Microphthalmia Transcription Factor Research Articles

Tokishakuyakusan alleviates ultraviolet-induced skin pigmentation by decreasing the expression of melanogenesis-related enzymes

Ethnopharmacological relevanceTokishakuyakusan (TSS), a traditional Kampo medicine, can effectively alleviate symptoms unique to women, such as menstrual pain and menopausal symptoms, and this effect is believed to be related to its ability to increase the secretion of female hormones. TSS is also believed to be effective against skin pigmentation. However, no studies have examined the effect of TSS on pigmentation. Aim of the studyIn this study, we conducted basic research to determine the effects of TSS on pigmentation. Materials and methodsFemale HRM-2 mice were given free access to a normal diet or a TSS-containing diet for 7 weeks. For 3 weeks starting from the 4th week of treatment, the back of the skin was irradiated with ultraviolet (UV) light, and the melanin level was measured. The expression levels of melanogenesis-related genes and inflammatory markers in the skin were analyzed. ResultsThe melanin level in the skin of the mice exposed to UV radiation was approximately three times greater than that in the skin of the mice in the non-UV-irradiated group, confirming pigmentation due to UV irradiation. The protein expression levels of tyrosinase (Tyr), tyrosinase-related protein-1 (Tyrp1), and dopachrome tautomerase (Dct), which are important for melanin production, were significantly greater in the UV irradiation group than in the non-UV irradiation group. In contrast, the amount of skin melanin in the mice treated with TSS was significantly lower than that in the UV-irradiated group, and the expression levels of melanogenesis-related enzymes were also lower. Furthermore, TSS significantly decreased the expression of microphthalmia transcription factor (Mitf), a transcription factor for melanogenesis-related enzymes, and the inflammatory cytokines interleukin-1β and interleukin-6. ConclusionsTSS inhibits melanin production in melanocytes by suppressing the increase in the expression of melanogenesis-related enzymes caused by UV irradiation. These findings suggested that this effect of TSS is exerted through the combined regulation of MITF expression and anti-inflammatory responses.

Clinical Characteristics and Outcomes of TFE3-Rearranged/TFEB-Altered Renal Cell Carcinoma with Systemic Therapies, Including Tyrosine Kinase Inhibitors or Immune Checkpoint Inhibitors: An Observational Study

Purpose: <i>TFE3</i>-rearranged/<i>TFEB</i>-altered renal cell carcinoma (RCC) is a rare subtype of RCC. Due to its rarity, there is an unmet medical need for effective therapies in advanced settings. The study aims to investigate the clinical and histopathological characteristics of patients with microphthalmia transcription factor family/ transcription factor E (MiTF/TFE) translocation RCC and the clinical outcomes of systemic therapies, including tyrosine kinase inhibitors (TKIs) or immune checkpoint inhibitors (ICIs).Materials and Methods: This was a single-center, retrospective study. We identified 32 eligible patients among a total of 37 patients diagnosed with MiTF/TFE translocation RCC between January 2004 and September 2021, and the study included 9 patients who were treated with systemic therapies. We collected data on clinical characteristics, targeted sequencing, and clinical outcomes.Results: The median age of the 32 patients was 45.5 years. Histologically, 26 patients (81.3%) had <i>TFE3</i>-rearranged RCC, and only 1 patient (3.1%) had <i>TFEB</i>-altered RCC. Curative or cytoreductive nephrectomy was performed in all 27 patients (84.4%), and 4 patients (12.6%) were diagnosed with metastatic disease at the time of the initial diagnosis. Nine patients (28.1%) were treated with systemic therapy with TKIs, 2 (6.3%) of whom received simultaneous TKI and ICI treatment. The response to systemic therapy (TKI or ICI) and duration of response ranged from complete response to progressive disease. Excluding 1 patient who was treated with a TKI in the adjuvant setting, the overall response rate in 8 metastatic patients was 50% and the complete response rate was 37.5%. The median follow-up period was 29 months. The median progressionfree survival was 21 months, median overall survival was not achieved, and 2 deaths occurred.Conclusions: Our findings suggest that TKI for treatment for metastatic <i>TFE3</i>-rearranged RCC is efficacious, with an overall response rate of 50% and a median progression-free survival of 21 months.

Abstract 3020: The tumor suppressive role of methylthioadenosine phosphorylase in melanoma

Abstract Methylthioadenosine phosphorylase (MTAP) is an enzyme that is expressed in virtually all tissues but lost in many cancers. MTAP deficiency can be due to either deletion of the MTAP gene or methylation of the MTAP promoter. In normal cells, MTAP catalyzes the conversion of methylthioadenosine (MTA), produced during polyamine biosynthesis, to adenine and 5-methylthioribose-1-phosphate, which is subsequently converted to methionine. MTAP deficiency results in intracellular accumulation of MTA. Although recent genome wide association studies (GWAS) have associated the MTAP locus with melanoma risk, the molecular mechanisms linking MTAP loss to increased tumorigenesis are not yet fully understood. In this study, we hypothesized that loss of MTAP and the resulting accumulation of MTA would dysregulate microphthalmia transcription factor (MITF), a master transcriptional regulator of melanocytes that is also an amplified or mutated oncogene in melanoma. We identify a novel signaling mechanism in which MTA accumulation from MTAP deficiency aberrantly induces MITF expression via inhibition of the cAMP phosphodiesterase PDE4D3 in melanocytes. Inhibition of PKA abolishes this MITF induction, suggesting that the PKA pathway is hyperactivated upon MTAP deficiency. Downregulation of MTAP leads to increased proliferation of melanoma cells in vitro and increased melanoma tumor growth in vivo. We also show that MTAP deficient melanoma cells are more resistant to the BRAF inhibitor vemurafenib and observe MTAP downregulation in BRAF inhibitor resistant melanoma clinical specimens. Furthermore, MTAP downregulation via decreased expression and/or genomic loss corresponds to significantly worse outcomes in early-stage cutaneous melanomas from multiple datasets, thereby identifying a clinically important sub-category of “thin-lethal” tumors. Citation Format: Jennifer J. Hsiao, Pedro Andreu-Perez, Lajos V. Kemény, Miroslav Hejna, Jérémie Nsengimana, Jennifer Allouche, Abdullah Al Emran, D. Timothy Bishop, Göran B. Jönsson, Julia Newton-Bishop, Jun Song, David E. Fisher. The tumor suppressive role of methylthioadenosine phosphorylase in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3020.

Mitf regulates gene expression networks implicated in B cell homeostasis, germinal center responses, and tolerance.

The microphthalmia transcription factor Mitf has been shown to regulate B cell activation and tolerance. However, the underlying B cell-specific mechanisms responsible, and those that distinguish Mitf from closely related Mitf/TFE (MiT) transcription factors Tfe3, Tfeb, and Tfec, remain obscure. Two complementary mouse models of Mitf and MiT deficiency were used: the Mitfmi-vga9/mi-vga9 systemic loss-of-function mutation, and B-cell specific MiT family inactivation via transgenic expression of a trans-dominant negative (TDN) protein (TDN-B). These models were employed to identify MiT family candidate target genes and pathways. Both models displayed spontaneous splenomegaly coincident with elevated plasma cell numbers, autoantibody titers, and proteinuria. These abnormalities appeared dependent on T helper cells, but independent of other non-B cell intrinsic effects of systemic Mitf inactivation. MiT inactivation in B cells augmented aspects of lupus-like autoimmune disease on the C57BL/6-Faslpr/lpr background. In both models, RNAseq of ex vivo resting B cells showed transcriptional upregulation of genes that control cell cycle, germinal center responses, and plasma cell differentiation. Among the genes strongly upregulated in both models were Socs6, Isp53 (Baiap1), S1pR2, and IgG2b/c. Mitf null B cells, but not TDN-B cells, showed evidence of type I interferon dysregulation. These studies clarify Mitf's role as 1) a key regulator of a B cell intrinsic germinal center program that influences self-tolerance through novel target genes, and 2) a regulator of systemic inflammatory processes that can impact the B cell microenvironment. This distinction of Mitf's function from that of related MiT transcription factors advances our understanding of B cell regulation and autoimmunity.

Renal cell carcinoma in the contralateral kidney with TFE3 gene translocation following chemotherapy for childhood nephroblastoma: A case report and literature review

Key Clinical MessageRenal cell carcinoma as a secondary malignant neoplasm is relatively rare; however, the possibility of secondary renal cell carcinoma following chemoradiotherapy for childhood nephroblastoma should be considered.AbstractThe occurrence of secondary renal cell carcinoma (RCC) following chemoradiotherapy for nephroblastoma is relatively rare, especially in microphthalmia transcription factor family translocation renal cell carcinoma. A 13‐year‐old Japanese male was referred to our department for treatment of a right kidney mass. The patient had undergone open left nephrectomy and adjuvant chemotherapy for nephroblastoma, 12 years before. Diagnostic imaging revealed a tumor in the right kidney and a lesion suspected to be metastasis in the left eighth rib. Chromophobe RCC or translocation RCC was suspected from the imaging pattern. TNM classification was cT1aN0M1, and the clinical stage was IV. Partial nephrectomy by robot‐assisted surgery for the right renal tumor and resection of the left eighth rib were performed. Pathologically, the renal tumor was diagnosed as translocation RCC, and the rib lesion demonstrated no evidence of malignancy. We are currently undergoing imaging follow‐up and the patient has been recurrence‐free for 15 months. In this study, we present a rare case of secondary translocation RCC after successful treatment of nephroblastoma.

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

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

Effects of a Standardized Hydrogenated Extract of Curcumin (Curowhite™) on Melanogenesis: A Pilot Study

The stimulation of melanogenesis by novel natural products is desirable for cosmetic applications such as skin tanning, anti-greying, and clinical use for treating vitiligo and leukoderma disorders. Microphthalmia transcription factor (MITF) is a central transcription factor that controls the expression of tyrosinase, which is a key enzyme responsible for catalyzing the rate-limiting processes of melanin production. Tetrahydrocurcuminoids (THCr), which mostly consist of tetrahydrocurcumin (THC), are a colorless bioactive mixture derived from curcuminoids that are extracted from the Curcuma longa plant. THCr has been reported to exhibit superior properties, including antioxidant and anti-inflammatory effects. Our previous study reported the greater melanogenesis-stimulating effects of purified THC, compared to hexahydrocurcumin (HHC) or octahydrocurcumin (OHC). Curowhite™ (CW) is a proprietary extract that consists of 25% hydrogenated curcuminoids (mixture of THCr, hexahydrocurcuminoids, and octahydrocurcuminoids) encapsulated in a β-cyclodextrin (βCyD) excipient. The encapsulation of THCr in a suitable excipient, such as the widely popular cyclodextrins, helps to enhance the stability, solubility, and bioavailability of the THCr. CW is marketed as a nutraceutical with GRAS status and is safe when administered orally, as shown in vivo studies. However, the impact of CW on melanogenesis remains unexplored. Herein, the impact of CW on melanogenesis were investigated using B16F10 and MNT-1 cells. Our findings show that CW is markedly cytotoxic to B16F10 cells without affecting the cellular melanin content. However, in MNT-1 cells, CW significantly stimulated intracellular melanin content over the concentration range (20–60 µg/mL) with increased dendrite formation while being nontoxic to MNT-1 cells or HaCaT cells after a 5-day treatment. Examination of the effects of the excipient βCyD on cytotoxicity and melanogenesis confirmed that the excipient had no contribution to the biological impacts that were found to be exclusively attributable to the encapsulated mixture (THCr). The mechanisms of CW’s promelanogenic effects in MNT-1 cells were found to be related, at least in part, to an increase in tyrosinase and MITF protein levels, as CW did not alter tyrosinase activity in MNT-1 cells. Moreover, CW exhibited antioxidant activity as obtained through DPPH radical scavenging assay. Together, the findings of this pilot study indicate that CW might hold an exciting avenue as a pro-pigmenting nutraceutical for treating hypopigmentation disorders, the detailed mechanisms of which warrant further exploration. Moreover, future investigations are necessary to examine CW’s effects on melanogenesis in normal human melanocytes and in vivo studies.

A novel VCP::TFE3 gene fusion resulting from t(X;9)(p11.23;p13.3) chromosome translocation in TFE3 rearranged renal cancer cell carcinoma.

Renal cell carcinoma (RCC) with rearrangement of transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3; TFE3-rearranged RCC) at Xp11.2 is a rare tumor entity but the most frequent among the microphthalmia transcription factor family translocation RCCs. Here, we report the identification of a new VCP::TFE3 fusion gene as the result of a t(X;9)(p11.23;p13.3) translocation identified by whole transcriptome sequencing. No other relevant molecular alteration was identified by whole exome sequencing. This case showed typical morphological features of TFE3-rearranged RCC with positive TFE3 immunostaining and positive TFE3 break-apart fluorescence in situ hybridization. MET was also overexpressed on immunohistochemistry. The patient had metastatic disease and was treated by surgery and five lines of therapy, including 24 months of stable disease on the mesenchymal epithelial transition (MET) inhibitor cabozantinib, with an overall survival of 7 years. In addition to expanding the spectrum of TFE3 rearrangement partners, this report highlights the complexity of these tumors and supports the development of translational programs in renal cancer.

The oncogenic and immunological roles of histidine triad nucleotide-binding protein 1 in human cancers and their experimental validation in the MCF-7 cell line.

Histidine triad nucleotide binding protein 1 (HINT1) is a haplo-insufficient tumor suppressor gene that plays a significant role in cell proliferation and survival. However, to date, no systematic pan-cancer analysis has been conducted to explore its function in prognosis, and its oncogenic and immunological roles. We also analyzed the role of HINT1 in breast cancer (BC) progression in vitro. An analysis of the HINT1 expression pattern was performed using the TIMER database. The infiltration of immune cells into several cancer types was also studied using the Xena Shiny tool. To determine the relationship between stemness and the expression of HINT1 mRNA, the Spearman correlation test was used with the SangerBox tool. The correlation between HINT1 and functional states in various cancers was determined from the CancerSEA database. The potential role of HINT1 in BC oncogenesis was also investigated by Western blot and Annexin V/PI assays. The Cancer Genome Atlas pan-cancer data analysis suggested that HINT1 was extensively altered in most tumor tissues but not in most adjacent normal tissues. A high expression of HINT1 was associated with the decreased infiltration of cluster of differentiation (CD)4+ T cells. Importantly, increased HINT1 expression was also associated with a large majority of tumors with high stemness and lower stromal, immune, and estimate scores. Further, the expression of HINT1 was significantly associated with the tumor mutational burden (TMB) and microsatellite instability (MSI) in certain tumor types. Finally, HINT1 overexpression was found to impair BC progression by promoting cell apoptosis. HINT1 upregulation also reduced the expression of microphthalmia transcription factor (MITF) and β-catenin in BC Michigan Cancer Foundation-7 (MCF-7) cells, and the phosphorylation of protein kinase B (p-Akt). The present study showed that HINT1 plays an oncogenic role in various cancers and could also be used as a biomarker for BC.

A unique hyperdynamic dimer interface permits small molecule perturbation of the melanoma oncoprotein MITF for melanoma therapy.

Microphthalmia transcription factor (MITF) regulates melanocyte development and is the "lineage-specific survival" oncogene of melanoma. MITF is essential for melanoma initiation, progression, and relapse and has been considered an important therapeutic target; however, direct inhibition of MITF through small molecules is considered impossible, due to the absence of a ligand-binding pocket for drug design. Here, our structural analyses show that the structure of MITF is hyperdynamic because of its out-of-register leucine zipper with a 3-residue insertion. The dynamic MITF is highly vulnerable to dimer-disrupting mutations, as we observed that MITF loss-of-function mutations in human Waardenburg syndrome type 2 A are frequently located on the dimer interface and disrupt the dimer forming ability accordingly. These observations suggest a unique opportunity to inhibit MITF with small molecules capable of disrupting the MITF dimer. From a high throughput screening against 654,650 compounds, we discovered compound TT-012, which specifically binds to dynamic MITF and destroys the latter's dimer formation and DNA-binding ability. Using chromatin immunoprecipitation assay and RNAsequencing, we showed that TT-012 inhibits the transcriptional activity of MITF in B16F10 melanoma cells. In addition, TT-012 inhibits the growth of high-MITF melanoma cells, and inhibits the tumor growth and metastasis with tolerable toxicity to liver and immune cells in animal models. Together, this study demonstrates a unique hyperdynamic dimer interface in melanoma oncoprotein MITF, and reveals a novel approach to therapeutically suppress MITF activity.

Proteogenomic characterization of MiT family translocation renal cell carcinoma

Microphthalmia transcription factor (MiT) family translocation renal cell carcinoma (tRCC) is a rare type of kidney cancer, which is not well characterized. Here we show the comprehensive proteogenomic analysis of tRCC tumors and normal adjacent tissues to elucidate the molecular landscape of this disease. Our study reveals that defective DNA repair plays an important role in tRCC carcinogenesis and progression. Metabolic processes are markedly dysregulated at both the mRNA and protein levels. Proteomic and phosphoproteome data identify mTOR signaling pathway as a potential therapeutic target. Moreover, molecular subtyping and immune infiltration analysis characterize the inter-tumoral heterogeneity of tRCC. Multi-omic integration reveals the dysregulation of cellular processes affected by genomic alterations, including oxidative phosphorylation, autophagy, transcription factor activity, and proteasome function. This study represents a comprehensive proteogenomic analysis of tRCC, providing valuable insights into its biological mechanisms, disease diagnosis, and prognostication.

Requirement of Zebrafish Adcy3a and Adcy5 in Melanosome Dispersion and Melanocyte Stripe Formation.

cAMP-PKA signaling plays a pivotal role in melanin synthesis and melanosome transport by responding to the binding of the α-melanocyte-stimulating hormone (α-MSH) to melanocortin-1 receptor (MC1R). Adenylate cyclases (ADCYs) are the enzymes responsible for the synthesis of cAMP from ATP, which comprises nine transmembrane isoforms (ADCYs 1-9) and one soluble adenylate cyclase (ADCY 10) in mammals. However, little is known about which and how ADCY isoforms regulate melanocyte generation, melanin biosynthesis, and melanosome transport in vivo. In this study, we have generated a series of single and double mutants of Adcy isoforms in zebrafish. Among them, adcy3a-/- and adcy5-/- double mutants cause defects in melanosome dispersion but do not impair melanoblast differentiation and melanocyte regeneration during the embryonic or larval stages. Activation of PKA, the main effector of cAMP signaling, significantly ameliorates the defects in melanosome dispersion in adcy3a-/- and adcy5-/- double mutants. Mechanistically, Adcy3a and Adcy5 regulate melanosome dispersion by activating kinesin-1 while inhibiting cytoplasmic dynein-1. In adult zebrafish, Adcy3a and Adcy5 participate in the regulation of the expression of microphthalmia transcription factor (Mitfa) and melanin synthesis enzymes Tyr, Dct, and Trp1b. The deletion of Adcy3a and Adcy5 inhibits melanin production and reduces pigmented melanocyte numbers, causing a defect in establishing adult melanocyte stripes. Hence, our studies demonstrate that Adcy3a and Adcy5 play essential but redundant functions in mediating α-MSH-MC1R/cAMP-PKA signaling for regulating melanin synthesis and melanosome dispersion.

Impact of KIT Editing on Coat Pigmentation and Fresh Meat Color in Yorkshire Pigs.

The white coat color of Yorkshire pigs is caused by the dominant white I allele, which has been associated with at least one copy of the 450-kb duplication encompassing the entire KIT gene and a splice mutation (G > A) at the first base of intron 17. The splice mutation in KIT has an adverse effect on pigmentation in mice. Therefore, removing the 450 kb duplications harboring the KIT copy with splice mutations is expected to affect Yorkshire pig pigmentation. In this study, we describe the use of a Yorkshire pig kidney cell strain with the I?/IBe-ed genotype, previously created by CRISPR-Cas9, as donor cells for somatic cell nuclear transfer to generate gene-edited Yorkshire pigs. The removal of the 450 kb duplications harboring the KIT copy with splice mutation did not alter the white coat color of Yorkshire pigs, which was confirmed by the absence of fully mature melanocytes and melanin accumulation in the hair follicles. Except for the improved transcription of tyrosinase, and slight increase in microphthalmia transcription factor and tyrosinase-related protein 1 protein expression, there was no significant impact of the removal of splice mutations on genes and signaling pathways (PI3K/AKT) involved in melanogenesis. However, the removal of the 450 kb duplications harboring the KIT copy with splice mutation substantially improved fresh meat color accompanied by significantly increased red blood cell number, which merits further investigation. Our study provides new insights into the role of structural mutations of the KIT gene in the formation of white coat color and erythropoiesis in Yorkshire pigs.

Discovery of Pinostrobin as a Melanogenic Agent in cAMP/PKA and p38 MAPK Signaling Pathway.

Melanogenesis is the process of melanin synthesis to protect the skin against ultraviolet radiation and other external stresses. The loss of skin pigmentation is closely related to depigmented skin disorders. The melanogenic effects of pinostrobin, an active flavanone found in honey, were evaluated. B16F10 cells were used for melanin content, tyrosinase activity, and the expression of melanogenesis-related markers. Moreover, computational simulations were performed to predict docking and pharmacokinetics. Pinostrobin increased melanin levels and tyrosinase activity by stimulating the expression of melanogenic regulatory factors including tyrosinase, tyrosinase-related protein (TRP) 1 and microphthalmia transcription factor (MITF). Specifically, the phosphorylation of cAMP response element binding (CREB) involved in the MITF activation was augmented by pinostrobin. Moreover, the compound upregulated the β-catenin by cAMP/PKA-mediated GSK-3β inactivation. Co-treatment with a PKA inhibitor, inhibited melanin production, tyrosinase activity, and expression of MITF, p-CREB, p-GSK-3β and p-β-catenin, demonstrating that pinostrobin-stimulated melanogenesis was closely related to cAMP/PKA signaling pathway. Furthermore, the combination of pinostrobin and a specific p38 inhibitor, showed that MITF upregulation by pinostrobin was partly associated with the p38 signaling pathway. Docking simulation exhibited that the oxygen group at C-4 and the hydroxyl group at C-5 of pinostrobin may play an essential role in melanogenesis. In silico analysis revealed that pinostrobin had the optimal pharmacokinetic profiles including gastrointestinal absorption, skin permeability, and inhibition of cytochrome (CYP) enzymes. From the present results, it might be suggested that pinostrobin could be useful as a potent and safe melanogenic agent in the depigmentation disorder, vitiligo.

Microphthalmia Transcription Factor almost Thirty Years after: Its Role in Melanogenesis and its Plant-Derived Inhibitors

It has been almost thirty years since the first publication on microphthalmia-associated transcription factor (MITF) in 1993. MITF, which plays an important role in the melanogenesis process, is an interesting target for melanoma therapy, due to its associates with melanoma survival. MITF promotes melanoma cell proliferation, whereas the sustained suppression of MITF expression causes aging. MITF contributes to differentiation, which involves breaking out of the cell cycle and triggering a melanogenesis, and this function appears to often persist during melanoma development given the frequently observed high pigmented lesions, even in the late stages of melanoma. Several drugs that could inhibit MITF e.g. histone deacetylase inhibitors, such as sodium butyrate and trichostatin A, have been proven could suppress M-MITF expression in melanoma cells. H1-receptor antagonists, particularly loratadine, could downregulate MITF and tyrosinase in melanocytes. Some plants can inhibit MITF e.g Gentiana veitchiorum Hemsl., Thymelaea hirsuta, Argania spinosa L. In this review, we update the information about MITF and describe the mechanism of its inhibitors in preventing melanogenesis.

