Thymic carcinosarcoma with melanocytic differentiation in a dog.

Carcinosarcomas are very rare tumors in dogs. Although carcinosarcomas with melanocytic differentiation arising from organs other than the thymus have been described in humans, this type of tumor has not been reported in dogs in any part of the body. We observed such a tumor in the cranial mediastinum of an 11-year-old spayed female dachshund. The dog was admitted to the clinic because of coughing, sporadic regurgitation, and dyspnea. Thoracic ultrasonography and computed tomography revealed a large mediastinal mass that was surgically removed via sternotomy. The tumor was of thymic origin and demonstrated 3 distinct components: an epithelial component positive for pancytokeratin (AE1/AE3) and high molecular weight cytokeratin (CK5/CK6) with some cystic spaces; a mesenchymal component positive for vimentin; and in association with the epithelial part, a minor melanocytic component positive for Melan A. Histologic metastasis of the epithelial and melanocytic components was present within a tracheobronchial lymph node. The dog died 105 d after surgery, after an episode of acute dyspnea. Key clinical message: To the authors' knowledge, this is the first report of thymic carcinosarcoma with melanocytic differentiation.

Carcinosarcome thymique avec différenciation mélanocytaire chez un chienLes carcinosarcomes sont des tumeurs très rares chez le chien. Bien que des carcinosarcomes avec différenciation mélanocytaire provenant d'organes autres que le thymus aient été décrits chez l'homme, ce type de tumeur n'a été rapporté chez le chien dans aucune partie du corps. Nous avons observé une telle tumeur dans le médiastin cránien d'une femelle teckel stérilisée de 11 ans. Le chien a été admis à la clinique en raison de toux, de régurgitations sporadiques et de dyspnée. L'échographie thoracique et la tomodensitométrie ont révélé une masse médiastinale importante qui a été retirée chirurgicalement par sternotomie. La tumeur était d'origine thymique et présentait 3 composantes distinctes : une composante épithéliale positive pour la pancytokératine (AE1/AE3) et la cytokératine de haut poids moléculaire (CK5/CK6) avec quelques espaces kystiques; un composant mésenchymateux positif à la vimentine; et en association avec la partie épithéliale, un composant mélanocytaire mineur positif pour Melan A. Des métastases histologiques des composants épithéliaux et mélanocytaires étaient présentes dans un ganglion lymphatique trachéobronchique. Le chien est décédé 105 jours après l'intervention chirurgicale, à la suite d'un épisode de dyspnée aiguë.Message clinique clé :À la connaissance des auteurs, il s'agit du premier cas de carcinosarcome thymique avec différenciation mélanocytaire.(Traduit par Dr Serge Messier).

Establishment of a promising vitiligo mouse model for pathogenesis and treatment studies.

AIMS: Vitiligo is a chronic dermatological condition characterized by the progressive loss of melanocytes, for which traditional therapy has shown limited efficacy. This study aimed to establish a vitiligo model with easy operability, high repeatability, and stable depigmentation to provide a foundation for studying the pathogenesis and developing novel therapies for vitiligo. METHODS: (1) Establishing vitiligo model: Firstly, deliver B16F10 cells to the back skin of C57BL/6 J via intradermal injection (day 0), and the CD4 depletion antibody was injected intraperitoneally on day 4 and 10. Secondly, the melanoma was surgically removed on day 12. Thirdly, CD8 antibody was administered intraperitoneally every fourth day till day 30. (2) Identification of vitiligo model: H&E staining, immunohistochemistry, and immunofluorescence were used to detect the melanocytes. The melanin was detected by transmission electron microscopy (TEM), Lillie ferrous sulfate staining and L-DOPA staining. RESULTS: (1) The back skin and hair began to appear white on day 30. Melanin loss reached peak on day 60; (2) Hematoxylin and eosin (H&E) staining, immunohistochemistry and immunofluorescence results showed melanocytes were reduced. L-DOPA staining, Lillie ferrous sulfate staining and TEM results showed that melanin decreased in the epidermis. CONCLUSION: We successfully establishment a vitiligo mouse model which can be more capable to simulate the pathogenesis of human vitiligo and provide an important basis for the study of pathogenesis and therapy of vitiligo.

