Primary Human Epidermal Melanocytes Research Articles

Comparative evaluation of six commercial adult toothpaste formulations reveals cytotoxicity and altered functions in a human oral melanocyte model: an in vitro study.

This study aims to compare six commercial adult toothpaste (labeled as A, B, C, D, E, and F) for cytotoxicity and melanocyte function alterations in vitro using primary human epidermal melanocytes from a Caucasian donor (HEMn-LP cells) as a model of oral melanocytes. Cells were incubated with toothpaste extracts (50% w/v) in culture media at dilutions (1:25, 1:50, 1:100, 1:200, 1:500, 1:800, and 1:1000) for 24h. MTS and LDH assays were used to assess cytotoxicity. The effects of noncytotoxic toothpaste concentrations on melanocyte functional endpoints were then examined using spectrophotometric methods. All toothpaste showed concentration-dependent cytotoxicity that was heterogeneous across toothpaste containing SLS detergent. IC50 values of cytotoxicity followed the order: A = E > C > B > D > F. To compare toothpaste, they were tested at 1:800 and 1:1000 dilutions that were noncytotoxic after 24h. None of the toothpaste affected cellular melanin production. However, toothpaste A, C, and D suppressed tyrosinase activity at both dilutions, while toothpaste B suppressed tyrosinase activity only at 1:800 dilution. Toothpaste A, C, E, and F elevated ROS production at 1:800 dilution, with no change at 1:1000 dilution. Toothpaste has a heterogeneous effect on melanocytes. Toothpaste B, E, and F at 1:1000 dilution were the safest as they did not alter melanocyte functions at this dilution, although toothpaste F is the least cytotoxic of these. Future studies are necessary to expand these results in a physiological environment of oral tissue. The findings of this study provide novel insight into the biocompatibility studies of toothpaste on oral melanocytes. They can aid dental practitioners and consumers in selecting noncytotoxic toothpaste that do not contribute to ROS generation by melanocytes in the oral cavity or lead to cytotoxicity and impaired cellular function.

Exploring the effect of butyric acid, a metabolite from periodontopathic bacteria, on primary human melanocytes: An in vitro study

Effects of butyric acid, a bacterial metabolite implicated in periodontitis progression, have never been examined on oral melanocytes. Herein, primary human epidermal melanocytes were used as a model for oral melanocytes. Results show the adverse effects of butyric acid (sodium butyrate; NaB) on them, which comprise marked cytotoxicity at higher concentrations (>1 mM) and robust differentiation at lower nontoxic concentrations. NaB did not alter MITF protein levels; however, it stimulated tyrosinase protein synthesis and inhibited tyrosinase activity, with no changes in cellular melanin. NaB did not affect oxidative stress, although it induced significant levels of the pro-inflammatory cytokine IL-6.

In Vitro Study of the Effects of Five Chemically Modified Tetracycline (CMT) Analogs on Human Epidermal Melanogenesis: Potential as Novel Anti-Melanogenic Agents

Treatment of hyperpigmented skin disorders by novel drug candidates without side effects remains an ongoing area of research. Chemically modified tetracyclines (CMTs) are a group of nonantimicrobial tetracycline drugs that have been shown to possess multiple pharmacological activities. We have previously documented the anti-melanogenic effects of CMT-3 and its 9-amino derivative, CMT-308. Herein, we have extended our analysis to evaluate other CMT analogs, namely CMT-1, CMT-4, CMT-5, CMT-6, and CMT-8, for their impact on melanogenesis using primary human epidermal melanocytes (HEMn-DP cells). CMT analogs were screened using a tetrazolium-based assay to identify nontoxic concentration ranges that were further used to analyze the effects of CMTs on cellular melanin content and morphology (via quantitation of dendricity). Cellular tyrosinase (TYR) activity and levels of melanogenesis proteins, TYR, and microphthalmia transcription factor (MITF) were also evaluated to elucidate the mechanisms underlying their effects on melanogenesis. The findings demonstrated that exposure to CMT-8 resulted in notable cytotoxic effects at concentrations >10 µM; hence, all five analogs were further evaluated and compared at 10 µM. None of the five CMT analogs exhibited any impact on intracellular melanin in HEMn-DP cells at the concentration of 10 µM. However, CMT-1, CMT-4, and CMT-8 robustly suppressed dendricity parameters in HEMn-DP cells, while CMT-5 and CMT-6 showed no effect, suggesting that only a subset of CMT analogs can attenuate melanocyte dendricity. Moreover, the analog CMT-5, which has β-diketone blocked, was ineffective, thus confirming the role of this moiety in suppressing dendrite formation. CMT-1 and CMT-8 did not affect cellular tyrosinase activity, while CMT-4 suppressed TYR activity at 10 µM. The capacity of CMT-4 and CMT-8 to suppress dendricity was partly associated with their ability to downregulate MITF protein levels, while CMT-1 had no effect on MITF but suppressed TYR protein levels. The results of this study indicate that CMT-1, CMT-4, and CMT-8 merit further investigation using in vivo studies as potential drug candidates for the treatment of hyperpigmentation disorders.

7-desacetoxy-6,7-dehydrogedunin discovered by high-throughput screening system suppresses melanogenesis through ATP-P2X7 signaling inhibition

BackgroundHyperpigmented skin disorders such as melasma and lentigo are common photoaging diseases that cause cosmetic problems. The pigmentation is usually exacerbated by ultraviolet (UV) radiation, and various factors and pathways are involved in UV-mediated melanogenesis. Adenosine 5′-triphosphate (ATP), a well-known molecular unit of intracellular energy, is also regarded as a mediator of UV-mediated melanogenesis via the P2X7 purinergic receptor. ObjectiveTo discover natural substances with an anti-melanogenic effect through inhibition of ATP-P2X7 axis by high-throughput screening (HTS). MethodsAmong natural compounds provided by the Korea Chemical Bank, chemical compounds with a P2X7 inhibiting effect were screened through an HTS system. Then the selected compounds were verified for their anti-melanogenic effect after treating primary human epidermal melanocytes (PHEMs) with and without ATP. The expression of MITF, tyrosinase, and PMEL/gp100 was analyzed by Western blot, and melanin content was measured as 405 nm absorbance. ResultsAmong 962 natural compounds, 58 showed greater than 80% suppression of YO-PRO-1 fluorescence, representing P2X7 activity. Among them, considering cell viability, chemical stability, and availability, 7-desaxacetoxy-6,7-dehydrogedunin (7DG), a limonoid natural compound, was selected. The expression of MITF, tyrosinase, and PMEL/gp100; tyrosinase enzyme activity; and melanin content, which were increased by ATP treatment were abrogated by 7DG. Even when 7DG was treated in PHEMs without addition of ATP, tyrosinase expression and melanin content were significantly decreased. Hypopigmenting effect of 7DG was confirmed in ex vivo culture of human skins. Conclusions7DG has an anti-melanogenic effect through ATP-P2X7 pathway inhibition and could be a potential skin whitening material.

Hydrogen Sulfide Promotes Cell Proliferation and Melanin Synthesis in Primary Human Epidermal Melanocytes

Background/Aim: Hydrogen sulfide (H₂S) has been found to act as a physiological intercellular messenger to regulate cell survival. In this study, we evaluated whether H₂S could promote cell proliferation and melanin synthesis in human epidermal melanocytes (HEMs). Methods: Primary HEMs were cocultured with sodium hydrosulfide (NaHS, the most widely used H₂S donor) or endogenously overexpressed with cystathionine-γ-lyase (CSE) gene, which is the most predominant H₂S-producing enzyme. Then, cell viability, intracellular melanin content, tyrosinase (TYR) activity, and expression of microphthalmia-associated transcription factor (MITF), TYR, together with TYR-related protein 1 (TRP-1) in both transcript and protein levels, were detected. Results: We first confirmed that NaHS (10–100 μm) increased cell viability, intracellular melanin content, and TYR activity in a dose-dependent manner. Then, we found that endogenous H₂S production also promoted cell proliferation, intracellular melanin content, and TYR activity. In addition, we observed the mRNA and protein expression of MITF, TYR, and TRP-1 was significantly up-regulated after NaHS treatment and CSE gene transfection. Conclusions: This study demonstrates that H₂S promotes cell proliferation and melanin synthesis in HEMs, which indicates pharmacologic regulation of H₂S may be potential treatment for skin disorders caused by loss of melanocytes or dysfunction of melanogenesis.

