shown in Fig. 6, 5-HT induced the cells migration, and IFN-γ inhibited this effect.

Fig. 6. IFN-γ Cooperated with 5-HT to Induce NHEM Migration

NHEM were seeded in the upper chamber of transwell. The bottom chamber was filled with MCDB153 supplemented with 50 µM of 5-HT, 5 ng/mL of IFN-γ and (50 µM of 5-HT+5 ng/mL of IFN-γ). After 24 h, the migrated NHEMs were quantified. Scale bar, 50 µm. ** p<0.01, compared with control; ##p<0.01, compared with 5-HT group.

DISCUSSION

Visible pigmentation of the skin, hair, and eyes depends primarily on the functions of melanocytes, which are the very minor population of cells specializing in synthesis and distribution of the pigmented biopolymer melanin. Cutaneous melanin pigment plays a critical role in camouflage, mimicry, social communication, and protection against harmful effects of solar radiation.23) The crucial role of skin pigmentation as a natural sun screen is to absorb ultraviolet (UV) radiation, which is the most significant factor influencing the human skin pigmentation process. As a direct effect of UV, especially UVA, immediate pigment darkening occurs within minutes and persists for several hours followed by persistent pigment darkening, which occurs within several hours and lasts for several days.13) At the same time, ultraviolet illuminate may cause some dermatitis diseases, and the most common is inflammation with the symptoms of redness, warmth, swelling and pain. Acute and chronic inflammation due to the inflammatory cytokines, may closely relate to pigmentary changes, either hypopigmentation or hyperpigmentation.18,24,25) Beyond that, in the case of vitiligo, an immune response is thought to be responsible for the destruction of normal melanocytes.26)

UV radiation causes skin pigmentation partly by inflammation, which is mediated by chemokines and cytokines secreted by cells neighboring melanocytes including keratinocytes, fibroblasts, or inflammatory cells in the skin. As reported, IFN-γ plays a role in inflammation-associated pigmentary changes in vivo. More and more researches have been done to elucidate the potential mechanisms by which IFN-γ involved in the regulation of melanogenesis. Because of the multidirectional nature and heterogeneous character of the melanogenesis modifying mechanisms, whose controlling factors are not organized into simple linear sequences, but a multidimensional network instead, with extensive functional overlapping with connections arranged both in series and in parallel. How the IFN-γ-mediated hypopigmentation delicately balancing skin pigmentation and its implications in disease pathophysiology have drawn researchers’ attention.

Skin is the largest body organ working as a biological barrier, which separate the internal milieu from noxious external environmental factors to maintain local and systemic homeostasis.1,27) The classical neuroendocrine axes such as the cutaneous HPA and hypothalamic-thyroid axis (HPT) exist in the skin, making the skin be an important peripheral neuro-endocrine-immune organ.23,28) Given their common embryonic origins, it is not surprising that skin shares numerous mediators with the nervous and endocrine system.1,11,29,30) 5-HT is a recognized neurotransmitter and molecular signal that distributes brain-wide with particular presence in hippocampus. It has been proved that the mammalian skin cells have the capability to produce and metabolize 5-HT whose cutaneous effects are mediated by the interactions with 5-HT receptors.12,13,31) 5-HT receptors are widely detected on mammalian melanocytes and dermal fibroblasts.32) Accordingly, the relationship between skin pigmentation and 5-HT are complicated. For example, 5-HT dose-dependently inhibits melanin production and tyrosinase activity in human SK-MEL-188 melanoma cells.7) 5-HT stimulates proliferation of melanocytes in a medium deprived of growth factors, whereas it inhibits cell growth in the presence of growth factors.14) So here in this study, the effects of IFN-γ together with 5-HT on melanogenesis were investigated.