Ritlecitinib, a JAK3/TEC family kinase inhibitor, stabilizes active lesions and repigments stable lesions in vitiligo.

The efficacy of ritlecitinib, an oral JAK3/TEC family kinase inhibitor, on active and stable lesions was evaluated in patients with active non-segmental vitiligo in a phase 2b trial (NCT03715829). Patients were randomized to placebo or daily ritlecitinib 50 mg (with or without 4-week 100-mg or 200-mg loading dose), 30 mg, or 10 mg for 24 weeks. Active lesions showed greater baseline expression of inflammatory/immune markers IFNG and CCL5, levels of CD103, and T-cell infiltrates than stable lesions. Patients with more active than stable vitiligo lesions showed higher baseline serum levels of CXCL9 and PD-L1, while patients with more stable than active lesions showed higher baseline serum levels of HO-1. At Week 24, ritlecitinib 50 mg significantly stabilized mean percent change from baseline in depigmentation extent in both active lesions and stable lesions vs. placebo-response, with stable lesions showing greater repigmentation. After 24 weeks of treatment, ritlecitinib 50 mg increased expression of melanocyte markers in stable lesions, while Th1/Th2-related and co-stimulatory molecules decreased significantly in both stable and active lesions. Serum from patients with more active than stable lesions showed decreased levels of ICOS and NK cell activation markers. These data, confirmed at transcription/protein levels, indicate that stable lesion repigmentation occurs early with ritlecitinib, while active lesions require stabilization of inflammation first. ClinicalTrials.gov: NCT03715829.

Analysis of granulysin expression in vitiligo and halo-nevus.

Vitiligo and halo nevus are immune-mediated skin diseases that have a similar pathogenesis and involve cellular cytotoxicity mechanisms that are not yet fully understood. In this study, we investigated the expression patterns of the cytolytic molecule granulysin (GNLY) in different cytotoxic cells in skin samples of vitiligo and halo nevus. Skin biopsies were taken from perilesional and lesional skin of ten vitiligo patients, eight patients with halo nevus and ten healthy controls. We analysed the expression of GNLY by immunohistochemistry in CD8+ and CD56+ NK cells. A significantly higher accumulation of GNLY+, CD8+ GNLY+ and fewer CD56+ GNLY+ cells was found in the lesional skin of vitiligo and halo nevus than in the healthy skin. These cells were localised in the basal epidermis and papillary dermis, suggesting that GNLY may be involved in the immune response against melanocytes. Similarly, but to a lesser extent, upregulation of GNLY+ and CD8+ GNLY+ cells was observed in the perilesional skin of vitiligo and halo nevus compared to healthy controls. In this study, we demonstrated for the first time an increased expression of CD8+ GNLY+ T lymphocytes and CD56+ GNLY+ NK cells in lesions of vitiligo and halo nevus, indicating the role of GNLY in the pathogenesis of both diseases.

Analysis of Lipoprotein Signaling in Iris Melanocytes.

Lipids are compounds involved in many biologic functions including cell structure, metabolism, energy storage and are involved in signaling. A prominent lipid in these functions is cholesterol. Cholesterol also plays a part in the signaling of melanocytes, which contain melanosomes. The maturation of these melanosomes happens during melanocyte growth. The deficit of melanogenesis or melanosome maturation is associated with ocular albinism in the eye. Aberrations of melanosome maturation are also associated with pigment dispersion syndrome. Albinism and pigment dispersion manifestations are systemic. Both melanogenesis and melanocyte maturation are affected by cholesterol metabolism. Cholesterol signaling is a part of many pathways in the body, and evaluating these signals can have implications in systemic disease processes of melanogenesis and melanosome maturation, like ocular albinism and pigment dispersion. Cholesterol is carried by lipoprotein particles. Low-density lipoprotein (LDL) is usually the transport vehicle for cholesterol to reach tissues and organelles. The LDL uptake on cells often sends out a cascade of internal signaling within the cells. We describe here LDL signaling related to lipase activity changes using enzymatic methods with a kit. We describe analyses of cholesterol esters and free cholesterol with liquid chromatography and gas chromatography with or in tandem with mass spectrometry (GC-MS and LC-MS/MS). These analyses will provide insight into melanosome maturation and melanogenesis. The methods described here are applicable to all melanocytes within the body of a model mammalian organism.