RNA-seq Reveals Dysregulation of Novel Melanocyte Genes upon Oxidative Stress: Implications in Vitiligo Pathogenesis

Oxidative stress is known to induce melanocyte death, but the underlying mechanisms are incompletely understood. To identify oxidative stress-induced global gene expression changes in melanocytes, we treated PIG1 melanocytes with H2O2 in a dose- and time-dependent manner and performed RNA-seq. This approach allowed us to capture the events occurring early as well as late phase after treatment with H2O2. Our bioinformatics analysis identified differentially expressed genes involved in various biological processes of melanocytes which are known to contribute to the vitiligo development, such as apoptosis, autophagy, cell cycle regulation, cell adhesion, immune and inflammatory responses, melanocyte pluripotency, and developmental signaling such as WNT and NOTCH pathways. We uncovered several novel genes that are not previously described to be involved in melanocytic response to stress nor in vitiligo pathogenesis. Quantitative PCR and western blot analysis of selected proteins, performed on PIG1 and primary human epidermal melanocytes, confirmed the RNA-seq data. Interestingly, we discovered an aberrant regulation of several transcription factors that are involved in diabetes, neurological, and psychiatric diseases, all of which are comorbid conditions in patients with vitiligo. Our results may lead to a better understanding of the molecular mechanisms underlying vitiligo pathogenesis and help developing new drug targets for effective treatment.

803 NB-UVB induces melanocyte migration on a collagen IV-coated surface through the activation of the P53/miR211/MMP9 axis

Vitiligo is the most commonly acquired depigmented disorder of the skin that is characterized by the destruction of melanocytes (MCs) resulting in achromic macules. Although narrow-band UVB (NB-UVB) has been clinically proven to be an effective therapeutic option in the treatment of vitiligo, the mechanism by which how NB-UVB induces repigmentation in vitiligo has not been clearly elucidated. In this study, we examined the role of P53/miR211/MMP9 in the migration of MCs upon NB-UVB exposure. The primary human epidermal MCs were cultured from human foreskin. The mRNA and protein levels of P53, TRPM1, and MMP9 were measured using qPCT and western blotting assay. Transwell ®-collagen IV-coated membrane inserts were used for measuring the MCs migration ability. The migration ability was also examined in the MCs transfected with hsa-mir211 mimic and lentivirus-P53 in the presence or absence of NB-UVB irradiation. The results showed that in NB-UVB-exposed MCs, there was a significant increase in P53 and MMP9 levels alongside a decrease in TRPM1 and miR211 levels. We also found that the migration ability and MMP9 level of MCs transfected with hsa-miR211 mimic or/ lentivirus-P53 were significantly changed as compared with controls. Collectively ， our results indicate that the P53/miR211/MMP9 axis plays a critical role in facilitating the migration of MCs exposed to NB-UVB irradiation, which may serve as a provocative target for promoting vitiligo repigmentation. (This study was supported by grant from the National Natural Science Foundation of China, NSFC#: 81573028)

Luteolin 7-Sulfate Attenuates Melanin Synthesis through Inhibition of CREB- and MITF-Mediated Tyrosinase Expression.

Antioxidants with antimelanogenic activity are potentially useful for the attenuation of skin hyperpigmentation disorders. In a previous study, luteolin 7-sulfate isolated from Phyllospadix iwatensis Makino, a marine plant, was shown to inhibit cellular melanin synthesis. The aim of the present study was to examine its action mechanism, focusing on the regulation of tyrosinase (TYR) expression in cells. Cell-based assay was undertaken using murine melanoma B16-F10 cells and primary human epidermal melanocytes (HEMs). Luteolin 7-sulfate showed lower toxicity compared to luteolin in B16-F10 cells. At the non-toxic concentration ranges, luteolin 7-sulfate attenuated melanin synthesis, stimulated by α-melanocyte-stimulating hormone or forskolin. Luteolin 7-sulfate attenuated forskolin-induced microphthalmia-associated transcription factor (MITF) and TYR expressions at the mRNA and protein levels in B16-F10 cells. It also attenuated the phosphorylation of cAMP-responsive element binding protein (CREB) stimulated by forskolin. Luteolin 7-sulfate also attenuated melanin synthesis in primary HEMs. This study demonstrates that luteolin 7-sulfate attenuates TYR gene expression through the intervention of a CREB- and MITF-mediated signaling pathway, leading to the decreased melanin synthesis.

Critical Role of ATP-P2X7 Axis in UV-Induced Melanogenesis

Purinergic signaling participates in skin physiology and pathology, such as hair growth, wound healing, inflammation, pain, and skin cancer. However, few studies have investigated the involvement of purinergic signaling in skin pigmentation. This study demonstrated that extracellular adenosine 5'-triphosphate (ATP) released from keratinocytes by UVB radiation promotes melanin production in primary human epidermal melanocytes and exvivo skin cultures. Intracellular calcium ion and protein kinase C/CREB signaling contributed to ATP-mediated melanogenesis. Also, P2X7 receptor was proven to play a pivotal role in ATP-mediated melanogenesis because P2X7 receptor blockade abrogated ATP-induced melanin production. In addition, MNT1 cells with P2X7 receptor knockout using CRISPR/Cas9 system did not show any increase in MITF expression when co-cultured with UV-irradiated keratinocytes compared to MNT1 cells with intact P2X7 receptor, which showed increased expression of MITF. In conclusion, our results indicate that the extracellular ATP-P2X7 signaling axis is an adjunctive mechanism in UV-induced melanogenesis. Furthermore, ATP-induced purinergic signaling in melanocytes may alter skin pigmentation.

A chemical compound from fruit extract of Juglans mandshurica inhibits melanogenesis through p-ERK-associated MITF degradation

BackgroundTrials for regulation of abnormal hyperpigmentation from the use of natural products have been going on for years. Leaf and root extracts from Juglans mandshurica are reported to function as antioxidants and to suppress allergic dermatitis. However, studies evaluating its fruit extract and the chemical compounds from the fruit extract are lacking in dermatology fields, including melanogenesis. PurposeThe aim of this study is to understand the effect of the fruit extract from J. mandshurica on pigmentation and to search for specific chemical compounds that affect melanogenesis. MethodsAfter screening out any anti-melanotic effects of the fruit extract from J. mandshurica in B16F10 melanoma cells, three major phenolic compounds isolated from the fruit extract were tested by western blot analysis for expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. Their effect on B16F10 melanoma cells with regard to melanogenesis was also confirmed in primary human epidermal melanocytes (PHEMs). PD98059 was tested to observe the compounds’ signaling role in the extracellular signal-regulated protein kinase (ERK)-pathway. ResultsFruit extract from J. mandshurica showed anti-melanotic effects in B16F10 melanoma cells. After chemical compounds were isolated from the fruit extracts, three phenolic compounds were evaluated for anti-melanotic effects. 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-propanediol (compound 1) showed the highest suppression effect among the three compounds. In B16F10 melanoma cells and PHEMs, reduced melanin contents were observed after treatment with the compound (1). Experiments using a blocker of ERK showed that the inhibitory effect of the compound (1) on melanogenesis was dependent on ERK-associated MITF degradation. ConclusionA chemical constituent of Juglans mandshurica Maxim. induces an inhibitory mechanism to melanogenesis. It has the potential to become a whitening agent in the medical field, though this requires further clinical investigation.

Toll-like receptors 2 and 3 enhance melanogenesis and melanosome transport in human melanocytes.

Because little is known about how the innate immune response influences skin pigmentation, we examined whether Toll-like receptor (TLR) agonists participate in melanogenesis and melanosome transportation. We observed that TLR2/2 agonist HKLM and TLR3 agonist Poly(I:C) increased the amount of extracellular melanin from primary human epidermal melanocytes. HKLM, but not Poly(I:C), increased the melanogenic genes such as tyrosinase and dopachrome tautomerase. Poly(I:C) increased the expression of Rab27A, a molecule that facilitates melanosome transport to perimembranous actin filament. UVB irradiation induced Rab27A and melanosome transportation in a similar manner of Poly(I:C). SiRNA for TLR3 or Rab27A suppressed the perimembranous accumulation of Gp100-positive vesicles in melanocytes and decreased melanin transfer to neighboring keratinocytes induced by both Poly(I:C) and UVB. These results suggest that the microenvironment in the epidermis and innate immune stimuli, such as microbiome and ultraviolet represented here by TLR2 and TLR3 agonists, could affect the melanogenesis in human melanocytes.

Untargeted lipidomic analysis of primary human epidermal melanocytes acutely and chronically exposed to UV radiation.

Ultraviolet (UV) radiation present in sunlight has been related to harmful effects on skin such as premature aging and skin cancer. In order to study the effects of UV radiation on skin, many investigations have been carried out at transcriptomic and proteomic levels. However, studies on the effects of UV radiation on lipid composition are scarce. In this work, primary cultures of melanocytes were exposed to UV radiation in a similar UVA/UVB ratio to that found in solar light. The UV exposure was carried out twice a week and different endpoints were investigated at 0.5 (acute exposure), 1.5 and 3 weeks. As a result, dendrite formation and a progressive reduction in cell viability were observed. Also, cell cycle arrest and a reduced E-cadherin content were detected at 0.5 and 1.5 weeks. In the second stage of the study, lipid extracts of melanocytes were analysed by liquid chromatography coupled to mass spectrometry (LC-MS) and subjected to an untargeted lipidomic approach using the ROIMCR chemometric method. Among the most important changes observed under UV irradiation, lipid raft components such as sphingomyelins and GM3 gangliosides as well as other signalling molecules such as phosphatidylinositols decreased progressively with time. These modifications indicated strong effects on important functions such as cell signalling and recognition. In contrast, triacylglycerol species, associated with energy storage, increased progressively, which could be interpreted as a survival mechanism under adverse conditions. Further studies are needed to better understand the functional implications of the changes observed.

Induction of retinal-dependent calcium influx in human melanocytes by UVA or UVB radiation contributes to the stimulation of melanosome transfer.

The transfer of melanosomes from melanocytes to neighbouring keratinocytes is critical to protect the skin from the deleterious effects of ultraviolet A (UVA) and ultraviolet B (UVB) irradiation; however, the initial factor(s) that stimulates melanosome transfer remains unclear. In this study, we investigated the induction of retinal-dependent calcium (Ca2+ ) influx in melanocytes (MCs) by UVA or UVB irradiation and the effect of transient receptor potential cation channel subfamily M member 1 (TRPM1) (melastatin1)-related Ca2+ influx on melanosome transfer. Primary human epidermal MCs were exposed to physiological doses of UVB or UVA light and loaded with a calcium indicator Fluo-4 dye. The change of intracellular calcium of MCs was monitored using a two-photon confocal fluorescence microscopy. MCs were co-cultured with human epidermal keratinocytes (KCs) in the absence or presence of voriconazole (a TRPM1 blocker) or calcium chelators. MCs were also transfected with TRPM1 siRNA for silencing the expression of TRPM1 gene. The melanosome transfer in the co-cultured cells was quantitatively analysed using flow cytometry and was further confirmed by immunofluorescent double-staining. The protein levels and distributions of TRPM1, OPN3 and OPN5 in MCs were measured by Western blotting or immunofluorescent staining. The retinal-dependent Ca2+ influx of UVA-exposed melanocytes differed greatly from that of UVB-exposed melanocytes in the timing-phase. The protein expression of TRPM1 in mono- and co-cultured MCs was dose-dependently up-regulated by UVA and UVB. TRPM1 siRNA-mediated knockdown and the blockage of TRPM1 channel using a putative antagonist (voriconazole) significantly inhibited melanosome transfer in co-cultures following UVA or UVB exposure. The distinct time-phases of Ca2+ influx in MCs induced by UVA or UVB contribute to the consecutive stimulation of melanosome transfer, thereby providing a potent photoprotection against harmful UV radiation.

Keap1 knockdown in melanocytes induces cell proliferation and survival via HO-1-associated β-catenin signaling

BackgroundNrf2-Keap1 signaling pathway protects cells against photo-oxidative stress. Yet in recent works, its role in melanogenesis together with cell protection functions against oxidative stress has been gaining interest. However, its effect on melanogenesis still has contradictory results from different studies. ObjectiveThe aims of our study were to investigate the effect of Keap1 silencing in melanocyte on melanogenesis and its associated mechanism. MethodsPrimary human epidermal melanocytes and melan-a cell line were used for this experiment. RNA sequencing was done to identify genes involved in melanocyte biology using Keap1 knockdown through siRNA techniques. And melanogenesis and the expression of melanogenesis-associated molecules were evaluated in Keap1 silenced melanocyte to examine the effects of Keap1 on melanogenesis, melanocyte growth, and related pathways. ResultsRNA-sequencing data revealed that Keap1 knockdown in primary human epidermal melanocytes (PHEMs) induced cell survival-related gene expression. Additionally, siRNA-mediated inhibition of Keap1 led to upregulation of MITF and melanogenesis-associated molecules along with Nrf2 activation in PHEMs. HO-1, a major gene that is upregulated in RNA-sequencing using Keap1-silenced PHEMs, protected melanocytes against H2O2-induced cell death and upregulated MITF and β-catenin expression. Further, increased expression of melanogenesis-associated molecules after Keap1 silencing was validated to occur through HO-1-associated β-catenin activation in a Keap1 and HO-1 double knockdown experiment. ConclusionThis work suggests that Keap1 silencing in melanocytes induced melanogenesis and the expression of melanogenesis-associated molecules through HO-1-associated β-catenin activation. Keap1 downregulation in melanocytes is important for cell proliferation and survival.

