Melanosome Transport Research Articles

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.

BMP-2 regulates the expression of myosin Va via smad in melan-a melanocyte.

Myosin Va (Myo Va) is one of three protein complexes involved in melanosome transport. In this study, we identified BMP-2 as an up-regulator of Myo Va expression using 2-methyl-naphtho[1,2,3-de]quinolin-8-one (MNQO). Our results showed that MNQO reduced the mRNA and protein expression of Myo Va and BMP-2 in melanocytes. Knockdown of BMP-2 by siRNA also affected Myo Va mRNA and protein expression, confirming that MNQO regulates Myo Va through BMP-2. Furthermore, phosphorylation of Smad1/5/8 by BMP2 treatment confirmed that the BMP-2/Smad signaling pathway regulates Myo Va expression in Melan-a melanocytes. Smad-binding elements were found in the Myo Va promoter and phosphorylated Smad1/5/8 bind directly to the Myo Va promoter to activate Myo Va transcription and BMP-2 enhances this binding. These findings provide insight into a new role for BMP-2 in Melan-a melanocytes and a mechanism of regulation of Myo Va expression that may be beneficial in the treatment of albinism or hyperpigmentation disorders.

Melanophilin regulates dendritogenesis in melanocytes for feather pigmentation

Limited studies using animal models with a few natural mutations in melanophilin (Mlph) provided partial functions of Mlph in melanosome trafficking. To investigate cellular functions of Mlph, especially ZnF motif of Mlph, we analyzed all three Mlph knockout (KO) quail lines, one and two base pair (bp) deletions as models for total KO, and three bp deletion causing deletion of one Cysteine (C84del) in the ZnF motif. All quail lines had diluted feather pigmentation with impaired dendritogenesis and melanosome transport in melanocytes. In vitro studies revealed capability of binding of the ZnF motif to PIP3, and impairment of PI3P binding and mislocalization of MLPH proteins with ZnF motif mutations. The shortened melanocyte dendrites by the C84del mutation were rescued by introducing WT Mlph in vitro. These results revealed the diluted feather pigmentation by Mlph mutations resulted from congregation of melanosomes in the cell bodies with impairment of the dendritogenesis and the transport of melanosomes to the cell periphery.

Genome-wide association study (GWAS) and novel prognostic markers in metastatic colorectal cancer (mCRC).

174 Background: Few genome-wide investigations have examined germline variants associated with mCRC outcomes following primary treatment. Here, we performed a GWAS meta-analysis to explore the impact of germline genetic variation on progression-free survival (PFS) and overall survival (OS) in four clinical trials of mCRC. Methods: The GWAS comprised 1,324 mCRC patients (pts) enrolled in four randomized Phase II/III trials for first line treatment: FIRE-3 (FOLFIRI-bevacizumab [bev]; FOLFIRI-cetuximab[cet]), MAVERICC (FOLFIRI-bev; FOLFOX6-bev), TRIBE (FOLFIRI-bev; FOLFOXIRI-bev), and TRIBE2 (mFOLFOX6-bev; FOLFOXIRI-bev). DNA isolated from blood was genotyped using the OncoArray and imputed against the Haplotype Reference Consortium panel. In each trial arm, associations between SNPs and PFS and OS were assessed using an additive coding in a Cox proportional hazard framework, adjusting for trial specific patient characteristics and principal components. Meta-analysis was carried out using an inverse-variance weighted fixed effect model. All reported GWAS p-values were adjusted for genomic inflation. To further investigate significant hits, we evaluated associations between gene expression measured from FFPE tumor samples and PFS and OS in 433 pts enrolled in an independent randomized first-line Phase III trial, CALGB/SWOG 80405. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated in multivariable Cox models for expression levels in the top (Q4, high) vs the bottom quartile (Q1, low). Results: Two SNPs in the MLPH intronic region and one SNP in the TYR intronic region surpassed the GWAS significance threshold for PFS and OS, respectively. The top ranked SNPs associated with PFS and OS, rs13409241 (A>G, PFS HR: 1.35, 95% CI [1.21, 1.51], P=4.34E-8) and rs10765199 (T>A, OS HR: 1.45, 95%CI [1.27, 1.65], P=4.69E-8), showed similar effects across all eight treatment arms ( I2 = 0% and 38.5%, respectively). No significant heterogeneity variant was found. In the CALGB/SWOG 80405 cohort, pts with MLPH-low tumors (N=109) showed significantly longer PFS (median 14.8 vs 7.6 months, low vs. high (as reference) adjusted HR 0.53, 95% CI [0.38, 0.74], P=2.1E-4) and OS (median 35.8 vs. 18.2 months, adjusted HR 0.65, 95% CI [0.46, 0.92], P=0.015) compared to MLPH-high (N=107). No significant associations for TYR expression and outcomes were found. MLPH and TYR are involved in melanosome transport and melanogenesis. Melanin has been shown to reduce vitamin D levels, which can affect CRC prognosis. MLPH has also been reported to accelerate epithelial-mesenchymal transition and metastasis in lung and prostate cancers. Conclusions: These results suggest that germline genetic variation in MLPH and TYR may affect mCRC prognosis via a mechanism that involves melanin and possibly the epithelial-mesenchymal transition. Note: Authors Millstein and Lenz contributed equally to this work.