TFAP2 paralogs facilitate chromatin access for MITF at pigmentation and cell proliferation genes.

In developing melanocytes and in melanoma cells, multiple paralogs of the Activating-enhancer-binding Protein 2 family of transcription factors (TFAP2) contribute to expression of genes encoding pigmentation regulators, but their interaction with Microphthalmia transcription factor (MITF), a master regulator of these cells, is unclear. Supporting the model that TFAP2 facilitates MITF’s ability to activate expression of pigmentation genes, single-cell seq analysis of zebrafish embryos revealed that pigmentation genes are only expressed in the subset of mitfa-expressing cells that also express tfap2 paralogs. To test this model in SK-MEL-28 melanoma cells we deleted the two TFAP2 paralogs with highest expression, TFAP2A and TFAP2C, creating TFAP2 knockout (TFAP2-KO) cells. We then assessed gene expression, chromatin accessibility, binding of TFAP2A and of MITF, and the chromatin marks H3K27Ac and H3K27Me3 which are characteristic of active enhancers and silenced chromatin, respectively. Integrated analyses of these datasets indicate TFAP2 paralogs directly activate enhancers near genes enriched for roles in pigmentation and proliferation, and directly repress enhancers near genes enriched for roles in cell adhesion. Consistently, compared to WT cells, TFAP2-KO cells proliferate less and adhere to one another more. TFAP2 paralogs and MITF co-operatively activate a subset of enhancers, with the former necessary for MITF binding and chromatin accessibility. By contrast, TFAP2 paralogs and MITF do not appear to co-operatively inhibit enhancers. These studies reveal a mechanism by which TFAP2 profoundly influences the set of genes activated by MITF, and thereby the phenotype of pigment cells and melanoma cells.

Silencing of Rab23 by siRNA inhibits ultraviolet B-induced melanogenesis via downregulation of PKA/CREB/MITF.

Recent investigations have shown that the Rab family of GTPases is associated with all aspects of melanogenesis. However, the effect of Rab23, which localizes to the plasma membrane and regulates the endocytic pathway within eukaryotic cells, in melanogenesis has not been reported. To understand the role of Rab23 in UVB-induced melanogenesis, we evaluated changes in the level of melanin, activity of tyrosinase and levels of melanogenesis-related proteins such as microphthalmia transcription factor and tyrosinase-related protein-1 (TRP-1) and the melanosome transport-related protein complex Rab27a-melanophilin-myosin Va after the downregulation of Rab23 in B16F10 and SK-MEL-2 cells with or without UVB irradiation. Our results showed that downregulating Rab23 reduced the melanin level and tyrosinase activity and inhibited the expression of proteins involved in UVB-induced melanogenesis. Rab23 colocalized with mature melanosomes marked with TRP-1. Furthermore, downregulating Rab23 induced the abnormal accumulation of melanosomes around the nucleus. We demonstrated that the downregulation of Rab23 inhibited melanin synthesis and melanosome transport by decreasing the PKA/CREB/MITF pathway, which is the key regulator of UVB-induced melanogenesis.

Mouse microphthalmia-associated transcription factor (Mitf) mutations affect the structure of the retinal vasculature.

Mice carrying pathogenic variants in the microphthalmia transcription factor (Mitf) gene show structural and functional changes in the retina and retinal pigment epithelium. The purpose of this study was to assess the vascular changes in Mitf mice carrying pathogenic variants by determining their retinal vessel diameter. Mice examined in this study were: B6-Mitfmi-vga9/+ (n = 6), B6-Mitfmi-enu22(398) /Mitfmi-enu22(398) (n = 6) and C57BL/6J wild type mice (n = 6), all 3 months old. Fundus images were taken with a Micron IV camera after intraperitoneal injection of fluorescein salt. Images were adjusted to enhance contrast and a custom written MATLAB program used to extract the mean vascular diameter at a pre-defined distance from the optic disc. The number of vessels, mean diameter and mean total diameter were examined. The mean diameter of retinal veins in Mitfmi-enu22(398) /Mitfmi-enu22(398) mice was 18.8% larger than in wild type (p = 0.026). No differences in the meandiameter of the retinal arteries were found between the genotypes. Mitfmi-enu22(398) /Mitfmi-enu22(398) mice have 17.2% more retinal arteries (p = 0.026), and 15.6% more retinal veins (p = 0.041) than wild type. A 24.8% increase was observed in the mean combined arterial diameter in mice with the Mitfmi-enu22(398)/ Mitfmi-enu22(398) compared to wild type mice (p = 0.024). A 38.6% increase was found in the mean combined venular diameter in mice with the Mitfmi-enu22(398) /Mitfmi-enu22(398) pathogenic variation as compared to wild type (p = 0.004). The mean combined retinal venular diameter in the Mitfmi-vga9/+ mice was 17.8% larger than in wild type (p = 0.03). An increase in vascularization of the retina in Mitfmi-enu22(398) /Mitfmi-enu22(398) mice was found, indicating an increased demand for blood flow to the retina.

Mohs Micrographic Surgery for Melanoma: A Convincing Treatment Modality.

Mohs micrographic surgery (MMS) is the gold standard for treating keratinocyte carcinomas in specific locations or due to other tumor-specific factors, given its comprehensive margin assessment, tissue sparing, and high cure rate. The use of MMS for treating invasive melanoma with intraoperative immunostains is still a subject of debate. Herein, a literature review supports the following recommendations. In cosmetically or functionally demanding sites such as head, neck, hands, feet, and pretibial leg, we recommend MMS for MMIS and all thin melanomas measuring <0.8 mm Breslow depth. Additionally, on the head and neck, all superficial spreading and lentigo maligna melanoma (LMM) histologic subtypes <0.8 mm Breslow depth should be treated with MMS. We recommend using melanoma antigen recognized by T cells 1 (MART-1) immunostain intraoperatively given its high sensitivity. However, microphthalmia transcription factor (MITF) and Sry-related HMG-Box gene 10 (SOX10) are useful adjuncts for difficult cases.

GIF-2209, an Oxindole Derivative, Accelerates Melanogenesis and Melanosome Secretion via the Modification of Lysosomes in B16F10 Mouse Melanoma Cells.

Melanogenesis and melanosome secretion are regulated by several mechanisms. In this study, we found that the oxindole derivative GIF-2209 accelerated melanogenesis associated with the discrimination in the expression and intracellular distributions of two melanogenic enzymes, tyrosinase (TYR) and tyrosinase-related protein-1 (TYRP-1). GIF-2209 upregulated the expression of TYR via a microphthalmia transcription factor (MITF)-independent mechanism, leading to high expression of protein. In contrast, GIF-2209 did not alter the mRNA levels of TYRP-1 and suppressed its protein levels. GIF-2209 induced the dissociation of TYR from TYRP-1 but did not alter the association between TYR and CD63, a melanosome and lysosome marker. The protein levels of CD63 were also upregulated by GIF-2209. GIF-2209 induced lysosome expansion and redistribution in all areas of the cytosol, accompanied by autophagy acceleration (upregulation of LC3BII protein levels and downregulation of p62 protein levels). In addition, GIF-2209 stimulated the secretion of melanosomes containing high levels of TYR, TYRP-1, and CD63 proteins. The GIF-2209 mediated melanosome secretion was sensitive to the lysosome inhibitor chloroquine. These results suggest that GIF-2209 may activate lysosomal functions with TYR gene expression, while it accelerates melanosome secretion, which finally leads to the depletion of intracellular melanogenic enzyme, especially TYRP-1 protein.