Interference with sphingosine kinase-1 reduces the hydrogen peroxide-induced oxidative stress damage in melanocytes through four and a half LIM domains 2.

Vitiligo is featured by manifestation of white maculae and primarily results from oxidative stress. Sphingosine kinase-1 (SPHK1) participates in oxidative stress. This paper was devised to explore the role of SPHK1 in vitiligo and to disclose the mechanism. PIG1 cell viability was appraised utilizing cell counting kit-8 assay while Western blot detected SPHK1 and four and a half LIM domains 2 (FHL2). The transduction efficacy of small interfering RNA (siRNA)-SPHK1, siRNA-FHL2 and pcDNA3.1 plasmid overexpressing FHL2 (Ov-FHL2) was checked using Western blot. Flow cytometry detected cell apoptotisis. Western blot detected mitochondrial cytochrome c (Mit-Cyt-c) and cytosolic cytochrome c (Cyto-Cyt-c). Dichloro-dihydro-fluorescein diacetate (DCFH-DA) detected reactive oxygen species (ROS) activity while oxidative stress markers were evaluated using corresponding assay kits. SPHK1 expression was discovered to be increased in hydrogen peroxide (H2O2)-challenged PIG1 cells and SPHK1 interference alleviated H2O2-challenged viability damage, apoptosis, oxidative stress and FHL2 expression in PIG1 cells. FHL2 depletion could suppress viability damage, apoptosis and oxidative stress in H2O2-challenged PIG1 cells. Rescue experiments demonstrated that the suppressive impacts of SPHK1 deficiency on PIG1 cell viability, apoptosis and oxidative stress induced by H2O2 were offset by FHL2 overexpression. Collectively, SPHK1 knockdown protected against vitiligo via the regulation of FHL2.

Melanin accumulation in acanthotic seborrheic keratosis: Reduced proliferation and early differentiation of keratinocytes and increased number of melanocytes.

Seborrheic keratosis (SK) is a common benign tumour, often associated with hyperpigmentation. To investigate the mechanism of melanin accumulation in SK, we have conducted comprehensive gene expression and histological analyses. We obtained five pairs of skin samples, including non-lesional and SK samples, from the backs of three male Japanese participants aged 40-59 years. To examine melanocytes and keratinocytes in SK, three pairs of skin samples were separated by laser capture microdissection into the basal layer and the other layer in the epidermis. We performed a comprehensive gene expression analysis to identify differentially expressed genes between non-lesional and SK skin, followed by gene ontology and pathway analysis. We found abnormal morphogenesis and cell proliferation in the basal layer, along with increased immune response and impaired cell differentiation and metabolism in the other layer of SK. We focused on cell proliferation and differentiation, as these are directly associated with melanin accumulation. Immunohistochemical analyses of Ki67, keratin 10, and keratin 14 demonstrated the decreases in the proliferation and early differentiation of the epidermis. Contrarily, no significant changes were observed in terminal differentiation markers, filaggrin and loricrin. Although the number of melanocytes was higher in SK than in non-lesional skin, melanogenic activity showed no difference. These results indicated that melanin accumulation in SK is caused by delayed melanin excretion due to reduced turnover around the basal and spinous layers of the epidermis and melanin production due to an increased number of melanocytes. Our findings provide new insights for therapeutic approaches in SK.

Exosomes Derived from Hypertrophic Scar Fibroblasts Suppress Melanogenesis in Normal Human Epidermal Melanocytes.

Post-burn hypertrophic scars often exhibit abnormal pigmentation. Exosomes play important roles in maintaining normal physiological homeostasis and in the pathological development of diseases. This study investigated the effects of the exosomes derived from hypertrophic scar fibroblasts (HTSFs) on melanocytes, which are pigment-producing cells. Normal fibroblasts (NFs) and HTSFs were isolated and cultured from normal skin and hypertrophic scar (HTS) tissue. Both the NF- and HTSF-exosomes were isolated from a cell culture medium and purified using a column-based technique. The normal human epidermal melanocytes were treated with both exosomes at a concentration of 100 µg/mL at different times. The cell proliferation, melanin content in the medium, apoptotic factors, transcription factors, melanin synthesis enzymes, signaling, signal transduction pathways, and activators of transcription factors (STAT) 1, 3, 5, and 6 were investigated. Compared with the Dulbecco's phosphate-buffered saline (DPBS)-treated controls and NF-exosomes, the HTSF-exosomes decreased the melanocyte proliferation and melanin secretion. The molecular patterns of apoptosis, proliferation, melanin synthesis, Smad and non-Smad signaling, and STATs were altered by the treatment with the HTSF-exosomes. No significant differences were observed between the DPBS-treated control and NF-exosome-treated cells. HTSF-derived exosomes may play a role in the pathological epidermal hypopigmentation observed in patients with HTS.

