MITF Research Articles

Dietary flavonoid myricetin 3-O-galactoside suppresses α-melanocyte stimulating hormone-induced melanogenesis in B16F10 melanoma cells by regulating PKA and ERK1/2 activation.

Melanogenesis is the process where skin pigment melanin is produced through tyrosinase activity. Overproduction of melanin causes skin disorders such asfreckles, spots, and hyperpigmentation. Myricetin 3-O-galactoside (M3G) is a dietary flavonoid with reported bioactivities. M3G was isolated from Limonium tetragonum and its anti-melanogenic properties were investigated inα-melanocyte stimulating hormone-stimulated B16F10 melanoma cells. The invitro anti-melanogenic capacity of M3G was confirmed by inhibited tyrosinase and melanin production. M3G-mediated suppression of melanogenic proteins, tyrosinase, microphthalmia-associated transcription factor (MITF), and tyrosinase-related proteins (TRP)-1 and TRP-2, were confirmed by mRNA and protein levels, analyzed by RT-qPCR and Western blot, respectively. Furthermore, M3G suppressed Wnt signaling through the inhibition of PKA phosphorylation. M3G also suppressed the consequent phosphorylation of CREB and nuclear levels of MITF. Analysis of MAPK activation further revealed that M3G increased the activation of ERK1/2 while p38 and JNK activation remained unaffected. Results showed that M3G suppressed melanogenesis in B16F10 cells by decreasing tyrosinase production and therefore inhibiting melanin formation. A possible action mechanism was the suppression of CREB activation and upregulation of ERK phosphorylation which might cause the decreased nuclear levels of MITF. In conclusion, M3G was suggested to be a potential nutraceutical with anti-melanogenic properties.

A de novo 2.3 kb structural variant in MITF explains a novel splashed white phenotype in a Thoroughbred family.

Splashed white in horses is characterized by extensive white patterning on the legs, face and abdomen and may be accompanied by deafness. To date, seven variants in microphthalmia-associated transcription factor (MITF) and two variants in Paired Box 3 (PAX3) have been identified to explain this phenotype. A splashed white Thoroughbred stallion, whose sire and dam were not patterned, was hypothesized to have a de novo variant leading to his white coat pattern. A whole-genome sequencing candidate gene approach identified two single nucleotide variants (SNVs) in SOX10, four SNVs in MITF and a 2.3 kb deletion in MITF with the alternative allele present in this stallion but absent in the other 18 horses analyzed. All six SNVs were annotated as modifiers and were not further considered. The deletion in MITF (NC_009159.3:g.21555811_21558139delinsAAAT) encompasses exon 9 encoding a part of the helix-loop-helix domain required for DNA binding. Sanger sequencing and parentage testing confirmed that this deletion was a de novo mutation of maternal origin. Consistent with the published nomenclature, we denote this likely causal variant as SW8. Genotyping three of this stallion's offspring identified SW8 only in the nearly all-white foal that was confirmed deaf by brainstem auditory evoked response testing. This foal was also a compound heterozygote for dominant white variants (W20/W22), but to date, W variants alone have not been connected to deafness. SW8 marks the fourth de novo MITF variant in horses reported to cause white patterning. The link between deafness and all MITF variants with and without other variants impacting melanocyte development and function needs to be further explored.

Potential role of the cell-penetrating peptide-conjugated soluble N-ethylmaleimide-sensitive factor attachment protein receptor motif of vesicle-associated membrane protein 2-patterned peptide in novel cosmeceutical skin product development.