Molecular understanding of the therapeutic potential of melanin inhibiting natural products.

With the development of society and the improvement of people's living standards, there is an increasing demand for melanin-inhibiting products that prioritize health, safety, and efficacy. Therefore, the development of natural products that can safely and efficiently inhibit melanin synthesis is of great social significance and has significant market potential. In this paper, by reviewing the literature reported in recent years, we summarized the natural products with inhibition of melanin synthesis effects that have been put into or not yet put into the market, and classified them according to the chemical groups of their compounds or the extraction methods of the natural products. Through the summary analysis, we found that these compounds mainly include terpenoids, phenylpropanoids, flavonoids and so on, while the natural product extracts mainly include methanol extracts, ethanol extracts, and aqueous extracts. Their main inhibition of melanin synthesis mechanisms include: (1) direct inhibition of tyrosinase activity; (2) down-regulation of the α-MSH-MC1R, Wnt, NO, PI3K/Akt and MAPK pathways through the expression of MITF and its downstream genes TYR, TRP-1, and TRP-2; (3) antioxidant; (4) inhibition of melanocyte growth through cytotoxicity; (5) inhibition of melanosome production and transport. This paper provides an in-depth discussion on the research progress of whitening natural products and their market value. The aim is to offer guidance for future research and development of natural skin whitening products.

Tribuloside acts on the PDE/cAMP/PKA pathway to enhance melanogenesis, melanocyte dendricity and melanosome transport

Ethnopharmacological relevanceTribuloside, a natural flavonoid extracted from Chinese medicine Tribulus terrestris L., has shown potent efficacy in treating various diseases. In China, the fruits of Tribulus terrestris L. have long been utilized for relieving headache, dizziness, itchiness, and vitiligo. Water-based extract derived from Tribulus terrestris L. can enhance melanogenesis in mouse hair follicle melanocytes by elevating the expression of α-melanocyte stimulating hormone (α-MSH) and melanocortin-1 recepter (MC-1R). Nevertheless, there is a lack of information regarding the impact of tribuloside on pigmentation in both laboratory settings and living organisms. Aim of the studyThe present research aimed to examine the impact of tribuloside on pigmentation, and delve into the underlying mechanism. Materials and methodsFollowing the administration of tribuloside in human epidermal melanocytes (HEMCs), we utilized microplate reader, Masson-Fontana ammoniacal silver stain, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to measure melanin contents, dendrite lengths, melanosome counts; L-DOPA oxidation assay to indicate tyrosinase activity, Western blotting to evaluate the expression of melanogenic and associated phosphodiesterase (PDE)/cyclic adenosine monophosphate (cAMP)/cyclic-AMP dependent protein kinase A (PKA) pathway proteins. A PDE-Glo assay to verify the inhibitory effect of tribuloside on PDE was also conducted. Additionally, we examined the impact of tribuloside on the pigmentation in both zebrafish model and human skin samples. ResultsTribuloside had a notable impact on the production of melanin in melanocytes, zebrafish, and human skin samples. These functions might be attributed to the inhibitory effect of tribuloside on PDE, which could increase the intracellular level of cAMP to stimulate the phosphorylation of cAMP-response element binding (CREB). Once activated, it induced microphthalmia-associated transcription factor (MITF) expression and increased the expression of tyrosinase, Rab27a and cell division cycle protein 42 (Cdc42), ultimately facilitating melanogenesis, melanocyte dendricity, and melanin transport. ConclusionTribuloside acts on the PDE/cAMP/PKA pathway to enhance melanogenesis, melanocyte dendricity, and melanosome transport; meanwhile, tribuloside does not have any toxic effects on cells and may be introduced into clinical prescriptions to promote pigmentation.

The inhibition of VDAC1 oligomerization promotes pigmentation through the CaMK–CRTCs/CREB–MITF pathway

The voltage-dependent anion channel 1 (VDAC1) forms an oligomeric structure on the mitochondrial outer membrane, which plays critical roles in many physiological processes. Research studies have demonstrated that the knockout of VDAC1 increases pigment content and up-regulates the expression of melanogenic genes. Due to its involvement in various physiological processes, the depletion of VDAC1 has significant detrimental effects on cellular functions and the inhibition of VDAC1 oligomerization has recently emerged as a promising strategy for the treatment of several diseases. In this study, we found that VDAC1 oligomerization inhibitors, VBIT-12 and NSC-15364, promote melanogenesis, dendrite formation and melanosome transport in human epidermal melanocytes (HEMCs). Mechanistically, treatment of HEMCs with an oligomerization inhibitor increased the level of cytoplasmic calcium ions, which activated calcium-calmodulin dependent protein kinase (CaMK) and led to the phosphorylation of CREB and the nuclear translocation of CREB-regulated transcription coactivators (CRTCs). Subsequently, CRTCs, p-CREB and CREB-binding protein (CBP) in the nucleus cooperatively recruit the transcription machinery to initiate the transcription of MITF thus promoting pigmentation. Importantly, our study also demonstrates that VDAC1 oligomerization inhibitors increase pigmentation in zebrafish and in human skin explants, highlighting their potential as a therapeutic strategy for skin pigmentation disorders.

Melanophilin Polymorphism in Ferrets of Different Color.

In mammals, the main contribution to the variability of pigmentation is made by two groups of genes directly related to the metabolic pathways of pigment synthesis and controlling the transport of melanosomes in melanocytes to keratinocytes. In order to identify the genetic basis of pigmentation variants, the nucleotide sequences of the melanophilin gene were compared in two groups of ferrets-silver-colored and wild-type animals-using sequencing of 16 exons. In carriers of silver color, a single nucleotide deletion was detected in the 9th exon, leading to a shift in the reading frame and the formation of a stop codon downstream. The protein encoded by the mutant allele is almost completely devoid of the C terminal domain of the protein responsible for the contact of melanosomes with actin during their moving to the periphery of melanocytes, but it retains the leading domain involved in the formation of melanosomes. The combination of the preservation of the N domain and the defect of the C domain of the mutant protein for the first time makes it possible to explain the incomplete dominance of the wild-type protein in heterozygotes.

Unveiling genetics of non-syndromic albinism using whole exome sequencing: A comprehensive study of TYR, TYRP1, OCA2 and MC1R genes in 17 families

BackgroundOculocutaneous albinism (OCA) is a group of skin depigmentation disorders. Clinical presentation of OCA includes defects in melanocyte differentiation, melanin biosynthesis, and melanosome maturation and transport. ObjectivesA molecular diagnostics study of families presenting oculocutaneous albinism. MethodsIn this study, 17 consanguineous OCA families consisting of 93 patients were investigated. Whole Exome Sequencing (WES) of the index patient in each family were performed. Short listed variants of WES were Sanger validated for Mendelian segregation in obligate carriers and other available family members. Variant prioritization and pathogenicity were classified as per the criteria of American College Medical Genetics and Genomics (ACMG). Comparative computational modelling was performed to predict the potential damaging effect of the altered proteins. Results15 pathogenic variations: c.132 T > A, c.346C > T, c.488C > G, c.1037G > A in TYR, c.1211C > T, c.1441G > A, c.1706_1707insT, c.2020C > G, c.2402G > C, c.2430del, in OCA2, c.1067G > A in TYRP1 and c.451C > T, c.515G > T, c.766C > T, c.917G > A in MC1R genes were identified. Three variants in OCA2 gene were characterized: c.1706_1707insT, c.2430del, and c.2402G > C, all of which were not reported before in OCA families. ConclusionA few studies focusing on mutation screening of OCA patients have been reported before; however, this study has uniquely presents the Pakhtun ethnic population residing on the North-Western boarder. It explains that TYR, OCA2, TYRP1, and MC1R variations lead to non-syndromic OCA phenotype The overlapping phenotypes of OCA can precisely be diagnosed for its molecular pathogenicity using WES. This study recommends WES as a first-line molecular diagnostic tool, and provides a basis for developing customized genetic tests i.e. pre-marital screening to reduce the disease burden in the future generations.

Anti-melanogenic effect of exosomes derived from human dermal fibroblasts (BJ-5ta-Ex) in C57BL/6 mice and B16F10 melanoma cells.

Exosomes are involved in intercellular communication by transferring cargo between cells and altering the specific functions of the target cells. Recent studies have demonstrated the therapeutic effects of exosomes in several skin diseases. However, understanding of the effects of exosomes on anti-pigmentation is limited. Therefore, we investigated whether BJ-5ta exosomes (BJ-5ta-Ex) derived from human foreskin fibroblasts regulate melanogenesis and delineated the underlying mechanism. Interestingly, treatment with BJ-5ta-Ex induced decreased melanin content, tyrosinase (TYR) activity, and expression of melanogenesis-related genes, including microphthalmia-related transcription factor (MITF), TYR, tyrosinase-related protein-1 (TRP1), and tyrosinase-related protein-2 (TRP2). In addition, BJ-5ta-Ex downregulated the cAMP/PKA and GSK-3β/β-catenin signaling pathways and upregulated the MAPK/ERK signaling pathway. Notably, treatment with BJ-5ta-Ex inhibited α-melanocyte-stimulating hormone-induced melanosome transport and decreased the expression of key proteins involved in melanosome transport, namely, rab27a and melanophilin (MLPH). To further confirm the depigmenting effects of BJ-5ta-Ex, we conducted experiments using a three-dimensional reconstituted human full skin model and ultraviolet B (UVB)-irradiated mouse model. Treatment with BJ-5ta-Ex improved tissue brightness and reduced the distribution of melanosomes. In UVB-irradiated mouse ears, BJ-5ta-Ex reduced the number of active melanocytes and melanin granules. These results demonstrate that BJ-5ta-Ex can be useful for the clinical treatment of hyperpigmentation disorders.

Oxidative and Endoplasmic Reticulum Stress Represent Novel Therapeutic Targets for Choroideremia.

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy, affecting the photoreceptors, retinal pigment epithelium (RPE) and choroid, with no approved therapy. CHM is caused by mutations in the CHM gene, which encodes the ubiquitously expressed Rab escort protein 1 (REP1). REP1 is involved in prenylation, a post-translational modification of Rab proteins, and plays an essential role in intracellular trafficking. In this study, we examined oxidative and endoplasmic reticulum (ER) stress pathways in chmru848 zebrafish and CHMY42X patient fibroblasts, and screened a number of neuroprotectants for their ability to reduce stress. The expression of the oxidative stress markers txn, cat and sod3a, and the ER stress markers bip, atf4 and atf6, were dysregulated in chmru848 fish. The expression of SOD2 was also reduced in CHMY42X fibroblasts, along with reduced BIP and increased CHOP expression. The lack of REP1 is associated with defects in vesicular trafficking, photoreceptor outer segment phagocytosis and melanosome transport, leading to increased levels of stress within the retina and RPE. Drugs targeting oxidative and ER stress pathways represent novel therapeutic avenues.

A phase Ib study of endogenous T cell therapy using SLC45A2-specific CD8 T cells for patients with metastatic uveal melanoma.

9588 Background: Overall survival (OS) for patients (pts) with advanced uveal melanoma (UM) is poor compared with outcomes in cutaneous melanoma. Roughly half of all pts with UM will develop distant metastatic disease despite effective treatment of the primary tumor. Antigen-specific endogenous T cells (ETC) can be expanded and infused successfully in pts with many different types of cancer. Our group has identified epitopes of SLC45A2, a melanosomal transport protein, which are highly expressed in UM and are present at low levels in normal melanocytes. In vitro, we showed that cytotoxic T cells against SLC45A2 were able to kill HLA-matched UM cell lines. In vivo, we hypothesize that infused ETC can traffic to tumor sites. Methods: Between 6/2017 and 12/2022, we conducted a single-center, IRB-approved, first-in-human phase 1b dose escalation study of ETC targeting SLC45A2 in pts with metastatic UM (NCT03068624). Eligible pts with metastatic UM who express HLA-A*02:01 or A*24:02 underwent apheresis to collect peripheral T cells which were then selected and expanded (Turnstile 1). For Turnstile 2, conditioning with low-dose cyclophosphamide (300 mg/m2) occurred on Day -2. For pts in the radiation cohort, radiation occurred between Day -7 and Day -1. Infusion of ETC was done via hepatic arterial or central venous catheter on Day 0 followed by low dose subcutaneous interleukin-2 (IL-2) twice daily for 14 days +/- ipilimumab. The study utilized a 3+3 design with a starting dose level of 3.3 x 109 cells/m2 of ETC alone. The primary objective was to evaluate the safety and tolerability of infusing ETC targeting SLC45A2. Secondary objectives were to evaluate the in vivo persistence and anti-tumor efficacy of this regimen. Results: With a median age of 58 years (30-77), 34 pts were enrolled, 16 (47%) men and 18 (53%) women. 33 (97%) pts underwent apheresis and 11 (32%) received infusion of ETC. One dose limiting toxicity (DLT) thought to be unrelated to treatment or underlying cancer (cardiac disorder Grade 5) occurred at the starting dose level with no additional DLTs. The maximum tolerated dose was established as 1 x 1010 cells/m2. The most common treatment-related grade 3 and 4 toxicities were lymphocyte count decrease 9 (82%), hyponatremia 1 (9%), and hypophosphatemia 1 (9%). Per immune-related response criteria (irRC), 4 (36%) pts had stable disease (SD), 6 (55%) had progression of disease (PD), and 1 (9%) was not evaluable for best response. Median overall survival (OS) was 8.9 weeks (wks). OS was 91% at 4 wks, 55% at 8 wks and 46% at 13 wks. Median progression-free survival was 5.9 wks. In the 4 patients with stable disease, the median duration of SD was 5.7 months. Conclusions: ETC targeting SLC45A2 is safe and well-tolerated in pts with metastatic UM. Further analyses are underway to evaluate T cell trafficking and persistence. Clinical trial information: NCT03068624 .

Phosphorylation of glucocorticoid receptor induced by 16-kauren-2-beta-18, 19-triol decreases expression of Melanophilin through JNK signalling.

16-kauren-2-beta-18, 19-triol (16-kauren) is a natural diterpenoid substance derived from Asteraceae psiadia punctulata, a small tropical shrub in Africa and Asia, and it can reduce Mlph expression without affecting the expression of Rab27a and MyoVa in melanocytes. Melanophilin (Mlph) is an important linker protein in the melanosome transport process. However, the signal transduction pathway for the regulation of Mlph expression has not been fully established. We examined the mechanism of 16-kauren on Mlph expression. Murine melan-a melanocytes were used for in vitro analysis. Western blot analysis, quantitative real-time polymerase chain reaction, and luciferase assay were performed. The inhibition of Mlph expression by 16-kauren-2β-18,19-triol (16-kauren) occurs through the JNK signal and is reversed following glucocorticoid receptor (GR) activation by dexamethasone (Dex). Especially, 16-kauren activates JNK and c-jun signalling, part of the MAPK pathway, with subsequent Mlph repression. When the JNK signal is weakened by siRNA, the inhibition of Mlph expression by 16-kauren was not seen. JNK activation by 16-kauren induces GR phosphorylation, which leads to Mlph repression. These results demonstrate that 16-kauren regulates Mlph expression through the phosphorylation of GR via the JNK signalling pathway.

Inhibition of Melanosome Transport by Inducing Exon Skipping in Melanophilin.

Exon skipping is an efficient technique to inhibit specific gene expression induced by a short-sequence peptide nucleic acid (PNA). To date, there has been no study on the effects of PNA on skin pigmentation. In melanocytes, the tripartite complex is responsible for the transport of mature melanosomes from the nucleus to the dendrites. The tripartite complex is composed of Rab27a, Mlph (Melanophilin), and Myosin Va. Defects in the protein Mlph, a melanosome transport-related protein, are known to cause hypopigmentation. Our study shows that Olipass peptide nucleic acid (OPNA), a cell membrane-permeable PNA, targets exon skipping in the Mlph SHD domain, which is involved in Rab27a binding. Our findings demonstrate that OPNA induced exon skipping in melan-a cells, resulting in shortened Mlph mRNA, reduced Mlph protein levels, and melanosome aggregation, as observed by microscopy. Therefore, OPNA inhibits the expression of Mlph by inducing exon skipping within the gene. These results suggest that OPNA, which targets Mlph, may be a potential new whitening agent to inhibit melanosome movement.

Cryptochrome 1 activation inhibits melanogenesis and melanosome transport through negative regulation of cAMP/PKA/CREB signaling pathway.

Cutaneous pigmentation was recently shown to be an event regulated by clock proteins. Cryptochrome (CRY) is a key protein composing the feedback loop of circadian clock, however, the function of CRY in melanocytes remains unclear. Here, we found that KL001, a synthetic small molecule modulator of CRY1, inhibited melanin synthesis, as well as reduced melanocyte dendrite elongation and melanosome transport. In addition, the dominant role of CRY1 in KL001-induced anti-melanogenesis was revealed by small interfering RNA transfection. Cellular tyrosinase activity and expression level of melanogenic proteins, including tyrosinase, TRP-1, TRP-2, and transport proteins like Rab27a, Cdc42 and Myosin Va induced by α-MSH were remarkably reversed after KL001 treatment. Mechanistically, CRY1 activation inhibited melanogenesis through CREB-dependent downregulation of MITF and CREB phosphorylation was mediated by classical cAMP/PKA pathway. In addition, the other CRY1 activator, KL044 also suppressed cAMP/PKA/CREB pathway and inhibited melanogenesis. Finally, anti-melanogenic efficacy of KL001 was confirmed by determination of melanin contents in UVB-tanning model of brown guinea pigs, which indicated that targeting CRY1 activity, via topical application of small molecule activator, can be utilized therapeutically to manage human pigmentary disorders.

Senescent melanocytes driven by glycolytic changes are characterized by melanosome transport dysfunction.

Rationale: Senescent melanocytes accumulate in photoaged skin and are closely related to skin aging. A better understanding of the molecular characteristics of senescent melanocytes may be the key to controlling skin aging. Methods: We have developed an in vitro model of senescence in melanocytes using UV irradiation and investigated the functional characteristics and molecular mechanisms underlying senescence in UV-irradiated melanocytes. Results: We have highlighted that in vitro senescent melanocytes are characterized by melanosome transport dysfunction resulting in melanin accumulation. The defective melanosome transport was confirmed with the ultrastructural characterization of both in vitro UV-induced senescent melanocytes and in vivo melanocytes of hypopigmented aging skin. A single-cell transcriptomic analysis revealed that the glycolytic metabolism pathway appeared to be significantly upregulated in most senescent phenotypes. Furthermore, the inhibition of glycolysis by pharmacological compounds mitigates the pro-aging effects of melanocytes senescence, suggesting that alterations in cellular glucose metabolism act as a driving force for senescence in melanocytes. Conclusion: These results demonstrate that senescent melanocytes are characterized by glycolytic metabolism changes and a defective melanosome transport process, which may be related to impaired mitochondrial function, highlighting the importance of metabolic reprogramming in regulating melanocyte senescence.

Membrane translocation of vinculin after UVA exposure facilitates melanosome trafficking.

Skin pigmentation is among the defenses against ultraviolet (UV) radiation. During formation of skin pigmentation, melanosomes that are transported to the cell membrane and released are internalized by keratinocytes. We here examined whether vinculin, the origin of actin fibers, is involved in this intracellular transport of melanosomes by using melanocytes with suppressed vinculin expression. Using fluorescence immunostaining, the migration of vinculin to the cell membrane due to exposure to 365-nm LED light was examined. The intracellular distribution of melanosomes after irradiation was weighted toward the pericellular region compared with non-irradiated cells. With the suppression of vinculin expression, the amount of extracellularly released melanin decreased. We conclude that the membrane migration of vinculin after UVA exposure is involved in the intracellular transport of melanosomes.

516 Dynein is involved in the movement, distribution, acidity, and transfer of melanosomes

Skin pigmentation depends on cellular processes including biogenesis, transport, maturation, and transfer of melanosomes to keratinocytes. However, the mechanisms associated with the fine spatial and temporal controls to ensure adequate pigmentation remain unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for the transport, acidity regulation and efficient transfer of melanosomes to keratinocytes. In Mus musculus melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional movement, leading to their peripheral localization in the cell.

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.

Rab32/38-Dependent and -Independent Transport of Tyrosinase to Melanosomes in B16-F1 Melanoma Cells.

B16-F1 melanoma cells have often been used as a model to investigate melanogenesis, but the evidence that melanosome biogenesis and transport occur by the same mechanisms in normal melanocytes and B16-F1 cells is insufficient. In this study, we established knockout B16-F1 cells for each of several key factors in melanogenesis, i.e., tyrosinase (Tyr), Hps4, Rab27A, and Rab32·Rab38 (Rab32/38), and then compared their phenotypes with the phenotypes of corresponding mutant mouse melanocyte cell lines, i.e., melan-c, melan-le, melan-ash, and Rab32-deficient melan-cht cells, respectively. The results showed that Tyr and Rab27A are also indispensable for melanin synthesis and peripheral melanosome distribution, respectively, in B16-F1 cells, but that Hps4 or its downstream targets Rab32/38 are not essential for Tyr transport in B16-F1 cells, suggesting the existence of a Rab32/38-independent Tyr transport mechanism in B16-F1 cells. We then performed comprehensive knockdown screening of Rab small GTPases and identified Rab10 and Rab24, previously uncharacterized Rabs in melanocytes, as being involved in Tyr transport under Rab32/38-null conditions. Our findings indicate a difference between the Tyr transport mechanism in melanocytes and B16-F1 cells in terms of Rab32/38-dependency and a limitation in regard to using melanoma cells as a model for melanocytes, especially when investigating the mechanism of endosomal Tyr transport.