Discovery of anti-melanogenic components in persimmon (Diospyros kaki) leaf using LC-MS/MS-MN, AlphaFold2-enabled virtual screening and biological validation.

Persimmon (Diospyros kaki) leaf is widely used as a tea substitute in East Asia, offering potential health benefits. Although studies have highlighted their effects on hyperpigmentation disorders, the active components remain unidentified. This study introduces a novel approach combining LC-MS/MS-based molecular networking with AlphaFold2-enabled virtual screening to expedite the identification of bioactive components in persimmon leaf. A total of 105 compounds were identified by MS/MS analysis. Further, virtual screening identified five flavonoids with potential anti-melanogenic properties. Bioassays confirmed myricetin, quercetin, and kaempferol inhibited melanogenesis in human melanocytes in a dose-dependent manner. Biolayer interferometry assays revealed strong binding affinity between these flavonols and hsTYR, with KD values of 23.26 ± 11.77 for myricetin, 12.43 ± 0.37 for quercetin, and 14.99 ± 3.80 µM for kaempferol. Molecular dynamics simulations provided insights into the binding interactions of these flavonols with hsTYR, particularly highlighting the essential role of the 3-OH group on the C-ring. This study elucidates the bioactive components responsible for the anti-melanogenic effects of persimmon leaf, supporting their use in product development.

A Nude Mouse Model of Xenografted Hypertrophic Scar Cells to Test Therapeutics in the Skin.

BACKGROUND: Existing animal models for testing therapeutics in the skin are limited. Mouse and rat models lack similarity to human skin in structure and wound healing mechanism. Pigs are regarded as the best model with regards to similarity to human skin; however, these studies are expensive, time-consuming, and only small numbers of biologic replicates can be obtained. In addition, local-regional effects of treating wounds that are closely adjacent to one-another with different treatments make assessment of treatment effectiveness difficult in pig models. Therefore, here, a novel nude mouse model of xenografted porcine hypertrophic scar (HTS) cells was developed. This model system was developed to test if supplying hypo-pigmented cells with exogenous alpha melanocyte stimulating hormone (α-MSH) will reverse pigment loss in vivo. METHODS: Dyschromic HTSs were created in red Duroc pigs. Epidermal scar cells (keratinocytes and melanocytes) were derived from regions of hyper-, hypo-, or normally pigmented scar or skin and were cryopreserved. Dermal fibroblasts (DFs) were isolated separately. Excisional wounds were created on nude mice and a grafting dome was placed. DFs were seeded on day 0 and formed a dermis. On day 3, epidermal cells were seeded onto the dermis. The grafting dome was removed on day 7 and hypo-pigmented xenografts were treated with synthetic α-MSH delivered with microneedling. On day 10, the xenografts were excised and saved. Sections were stained using hematoxylin and eosin hematoxylin and eosin (H&E) to assess xenograft structure. RNA was isolated and quantitative real-time polymerase chain reaction (qRT-PCR) was performed for melanogenesis-related genes TYR, TYRP1, and DCT. RESULTS: The seeding of HTSDFs formed a dermis that is similar in structure and cellularity to HTS dermis from the porcine model. When hyper-, hypo-, and normally-pigmented epidermal cells were seeded, a fully stratified epithelium was formed by day 14. H&E staining and measurement of the epidermis showed the average thickness to be 0.11 ± 0.07 µm vs. 0.06 ± 0.03 µm in normal pig skin. Hypo-pigmented xenografts that were treated with synthetic α-MSH showed increases in pigmentation and had increased gene expression of TYR, TYRP1, and DCT compared to untreated controls (TYR: 2.7 ± 1.1 vs. 0.3 ± 1.1; TYRP1: 2.6 ± 0.6 vs. 0.3 ± 0.7; DCT 0.7 ± 0.9 vs. 0.3 ± 1-fold change from control; n = 3). CONCLUSIONS: The developed nude mouse skin xenograft model can be used to study treatments for the skin. The cells that can be xenografted can be derived from patient samples or from pig samples and form a robust dual-skin layer containing epidermis and dermis that is responsive to treatment. Specifically, we found that hypo-pigmented regions of scar can be stimulated to make melanin by synthetic α-MSH in vivo.