This study aimed to investigate and verify the effect of cell-penetrating peptide (CPP)-conjugated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) motif of vesicle-associated membrane protein 2 (VAMP2)-patterned peptide (INCI name: Acetyl sh-Oligopeptide-26 sh-Oligopeptide-27 SP, trade name: M.Biome-BT) on improving skin function in vitro. The cytotoxicity of CPP-conjugated SNARE motif of VAMP2-patterned peptide (CVP) was investigated using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay against B16-F10 cells and human dermal fibroblasts (HDFs) and a reconstructed skin irritation test. The anti-wrinkle activity of M.Biome-BT was determined by assessing the release of norepinephrine and dopamine in PC-12 cells via ELISA. The skin-whitening effects of CVP were assessed in B16-F10 cells by measuring the intra- and extracellular melanin contents and expression levels of melanin production-related genes, such as microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and TRP-2. CVP is not cytotoxic to B16-F10 cells and HDFs, and no skin irritation was observed. CVP treatment considerably diminished K+ -induced norepinephrine and dopamine secretion compared with the non-treated control group (62% and 40%, respectively). Additionally, the inhibition ability of CVP on norepinephrine and dopamine release was comparable to that of botulinum neurotoxin type A (BoNT/A). CVP also increased intracellular melanin content in a dose-dependent manner, whereas extracellular melanin content decreased (76%-85%). However, CVP treatment did not affect the mRNA expression of MITF, TYR, TRP-1, and TRP-2. These results suggest that CVP does not inhibit melanin production; however, it may induce a whitening effect by inhibiting melanin transport. Taken together, our findings indicate that CVP could be used as an active and safe cosmeceutical ingredient for antiaging applications.

A20 ameliorates advanced glycation end products-induced melanogenesis by inhibiting NLRP3 inflammasome activation in human dermal fibroblasts

BackgroundAdvanced glycation end products (AGEs) promote melanogenesis through activating NLRP3 inflammasome in fibroblasts. Although A20 has been highlighted to inhibit NLRP3 inflammasome activation, its roles and mechanisms remain elusive in photoaging-associated pigmentation. ObjectivesTo determine the significance of fibroblast A20 in AGEs-induced NLRP3 inflammasome activation and pigmentation. MethodsThe correlation between A20 and AGEs or melanin was studied in sun-exposed skin and lesions of melasma and solar lentigo. We then investigated A20 level in AGEs-treated fibroblast and the effect of fibroblast A20 overexpression or knockdown on AGEs-BSA-induced NLRP3 inflammasome activation and pigmentation, respectively. Finally, the severity of NLRP3 inflammasome activation and pigmentation was evaluated after mice were injected intradermally with A20-overexpression adeno-associated virus and AGEs-BSA. ResultsDermal A20 expression was decreased and exhibited negative correlation with either dermal AGEs deposition or epidermal melanin level in sun-exposed skin and pigmentary lesions. Moreover, both AGEs-BSA and AGEs-collagen robustly decreased A20 expression via binding to RAGE in fibroblasts. Further, A20 overexpression or depletion significantly decreased or augmented AGEs-BSA-induced activation of NF-κB pathway and NLRP3 inflammasome and IL-18 production and secretion in fibroblasts, respectively. Importantly, fibroblast A20 potently repressed AGEs-BSA-stimulated melanin content，tyrosinase activity，and expression of microphthalmia-associated transcription factor and tyrosinase in melanocytes. Particularly, fibroblast A20 significantly abrogated AGEs-BSA-promoted melanogenesis in ex vivo skin and mouse models. Additionally, fibroblast A20 inhibited AGEs-BSA-activated MAPKs in melanocytes and the epidermis of ex vivo skin. ConclusionsFibroblast A20 suppresses AGEs-stimulate melanogenesis in photoaging-associated hyperpigmentation disorders by inhibiting NLRP3 inflammasome activation.

GREB1 isoform 4 is specifically transcribed by MITF and required for melanoma proliferation

Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5’-upstream region of GREB1 exon 19 are necessary for GREB1 Is4 transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENflox melanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

Inhibitory effect of Cyrtomium falcatum on melanogenesis in α-MSH-stimulated B16F10 murine melanoma cells

Objective: To explore the anti-melanogenic potential of Cyrtomium falcatum. Methods: The effects of Cyrtomium falcatum crude extract and its solvent fractions on tyrosinase activity, melanin content, and the expressions of melanogenesis-related genes and proteins were analyzed in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 cells. Results: α-MSH treatment significantly increased tyrosinase activity, and extracellular and intracellular melanin content, as well as the expression levels of tyrosinase, microphthalmia-associated transcription factor (MITF), tyrosinase-related protein (TRP)-1, and TRP-2 in B16F10 cells. Treatment with Cyrtomium falcatum crude extract and its solvent fractions reduced tyrosinase activity and extracellular and intracellular melanin content and downregulated the expression levels of tyrosinase, MITF, TRP-1, and TRP-2 in a dose-dependent manner. Conclusions: Cyrtomium falcatum has potential anti-melanogenesis effects and can be used as a potential source material in cosmeceutical industry for the research and development of novel lead molecules with whitening properties.

Two Novel Variants in MITF and PAX3 Associated With Splashed White Phenotypes in Horses.

Mutations causing depigmentation are relatively common in Equus caballus (horse). Over 40 alleles in multiple genes are associated with increased white spotting (as of February 2023). The splashed white phenotype, a coat spotting pattern described as appearing like the horse has been splashed with white paint, was previously associated with variants in the PAX3 and MITF genes. Both genes encode transcription factors known to control melanocyte migration and pigmentation. We report two novel mutations, a stop-gain mutation in PAX3 (XM_005610643.3:c.927C>T, ECA6:11,196,181, EquCab3.0) and a missense mutation in a binding domain of MITF (NM_001163874.1:c.993A>T, ECA16:21,559,940, EquCab3.0), each with a strong association with increased depigmentation in Pura Raza Española horses (P=1.144E-11, N=30, P=4.441E-16, N=39 respectively). Using a quantitative method to score depigmentation, the PAX3 and MITF mutations were found to have average white scores of 25.50 and 24.45, respectively, compared to the average white coat spotting score of 1.89 in the control set. The functional impact for each mutation was predicted to be moderate to extreme (I-TASSER, SMART, Variant Effect Predictor, SIFT). We propose to designate the MITF mutant allele as Splashed White 9 and the PAX3 mutant allele as Splashed White 10 per convention.

Gynostemma pentaphyllum Makino extract induces hair growth and exhibits an anti-graying effect via multiple mechanisms.

In traditional Asian medicine, Gynostemma pentaphyllum Makino leaf extract (Gp) is used to treat aging, metabolic syndrome, diabetes, and neurodegenerative diseases. Hair loss and hair-graying are common phenomena that haunt everyone. However, whether Gp activities on inhibition of hair loss and getting gray have been rarely studied. Study the Gp activity and mechanism by in vivo and in vitro experiments to explore its application on hair health. In the present study, we determined the effects of Gp on the expression of hair growth-related genes and proliferation of human dermal papilla cells (hDPCs). Furthermore, Gp was topically applied to the hair-shaved skin of male C57BL/6 mice, and the histological profile of the skin was studied. Because emotional stress may lead to melanocyte disappearance, norepinephrine-exposed mice B16 melanocytes were treated with Gp to elucidate the anti-hair graying capacity of Gp in response to this stress type. Gp stimulated the proliferation of hDPCs and the Wnt signaling pathways associated with hair growth; furthermore, the expression of the hair loss-related gene transforming growth factor-β1 was suppressed. Gp treatment significantly increased the size of hair follicles in the treated mice and stimulated them. Moreover, Gp not only increased melanin synthesis but also tyrosinase activity in B16 cells. Quantitative real-time polymerase chain reaction revealed that Gp increased melanin synthesis by increasing the expression of tyrosine-related protein-1, tyrosine-related protein-2, tyrosinase, and microphthalmia-associated transcription factor. Our study provides preclinical evidence regarding the potential of Gp as a promising hair growth and anti-graying agent.

Sargassum fusiforme Extract Induces Melanogenesis through the cAMP/PKA/CREB Signaling Pathway

The aim of this study was to investigate the effect of Sargassum fusiforme extract (SFE) on melanogenesis and its mechanism both in vitro and ex vivo. The melanogenic-inducing effect of SFE was evaluated using a melanin contents assay and a cellular tyrosinase activity assay. To investigate whether SFE could protect melanocytes against oxidative stress, hydrogen peroxidase was used. The molecular mechanism underlying the effect of SFE on melanogenesis was determined via Western blot analysis of tyrosinase, a microphthalmia-associated transcription factor (MITF), and a phosphorylated cAMP response element-binding protein (p-CREB) expression. The degree of pigmentation in a 3D skin model was determined by measuring the L* values. Contents of melanin in ex vivo human hair follicles were evaluated via Fontana–Masson staining. SFE significantly increased melanin contents and cellular tyrosinase activity in human epidermal melanocytes. SFE also increased the phosphorylation of CREB and the protein levels of tyrosinase and MITF. Moreover, SFE attenuated oxidative stress-induced cytotoxicity and depigmentation. Finally, the melanogenesis promoting effect of SFE was confirmed in both a 3D skin model and ex vivo human hair follicles. These findings suggest that SFE can induce melanogenesis via the cAMP/PKA/CREB signaling pathway in human epidermal melanocytes through its hyperpigmentation activity.

Atractylodes lancea rhizome derived exosome-like nanoparticles prevent alpha-melanocyte stimulating hormone-induced melanogenesis in B16-F10 melanoma cells

Aberrant melanin overproduction can significantly impact an individual's appearance and cause mental and psychological distress. Current inhibitors of melanin production exert harmful side effects due to inadequate selectivity; thus a need to develop more selective melanin synthesis inhibitors is necessary. Extracellular vesicles are important agents of intercellular signalling in prokaryotes and eukaryotes. Recently, plant-derived nanoparticles, similar to mammalian exosomes, have attracted attention for their use in health research. In this study, to investigate the potential of plant-derived exosome-like nanoparticles (ELNs) as inhibitors of melanin production, we used hot water to extract ELNs from the rhizome of Atractylodes lancea (A-ELNs). The size of A-ENLs ranged from 34 to 401 nm and carried three microRNA: ath-miR166f, ath-miR162a-5p, and ath-miR162b-5p. These A-ENLs were applied to B16-F10 melanoma cells treated with α-melanocyte-stimulating hormone (α-MSH). After A-ELNs were taken up by B16-F10 cells, their melanin levels were significantly reduced. Furthermore, A-ELNs significantly reduced tyrosinase activity in B16-F10 cells and mRNA expression of microphthalmia-associated transcription factor (Mitf), tyrosinase, tyrosinase-related protein 1, and DOPA chrome tautomerase. These results suggest that A-ELN suppresses melanogenic enzymes expression by downregulating Mitf, thereby inhibiting melanin synthesis. Hence, A-ELN can be developed into a novel topical drug after additional studies and optimization.

Abstract B010: Germline susceptibility to renal cell carcinoma and implications for genetic screening

Abstract Background: Genetic susceptibility to renal cell carcinoma (RCC) remains poorly understood and limited to specific hereditary cancer syndromes. Whereas few studies have investigated rare germline variants in specific populations, the majority of them have not taken into account differences between histological subtypes of RCC. Objective: To identify common risk-genes for RCC within the Canadian population, investigate their association to clinical annotations and outcomes, and compare gene-burden among RCC patients from various countries. Methods: We conducted targeted DNA sequencing of 19 RCC-related and 27 cancer predisposition genes for 960 RCC patients (759 with clear cell and 201 with non-clear cell RCC) from Canada. We identified genes enriched in rare germline pathogenic variants (PVs) in RCC compared to a cancer-free control population (gnomAD, n=118148). Gene associations to RCC subtypes and clinical annotations were examined using Firth’s logistic regression models and Fisher’s exact tests, respectively. In addition, we compared prevalence of PVs in Canadian patients to those in previous studies from Japan (n=1632), the UK (n=1336) and the USA (n=254). Furthermore, we evaluated the performance of current criteria for RCC genetic screening for including patients with PVs in Canada, the US, and the UK. Results: We identified 39 germline PVs in 56 RCC patients from the Canadian cohort. Compared to cancer-free controls, PVs in MITF (OR: 212.3, 95% CI: 4.0-6.8, p= 1.44 × 10−9), CHEK2 (OR: 4.8, 95% CI: 1.0-2.1, p=3.94 × 10−5), and ATM (OR: 4.5, 95% CI: 0.7-2.2, p=0.016) genes were significantly enriched in patients with clear cell RCC, whereas PVs in FH (OR: 215.1, 95% CI: 4.2-6.4, p=6.14 × 10−9) were enriched in patients with non-clear cell RCC. We observed an association between PVs in DNA repair genes BRCA1, BRCA2 and ATM with the presence of metastasis in ccRCC (p=0.004) and RCC overall (p=0.003). Comparisons of gene-burden to other populations showed an enrichment for TP53 in RCC patients from Japan, while RCC patients from Canada showed an enrichment for CHEK2 and ATM. RCC patients from the US showed an enrichment for germline PVs in FH in comparison to Canadians. Notably, our analysis reveals that current criteria for referral to genetic screening for hereditary renal cancer fail to include the majority (73%) of patients harboring rare germline PVs in risk genes for RCC, both in Canada and globally. Conclusions: MITF, CHEK2, ATM, and FH were identified as risk-genes for RCC within the Canadian population, while germline mutations in BRCA1/2 and ATM are associated with risk of metastasis. Globally, clinical guidelines for genetic screening in RCC fail to identify up to 80% of patients with rare germline PVs. Citation Format: Kate I. Glennon, Mikiko Endo, Yoshiaki Usui, Yusuke Iwasaki, Rodney H. Breau, Anil Kapoor, Simon Tanguay, Yukihide Momozawa, Yasser Riazalhosseini. Germline susceptibility to renal cell carcinoma and implications for genetic screening [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B010.

Modeling of pigmentation disorders associated with MITF mutation in Waardenburg syndrome revealed an impaired melanogenesis pathway in iPS-derived melanocytes.

Waardenburg Syndrome (WS) is a rare genetic disorder that leads to congenital hearing loss and pigmentation defects. Microphthalmia-associated transcription factor (MITF) is one of its significant pathogenic genes. Despite the comprehensive investigation in animal models, the pathogenic mechanism is still poorly described in humans due to difficulties accessing embryonic tissues. In this work, we used induced pluripotent stem cells derived from a WS patient carrying a heterozygous mutation in the MITF gene c.626A>T (p.His209Leu), and differentiated toward melanocyte lineage, which is the most affected cell type involved in WS. Compared with the wild-type cell line, the MITFmut cell line showed a reduced expression of the characteristic melanocyte-related genes and a lesser proportion of mature, fully pigmented melanosomes. The transcriptome analysis also revealed widespread gene expression changes at the melanocyte stage in the MITFmut cell line. The differentially expressed genes were enriched in melanogenesis and cell proliferation-related pathways. Interestingly, ion transport-related genes also showed a significant difference in MITFmut -induced melanocytes, indicating that the MITF mutant may lead to the dysfunction of potassium channels and transporters produced by intermediate cells in the cochlea, further causing the associated phenotype of deafness. Altogether, our study provides valuable insights into how MITF mutation affects WS patients, which might result in defective melanocyte development and the related phenotype based on the patient-derived iPSC model.

Role of microphthalmia-associated transcription factor in uveal melanoma and its association with clinicopathological parameters.

153 Background: High melanin content could lead to high metastatic potential in Uveal Melanoma (UM). In the Asian population, Microphthalmia-Associated Transcription Factor (MITF) regulates the melanogenesis pathway and activates Silver Protein (SILV) for eumelanin synthesis. MITF also stimulates the hypoxia–inducible factor 1-alpha (HIF-1α) gene that regulates hypoxia around the tumor microenvironment. Although their oncogenic potential has been observed in a variety of malignancies, MITF and SILV have not been investigated in UM in the Asian population. Therefore, our study aimed to detect the prognostic significance of MITF, SILV, and HIF1-α gene/protein expression levels in UM patients. Methods: Immunohistochemistry of MITF, SILV & HIF-1 α was performed on 82 UM tissue samples. Western blot of all three markers was carried out on representative cases. The mRNA expression level was done by qRT-PCR in 70 fresh UM cases. Cox proportional hazard model and log-rank test were used to determine the prognostic outcome of these markers. Results: MITF and SILV proteins were expressed in 56% and 70% of cases, respectively. High pigmentation, BAP1 loss, and HIF-1α expression were statistically correlated with MITF and SILV protein expression. Upregulation of both genes was found in more than 50% of cases at the mRNA level. This was also statistically significant with high pigmentation and HIF-1α mRNA expression. High expression of MITF protein showed reduced disease-free survival. Conclusions: Our study highlighted the fact that MITF and SILV could be potential biomarkers in UM development. Hence, it might play a key role in future therapeutic target.

Histone acetylation regulates BMMCs recognition of foot-and-mouth disease virus-like particles.

Foot-and-mouth disease (FMD) is one of the most economically and socially devastating diseases affecting animal agriculture worldwide. Foot-and-mouth disease virus (FMDV) virus-like particles (VLPs) have been widely studied as a candidate vaccine. Mast cells (MCs) are highly versatile innate immunity cells that perform various functions in regulating innate and adaptive immune responses. Recently, we found that MCs can recognize recombinant FMDV VP1-VP4 protein to produce various cytokines with differential expression, suggesting that this may be epigenetically regulated. In this study, we evaluated the effect of trichostatin A (TSA), a histone deacetylase inhibitor, on bone marrow-derived mast cells (BMMCs) recognition of FMDV-VLPs in vitro. BMMCs can recognize FMDV-VLPs via mannose receptors (MRs) and resulted in enhanced expression and secretion of tumour necrosis factor α (TNF-α) and interleukin (IL)-13. Nevertheless, BMMCs recognition of FMDV-VLPs to secrete IL-6 was irrelevant to MRs, and MRs may play a negative regulation for IL-10 secretion. Pre-treatment with TSA caused decreased expression of IL-6, TNF-α and IL-13, and increased expression of IL-10. Furthermore, the expression of nuclear factor-kappa B (NF-κB) was supressed in TSA treated BMMCs, suggesting histone acetylation may alter NF-κB expression to influence the TNF-α and IL-13 secretion. Pre-treatment with TSA had no influence on the expression of microphthalmia-associated transcription factor (MITF) and GATA-2. These data therefore suggest that altered histone acetylation regulates the immune responses induced by BMMCs recognition of FMDV-VLPs, providing an understanding and theory basis for the prevention and control of FMD based MCs.

Inhibitory Effects of Polyphenol- and Flavonoid-Enriched Rice Seed Extract on Melanogenesis in Melan-a Cells via MAPK Signaling-Mediated MITF Downregulation.

Melanin production is an important process that prevents the host skin from harmful ultraviolet radiation; however, an overproduction of melanin results in skin diseases. In the present study, we determined the antioxidative and anti-melanogenic activities of polyphenol- and flavonoid-enriched rice seed extracts in melan-a cells. The polyphenol and flavonoid content of Hopum (HP) and Sebok (SB) rice seed extracts was measured. The antioxidant capacity was determined using the ABTS radical scavenging method. SB contained high amounts of polyphenols and flavonoids, which significantly increased antioxidative activity compared with HP. Various concentrations of these extracts were evaluated in a cytotoxicity using melan-a cells. At 100 µg/mL, there was no significant difference for all treatments compared with untreated cells. Therefore, 100 µg/mL was selected as a concentration for the further experiments. SB significantly suppressed the phosphorylation/activation of p-38 MAPK, increased the expression of phosphorylated ERK 1/2 and Akt, and downregulated the microphthalmia-associated transcription factor (MITF). This resulted in decreased levels of tyrosinase and tyrosinase-related protein-1 and -2. These results indicate the potential of polyphenol- and flavonoid-enriched rice seed as a treatment for hyperpigmentation.

Citrulluside T, Isolated from the Citrullus lanatus Stem, Inhibits Melanogenesis in α-MSH-Induced Mouse B16F10 Cells

With the increasing number of cosmetic consumers emphasizing value consumption and sustainability, upcycling has gained attention as a solution to agricultural by-products, which are the main culprits of environmental problems. In this study, we isolated citrulluside T with whitening activity from discarded Citrullus lanatus stems and investigated the anti-melanogenic effect of citrulluside T and the underlying mechanisms. We found that citrulluside T did not exhibit cytotoxicity up to a concentration of 90 μM and significantly reduced the melanin content and intracellular tyrosinase activity in B16F10 cells. In addition, citrulluside T inhibited the expression of melanogenic enzymes such as tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2, as well as melanin synthesis via cAMP-dependent protein kinase (PKA)/cAMP response element-binding protein (CREB)-mediated downregulation of microphthalmia-associated transcription factor (MITF), a key transcription factor in melanogenesis. Furthermore, we found that citrulluside T exerted its anti-melanogenic effect by downregulating the β-catenin protein and upregulating phosphorylated β-catenin. Finally, we confirmed that citrulluside T was safe for skin through skin irritation tests on 33 subjects, suggesting its applicability as a protective agent against hyperpigmentation for topical applications such as cosmetics and ointments.

Novel Insights on 6:6 Perfluoroalkyl Phosphonic Acid-Induced Melanin Synthesis Disorders Leading to Pigmentation in Tadpoles.

As alternatives to traditional per- and polyfluoroalkyl substances, perfluoroalkyl phosphonic acids (PFPiAs) are widely present in aquatic environments and can potentially harm aquatic organisms. Pigmentation affects the probability of aquatic organisms being preyed on and serves as an important toxic endpoint of development, but little is known about the impacts of PFPiAs on the development of aquatic organisms. In this study, Xenopus laevis embryos were exposed to 6:6 PFPiA (1, 10, and 100 nM) for 14 days. The developed tadpoles exhibited evident pigmentation with increased melanin particle size and density on the skin. Pathological and behavioral experiments revealed that the retinal layers became thinner, reducing the photosensitivity and disturbing the circadian rhythm of the tadpoles. Compared to the control group, the exposed tadpoles showed higher levels but less changes of melanin throughout the light/dark cycle, as well as distinct oxidative damage. Consequently, the expression level of microphthalmia-associated transcription factor (MITF), a key factor inducing melanin synthesis, increased significantly. Molecular docking analysis suggested that 6:6 PFPiA forms strong interactions in the binding pocket of MITF, implying that it could activate MITF directly. The activation of MITF ultimately promotes melanin synthesis, resulting in pigmentation on tadpoles.

Progressive Cone-Rod Dystrophy and RPE Dysfunction in Mitfmi/+ Mice.

Mutations in the mouse microphthalmia-associated transcription factor (Mitf) gene affect retinal pigment epithelium (RPE) differentiation and development and can lead to hypopigmentation, microphthalmia, deafness, and blindness. For instance, an association has been established between loss-of-function mutations in the mouse Mitf gene and a variety of human retinal diseases, including Waardenburg type 2 and Tietz syndromes. Although there is evidence showing that mice with the homozygous Mitfmi mutation manifest microphthalmia and osteopetrosis, there are limited or no data on the effects of the heterozygous condition in the eye. Mitf mice can therefore be regarded as an important model system for the study of human disease. Thus, we characterized Mitfmi/+ mice at 1, 3, 12, and 18 months old in comparison with age-matched wild-type mice. The light- and dark-adapted electroretinogram (ERG) recordings showed progressive cone-rod dystrophy in Mitfmi/+ mice. The RPE response was reduced in the mutant in all age groups studied. Progressive loss of pigmentation was found in Mitfmi/+ mice. Histological retinal sections revealed evidence of retinal degeneration in Mitfmi/+ mice at older ages. For the first time, we report a mouse model of progressive cone-rod dystrophy and RPE dysfunction with a mutation in the Mitf gene.

Histopathological differences between vitiligo and lichen sclerosus et atrophicus using quantitative immunohistochemical analysis.

Lichen sclerosus et atrophicus (LS) is rare skin condition characterized by the presence of whitish patches primarily affecting the genital and perianal areas, though it can occur other parts of the body. LS may result in skin depigmentation without textural changes and should be differentiated from vitiligo. However, the histopathological features of hypopigmentation during vitiligo and LS have rarely been compared and have not been precisely described using quantitative immunohistochemical analysis. This study, therefore, aimed to investigate and compare the pigmentary characteristics of LS and vitiligo lesions using histochemical and immunohistochemical staining. We included 31 and 46 patients diagnosed with LS and vitiligo, respectively, at Ajou University Hospital between March 2009 and March 2020 in this study. Their medical charts and skin biopsy specimens were retrospectively reviewed. Additionally, Fontana-Masson staining for melanin and immunohistochemical staining for Melan-A, NKI/beteb, tyrosinase, and microphthalmia-associated transcription factor was performed. The melanin content, as well as the number of melanocytes was, in general, significantly higher in the epidermis of patients in the LS group compared with that in the vitiligo group. However, 22.6% of LS tissues showed less melanin pigmentation, 25.8% of LS specimens exhibited a lower number of melanocytes, and 29.0% of LS specimens demonstrated less melanocyte activity when compared with the average of vitiligo specimens. As lower melanin pigmentation and the near absence number of melanocytes were also observed in several LS specimens, both the clinical and histological findings must be comprehensively reviewed to differentiate vitiligo from LS.

EGCG, GCG, TFDG, or TSA Inhibiting Melanin Synthesis by Downregulating MC1R Expression

Without affecting cell viability, epigallocatechin gallate (EGCG), gallocatechin gallate (GCG), theaflavine-3,3′-digallate (TFDG), or theasinensin A (TSA) have been found to effectively reduce intracellular melanin content and tyrosinase (TYR) activity. However, studies on the anti-melanogenic mechanism of the above samples remain weak, and the activities of these samples in regulating melanogenesis at the molecular level lack comparison. Using B16F10 cells with the α-melanocyte-stimulating hormone (α-MSH) stimulation and without the α-MSH stimulation as models, the effects of EGCG, GCG, TFDG, or TSA on cell phenotypes and expression of key targets related to melanogenesis were studied. The results showed that α-MSH always promoted melanogenesis with or without adding the four samples. Meanwhile, the anti-melanogenic activities of the four samples were not affected by whether the α-MSH was added in the medium or not and the added time of the α-MSH. On this basis, the 100 µg/mL EGCG, GCG, TFDG, or TSA did not affect the TYR catalytic activity but inhibited melanin formation partly through downregulating the melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), and the TYR family. The downregulation abilities of catechins on the TYR family and MITF expression were stronger than those of dimers at both the transcription and translation levels, while the ability of dimers to downregulate the MC1R expression was stronger than that of catechins at both the transcription and translation levels to some extent. The results of molecular docking showed that these four samples could stably bind to MC1R protein. Taken together, this study offered molecular mechanisms for the anti-melanogenic activity of the EGCG, GCG, TFDG, and TSA, as potential effective components against the UV-induced tanning reactions, and a key target (MC1R) was identified.