YAP prevents senescence of dermal fibroblast and inhibits melanogenesis via paracrine effect of DKK1.

Senile skin hyperpigmentation displays remarkable histopathological features of dermal aging. The crosstalk between melanocytes and dermal fibroblasts plays crucial roles in aging-related pigmentation. While senescent fibroblasts can upregulate pro-melanogenic factors, the role of anti-melanogenic factors, such as dickkopf1 (DKK1), and the upstream regulatory mechanism during aging remain obscure. This study investigated the roles of yes-associated protein (YAP) and DKK1 in the regulation of dermal fibroblast senescence and melanogenesis. Our findings demonstrated decreased YAP activity and DKK1 levels in intrinsic and extrinsic senescent fibroblasts. YAP depletion induced fibroblast senescence and downregulated the expression and secretion of DKK1, whereas YAP overexpression partially reversed the effect. The transcriptional regulation of DKK1 by YAP was supported by dual-luciferase reporter and chromatin immunoprecipitation assays. Moreover, YAP depletion in fibroblasts upregulated Wnt/ß-catenin in melanocytes and stimulated melanogenesis, which was partially rescued by the re-supplementation of DKK1. Conversely, overexpression of YAP in senescent fibroblasts decreased Wnt/ß-catenin levels in melanocytes and inhibited melanogenesis. Additionally, reduced levels of YAP and DKK1 were verified in the dermis of solar lentigines. These findings suggest that, during skin aging, epidermal pigmentation may be influenced by YAP in the dermal microenvironment via the paracrine effect of DKK1.

Anti-Melanogenic Effects of Takifugu flavidus Muscle Hydrolysate in B16F10 Melanoma Cells and Zebrafish.

Abnormal melanogenesis can lead to hyperpigmentation. Tyrosinase (TYR), a key rate-limiting enzyme in melanin production, is an important therapeutic target for these disorders. We investigated the TYR inhibitory activity of hydrolysates extracted from the muscle tissue of Takifugu flavidus (TFMH). We used computer-aided virtual screening to identify a novel peptide that potently inhibited melanin synthesis, simulated its binding mode to TYR, and evaluated functional efficacy in vitro and in vivo. TFMH inhibited the diphenolase activities of mTYR, reducing TYR substrate binding activity and effectively inhibiting melanin synthesis. TFMH indirectly reduced cAMP response element-binding protein phosphorylation in vitro by downregulating melanocortin 1 receptor expression, thereby inhibiting expression of the microphthalmia-associated transcription factor, further decreasing TYR, tyrosinase related protein 1, and dopachrome tautomerase expression and ultimately impeding melanin synthesis. In zebrafish, TFMH significantly reduced black spot formation. TFMH (200 µg/mL) decreased zebrafish TYR activity by 43% and melanin content by 52%. Molecular dynamics simulations over 100 ns revealed that the FGFRSP (T-6) peptide stably binds mushroom TYR via hydrogen bonds and ionic interactions. T-6 (400 µmol/L) reduced melanin content in B16F10 melanoma cells by 71% and TYR activity by 79%. In zebrafish, T-6 (200 µmol/L) inhibited melanin production by 64%. TFMH and T-6 exhibit good potential for the development of natural skin-whitening cosmetic products.

Exploring the Influence of Cold Plasma on Epidermal Melanogenesis In Situ and In Vitro.

Epidermal melanin synthesis determines an individual's skin color. In humans, melanin is formed by melanocytes within the epidermis. The process of melanin synthesis strongly depends on a range of cellular factors, including the fine-tuned interplay with reactive oxygen species (ROS). In this context, a role of cold atmospheric plasma (CAP) on melanin synthesis was proposed due to its tunable ROS generation. Herein, the argon-driven plasma jet kINPen® MED was employed, and its impact on melanin synthesis was evaluated by comparison with known stimulants such as the phosphodiesterase inhibitor IBMX and UV radiation. Different available model systems were employed, and the melanin content of both cultured human melanocytes (in vitro) and full-thickness human skin biopsies (in situ) were analyzed. A histochemical method detected melanin in skin tissue. Cellular melanin was measured by NIR autofluorescence using flow cytometry, and a highly sensitive HPLC-MS method was applied, which enabled the differentiation of eu- and pheomelanin by their degradation products. The melanin content in full-thickness human skin biopsies increased after repeated CAP exposure, while there were only minor effects in cultured melanocytes compared to UV radiation and IBMX treatment. Based on these findings, CAP does not appear to be a useful option for treating skin pigmentation disorders. On the other hand, the risk of hyperpigmentation as an adverse effect of CAP application for wound healing or other dermatological diseases seems to be neglectable.

Measuring Laccase Activity and Melanin Production in Cryptococcus neoformans.

Melanin is a complex dark pigment synthetized by the phenoloxidase enzyme laccase in Cryptococcus neoformans. In vitro, this enzyme oxidizes exogenous catecholamines to produce melanin that may be secreted or incorporated into the fungal cell wall. This pigment has multiple roles in C. neoformans virulence during its interaction with different hosts and probably also in protecting fungal cells in the environment against predation and oxidative and radiation stresses, among others. However, it is important to note that laccase also has melanin-independent roles in C. neoformans interactions with host cells. In this chapter, we describe a quantitative laccase assay and a method for evaluating the kinetics of melanin production in C. neoformans colonies.

Free radical production induced by visible light in live fruit flies.

Visible light triggers free radical production in alive and intact Drosophila melanogaster. We exposed fruit flies to red (613-631 nm), green (515-535 nm), and blue (455-475 nm) light while we monitored changes in unpaired electron content with an electron spin resonance spectrometer (ESR/EPR). The immediate response to light is a rapid increase in spin content lasting approximately 10 s followed by a slower, linear increase for approximately 170 s. When the light is turned off, the spin population promptly decays with a similar time course, though never fully returning to baseline. The magnitude and time course of the spin production depends on the wavelength of the light. Initially, we surmised that eumelanin might be responsible for the spin change because of its documented ability for visible light absorption and its highly stable free radical content. To explore this, we utilized different fruit fly strains with varying eumelanin content and clarified the relation of melanin types with the spin response. Our findings revealed that flies with darker cuticle have at least three-fold more unpaired electrons than flies with yellow cuticle. However, to our surprise, the increase in unpaired electron population by light was not drastically different amongst the genotypes. This suggests that light-induced free radical production may not exclusively rely on the presence of black melanin, but may instead be dependent on light effects on quinone-based cuticular polymers.

Fast freezing inhibits melanin synthesis of melanocytes by modulating the Wnt/ß-catenin signalling pathway.

Skin hyperpigmentation is mainly caused by excessive synthesis of melanin; however, there is still no safe and effective therapy for its removal. Here, we found that the dermal freezer was able to improve UVB-induced hyperpigmentation of guinea pigs without causing obvious epidermal damage. We also mimic freezing stimulation at the cellular level by rapid freezing and observed that freezing treatments <2.5 min could not decrease cell viability or induce cell apoptosis in B16F10 and Melan-A cells. Critically, melanin content and tyrosinase activity in two cells were greatly reduced after freezing treatments. The dramatic decrease in tyrosinase activity was associated with the downregulation of MITF, TYR, TRP-1 and TRP-2 protein expression in response to freezing treatments for two cells. Furthermore, our results first demonstrated that freezing treatments significantly reduced the levels of p-GSK3ß and ß-catenin and the nuclear accumulation of ß-catenin in B16F10 and Melan-A cells. Together, these data suggest that fast freezing treatments can inhibit melanogenesis-related gene expression in melanocytes by regulating the Wnt/ß-catenin signalling pathway. The inhibition of melanin production eventually contributed to the improvement in skin hyperpigmentation induced by UVB. Therefore, fast freezing treatments may be a new alternative of skin whitening in the clinic in the future.

Unveiling the Potential of Ultrasonic-Assisted Ethanol Extract from Sargassum horneri in Inhibiting Tyrosinase Activity and Melanin Production in B16F10 Murine Melanocytes.

BACKGROUNDS: Melanogenesis, regulated by genetic, hormonal, and environmental factors, occurs in melanocytes in the basal layer of the epidermis. Dysregulation of this process can lead to various skin disorders, such as hyperpigmentation and hypopigmentation. Therefore, the present study investigated the effect of ultrasonic-assisted ethanol extract (SHUE) from Sargassum horneri (S. horneri), brown seaweed against melanogenesis in α-melanocyte-stimulating hormone (MSH)-stimulated B16F10 murine melanocytes. METHODS: Firstly, yield and proximate compositional analysis of the samples were conducted. The effect of SHUE on cell viability has been evaluated by using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. After that, the melanin content and cellular tyrosinase activity in α-MSH-stimulated B16F10 murine melanocytes were examined. Western blot analysis was carried out to investigate the protein expression levels of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP1), and tyrosinase-related protein-2 (TRP2). In addition, the effect of extracellular signal-regulated kinase (ERK) on the melanogenesis process was assessed via Western blotting. RESULTS: As per the analysis, SHUE contained the highest average yield on a dry basis at 28.70 ± 3.21%. The findings showed that SHUE reduced the melanin content and cellular tyrosinase activity in α-MSH-stimulated B16F10 murine melanocytes. Additionally, the expression levels of MITF, TRP1, and TRP2 protein were significantly downregulated by SHUE treatment in α-MSH-stimulated B16F10 murine melanocytes. Moreover, SHUE upregulated the phosphorylation of ERK and AKT in α-MSH-stimulated B16F10 murine melanocytes. In addition, experiments conducted using the ERK inhibitor (PD98059) revealed that the activity of SHUE depends on the ERK signaling cascade. CONCLUSION: These results suggest that SHUE has an anti-melanogenic effect and can be used as a material in the formulation of cosmetics related to whitening and lightening.

Comparative Analysis of Olive-Derived Phenolic Compounds' Pro-Melanogenesis Effects on B16F10 Cells and Epidermal Human Melanocytes.

Olive leaf contains plenty of phenolic compounds, among which oleuropein (OP) is the main component and belongs to the group of secoiridoids. Additionally, phenolic compounds such as oleocanthal (OL) and oleacein (OC), which share a structural similarity with OP and two aldehyde groups, are also present in olive leaves. These compounds have been studied for several health benefits, such as anti-cancer and antioxidant effects. However, their impact on the skin remains unknown. Therefore, this study aims to compare the effects of these three compounds on melanogenesis using B16F10 cells and human epidermal cells. Thousands of gene expressions were measured by global gene expression profiling with B16F10 cells. We found that glutaraldehyde compounds derived from olive leaves have a potential effect on the activation of the melanogenesis pathway and inducing differentiation in B16F10 cells. Accordingly, the pro-melanogenesis effect was investigated by means of melanin quantification, mRNA, and protein expression using human epidermal melanocytes (HEM). This study suggests that secoiridoid and its derivates have an impact on skin protection by promoting melanin production in both human and mouse cell lines.

Combination of 308-nm excimer laser and piperine promotes melanocyte proliferation, migration, and melanin content production via the miR-328/SFRP1 axis.

OBJECTIVE: Both piperine and a 308-nm excimer laser have significant curative effects on vitiligo. This study mainly explored the molecular mechanism of a 308-nm excimer combined with piperine in regulating melanocyte proliferation. METHODS: Epidermal melanocytes were cultured in piperine solution, and the cells were irradiated by an XTRAC excimer laser treatment system at 308-nm output monochromatic light. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were for detecting the expression levels of genes or proteins. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Transwell method was for assessing cell viability and migration capacity. The content of melanin was also detected. RESULTS: The combination of the 308-nm excimer laser and piperine enhanced the cell proliferation, migration, and melanin production of melanocytes and upregulated the level of miR-328, and restraint of miR-328 reversed the influence of the 308-nm excimer laser and piperine. Secreted frizzled-related protein 1 (SFRP1) is a direct target gene of miR-328, and miR-328 can inhibit the expression of SFRP1 and elevate the protein level of the Wnt/ß-catenin signaling pathway. CONCLUSION: The 308-nm excimer laser combined with piperine may be more efficient than piperine alone in the remedy of vitiligo, and the miR-328/SFRP1 and Wnt/ß-catenin pathways are participated in the proliferation, migration, and melanin synthesis of melanocytes.

Exploiting Principal Component Analysis (PCA) to reveal temperature, buffer and metal ions' role in neuromelanin (NM) synthesis by dopamine (DA) oxidative polymerization.

Neuromelanin (NM) plays a well-established role in neurological disorders pathogenesis; the mechanism of action is still discussed and the investigations in this field are limited by NM's complex and heterogeneous composition, insolubility, and low availability from human brains. An alternative can be offered by synthetic NM obtained from dopamine (DA) oxidative polymerization; however, a deep knowledge of the influence of both physicochemical parameters (T, pH, ionic strength) and other compounds in the reaction media (buffer, metal ions, other catecholamines) on DA oxidation process and, consequently, on synthetic NM features is mandatory to develop reliable NM preparation methodologies. To partially fulfill this aim, the present work focuses on defining the role of temperature, buffer and metal ions on both DA oxidation rate and DA oligomer size. DA oxidation in the specific conditions is monitored by UV-Vis spectroscopy and Principal Component Analysis (PCA) is run either on the raw spectra to model the background absorption increase, related to small DA oligomers formation, or on their first derivative to rationalize DA consumption. After having studied three case studies, 3-Way PCA is applied to directly evaluate the effect of temperature and buffer type on DA oxidation in the presence of different metal ions. Despite the proof-of-concept nature of the work and the number of compounds still to be included in the investigation, the preliminary results and the possibility to further expand the chemometric approach represent an interesting contribution to the field of in vitro simulation of NM synthesis.

Role of kinin receptors in skin pigmentation.

Previous studies have shown that all kinin system is constitutively expressed in the normal and inflamed skin, with a potential role in both physiological and pathological processes. However, the understanding regarding the involvement of the kinin system in skin pigmentation and pigmentation disorders remains incomplete. In this context, the present study was designed to determine the role of kinins in the Monobenzone (MBZ)-induced vitiligo-like model. Our findings showed that MBZ induces higher local skin depigmentation in kinin receptors knockout mice (KOB1R, KOB2R and KOB1B2R) than in wild type (WT). Remarkably, lower levels of melanin content and reduced ROS generation were detected in KOB1R and KOB2R mice treated with MBZ. In addition, both KOB1R and KOB2R show increased dermal cell infiltrate in vitiligo-like skin, when compared to WT-MBZ. Additionally, lack of B1R was associated with greater skin accumulation of IL-4, IL-6, and IL-17 by MBZ, while KOB1B2R presented lower levels of TNF and IL-1. Of note, the absence of both kinin B1 and B2 receptors demonstrates a protective effect by preventing the increase in polymorphonuclear and mononuclear cell infiltrations, as well as inflammatory cytokine levels induced by MBZ. In addition, in vitro assays confirm that B1R and B2R agonists increase intracellular melanin synthesis, while bradykinin significantly enhanced extracellular melanin levels and proliferation of B16F10 cells. Our findings highlight that the lack of kinin receptors caused more severe depigmentation in the skin, as well as genetic deletion of both B1/B2 receptors seems to be linked with changes in levels of constitutive melanin levels, suggesting the involvement of kinin system in crucial skin pigmentation pathways.

Ribosomal S6 kinase 2-forkhead box protein O4 signaling pathway plays an essential role in melanogenesis.

Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4's transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis.

Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system.

The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.

Molecular characterization of transcription factor CREB3L2 and CREB3L3 and their role in melanogenesis in Pacific oysters (Crassostrea gigas).

Colorful shells in mollusks are commonly attributable to the presence of biological pigments. In Pacific oysters, the inheritance patterns of several shell colors have been investigated, but little is known about the molecular mechanisms of melanogenesis and pigmentation. cAMP-response element binding proteins (CREB) are important transcription factors in the cAMP-mediated melanogenesis pathway. In this study, we characterized two CREB genes (CREB3L2 and CREB3L3) from Pacific oysters. Both of them contained a conserved DNA-binding and dimerization domain (a basic-leucine zipper domain). CREB3L2 and CREB3L3 were expressed highly in the mantle tissues and exhibited higher expression levels in the black-shell oyster than in the white. Masson-Fontana melanin staining and immunofluorescence analysis showed that the location of CREB3L2 protein was generally consistent with the distribution of melanin in oyster edge mantle. Dual-luciferase reporter assays revealed that CREB3L2 and CREB3L3 could activate the microphthalmia-associated transcription factor (MITF) promoter and this process was regulated by the level of cAMP. Additionally, we found that cAMP regulated melanogenic gene expression through the CREB-MITF-TYR axis. These results implied that CREB3L2 and CREB3L3 play important roles in melanin synthesis and pigmentation in Pacific oysters.

Newly identified peptide Nigrocin-OA27 inhibits UVB induced melanin production via the MITF/TYR pathway.

Melasma is a common skin disease induced by an increase in the content of melanin in the skin, which also causes serious physical and mental harm to patients. In this research, a novel peptide (Nigrocin-OA27) from Odorrana andersonii is shown to exert a whitening effect on C57 mice pigmentation model. The peptide also demonstrated non-toxic and antioxidant capacity, and can significantly reduce melanin content in B16 cells. Topical application effectively delivered Nigrocin-OA27 to skin's epidermal and dermal layers and exhibited significant preventive and whitening effects on the UVB-induced ear pigmentation model in C57 mice. The whitening mechanism of Nigrocin-OA27 may be related to reduced levels of the microphthalmia-associated transcription factor and the key enzyme for melanogenesis-tyrosinase (TYR). Nigrocin-OA27 also inhibited the catalytic activity by adhering to the active core of TYR, thereby reducing melanin formation and deposition. In conclusion, Nigrocin-OA27 may be a potentially effective external agent to treat melasma by inhibiting aberrant skin melanin synthesis.

NsdD, a GATA-type transcription factor is involved in regulation and biosynthesis of macromolecules melanin, pullulan, and polymalate in Aureobasidium melanogenum.

In this study, an NSDD gene, which encoded a GATA-type transcription factor involved in the regulation and biosynthesis of melanin, pullulan, and polymalate (PMA) in Aureobasidium melanogenum, was characterized. After the NSDD gene was completely removed, melanin production by the Δnsd mutants was enhanced, while pullulan and polymalate production was significantly reduced. Transcription levels of the genes involved in melanin biosynthesis were up-regulated while expression levels of the genes responsible for pullulan and PMA biosynthesis were down-regulated in the Δnsdd mutants. In contrast, the complementation of the NSDD gene in the Δnsdd mutants made the overexpressing mutants restore melanin production and transcription levels of the genes responsible for melanin biosynthesis. Inversely, the complementation strains, compared to the wild type strains, showed enhanced pullulan and PMA yields. These results demonstrated that the NsdD was not only a negative regulator for melanin biosynthesis, but also a key positive regulator for pullulan and PMA biosynthesis in A. melanogenum. It was proposed how the same transcriptional factor could play a negative role in melanin biosynthesis and a positive role in pullulan and PMA biosynthesis. This study provided novel insights into the regulatory mechanisms of multiple A. melanogenum metabolites and the possibility for improving its yields of some industrial products through genetic approaches.

Rapamycin vs TORin-1 or Gleevec vs Nilotinib: Simple chemical evolution that converts PAK1-blockers to TOR-blockers or vice versa?

Both PAK1 (RAC/CDC42-activating kinase 1) and TOR (Target of Rapamycin) are among the major oncogenic/ageing kinases. However, they play the opposite role in our immune system, namely immune system is suppressed by PAK1, while it requires TOR. Thus, PAK1-blockers, would be more effective for therapy of cancers, than TOR-blockers. Since 2015 when we discovered genetically that PDGF-induced melanogenesis depends on "PAK1", we are able to screening a series of PAK1-blockers as melanogenesis-inhibitors which could eventually promote longevity. Interestingly, rapamycin, the first TOR-inhibitor, promotes melanogenesis, clearly indicating that TOR suppresses melanogenesis. However, a new TOR-inhibitor called TORin-1 no longer suppresses immune system, and blocks melanogenesis in cell culture. These observations strongly indicate that TORin-1 acts as PAK1-blockers, instead of TOR-blockers, in vivo. Thus, it is most likely that melanogenesis in cell culture could enable us to discriminate PAK1-blockers from TORblockers.