Photoprotection by dietary phenolics against melanogenesis induced by UVA through Nrf2-dependent antioxidant responses

Dietary phenolics may play a protective role in UV-mediated skin pigmentation through their antioxidant and UV-absorbing actions. In this study, we investigated whether genetic silencing of Nrf2, regulating the transcription of antioxidant genes, affected melanogenesis in primary human epidermal melanocytes (HEMn) and B16F10 melanoma cells subjected to UVA (8J/cm2) exposure. Then, we explored the antimelanogenic actions of phenolics; caffeic acid (CA) and ferulic acid (FA) providing partial UVA protection; quercetin (QU) and rutin (RU) providing strong UVA protection and; avobenzone (AV), an efficient UVA filter, in association with modulation of Nrf2-mediated antioxidant defenses in response to UVA insults in B16F10 cells. Upon oxidative insults, Nrf2 silencing promoted melanogenesis in both HEMn and B16F10 cells irradiated with UVA. Stimulation of melanogenesis by UVA correlated with increased ROS and oxidative DNA damage (8-OHdG), GSH depletion as well as a transient downregulation of Nrf2 nuclear translocation and of Nrf2-ARE signaling in B16F10 cells. All test compounds exerted antimelanogenic effects with respect to their abilities to reverse UVA-mediated oxidative damage as well as downregulation of Nrf2 activity and its target antioxidants (GCLC, GST and NQO1) in B16F10 cells. In conclusion, defective Nrf2 may promote melanogenesis under UVA irradiation through oxidative stress mechanisms. Compounds with antioxidant and/or UVA absorption properties could protect against UVA-induced melanogenesis through indirect regulatory effect on Nrf2-ARE pathway.

Rnaset2 inhibits melanocyte outgrowth possibly through interacting with shootin1

BackgroundImpaired dendrite outgrowth of melanocytes is one of the reasons triggering vitiligo. RNASET2 was identified as one of the risk genes for vitiligo in a GWAS study conducted in the Chinese Han population. However, the role of Rnaset2 in the outgrowth of melanocytes is rarely studied. ObjectiveThis study is to investigate the effects of Rnaset2 in regulating the outgrowth of melanocytes and its interacting proteins. MethodsStress conditions (UV irradiation, hydrogen peroxide, and lipopolysaccharides) were applied to primary human epidermal melanocytes (HEMs) and epidermal keratinocytes (HEKs). HEKs with Rnaset2 overexpression were co-cultured with HEMs. Rnaset2 expression levels were detected by ELISA. HEMs, HEKs and A375 cells were treated with recombinant Rnaset2 protein and actin network was observed with fluorescence microscope. Cell migration assay was performed using nuclepore filters after incubating A375 cells with recombinant Rnaset2 protein. Human proteome microarray was performed to identify proteins interacting with Rnaset2. Co-immunoprecipitation was conducted to verify the results. ResultsOur results showed that after exposing to stress conditions, Rnaset2 expression and secretion by HEKs and HEMs were increased. Co-culture of HEKs and HEMs showed that outgrowth of HEMs was inhibited by Rnaset2 overexpression in HEKs. Additionally, human recombinant Rnaset2 protein treatment altered the actin network of HEMs, HEKs and A375 cells. The migration of A375 cells was also inhibited by human recombinant Rnaset2 protein treatment. Human proteome microarray identified shootin1, an important protein involved in axon outgrowth, as one of the interacting proteins of Rnaset2. Co-immunoprecipitation confirmed that Rnaset2 interacted with shootin1 in vitro. ConclusionRnaset2 inhibits melanocyte outgrowth through interacting with shootin1 and this effect may be associated with vitiligo pathogenesis. Rnaset2 may be a potential therapeutic target for vitiligo.

