MITF Research Articles

Melanocyte Density in the Diagnosis of Melanoma In Situ in Sun-Damaged Skin.

Histologic differentiation between melanoma in situ in chronically sun-damaged skin (CSDS) [lentigo maligna (LM)] and CSDS without malignancy is difficult because signs of melanocyte activation and proliferation are found in both. A potentially reliable and quantifiable criterion is melanocyte density (MD). Here, we evaluated whether and to what extent MD allows the distinction between LM and CSDS, which is particularly relevant for the evaluation of borderline cases and surgical margins.Articles assessing MD in LM and/or CSDS were evaluated in a systematic review. The results were categorized and compared according to staining. Cutoff values were included whenever stated.Twenty articles matched the selection criteria. Six hundred forty-four samples of CSDS and 227 samples of LM were considered. In each individual study, mean MD scores were higher for LM than for CSDS. However, looking at the overall study situation, it becomes clear that the data are very heterogeneous and show overlaps. Therefore, no reliable orientation value can be derived. Only 1 article defined a cutoff value.The data of MD in LM in contrast to CSDS were sparse, and a defined cutoff value was only mentioned in 1 article for microphthalmia-associated transcription factor, which cannot yet be generalized. Especially regarding the importance for the definition of surgical resection margins, this unsatisfactory data set highlights the need for further studies. More precise diagnostic criteria could spare some patients extensive and possibly disfiguring surgery.

The MITF/mir-579-3p regulatory axis dictates BRAF-mutated melanoma cell fate in response to MAPK inhibitors

Therapy of melanoma has improved dramatically over the last years thanks to the development of targeted therapies (MAPKi) and immunotherapies. However, drug resistance continues to limit the efficacy of these therapies. Our research group has provided robust evidence as to the involvement of a set of microRNAs in the development of resistance to target therapy in BRAF-mutated melanomas. Among them, a pivotal role is played by the oncosuppressor miR-579-3p. Here we show that miR-579-3p and the microphthalmia-associated transcription factor (MITF) influence reciprocally their expression through positive feedback regulatory loops. In particular we show that miR-579-3p is specifically deregulated in BRAF-mutant melanomas and that its expression levels mirror those of MITF. Luciferase and ChIP studies show that MITF is a positive regulator of miR-579-3p, which is located in the intron 11 of the human gene ZFR (Zink-finger recombinase) and is co-transcribed with its host gene. Moreover, miR-579-3p, by targeting BRAF, is able to stabilize MITF protein thus inducing its own transcription. From biological points of view, early exposure to MAPKi or, alternatively miR-579-3p transfection, induce block of proliferation and trigger senescence programs in BRAF-mutant melanoma cells. Finally, the long-term development of resistance to MAPKi is able to select cells characterized by the loss of both miR-579-3p and MITF and the same down-regulation is also present in patients relapsing after treatments. Altogether these findings suggest that miR-579-3p/MITF interplay potentially governs the balance between proliferation, senescence and resistance to therapies in BRAF-mutant melanomas.

MITF regulates the subcellular location of HIF1α through SUMOylation to promote the invasion and metastasis of daughter cells derived from polyploid giant cancer cells.

High concentrations of cobalt chloride (CoCl2) can induce the formation of polyploid giant cancer cells (PGCCs) in various tumors, which can produce daughter cells with strong proliferative, migratory and invasive abilities via asymmetric division. To study the role of hypoxia‑inducible factor (HIF) 1α in the formation of PGCCs, colon cancer cell lines Hct116 and LoVo were used as experimental subjects. Western blotting, nuclear and cytoplasmic protein extraction and immunocytochemical experiments were used to compare the changes in the expression and subcellular localization of HIF1α, microphthalmia‑associated transcription factor (MITF), protein inhibitor of activated STAT protein4 (PIAS4) and von Hippel‑Lindau disease tumor suppressor (VHL) after treatment with CoCl2. The SUMOylation of HIFα was verified by co‑immunoprecipitation assay. After inhibiting HIF1α SUMOylation, the changes in proliferation, migration and invasion abilities of Hct116 and LoVo were compared by plate colony formation, wound healing and Transwell migration and invasion. In addition, lysine sites that led to SUMOylation of HIF1α were identified through site mutation experiments. The results showed that CoCl2 can induce the formation of PGCCs with the expression level of HIF1α higher in treated cells than in control cells. HIF1α was primarily located in the cytoplasm of control cell. Following CoCl2 treatment, the subcellular localization of HIF1α was primarily in the nuclei of PGCCs with daughter cells (PDCs). After treatment with SUMOylation inhibitors, the nuclear HIF1α expression in PDCs decreased. Furthermore, their proliferation, migration and invasion abilities also decreased. After inhibiting the expression of MITF, the expression of HIF1α decreased. MITF can regulate HIF1α SUMOylation. Expression and subcellular localization of VHL and HIF1α did not change following PIAS4 knockdown. SUMOylation of HIF1α occurs at the amino acid sites K391 and K477 in PDCs. After mutation of the two sites, nuclear expression of HIF1α in PDCs was reduced, along with a significant reduction in the proliferation, migration and invasion abilities. In conclusion, the post‑translation modification regulated the subcellular location of HIF1α and the nuclear expression of HIF1α promoted the proliferation, migration and invasion abilities of PDCs. MITF could regulate the transcription and protein levels of HIF1α and participate in the regulation of HIF1α SUMOylation.

Revealing mitf functions and visualizing allografted tumor metastasis in colorless and immunodeficient Xenopus tropicalis

Transparent immunodeficient animal models not only enhance in vivo imaging investigations of visceral organ development but also facilitate in vivo tracking of transplanted tumor cells. However, at present, transparent and immunodeficient animal models are confined to zebrafish, presenting substantial challenges for real-time, in vivo imaging studies addressing specific biological inquiries. Here, we employed a mitf−/−/prkdc−/−/il2rg−/− triple-knockout strategy to establish a colorless and immunodeficient amphibian model of Xenopus tropicalis. By disrupting the mitf gene, we observed the loss of melanophores, xanthophores, and granular glands in Xenopus tropicalis. Through the endogenous mitf promoter to drive BRAFV600E expression, we confirmed mitf expression in melanophores, xanthophores and granular glands. Moreover, the reconstruction of the disrupted site effectively reinstated melanophores, xanthophores, and granular glands, further highlighting the crucial role of mitf as a regulator in their development. By crossing mitf−/− frogs with prkdc−/−/il2rg−/− frogs, we generated a mitf−/−/prkdc−/−/il2rg−/−Xenopus tropicalis line, providing a colorless and immunodeficient amphibian model. Utilizing this model, we successfully observed intravital metastases of allotransplanted xanthophoromas and migrations of allotransplanted melanomas. Overall, colorless and immunodeficient Xenopus tropicalis holds great promise as a valuable platform for tumorous and developmental biology research.

Exploring the potential of white birch sap: A natural alternative to traditional skin whitening agents with reduced side effects

Common tyrosinase (TYR) inhibitors used in cosmetics, such as hydroquinone, kojic acid, and arbutin, can cause side effects including erythema, skin peeling, and dryness. Therefore, the development of natural whitening agents that offer excellent permeability, minimal irritation, and high safety has become a primary focus in the field of TYR inhibitors. In this study, we demonstrate that White birch sap (WBS), within a safe concentration range, effectively reduces TYR activity and melanin content in both B16F10 mouse melanoma cells and zebrafish larvae. Importantly, WBS exhibits minimal irritation to neutrophils in fluorescent zebrafish and does not affect the behavior of adult zebrafish. Furthermore, WBS downregulates the gene expression levels of microphthalmia-associated transcription factor, TYR, tyrosinase-related protein-1, and tyrosinase-related protein-2 in B16F10 cells. In conclusion, our research confirms that WBS, a naturally derived substance, offers high safety and mild effects, making it a promising candidate for a skin-whitening agent.

Optimization, Metabolomic Analysis, Antioxidant Potential andDepigmenting Activity of Polyphenolic Compounds fromUnmature Ajwa Date Seeds (Phoenix dactylifera L.) Using Ultrasonic-Assisted Extraction.

This study sought to optimize the ultrasonic-assisted extraction of polyphenolic compounds from unmature Ajwa date seeds (UMS), conduct untargeted metabolite identification and assess antioxidant and depigmenting activities. Response surface methodology (RSM) utilizing the Box-Behnken design (BBD) and artificial neural network (ANN) modeling was applied to optimize extraction conditions, including the ethanol concentration, extraction temperature and time. The determined optimal conditions comprised the ethanol concentration (62.00%), extraction time (29.00 min), and extraction temperature (50 °C). Under these conditions, UMS exhibited total phenolic content (TPC) and total flavonoid content (TFC) values of 77.52 ± 1.55 mgGAE/g and 58.85 ± 1.12 mgCE/g, respectively, with low relative standard deviation (RSD%) and relative standard error (RSE%). High-resolution mass spectrometry analysis unveiled the presence of 104 secondary metabolites in UMS, encompassing phenols, flavonoids, sesquiterpenoids, lignans and fatty acids. Furthermore, UMS demonstrated robust antioxidant activities in various cell-free antioxidant assays, implicating engagement in both hydrogen atom transfer and single electron transfer mechanisms. Additionally, UMS effectively mitigated tert-butyl hydroperoxide (t-BHP)-induced cellular reactive oxygen species (ROS) generation in a concentration-dependent manner. Crucially, UMS showcased the ability to activate mitogen-activated protein kinases (MAPKs) and suppress key proteins including tyrosinase (Tyr), tyrosinase-related protein-1 and -2 (Trp-1 and -2) and microphthalmia-associated transcription factor (MITF), which associated melanin production in MNT-1 cell. In summary, this study not only optimized the extraction process for polyphenolic compounds from UMS but also elucidated its diverse secondary metabolite profile. The observed antioxidant and depigmenting activities underscore the promising applications of UMS in skincare formulations and pharmaceutical developments.

Absence of meningeal mast cells in the Mitf mutant mouse.

Mast cells (MCs) are located in the meninges of the central nervous system (CNS), where they play key roles in the immune response. MC-deficient mice are advantageous in delineating the role of MCs in the immune response in vivo. In this study, we illustrate that a mutation in microphthalmia-associated transcription factor (Mitf) affects meningeal MC number in a dosage-dependent manner. C57BL/6J Mitf null mice lack meningeal MCs completely, whereas heterozygous mice have on average 25% fewer MCs. Mitf heterozygous mice might be a valuable tool to study the role of MCs in the meninges.

The Antimelanogenic Activity of the Extract of Heukharang Lettuce (Lactuca sativa L.) Leaf

Heukharang (Lactuca sativa L.), the artificially developed lettuce cultivar for enhancement of lactucin content, exhibits antioxidant activities and sleep-promoting effects. However, potential of Heukharang as a raw material for the skin-whitening agent has not been investigated yet. This study evaluated the effects of Heukharang extract (HHE) on α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis in vitro and in vivo models. Our findings revealed that HHE (25–100 μg/mL) effectively inhibited α-MSH-induced melanin synthesis in both 2D and 3D cell culture environments without compromising the viability of the B16F10 murine melanoma cell line. Furthermore, HHE suppressed intracellular tyrosinase activity, along with both gene and protein expression of tyrosinase (TYR) and tyrosinase-related protein (TRP)-1 and TRP-2. HHE downregulated the mRNA level of microphthalmia-associated transcription factor (MITF) and the phosphorylation of cAMP response element-binding (CREB) protein. The antimelanogenic activity of HHE (50 and 100 μg/mL) was also confirmed using a zebrafish embryo model, highlighting its efficacy in inhibiting α-MSH-induced melanogenesis through the downregulating CREB/MITF/TYR signaling pathways. Moreover, our study demonstrated that a corresponding amount of lactucin (5–20 μM) to the HHE inhibited and regulated melanogenesis. These results collectively suggest that HHE, enriched with lactucin, holds promise as a potential skin-whitening agent for nutricosmetic industries.

Oncogenic fusions in renal cell carcinoma.

475 Background: Detecting actionable or diagnostic fusions in solid tumors may significantly alter clinical decisions, including therapy selection. Some fusions have matched drugs approved for use in the patient’s cancer, or in another cancer. Others have published evidence of benefit with particular drugs but are not included in guidelines, and some may represent an inclusion criterion for clinical trials. While diagnostic fusions may not have associated therapies, they are cancer-defining and can lead to re-classification of tumors. Here we describe actionable and diagnostic fusions we have observed in renal cell carcinoma (RCC). Methods: Tumor samples from patients diagnosed with RCC were analyzed using the OncoExTra assay, which identifies somatic mutations and gene fusions through tumor-normal, whole-exome DNA and whole-transcriptome RNA sequencing. All actionable fusions, defined as those with associated FDA-approved targeted therapies in any cancer type, those that made patients eligible for an active clinical trial, or those with evidence in guidelines or the literature for possible matched therapies in any cancer, are detectable using this assay. We examined samples from patients with RCC who had the OncoExtra assay performed between May 2018 and Mar 2023 and identified both actionable and diagnostic fusions. Results: Among 389 tumor samples from patients diagnosed with RCC, there were 11 (2.8%) actionable and 1 (0.3%) diagnostic fusion identified. The actionable fusions included 6 (1.5%) TFE3, 1 (0.3%) ALK, 1 (0.3%) EGFR, 1 (0.3%) ERBB4, 1 (0.3%) YAP and 1 (0.3%) FGFR2, and the diagnostic fusion was NAB2/STAT6. The 6 TFE3 oncogene fusions involved 4 different partner genes ( PRCC was the partner in 3 fusions). ALK, EGFR, ERBB and FGFR fusions are found across a variety of cancers, and all have matched FDA-approved drug therapies in at least one cancer type. For example, ALK fusions, including the observed EML4/ALK fusion, which is common in non-small cell lung cancer, have several matched ALK-inhibitor therapies. TFE3 (transcription factor binding to IGHM enhancer 3) gene fusions are a diagnostic genomic alteration in microphthalmia-associated transcription factor (MiT) translocation RCC, which is a rare but aggressive subtype of RCC. While TFE3 fusions do not have FDA-approved matched therapies in any cancer, they are known to result in PIK3K/AKT/mTOR and MET activation, suggesting inhibitors of these pathways may be considered in these patients. The NAB2/STAT6 diagnostic fusion is characteristic of solitary fibrous tumors, suggesting that the patient likely had this kind of tumor. Conclusions: Whole-transcriptome sequencing allowed the identification of actionable fusions with the potential to affect clinical decisions regarding therapy in 2.8% of RCC patient tumors. In addition, one patient was found to have a fusion that is characteristic of a solitary fibrous tumor, demonstrating the power of genomic profiling to aid diagnosis.

Ginsenoside Rf in wild ginseng adventitious roots extract inhibits melanogenesis via cAMP/PKA and NO/cGMP signalling pathways in α-melanocyte-stimulating hormone-stimulated B16F10 mouse melanoma cells and zebrafish

The scarcity of more effective wild ginseng has severely limited its use, culturing of adventitious roots from wild ginseng were its good substitute. In this study, we found ginsenoside Rf as the special component in adventitious roots extract significantly decreased melanin levels and tyrosinase activity in B16F10 cells and zebrafish, and suppressed the expression of microphthalmia-associated transcription factor and melanogenic enzymes in B16F10 cells. Notably, Rf treatment of B16F10 cells led to reduced cell levels of adenosine cyclic 3′, 5′-monophosphate (cAMP), nitric oxide (NO), and guanoside cyclic 3′, 5′-monophosphate (cGMP), and reduced activities of adenylate cyclase (AC), protein kinase A (PKA), guanylate cyclase (GC), and protein kinase G (PKG), which suggest Rf anti-melanogenic activity potentially involved inhibition of AC/cAMP/PKA and NO/GC/cGMP/PKG signalling pathway. This work provides experimental basis for skin-lightening effect of wild ginseng adventitious roots and their functional part.

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.

Thermodynamic Allosteric Switch-Actuated 3D DNA Nanomachine for Ultrasensitive Electrochemical/Fluorescent Dual-Mode Biosensing of a Transcription Factor.

Herein, we reported an innovative thermodynamic allosteric switch-actuated 3D DNA nanomachine for selective, sensitive, and accurate electrochemical (EC)/fluorescent (FL) dual-mode biosensing of a microphthalmia-associated transcription factor (MITF). The thermodynamic allosteric switch was ingeniously customized as a hairpin probe (HP) that was in dynamic equilibrium but rapidly interconverting conformations. At the "inactive state", the MITF-binding region and the switch part were "sequestered". Upon the introduction of MITF, an MITF-HP complex promptly formed, and the equilibrium of HP thermodynamically inclined from the "inactive state" toward the "active state" conformation. Immediately, the exposed switch on HP effectively actuated the 3D DNA nanomachine and synchronously produced the restriction site for Nb.BbvCI nicking endonuclease. After the autonomous conveying of the 3D DNA nanomachine by means of the high-efficiency circularly nicking endonuclease signal amplification (NESA), not only was MB-S1 in the supernatant used for FL measurements but also MB-SP/MNs/S2 in the precipitate was adapted for EC analysis, significantly improving the utilization of output products derived from the 3D DNA nanomachine. Accordingly, benefiting from the efficient DNA nanomachine signal amplification manner and the self-calibration function of a dual-mode bioassay, the constructed biosensor exhibits superior sensitivity and accuracy for MITF determination.

Multi-technology integrated network pharmacology-based study on phytochemicals, active metabolites, and molecular mechanism of Psoraleae Fructus to promote melanogenesis

Ethnopharmacological relevanceAccording to the Compendium of Materia Medica (Shizhen Li, Ming dynasty) and Welfare Pharmacy (Song dynasty), Psoraleae Fructus (PF), a traditional Chinese medicine (TCM) has a bitter taste and warm nature, which has the effect of treating spleen and kidney deficiency and skin disease. Although PF has been widely used since ancient times and has shown satisfactory efficacy in treating vitiligo, the active substances and the mechanism of PF in promoting melanogenesis remain unclear. Aim of the studyTo explore the active substances and action mechanisms of PF in promoting melanogenesis. Materials and methodsFirstly, UPLC-UV-Q-TOF/MS was used to characterize the components in PF extract and identify the absorption components and metabolites of PF after oral administration at usual doses in rats. Secondly, the active substances and related targets and pathways were predicted by network pharmacology and molecular docking. Finally, pharmacodynamic and molecular biology experiments were used to verify the prediction results. ResultsThe experimental results showed that 15 compounds were identified in PF extract, and 44 compounds, consisting of 8 prototype components and 36 metabolites (including isomers) were identified in rats' plasma. Promising action targets (MAPK1, MAPK8, MAPK14) and signaling pathways (MAPK signaling pathway) were screened and refined to elucidate the mechanism of PF against vitiligo based on network pharmacology. Bergaptol and xanthotol (the main metabolites of PF), psoralen (prototype drug), and PF extract significantly increased melanin production in zebrafish embryos. Furthermore, bergaptol could promote the pigmentation of zebrafish embryos more than psoralen and PF extract. Bergaptol significantly increased the protein expression levels of p-P38 and decreased ERK phosphorylation in B16F10 cells, which was also supported by the corresponding inhibitor/activator combination study. Moreover, bergaptol increased the mRNA expression levels of the downstream microphthalmia-associated transcription factor (MITF) and tyrosinase in B16F10 cells. Our data elucidate that bergaptol may promote melanogenesis by regulating the p-P38 and p-ERK signaling pathway. ConclusionsThis study will lay a foundation for discovering potential new drugs for treating vitiligo and provide feasible ideas for exploring the mechanism of traditional Chinese medicine.

Significant and Various Effects of ML329-Induced MITF Suppression in the Melanoma Cell Line.

To study the inhibitory effects on microphthalmia-associated transcription factor (MITF)-related biological aspects in malignant melanomas (MMs) in the presence or absence of the low-molecular MITF specific inhibitor ML329, cell viability, cellular metabolic functions, and three-dimensional (3D) spheroid formation efficacy were compared among MM cell lines including SK-mel-24, A375, dabrafenib- and trametinib-resistant A375 (A375DT), and WM266-4. Upon exposure to 2 or 10 μM of ML329, cell viability was significantly decreased in WM266-4, SK-mel-24, and A375DT cells, but not A375 cells, in a dose-dependent manner, and these toxic effects of ML329 were most evident in WM266-4 cells. Extracellular flux assays conducted using a Seahorse bioanalyzer revealed that treatment with ML329 increased basal respiration, ATP-linked respiration, proton leakage, and non-mitochondrial respiration in WM266-4 cells and decreased glycolytic function in SK-mel-24 cells, whereas there were no marked effects of ML329 on A375 and A375DT cells. A glycolytic stress assay under conditions of high glucose concentrations also demonstrated that the inhibitory effect of ML329 on the glycolytic function of WM266-4 cells was dose-dependent. In addition, ML329 significantly decreased 3D-spheroid-forming ability, though the effects of ML329 were variable among the MM cell lines. Furthermore, the mRNA expression levels of selected genes, including STAT3 as a possible regulator of 3D spheroid formation, KRAS and SOX2 as oncogenic-signaling-related factors, PCG1a as the main regulator of mitochondrial biogenesis, and HIF1a as a major hypoxia transcriptional regulator, fluctuated among the MM cell lines, possibly supporting the diverse ML329 effects mentioned above. The findings of diverse ML329 effects on various MM cell lines suggest that MITF-associated biological activities are different among various types of MM.

Putrescine Upregulates Melanogenesis Through Modulation of MITF Transcription Factor in B16F1 Mouse Melanoma Cells.

Ageing is a biochemical, metabolic and genetic physiological phenomenon. The suppression of melanin biosynthesis, evident in the greying of the hair, is a hallmark of ageing resulting from translation failure, reduced enzyme activity and cellular senescence. Putrescine, the smallest member of the polyamine family and an organic chemical, is present in living mammalian cells and plays a crucial role in regulating skin melanogenesis. Therefore, the purpose of this study is to explore the effect of putrescine on the signalling pathways of melanogenesis in melanoma cells. Melanin production capacity of putrescine was analysed using a tyrosinase activity assay. To assess the cell viability of B16F1 cells exposed to putrescine, a tetrazolium dye MTT assay was performed. The effect of putrescine on melanin synthesis in the presence of H2O2 was evaluated using various in vitro assays in B16F1 cells. The effect of putrescine on melanin production in B16F1 cells was determined using a specific melanin production assay. Gene expression was analysed using real-time polymerase chain reaction (RT-PCR). Furthermore, the effect of putrescine on the expression of proteins related to melanin production in the cells treated with H2O2 was analysed by immunofluorescence and Western blot analysis. Putrescine increased tyrosinase activity and showed no cytotoxicity in B16F1 cells. In addition, putrescine effectively scavenged H2O2, as shown by the reduction of intracellular H2O2 amounts in 2',7'-dichlorofluorescin diacetate analysis, and promoted melanin production in living cells. The stimulation of melanogenesis by putrescine was attributed to the increased expression of Mitf, Tyr, Trp-1 and Trp-2 genes. Immunofluorescence assays revealed that putrescine enhanced the expression of proteins associated with melanogenesis and upregulated TYR, TRP-1 and TRP-2 via the microphthalmia-associated transcription factor (MITF) and increased the expression of methionine sulfoxide reductases A (MSRA) and B (MSRB) in the cells treated with H2O2, effectively promoting melanogenesis. These results suggest that putrescine can be used to stimulate melanin synthesis. This is the first study to investigate the effect of putrescine on the signalling pathways of melanogenesis in B16F1 melanoma cells. The results confirm that putrescine can promote melanogenesis through the expression of TYR, TRP-1 and TRP-2 via the MITF in cells treated with H2O2. Putrescine can be used exclusively as a cosmetic product to prevent premature greying of hair.

A CRISPR-Cas12a-based electrochemical biosensor for the detection of microphthalmia-associated transcription factor.

Anovel electrochemical biosensor that combines the CRISPR-Cas12a system with a gold electrode is reportedfor the rapid and sensitive detection of microphthalmia-associated transcription factor (MITF). The biosensor consists of a gold electrode modified with DNA1, which contains the target sequence of MITF and is labeled with ferrocene, an electroactive molecule. The biosensor also includes hairpin DNA, which has a binding site for MITF and can hybridize with helper DNA to form a double-stranded complex that activates CRISPR-Cas12a. When MITF is present, it binds to hairpin DNA and prevents its hybridization with helper DNA, thus inhibiting CRISPR-Cas12a activity and preserving the DPV signal of ferrocene. When MITF is absent, hairpin DNA hybridizes with helper DNA and activates CRISPR-Cas12a, which cleaves DNA1 and releases ferrocene, thus reducing the DPV signal. The biosensor can detect MITF with high sensitivity (with an LOD of 8.14 fM), specificity, and accuracy in various samples, such as cell nuclear extracts and human serum. The biosensor can also diagnose and monitor melanocyte-related diseases and melanin production. This work provides a simple, fast, sensitive, and cost-effective biosensor for MITF detection and a valuable tool for applications in genetic testing, disease diagnosis, and drug screening.

Investigating the role and regulation of GPNMB in progranulin-deficient macrophages.

Progranulin (PGRN) is a holoprotein that is internalized and taken to the lysosome where it is processed to individual granulins (GRNs). PGRN is critical for successful aging, and insufficient levels of PGRN are associated with increased risk for developing neurodegenerative diseases like AD, PD, and FTD. A unifying feature among these diseases is dysregulation of peripheral immune cell populations. However, considerable gaps exist in our understanding of the function(s) of PGRN/GRNs in immune cells and their role in regulating central-peripheral neuroimmune crosstalk. One of the most upregulated genes and proteins in humans with GRN haploinsufficiency and in aged Grn knock-out (KO) mice is glycoprotein non-metastatic B (GPNMB) but its normal role within the context of immune crosstalk has not been elucidated. To address this gap, peritoneal macrophages (pMacs) from 5-to-6-month old WT and Grn KO mice were assessed for Gpnmb expression and stimulation-dependent cytokine release in the presence or absence of the Gpnmb extracellular domain (ECD). Cellular localization, as well as inhibition of, the microphthalmia-associated transcription factor (MITF) was assessed to determine its mechanistic role in Gpnmb overexpression in Grn KO pMacs. We observed an increase in GPNMB protein and mRNA as a result of insufficient progranulin in peripheral immune cells at a very early age relative to previous reports on the brain. Stimulation-dependent cytokine release was decreased in the media of Grn KO pMacs relative to WT controls; a phenotype that could be mimicked in WT pMacs with the addition og GPNMB ECD. We also found that MITF is dysregulated in Grn KO pMacs; however, its nuclear translocation and activity are not required to rescue the immune dysregulation of Grn KO macrophages, suggesting redundancy in the system. These findings highlight the fact that knowledge of early-stage disease mechanism(s) in peripheral populations may inform treatment strategies to delay disease progression at an early, prodromal timepoint prior to development of neuroinflammation and CNS pathology.

The Mutant Menage- Mit Family Translocation Renal Cell Carcinoma

Microphthalmia associated transcription factor (MiT) family translocation renal cell carcinoma is neoplasm which enunciates genetic fusions within members of MiT family of transcription factors, especially TFE3 gene and TFEB gene. Neoplasm frequently incriminates the adult population ≤ 35 years of age with an intense female predilection. Tumefaction exhibits a papillary, solid or alveolar pattern of tumour configuration and is composed of clear cells or non-cohesive, pseudo stratified, eosinophilic epithelial cells. Tumour cells are pervaded with voluminous cytoplasm and pleomorphic nuclei of advanced grade. Neoplastic cells are intensely immune reactive to TFE3 or TFEB, PAX8, cathepsin K, CD10, alpha methyacyl CoA racemase (AMACR), vimentin or E-cadherin. Tumour cells appear immune non-reactive to cytokeratin, epithelial membrane antigen (EMA), carbonic anhydrase IX(CAIX), CD45, calretinin and smooth muscle actin(SMA). MiT family translocation renal cell carcinoma requires segregation from neoplasms such as clear cell papillary renal cell carcinoma, clear cell renal cell carcinoma, epithelioid angiomyolipoma, chromophobe renal cell carcinoma or papillary renal cell carcinoma. Neoplasm may be appropriately alleviated by cogent manoeuvers as surgical extermination of the neoplasm.

Tranexamic Acid Ameliorates Skin Hyperpigmentation by Downregulating Endothelin-1 Expression in Dermal Microvascular Endothelial Cells.

Although reports suggest that tranexamic acid (TXA) has clinical benefits for melasma patients by oral, intralesional and topical treatment, the optimal route of TXA therapy and the underlying mechanism involved remain poorly defined. To compare the skin lightening effect between oral TXA and topical TXA and to dissect the molecular mechanisms using ultraviolet B (UVB)-induced hyperpigmentation mouse model, ex vivo cultured human skin explant, and cultured melanocytes (MCs) and endothelial cells. Melanin content and cluster of differentiation 31 (CD31)-positive cell numbers were measured in tail skins from UVB-irradiated mice treated by intragastral or topical TXA using immunofluorescent and Fontana-Masson staining. The conditioned medium (CM) was harvested from human umbilical vein endothelial cells treated with or without 3 mM TXA and was used to treat MCs for 48 hours. mRNA and protein levels of tyrosinase and microphthalmia-associated transcription factor were measured using quantitative real-time reverse transcription polymerase chain reaction and western blotting assays. HMB45- and CD31-positive cell numbers as well as melanin content were also examined in ex vivo cultured human skin explants. The hyperpigmented phenotype were significantly mitigated in UVB-irradiated tail skin plus intragastral TXA-treated mice compared with mice treated with UVB only or with UVB plus topical TXA. CD31-positive cell numbers correlated with the anti-melanogenic activity of TXA therapy. The data from cultured cells and skin tissues showed that suppression of endothelin-1 (ET-1) in vascular endothelial cells by TXA reduced melanogenesis and MC proliferation. Oral TXA outperforms topical TXA treatment in skin lightening, which contributes to suppression of ET-1 in dermal microvascular endothelial cells by TXA.

Interruption of p38MAPK-MSK1-CREB-MITF-M pathway to prevent hyperpigmentation in the skin.

Background: Melanocortin 1 receptor (MC1R), a receptor of α-melanocyte-stimulating hormone (α-MSH), is exclusively present in melanocytes where α-MSH/MC1R stimulate melanin pigmentation through microphthalmia-associated transcription factor M (MITF-M). Toll-like receptor 4 (TLR4), a receptor of endotoxin lipopolysaccharide (LPS), is distributed in immune and other cell types including melanocytes where LPS/TLR4 activate transcriptional activity of nuclear factor (NF)-κB to express cytokines in innate immunity. LPS/TLR4 also up-regulate MITF-M-target melanogenic genes in melanocytes. Here, we propose a molecular target of antimelanogenic activity through elucidating inhibitory mechanism on α-MSH-induced melanogenic programs by benzimidazole-2-butanol (BI2B), an inhibitor of LPS/TLR4-activated transcriptional activity of NF-κB. Methods: Ultraviolet B (UV-B)-irradiated skins of HRM-2 hairless mice and α-MSH-activated melanocyte cultures were employed to examine melanogenic programs. Results: Topical treatment with BI2B ameliorated UV-B-irradiated skin hyperpigmentation in mice. BI2B suppressed the protein or mRNA levels of melanogenic markers, such as tyrosinase (TYR), MITF-M and proopiomelanocortin (POMC), in UV-B-exposed and pigmented skin tissues. Moreover, BI2B inhibited melanin pigmentation in UV-B-irradiated co-cultures of keratinocyte and melanocyte cells and that in α-MSH-activated melanocyte cultures. Mechanistically, BI2B inhibited the activation of cAMP response element-binding protein (CREB) in α-MSH-induced melanogenic programs and suppressed the expression of MITF-M at the promoter level. As a molecular target, BI2B primarily inhibited mitogen-activated protein kinase (MAPK) kinase 3 (MKK3)-catalyzed kinase activity on p38MAPK. Subsequently, BI2B interrupted downstream pathway of p38MAPK-mitogen and stress-activated protein kinase-1 (MSK1)-CREB-MITF-M, and suppressed MITF-M-target melanogenic genes, encoding enzymes TYR, TYR-related protein-1 (TRP-1) and dopachrome tautomerase (DCT) in melanin biosynthesis, and encoding proteins PMEL17 and Rab27A in the transfer of pigmented melanosomes to the overlaying keratinocytes in the skin. Conclusion: Targeting the MKK3-p38MAPK-MSK1-CREB-MITF-M pathway was suggested as a rationale to inhibit UV-B- or α-MSH-induced facultative melanogenesis and as a strategy to prevent acquired pigmentary disorders in the skin.