First in vitro cell co-culture experiments using laser-induced high-energy electron FLASH irradiation for the development of anti-cancer therapeutic strategies.

Radiation delivery at ultrahigh dose rates (UHDRs) has potential for use as a new anticancer therapeutic strategy. The FLASH effect induced by UHDR irradiation has been shown to maintain antitumour efficacy with a reduction in normal tissue toxicity; however, the FLASH effect has been difficult to demonstrate in vitro. The objective to demonstrate the FLASH effect in vitro is challenging, aiming to reveal a differential response between cancer and normal cells to further identify cell molecular mechanisms. New high-intensity petawatt laser-driven accelerators can deliver very high-energy electrons (VHEEs) at dose rates as high as 1013 Gy/s in very short pulses (10-13 s). Here, we present the first in vitro experiments carried out on cancer cells and normal non-transformed cells concurrently exposed to laser-plasma accelerated (LPA) electrons. Specifically, melanoma cancer cells and normal melanocyte co-cultures grown on chamber slides were simultaneously irradiated with LPA electrons. A non-uniform dose distribution on the cell cultures was revealed by Gafchromic films placed behind the chamber slide supporting the cells. In parallel experiments, cell co-cultures were exposed to pulsed X-ray irradiation, which served as positive controls for radiation-induced nuclear DNA double-strand breaks. By measuring the impact on discrete areas of the cell monolayers, the greatest proportion of the damaged DNA-containing nuclei was attained by the LPA electrons at a cumulative dose one order of magnitude lower than the dose obtained by pulsed X-ray irradiation. Interestingly, in certain discrete areas, we observed that LPA electron exposure had a different effect on the DNA damage in healthy normal human epidermal melanocyte (NHEM) cells than in A375 melanoma cells; here, the normal cells were less affected by the LPA exposure than cancer cells. This result is the first in vitro demonstration of a differential response of tumour and normal cells exposed to FLASH irradiation and may contribute to the development of new cell culture strategies to explore fundamental understanding of FLASH-induced cell effect.

α-Chitosan and ß-Oligochitosan Mixtures-Based Formula for In Vitro Assessment of Melanocyte Cells Response.

Chitosan is a natural polymer with numerous biomedical applications. The cellular activity of chitosan has been studied in various types of cancer, including melanoma, and indicates that these molecules can open new perspectives on antiproliferative action and anticancer therapy. This study analyzes how different chitosan conformations, such as α-chitosan (CH) or ß-oligochitosan (CO), with various degrees of deacetylation (DDA) and molar mass (MM), both in different concentrations and in CH-CO mixtures, influence the cellular processes of SK-MEL-28 melanocytes, to estimate the reactivity of these cells to the applied treatments. The in vitro evaluation was carried out, aiming at the cellular metabolism (MTT assay), cellular morphology, and chitinase-like glycoprotein YKL-40 expression. The in vitro effect of the CH-CO mixture application on melanocytes is obvious at low concentrations of α-chitosan/ß-oligochitosan (1:2 ratio), with the cell's response supporting the hypothesis that ß-oligo-chitosan amplifies the effect. This oligochitosan mixture, favored by the ß conformation and its small size, penetrates faster into the cells, being more reactive when interacting with some cellular components. Morphological effects expressed by the loss of cell adhesion and the depletion of YKL-40 synthesis are significant responses of melanocytes. ß-oligochitosan (1.5 kDa) induces an extension of cytophysiological effects and limits the cell viability compared to α-chitosan (400-900 kDa). Statistical analysis using multivariate techniques showed differences between the CH samples and CH-CO mixtures.

Laccase-Treated Polystyrene Surfaces with Caffeic Acid, