MITF Protein Research Articles

Edible bird's nest peptide (EBNP) with high whitening activity: Sequences analysis, whitening activity characterization and molecular docking study

At present, more and more researchers found that peptides have strong physiological activity. In this study, the effects of Edible bird's nest peptide (EBNP) on whitening were investigated in vitro and in vivo. It was found that EBNP reduced melanin content and inhibited tyrosinase activity of B16 cells and zebrafish in a concentration-dependent manner. Additionally, EBNP significantly down-regulated the expression of MITF, TYR, TYRP1 and TYRP2 genes in B16 cells at 500 μg/mL (p < 0.05) and the expression of MITF and TYR genes in zebrafish at 50 μg/mL (p < 0.05). After the zebrafish was treated with EBNP (50 μg/mL), the expression of MITF and TYR genes was down-regulated by 61.51 % and 87.00 %, respectively. Molecular docking showed that peptide FRAF mainly bound to MITF, TYR, TYRP1 and TYRP2 proteins by hydrogen bonds and electrostatic interactions. These results suggested that EBNP was a promising whitening material for food, skin care and pharmaceutical industries.

Anti-Melanogenic Activity of Ethanolic Extract from Garcinia atroviridis Fruits Using In Vitro Experiments, Network Pharmacology, Molecular Docking, and Molecular Dynamics Simulation.

Melanin, the pigment responsible for human skin color, increases susceptibility to UV radiation, leading to excessive melanin production and hyperpigmentation disorders. This study investigated the ethanolic extract of Garcinia atroviridis fruits for its phenolic and flavonoid contents, antioxidant activity, and impact on melanogenesis pathways using qRT-PCR and Western blot analysis. Utilizing network pharmacology, molecular docking, and dynamics simulations, researchers explored G. atroviridis fruit extract's active compounds, targets, and pharmacological effects on hyperpigmentation. G. atroviridis fruit extract exhibited antioxidant properties, scavenging DPPH• and ABTS•+ radicals radicals and chelating copper. It inhibited cellular tyrosinase activity and melanin content in stimulated B16F10 cells, downregulating TYR, TRP-1, phosphorylated CREB, CREB, and MITF proteins along with transcription levels of MITF, TYR, and TRP-2. LC-MS analysis identified thirty-three metabolites, with seventeen compounds selected for further investigation. Network pharmacology revealed 41 hyperpigmentation-associated genes and identified significant GO terms and KEGG pathways, including cancer-related pathways. Kaempferol-3-O-α-L-rhamnoside exhibited high binding affinity against MAPK3/ERK1, potentially regulating melanogenesis by inhibiting tyrosinase activity. Stable ligand-protein interactions in molecular dynamics simulations supported these findings. Overall, this study suggests that the ethanolic extract of G. atroviridis fruits possesses significant antioxidant, tyrosinase inhibitory, and anti-melanogenic properties mediated through key molecular targets and pathways.

Germacrone, isolated from Curcuma wenyujin, inhibits melanin synthesis through the regulation of the MAPK signaling pathway.

Abnormal melanin synthesis causes hyperpigmentation disorders, such as chloasma, freckles, and melanoma, which are highly multiple and prevalent. There were few reports on the anti-melanogenic effect of Curcuma wenyujin Y.H. Chen et C. Ling, and the bioactive compound has not been elucidated as well. The study aims to investigate the anti-melanogenic effect of C. wenyujin, and identify the bioactive compound, and further explore its underlying mechanism. Our results showed that the Petroleum ether fraction extracted from C. wenyujin rhizome had a significant anti-melanogenic effect, and germacrone isolated from it was confirmed as the major bioactive compound. To our data, germacrone significantly inhibited tyrosinase (TYR) activity, reduced melanosome synthesis, reduced dendrites formation of B16F10 cells, and melanosome transport to keratinocytes. Moreover, germacrone effectively decreased the hyperpigmentation in zebrafish and the skin of guinea pigs in vivo. Western-blot analysis showed that germacrone down-regulated the expression of TYR, TRP-1, TRP-2, Rab27a, Cdc42, and MITF proteins via the activation of the MAPK signaling pathway. Taken together, germacrone is an effective bioactive compound for melanogenesis inhibition. Our studies suggest that germacrone may be considered a potential candidate for skin whitening.

Fast freezing inhibits melanin synthesis of melanocytes by modulating the Wnt/β-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-GSK3β and β-catenin and the nuclear accumulation of β-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/β-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.

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.

Melanogrit potentiates melanogenesis by escalating cellular tyrosinase activity and MITF levels via pERK inhibition.

Vitiligo is characterized by the development of white patches on the skin either due to the loss of functional melanocytes or perturbations in the melanogenesis pathway. In the present study, we investigated the therapeutic potential of herbo-mineral formulation, Melanogrit in neutralizing the white patches in the skin. The study utilized UPLC/MS-QToF technique to determine the diversified phytochemical profile in Melanogrit. The murine B16F10 cells when treated with Melanogrit underwent morphological changes, including increased angularity, enlarged cell size, and greater dendritic protrusions. To establish an equivalent model to study melanogenesis, we carefully optimized the dosage of α-melanocyte stimulating hormone (αMSH) in B16F10 cells as an alternative to using melanocyte-keratinocyte cocultures. The study determined a sub-optimal dose of αMSH (0.2 nM) in B16F10 cells that does not manifest any measurable effects on melanogenesis. In contrast, Melanogrit when used in conjunction with 0.2 nM αMSH, induced a dose-dependent increase in extracellular and intracellular melanin levels. Melanogrit transcriptionally up-regulated the decisive genes of the melanogenesis pathway, MITF, TYR, and TRP1, which was evident from the increased cellular tyrosine activity. Our findings also demonstrated that Melanogrit ameliorated the MITF protein levels by inhibiting pERK; notably without involving GSK3β in the process. Taken together, our findings strongly suggest that Melanogrit has the potential to stimulate melanogenesis, making it a promising candidate for clinical applications in the treatment of white skin patches that develop in vitiligo patients.

Syringetin Promotes Melanogenesis in B16F10 Cells.

Syringetin, an active compound present in red grapes, jambolan fruits, Lysimachia congestiflora, and Vaccinium ashei, is a dimethyl myricetin derivative which contains free hydroxyl groups at the C-2' and C-4' positions in ring B. Recent studies have revealed that syringetin possesses multiple pharmacological properties, such as antitumor, hepatoprotective, antidiabetic, antioxidative, and cytoprotective activities. To date, there has been no attempt to test the action of syringetin on melanogenesis. In addition, the molecular mechanism for the melanogenic effects of syringetin remains largely unknown. In this study, we investigated the effect of syringetin on melanogenesis in a murine melanoma cell line from a C57BL/6J mouse, B16F10. Our results showed that syringetin markedly stimulated melanin production and tyrosinase activity in a concentration-dependent manner in B16F10 cells. We also found that syringetin increased MITF, tyrosinase, TRP-1, and TRP-2 protein expression. Moreover, syringetin inhibited ERK and PI3K/Akt phosphorylation by stimulating p38, JNK, PKA phosphorylation levels, subsequently stimulating MITF and TRP upregulation, resulting in the activation of melanin synthesis. Furthermore, we observed that syringetin activated phosphorylation of GSK3β and β-catenin and reduced the protein level of β-catenin, suggesting that syringetin stimulates melanogenesis through the GSK3β/β-catenin signal pathway. Finally, a primary skin irritation test was conducted on the upper backs of 31 healthy volunteers to determine the irritation or sensitization potential of syringetin for topical application. The results of the test indicated that syringetin did not cause any adverse effects on the skin. Taken together, our findings indicated that syringetin may be an effective pigmentation stimulator for use in cosmetics and in the medical treatment of hypopigmentation disorders.

6-Methylcoumarin Promotes Melanogenesis through the PKA/CREB, MAPK, AKT/PI3K, and GSK3β/β-Catenin Signaling Pathways.

We investigated the effects of four coumarin derivatives, namely, 6-methylcoumarin, 7-methylcoumarin, 4-hydroxy-6-methylcoumarin, and 4-hydroxy-7-methylcoumarin, which have similar structures on melanogenesis in a murine melanoma cell line from a C57BL/6J mouse called B16F10. Our results showed that only 6-methylcoumarin significantly increased the melanin synthesis in a concentration-dependent manner. In addition, the tyrosinase, TRP-1, TRP-2, and MITF protein levels were found to significantly increase in response to 6-methylcoumarin in a concentration-dependent manner. To elucidate the molecular mechanism whereby 6-methylcoumarin-induced melanogenesis influences the melanogenesis-related protein expression and melanogenesis-regulating protein activation, we further assessed the B16F10 cells. The inhibition of the ERK, Akt, and CREB phosphorylation, and conversely, the increased p38, JNK, and PKA phosphorylation activated the melanin synthesis via MITF upregulation, which ultimately led to increased melanin synthesis. Accordingly, 6-methylcoumarin increased the p38, JNK, and PKA phosphorylation in the B16F10 cells, whereas it decreased the phosphorylated ERK, Akt, and CREB expressions. In addition, the 6-methylcoumarin activated GSK3β and β-catenin phosphorylation and reduced the β-catenin protein level. These results suggest that 6-methylcoumarin stimulates melanogenesis through the GSK3β/β-catenin signal pathway, thereby affecting the pigmentation process. Finally, we tested the safety of 6-methylcoumarin for topical applications using a primary human skin irritation test on the normal skin of 31 healthy volunteers. We found that 6-methylcoumarin did not cause any adverse effects at concentrations of 125 and 250 μM. Our findings indicate that 6-methylcoumarin may be an effective pigmentation stimulator for use in cosmetics and the medical treatment of photoprotection and hypopigmentation disorders.

Melanoma Clinical Decision Support System: An Artificial Intelligence-Based Tool to Diagnose and Predict Disease Outcome in Early-Stage Melanoma Patients.

This study set out to assess the performance of an artificial intelligence (AI) algorithm based on clinical data and dermatoscopic imaging for the early diagnosis of melanoma, and its capacity to define the metastatic progression of melanoma through serological and histopathological biomarkers, enabling dermatologists to make more informed decisions about patient management. Integrated analysis of demographic data, images of the skin lesions, and serum and histopathological markers were analyzed in a group of 196 patients with melanoma. The interleukins (ILs) IL-4, IL-6, IL-10, and IL-17A as well as IFNγ (interferon), GM-CSF (granulocyte and macrophage colony-stimulating factor), TGFβ (transforming growth factor), and the protein DCD (dermcidin) were quantified in the serum of melanoma patients at the time of diagnosis, and the expression of the RKIP, PIRIN, BCL2, BCL3, MITF, and ANXA5 proteins was detected by immunohistochemistry (IHC) in melanoma biopsies. An AI algorithm was used to improve the early diagnosis of melanoma and to predict the risk of metastasis and of disease-free survival. Two models were obtained to predict metastasis (including "all patients" or only patients "at early stages of melanoma"), and a series of attributes were seen to predict the progression of metastasis: Breslow thickness, infiltrating BCL-2 expressing lymphocytes, and IL-4 and IL-6 serum levels. Importantly, a decrease in serum GM-CSF seems to be a marker of poor prognosis in patients with early-stage melanomas.

α-Mangostin Suppresses Melanoma Growth, Migration, and Invasion and Potentiates the Anti-tumor Effect of Chemotherapy.

Purpose: Melanoma is a highly malignant tumor, which metastasizes and has poor prognosis in late-stage cancer patients. α-Mangostin possesses pharmacological properties, including antioxidant, anti-infective, and anticarcinogenic activities. We investigated α-Mangostin effect on melanoma growth, migration, and invasion and its possible molecular mechanism. Methods: Melanoma cells growth inhibition was determined by the colorimetric 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. Morphological changes of α-Mangostin-treated melanoma cells were evaluated by transmission electron microscopy and JC-1 staining. Cell apoptosis and cell cycle arrest were assessed by flow cytometry. The effect of α-Mangostin on tumor cells migration and invasion was observed by migration and invasion in vitro assay. Furthermore, the nude and C57BL/6 mouse subcutaneous melanoma models were used to evaluate the in vivo anti-tumor effect of α-Mangostin. Western blot and real time-PCR were performed to analyze the influence of α-Mangostin on some of the common signaling pathways in melanoma cell lines. Signaling pathways were further verified in dissected tumor tissues. Results: α-Mangostin inhibited in vitro melanoma cells proliferation, migration, and invasion of melanoma cells, induced cell cycle arrest in G0/G1 phase, and caused mitochondrial swelling and membrane depolarization, whereas it effectively suppressed melanoma growth in xenografted mice. In addition, α-Mangostin potentiated the in vitro and in vivo anti-tumor effects of cisplatin both in vitro and in vivo. Mechanistically, α-Mangostin down-regulated expression of RAS protein and mRNA, as well as phosphorylation of PI3K in A375, B16F10, M14 and SK-MEL-2 cells. MITF protein and mRNA were inhibited only in M14 cells. Conclusion: α-Mangostin suppresses melanoma cells growth, migration and invasion, and synergistically enhances the anti-tumor effect of chemotherapy, whose mechanism may be mediated through inhibiting Ras, PI3K and MITF.

Interaction between moxifloxacin and Mcl-1 and MITF proteins: the effect on growth inhibition and apoptosis in MDA-MB-231 human triple-negative breast cancer cells

BackgroundMicrophthalmia-associated transcription factor (MITF) activates the expression of genes involved in cellular proliferation, DNA replication, and repair, whereas Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing apoptosis. The objective of the present study was to verify whether the interaction between moxifloxacin (MFLX), one of the fluoroquinolones, and MITF/Mcl-1 protein, could affect the viability, proliferation, and apoptosis in human breast cancer using both in silico and in vitro models.MethodsMolecular docking analysis (in silico), fluorescence image cytometry, and Western blot (in vitro) techniques were applied to assess the contribution of MITF and Mcl-1 proteins in the MFLX-induced anti-proliferative and pro-apoptotic effects on the MDA-MB-231 breast cancer cells.ResultsWe indicated the ability of MFLX to form complexes with MITF and Mcl-1 as well as the drug’s capacity to affect the expression of the tested proteins. We also showed that MFLX decreased the viability and proliferation of MDA-MB-231 cells and induced apoptosis via the intrinsic death pathway. Moreover, the analysis of the cell cycle progression revealed that MFLX caused a block in the S and G2/M phases.ConclusionsWe demonstrated for the first time that the observed effects of MFLX on MDA-MB-231 breast cancer cells (growth inhibition and apoptosis induction) could be related to the drug’s ability to interact with MITF and Mcl-1 proteins. Furthermore, the presented results suggest that MITF and Mcl-1 proteins could be considered as the target in the therapy of breast cancer.Graphical abstract

The critical role of glutathione redox homeostasis towards oxidation in ermanin-induced melanogenesis

Vitiligo is a depigmented disease featured as diagnosis simplicity and cure difficulty. Its occurrence and development are associated with a variety of factors, including oxidative stress, heredity and immunity, etc. Existing drugs for the treatment of vitiligo are to reduce the death of melanocytes and induce pigment accumulation as the main treatment strategy. Ermanin, a member of the flavonoids, is extracted from bee glue which is wildly used to treat vitiligo in traditional Chinese medicine. Therefore, this article discusses the relationship between melanogenesis and glutathione redox homeostasis by ermanin via biochemical and free radical approaches in vivo and in vitro. In this study, we found that ermanin effectively increased the melanin content at the in vivo model (zebrafish). Moreover, the melanin levels at the in vitro models (B16F10 cells and primary melanocytes) were also increased significantly accompanied with a shift of glutathione redox homeostasis towards oxidation.Ermanin also significantly enhanced the activity of tyrosinase. Meanwhile, ermanin increased the expression levels of TYR, TRP-1, and DCT genes, while ROS accumulation and glutathione depletion mediated the accumulation of pigments caused by ermanin, which increased the production of pigments and regulated the expression mRNA levels of TYR and DCT genes. From the perspective of pigment production regulation pathways, western blot showed that the pigment accumulation caused by ermanin was closely related to the CREB-MITF pathways, it activated CREB, TYR, TRP-1, and DCT proteins. The use of CREB specific inhibitor 666-15 and MITF inhibitor ML329 confirmed that the pigment accumulation caused by ermanin was positively correlated with CREB and MITF proteins. Our findings revealed the potential mechanisms by which ermanin promoted the production of melanin through activated CREB-MITF signaling pathway and glutathione redox homeostasis towards oxidation function as a signal are beneficial to melanin production and will help develop novel therapeutic approaches for vitiligo.

Waardenburg syndrome type 2A in a large Iranian family with a novel MITF gene mutation

BackgroundThe characteristics of Waardenburg syndrome (WS) as a scarce heritable disorder are sensorineural hearing loss and deficits of pigmentation in the skin, hair, and eye. Here, clinical features and detection of the mutation in the MITF gene of WS2 patients are reported in a sizable Iranian family.MethodsA man aged 28-years represented with symptoms of mild unilateral hearing loss (right ear), complete heterochromia iridis, premature graying prior to 30 years of age, and synophrys. In this research, there was a sizable family in Iran comprising three generations with seven WS patients and two healthy members. Whole exome sequencing was applied for proband for the identification of the candidate genetic mutations associated with the disease. The detected mutation in proband and investigated family members was validated by PCR-Sanger sequencing.ResultsA novel heterozygous mutation, NM_198159.3:c.1026dup p.(Asn343Glufs*27), in exon 9 of the MITF gene co-segregated with WS2 in the affected family members. The variant was forecasted as a disease-causing variant by the Mutation Taster. According to the UniProt database, this variant has been located in basic helix-loop-helix (bHLH) domain of the protein with critical role in DNA binding.ConclusionsA frameshift was caused by a nucleotide insertion, c.1026dup, in exon 9 of the MITF gene. This mutation is able to induce an early termination, resulting in forming a truncated protein capable of affecting the normal function of the MITF protein. Helpful information is provided through an exactly described mutations involved in WS to clarify the molecular cause of clinical characteristics of WS and have a contribution to better genetic counseling of WS patients.

Deubiquitination of MITF-M Regulates Melanocytes Proliferation and Apoptosis.

Microphthalmia-associated transcription factor-M (MITF-M) is the key gene in the proliferation and differentiation of melanocytes, which undergoes an array of post-translation modifications. As shown in our previous study, deubiquitinase USP13 is directly involved in melanogenesis. However, it is still ambiguous that the effect of USP13-mediated MITF-M expression on melanocytes proliferation and apoptosis. Herein, we found that MITF-M overexpressing melanocytes showed high cell proliferation, reduced apoptosis, and increased melanin levels. Besides, melanin-related genes, TYR, DCT, GPNMB, and PMEL, were significantly up-regulated in MITF-M overexpressing melanocytes. Furthermore, Exogenous USP13 significantly upregulated the endogenous MITF-M protein level, downregulated USP13 significantly inhibited MITF-M protein levels, without altering MITF-M mRNA expression. In addition, USP13 upregulation mitigated the MITF-M degradation and significantly increased the half-life of MITF-M. Also, USP13 stabilized the exogenous MITF protein levels. In conclusion, the MITF-M level was regulated by USP13 deubiquitinase in melanocytes, affecting melanocytes proliferation and apoptosis. This study provides the theoretical basis for coat color transformation that could be useful in the development of the new breed in fur animals.

Novel Chemically Modified Curcumin (CMC) Derivatives Inhibit Tyrosinase Activity and Melanin Synthesis in B16F10 Mouse Melanoma Cells.

Skin hyperpigmentation disorders arise due to excessive production of the macromolecular pigment melanin catalyzed by the enzyme tyrosinase. Recently, the therapeutic use of curcumin for inhibiting tyrosinase activity and production of melanin have been recognized, but poor stability and solubility have limited its use, which has inspired synthesis of curcumin analogs. Here, we investigated four novel chemically modified curcumin (CMC) derivatives (CMC2.14, CMC2.5, CMC2.23 and CMC2.24) and compared them to the parent compound curcumin (PC) for inhibition of in vitro tyrosinase activity using two substrates for monophenolase and diphenolase activities of the enzyme and for diminution of cellular melanogenesis. Enzyme kinetics were analyzed using Lineweaver-Burk and Dixon plots and nonlinear curve-fitting to determine the mechanism for tyrosinase inhibition. Copper chelating activity, using pyrocatechol violet dye indicator assay, and antioxidant activity, using a DPPH radical scavenging assay, were also conducted. Next, the capacity of these derivatives to inhibit tyrosinase-catalyzed melanogenesis was studied in B16F10 mouse melanoma cells and the mechanisms of inhibition were elucidated. Inhibition mechanisms were studied by measuring intracellular tyrosinase activity, cell-free and intracellular α-glucosidase enzyme activity, and effects on MITF protein level and cAMP maturation factor. Our results showed that CMC2.24 showed the greatest efficacy as a tyrosinase inhibitor of all the CMCs and was better than PC as well as a popular tyrosinase inhibitor-kojic acid. Both CMC2.24 and CMC2.23 inhibited tyrosinase enzyme activity by a mixed mode of inhibition with a predominant competitive mode. In addition, CMC2.24 as well as CMC2.23 showed a comparable robust efficacy in inhibiting melanogenesis in cultured melanocytes. Furthermore, after removal of CMC2.24 or CMC2.23 from the medium, we could demonstrate a partial recovery of the suppressed intracellular tyrosinase activity in the melanocytes. Our results provide a proof-of-principle for the novel use of the CMCs that shows them to be far superior to the parent compound, curcumin, for skin depigmentation.

Diphlorethohydroxycarmalol inhibits melanogenesis via protein kinase A/cAMP response element-binding protein and extracellular signal-regulated kinase-mediated microphthalmia-associated transcription factor downregulation in α-melanocyte stimulating hormone-stimulated B16F10 melanoma cells and zebrafish.

Diphlorethohydroxycarmalol (DPHC) is a marine polyphenolic compound derived from brown alga Ishige okamurae. A previously study has suggested that DPHC possesses strong mushroom tyrosinase inhibitory activity. However, the anti-melanogenesis effect of DPHC has not been reported at cellular level. The objective of the present study was to clarify the melanogenesis inhibitory effect of DPHC and its molecular mechanisms in murine melanoma cells (B16F10) and zebrafish model. DPHC significantly inhibited tyrosinase activity and melanin content dose-dependently in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. This polyphenolic compound also suppressed the expression of phosphorylation of cAMP response element-binding protein (CREB) by attenuating phosphorylation of cAMP-dependent protein kinase A, resulting in decreased MITF expression levels. Furthermore, DPHC downregulated MITF protein expression levels by promoting the phosphorylation of extracellular signal-regulated kinase. It also inhibited tyrosinase, tyrosinase-related protein 1 (TRP-1), and TRP-2 in α-MSH stimulated B16F10 cells. In in vivo studies using zebrafish, DPHC also markedly inhibited melanin synthesis in a dose-dependent manner. These results demonstrate that DPHC can effectively inhibit melanogenesis in melanoma cells in vitro and in zebrafish in vivo, suggesting that DPHC could be applied in fields of pharmaceutical and cosmeceuticals as a skin-whitening agent. Significance of study: The present study showed for the first time that DPHC could inhibit a-MSH-stimulated melanogenesis via PKA/CREB and ERK pathway in melanoma cells. It also could inhibit pigmentation in vivo in a zebrafish model. This evidence suggests that DPHC has potential as a skin whitening agent. Taken together, DPHC could be considered as a novel anti-melanogenic agent to be applied in cosmetic, food, and medical industry.