5-HT and IFN-γ both played little parts in cell proliferation, treatment with 5-HT resulted in a dose-dependent increase of TYR activity and melanin contents in NHEM, while with IFN-γ obtained a decrease effect (Figs. 1A–C). These regulatory results were due to changes of the expression levels of TYR, TRP-1 and DCT (Fig. 1D). Considerable bodies of data indicated that the PKA signaling pathway contributes to the regulation of skin and hair pigmentation in humans and mice. PKA can be activated by the elevation of cellular cAMP.33) The activation of this pathway induced the phosphorylation of CREB, resulting in the expression of TYR, TRP-1, and DCT genes.34) As the data showed in Fig. 1F, 5-HT treatment for 120 min induced CREB phosphorylation, while IFN-γ has no obvious effect on CREB phosphorylation. Treated with 5-HT and IFN-γ together, IFN-γ could offset the pro-melanogenesis effect of 5-HT on NHEM and the intense of this neutralization was below the baseline level, shown in Figs. 3A and B. IFN-γ neutralized the up-regulation of 5-HT on TYR, TRP-1 and DCT (Fig. 3C). Melanosomes mature through four distinct morphological stages, and melanin synthesis in late-stage (III and IV) melanosomes. During this process, the selective targeting of integral membrane melanogenic enzymes, such as TYR and TRP-1, and other components that regulate enzyme activity are required.35) In our data, the cross-talk between IFN-γ and 5-HT made no obvious effect on DCT expression, being different from the effect on TYR and TRP-1 expressions. Skin cells express mRNA encoding 5-HT receptors, and more than one member was proved to take part in melanogenesis. The participations of 5-HT1A/2A receptor were examined by the usages of 5-HT1A receptor agonist 8-OH-DPAT, 5-HT2A receptor agonist DOI, 5-HT1A receptor antagonist WAY100635, and 5-HT2A receptor antagonist Ketanserin. The pro-melanogenesis of 8-OH-DPAT/DOI and the suppression of WAY100635/Ketanserin demonstrated the involvement of 5-HT1A/2A receptor in pigmentation (Figs. 2A, B). IFN-γ brought into its action by activating Stat-1 (Fig. 4C). Though 5-HT1A/2A receptor participated in melanogenesis process, IFN-γ played no role in 5-HT1A/2A receptor expressions (Fig. 4D). Working together with IFN-γ, pro-melanogenesis effect of both 8-OH-DPAT and DOI was reversed to be below control. Furthermore, neither WAY100635 nor Ketanserin exerted an effect on the inhibition of IFN-γ (Figs. 4A, B). Considering that some of the influences were observed after long-term treatment (5–7 d) of IFN-γ during skin inflammation, in present study, the processing time of IFN-γ was then extended to 7 d. In accordance with those reported in previous papers, our data also demonstrated that the prevention effect of IFN-γ was reversible after its removal, as shown in the second column of Figs. 5A and B. Nevertheless, the effect of cross-talk between 5-HT and IFN-γ on NHEM melanogenesis was irreversible. Neither treated with 5-HT for 5 d nor 12 d soever, pigmentation level did not recover after displacing the IFN-γ-containing medium (the sixth and seventh column in Figs. 5A, B). It has been reported that IFN-γ signaling mediates hypopigmentation of primary human melanocytes by arresting melanosome maturation. IFN-γ-induced these changes in melanogenesis are reversible.19) In our study, the results was consistent with the data report, comparing the first column with the second column in Figs. 5A and B. Treatment with 5-HT for 7 d or 12 d, its influence on the pigmentation has little difference (comparing the fourth column with the fifth in Figs. 5A, B). Treatment with 5-HT and IFN-γ together, the increase of melanogenesis induced by 5-HT was counteract by IFN-γ (comparing the fourth column with the sixth in Figs. 5A, B). That we were unexpected was additional 5 d stimulation by 5-HT not increasing the pigmentation anymore after 7 d co-treatment. Since IFN-γ had no effect on the expression level of 5-HT1A/2A receptor (Fig. 4D) and affected maturation of melanosome, we speculated that the recovery procedure generated some partial function of melanosome to the control level, but was relative slower to face up to other stimuli in this condition (comparing the fourth and fifth column with the seventh in Figs. 5A, B). In addition to the roles mentioned above, IFN-γ inhibited 5-HT to induce NHEM migration (Fig. 6). There exist an important functional association between melanocytes and keratinocytes. Many of the currently identified cytokines can be produced by keratinocytes and secreted to melanocytes to regulate their proliferation, differentiation and migration. In epidermal melanocytes, melanosomes are lysosome-related organelles that synthesize, store and transport melanin. Then mature melanosomes are transferred to surrounding keratinocytes, which is essential for skin and coat color. It is a unique and poorly understood biological process that melanosome transfer to keratinocytes, which involves whole organelle donation to another cell. Filopodia has been reported to conduit for melanosome transfer to keratinocytes.36) In this case, the mobility ability of melanocytes is particularly important for the connection between melanocytes and kerationocytes.

To be specific, 5-HT bound to its 5-HT1A and 5-HT2A receptor to activate PKA signaling pathway, and then, activity of the rate-limiting enzyme TYR was increased. Besides, the expressions of TYR, TRP-1 and DCT were up-regulated, especially the expression of TYR. 5-HT also induced the migration of melanocytes, which benefited for them receiving “messenger” proteins from its neighboring keratinocytes and establishing the cell–cell communication. In this way, 5-HT induced melanogenesis of melanocytes. But for IFN-γ the situation is different. IFN-γ increased phosphorylation status of Stat-1 protein, accordingly, decreased the expressions of TYR, TPR-1 and DCT in the subsequent steps. Among these pigmentation regulatory proteins, DCT showed a drastic decrease, which was closely related to the melanosome maturation. Our data and relevant literature both demonstrated that IFN-γ did not affect the CREB phosphorylation, which suggests that IFN-γ does not interfere with the c-AMP level.37) IFN-γ mediated hypopigmentation of melanocytes by arresting melanosome maturation. Looking above, IFN-γ inhibited basal and 5-HT-induced melanogenesis in NHEM. Since IFN-γ did not affect the expression of 5-HT1A/2A receptor, either the PKA signaling pathway, the cross-talk effect between 5-HT and IFN-γ may be due to the effect of IFN-γ on melanosome function and the global state of melanocytes. The suppression of IFN-γ on 5-HT-induced melanogenesis may further suggest the negative role of IFN-γ in inflammation-associated pigmentary changes. For the potential mechanisms, we carried on a bold speculation. The pigmentation of skin in mammals is driven by the creation of melanosomes in melanocytes, and the subsequent intercellular transfer of this organelle to keratinocytes. IFN-γ inhibited 5-HT to induce melanocytes migration. We speculate that this process affects melanosome transfer and adversely influences its regeneration recycling, the new emergence and the maturation. The normal cellular programming of melanocyte is likely to be impeded by IFN-γ signaling. Further mechanism of the action waits for our digging.

Acknowledgments

This work was supported by the National Youth Science Fund Project of China (81503176 and 81403030); the Natural Youth Science Foundation of Jiangsu Province (BK2014124); the Fundamental Research Funds for the Central Universities (JUSRP11566); the National Undergraduate Training Programs for Innovation and Entrepreneurship (201510295076).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES
 
© 2016 The Pharmaceutical Society of Japan
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