Transcriptomic and Physiological Analysis Reveal Melanin Synthesis-Related Genes and Pathways in Pacific Oysters (Crassostrea gigas).

Shell color is one of the shell traits of molluscs, which has been regarded as an economic trait in some bivalves. Pacific oysters (Crassostrea gigas) are important aquaculture shellfish worldwide. In the past decade, several shell color strains of C. gigas were developed through selective breeding, which provides valuable materials for research on the inheritance pattern and regulation mechanisms of shell color. The inheritance patterns of different shell colors in C. gigas have been identified in certain research; however, the regulation mechanism of oyster pigmentation and shell color formation remains unclear. In this study, we performed transcriptomic and physiological analyses using black and white shell oysters to investigate the molecular mechanism of melanin synthesis in C. gigas. Several pigmentation-related pathways, such as cytochrome P450, melanogenesis, tyrosine metabolism, and the cAMP signaling pathway were found. The majority of differentially expressed genes and some signaling molecules from these pathways exhibited a higher level in the black shell oysters than in the white, especially after L-tyrosine feeding, suggesting that those differences may cause a variation of tyrosine metabolism and melanin synthesis. In addition, the in vitro assay using primary cells from mantle tissue showed that L-tyrosine incubation increased cAMP level, gene and protein expression, and melanin content. This study reveals the difference in tyrosine metabolism and melanin synthesis in black and white shell oysters and provides evidence for the potential regulatory mechanism of shell color in oysters.

Transcriptional and post-translational regulation of MITF mediated by bHLH domain during the melanogenesis and melanocyte proliferation in Crassostrea gigas.

Melanocyte differentiation is orchestrated by the master regulator transcription factor MITF. However, its ability to discern distinct binding sites linked to effective gene regulation remains poorly understood. This study aims to assess how co-activator acetyltransferase interacts with MITF to modulate their related lysine action, thereby mediating downstream gene regulation, including DNA affinity, stability, transcriptional activity, particularly in the process of shell pigmentation. Here, we have demonstrated that the CgMITF protein can be acetylated, further enabling selective amplification of the melanocyte maturation program. Collaboration with transcriptional co-regulator p300 advances MITF dynamically interplay with downstream targeted gene promoters. We have established that MITF activation was partially dependent on the bHLH domain, which was well conserved across species. The bHLH domain contained conserved lysine residues, including K6 and K43, which interacted with the E-box motif of downstream targeted-genes. Mutations at K6 and K43 lead to a decrease in the binding affinity of the E-box motif. CgMITF protein bound to the E-box motif within the promoter regions of the tyrosinase-related genes, contributing to melanogenesis, and also interacted with the E-box motif within the TBX2 promoter regions, associated with melanocyte proliferation. We elucidated how the bHLH domain links the transcriptional regulation and acetylation modifications in the melanocyte development in C. gigas.

Melanin Inhibitory Effect of Tuber himalayense Isolated in Incheon, Korea.

There has been a growing interest in skin beauty and antimelanogenic products. Melanogenesis is the process of melanin synthesis whereby melanocytes are activated by UV light or hormone stimulation to produce melanin. Melanogenesis is mediated by several enzymes, such as tyrosinase (TYR), microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), and TRP-2. In this study, we investigated the effect of Tuber himalayense extract on melanin synthesis in α-melanocyte-stimulating hormone (α-MSH)-treated B16F10 melanoma cells. We confirmed that T. himalayense extract was not toxic to α-MSH-treated B16F10 melanoma cells and exhibited a significant inhibitory effect on melanin synthesis at concentrations of 25, 50, and 100 µg/ml. Additionally, the T. himalayense extract inhibited melanin, TRP-1, TRP-2, tyrosinase, and MITF, which are enzymes involved in melanin synthesis, in a concentration-dependent manner. Furthermore, T. himalayense extract inhibited the mitogen-activated protein kinase (MAPK) pathways, such as extracellular signal-regulated kinase-1/2 (ERK), c-Jun N-terminal kinase (JNK), and p38. Therefore, we hypothesized that various components of T. himalayense extract affect multiple factors involved in melanogenesis in B16F10 cells. Our results indicate that T. himalayense extract could potentially be used as a new material for preparing whitening cosmetics.