Analysis of genetic characteristics in two Chinese children of type Ⅱ Waardenburg syndrome

Objective: To screen and analyze the mutations of MITF gene in two children of type Ⅱ Waardenburg syndrome (WS2) from different families in Yunnan,China,and to explore the possible molecular pathogenesis. Methods: With informed consent, medical history collection, physical examinations, audiological evaluation, and high resolution computer tomography (HRCT) scan of temporal bone were performed on the two WS2 probands and their family members. Genomic DNA was extracted from peripheral blood of all individuals. The coding regions including all exons, part of introns and promoters of MITF, PAX3, SOX10, SNAI2, END3, ENDRB, and KITLG genes were sequenced by high-throughput sequencing. According to the results of high-throughput sequencing, pathogenic mutations detected in the probands and their parents were verified by Sanger sequencing. Results: The proband 1 carried c.641_643delGAA mutation in the 7th exon of MITF gene, which was a frame-shift mutation resulting in an amino acid change of p.214delR. It was a de novo mutation as the parents of proband 1 showed no variation on this site. The proband 2 carried heterozygous loss of the large fragment ranging from exon 1 to exon 9 of MITF gene, which defected the function of MITF protein. Conclusion: Genetic examinations provide important evidence for diagnosis of Waardenburg syndrome. Heterozygous mutation c.641_643delGAA and heterozygous loss of the large fragment ranging from exon 1 to exon 9 of MITF gene might be the molecular pathogenesis of the two WS2 probands in this study.

Serum levels of miRNA-21-5p in vitiligo patients and effects of miRNA-21-5p on SOX5, beta-catenin, CDK2 and MITF protein expression in normal human melanocytes

BackgroundEpigenetics of vitiligo was evaluated in few studies. In particular, the role of miR-21, a microRNA involved in various processes, including melanogenesis, was never investigated. ObjectiveEvaluation of serum levels of miR-21-5p in vitiligo patients and miR-21-5p effects on melanogenesis. MethodsWe measured serum levels of miR-21-5p in 40 patients affected by nonsegmental vitiligo and 40 sex- and age-matched healthy controls. Next, normal human melanocytes were transfected with miR-21-5p to study the effects of this microRNA, which targeted some proteins involved in melanogenesis pathway like SOX5, beta-catenin, cyclin-dependent kinase 2 (CDK2), and MITF. ResultsThe expression of miR-21-5p in vitiligo patients was 3.6–4454.4 fold (mean 990.4 ± 1397.9) higher than in controls. The relative expression of miR-21-5p was directly and significantly correlated with disease severity, defined by VASI (Vitiligo Area and Severity Index) score (Rho = 0.89, p = 10−7), but not other individual or clinical characteristics. In the second part of the study, a significant reduction of SOX5, beta-catenin and CDK2 protein expression and increase of MITF protein expression was observed in cultured melanocytes after 24 h trasfection with miR-21-5p. ConclusionAccording to literature, miR-21-5p upregulation and consequent SOX5 downregulation should upregulate melanogenesis, while vitiligo is characterized by skin depigmentation. Our results suggest that current knowledge of the pathogenesis of vitiligo is probably incomplete. Clinical manifestations could result from an altered balance between metabolic pathways with contrasting effects. In this view, miR-21-5p upregulation might be a tentative compensation mechanism.Further studies appear necessary to confirm and better understand our results and their importance.