UVA Phototransduction Drives Early Melanin Synthesis in Human Melanocytes

Exposure of human skin to solar ultraviolet radiation (UVR), a powerful carcinogen [1] comprising ~95% ultraviolet A (UVA) and ~5% ultraviolet B (UVB) at the Earth's surface, promotes melanin synthesis in epidermal melanocytes [2, 3], which protects skin from DNA damage [4, 5]. UVB causes DNA lesions [6] that lead to transcriptional activation of melanin-producing enzymes, resulting in delayed skin pigmentation within days [7]. In contrast, UVA causes primarily oxidative damage [8] and leads to immediate pigment darkening (IPD) within minutes, via an unknown mechanism [9, 10]. No receptor protein directly mediating phototransduction in skin has been identified. Here we demonstrate that exposure of primary human epidermal melanocytes (HEMs) to UVA causes calcium mobilization and early melanin synthesis. Calcium responses were abolished by treatment with G protein or phospholipase C (PLC) inhibitors or by depletion of intracellular calcium stores. We show that the visual photopigment rhodopsin [11] is expressed in HEMs and contributes to UVR phototransduction. Upon UVR exposure, significant melanin production was measured within one hour; cellular melanin continued to increase in a retinal- and calcium-dependent manner up to 5-fold after 24 hr. Our findings identify a novel UVA-sensitive signaling pathway in melanocytes that leads to calcium mobilization and melanin synthesis and may underlie the mechanism of IPD in human skin.

The redox antimalarial dihydroartemisinin targets human metastatic melanoma cells but not primary melanocytes with induction of NOXA-dependent apoptosis

Recent research suggests that altered redox control of melanoma cell survival, proliferation, and invasiveness represents a chemical vulnerability that can be targeted by pharmacological modulation of cellular oxidative stress. The endoperoxide artemisinin and semisynthetic artemisinin-derivatives including dihydroartemisinin (DHA) constitute a major class of antimalarials that kill plasmodium parasites through induction of iron-dependent oxidative stress. Here, we demonstrate that DHA may serve as a redox chemotherapeutic that selectively induces melanoma cell apoptosis without compromising viability of primary human melanocytes. Cultured human metastatic melanoma cells (A375, G361, LOX) were sensitive to DHA-induced apoptosis with upregulation of cellular oxidative stress, phosphatidylserine externalization, and activational cleavage of procaspase 3. Expression array analysis revealed DHA-induced upregulation of oxidative and genotoxic stress response genes (GADD45A, GADD153, CDKN1A, PMAIP1, HMOX1, EGR1) in A375 cells. DHA exposure caused early upregulation of the BH3-only protein NOXA, a proapototic member of the Bcl2 family encoded by PMAIP1, and genetic antagonism (siRNA targeting PMAIP1) rescued melanoma cells from apoptosis indicating a causative role of NOXA-upregulation in DHA-induced melanoma cell death. Comet analysis revealed early DHA-induction of genotoxic stress accompanied by p53 activational phosphorylation (Ser 15). In primary human epidermal melanocytes, viability was not compromised by DHA, and oxidative stress, comet tail moment, and PMAIP1 (NOXA) expression remained unaltered. Taken together, these data demonstrate that metastatic melanoma cells display a specific vulnerability to DHA-induced NOXA-dependent apoptosis and suggest feasibility of future anti-melanoma intervention using artemisinin-derived clinical redox antimalarials.

Interactions of Human Myosin Va Isoforms, Endogenously Expressed in Human Melanocytes, Are Tightly Regulated by the Tail Domain

Primary human epidermal melanocytes express six endogenous isoforms of the human actin-associated myosin Va motor protein, involved in organelle transport. As isoforms containing exon F are most abundant in melanocytes, we hypothesized that these isoforms probably have a melanocyte-specific function. To uncover the biologic role of the six isoforms we introduced enhanced green fluorescent protein (eGFP)-myosin Va tail constructs in human melanocytes. We found that the medial tail, undergoing alternative splicing, has to be expressed in combination with the globular tail in order to obtain clear colocalization with organelles. Our data show that isoforms lacking exon F but containing exon D are associated with vesicles near the Golgi area. Myosin Va isoforms containing exon F are able to colocalize with and influence melanosome distribution by indirect interaction with rab27a and direct interaction with melanophilin. These results indicate that the myosin Va medial tail domain provides the globular tail domain with organelle-interacting specificity.