CMT-308, a Nonantimicrobial Chemically-Modified Tetracycline, Exhibits Anti-Melanogenic Activity by Suppression of Melanosome Export

CMT-308 is a nonantimicrobial chemically-modified tetracycline (CMT), which we have previously shown exhibits antifungal activity and pleiotropic anti-inflammatory activities, including inhibition of the enzymatic activity of matrix metalloproteinases (MMPs). Based on its chemical structure, we hypothesized that CMT-308 could inhibit melanogenesis and might be a candidate for the treatment of skin hyperpigmentation disorders which occur due to unregulated melanin biosynthesis and/or transport. CMT-308 was first studied for any effects on activity of the enzyme tyrosinase in vitro using a purified preparation of mushroom tyrosinase; the mode of inhibition of the soluble fungal enzyme was evaluated by Lineweaver-Burk and Dixon plots as well as by non-linear least squares fitting. Next, the effects of CMT-308 were tested in mammalian cell cultures using B16F10 mouse melanoma cells and further validated in darkly-pigmented human melanocytes (HEMn-DP). Our results showed that micromolar concentrations of CMT-308 inhibited mushroom tyrosinase enzyme activity, using the first two substrates in the melanogenesis pathway (l-tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA)); CMT-308 inhibited mushroom tyrosinase primarily via a mixed mode of inhibition, with the major contribution from a competitive mode. In B16F10 cell cultures, CMT-308 (10 µM) significantly diminished total melanin levels with a selective reduction of extracellular melanin levels, under both basal and hormone-stimulated conditions without any cytotoxicity over a duration of 72 h. Studies of potential mechanisms of inhibition of melanogenesis in B16F10 cells showed that, in mammalian cells, CMT-308 did not inhibit intracellular tyrosinase activity or the activity of α-glucosidase, an enzyme that regulates maturation of tyrosinase. However, CMT-308 suppressed MITF protein expression in B16F10 cells and showed copper chelating activity and antioxidant activity in a cell-free system. The significantly lower extracellular melanin levels obtained at 10 µM indicate that CMT-308’s anti-melanogenic action may be attributed to a selective inhibition of melanosome export with the perinuclear aggregation of melanosomes, rather than a direct effect on the tyrosinase-catalyzed steps in melanin biosynthesis. These results were validated in HEMn-DP cells where CMT-308 suppressed dendricity in a fully reversible manner without affecting intracellular melanin synthesis. Furthermore, the capacity of CMT-308 to inhibit melanosome export was retained in cocultures of HEMn-DP and HaCaT. In summary, our results offer promise for therapeutic strategies to combat the effects of hyperpigmentation by use of CMT-308 at low micromolar concentrations.

MITF and TFEB cross-regulation in melanoma cells.

The MITF, TFEB, TFE3 and TFEC (MiT-TFE) proteins belong to the basic helix-loop-helix family of leucine zipper transcription factors. MITF is crucial for melanocyte development and differentiation, and has been termed a lineage-specific oncogene in melanoma. The three related proteins MITF, TFEB and TFE3 have been shown to be involved in the biogenesis and function of lysosomes and autophagosomes, regulating cellular clearance pathways. Here we investigated the cross-regulatory relationship of MITF and TFEB in melanoma cells. Like MITF, the TFEB and TFE3 genes are expressed in melanoma cells as well as in melanoma tumors, albeit at lower levels. We show that the MITF and TFEB proteins, but not TFE3, directly affect each other's mRNA and protein expression. In addition, the subcellular localization of MITF and TFEB is subject to regulation by the mTOR signaling pathway, which impacts their cross-regulatory relationship at the transcriptional level. Our work shows that the relationship between MITF and TFEB is multifaceted and that the cross-regulatory interactions of these factors need to be taken into account when considering pathways regulated by these proteins.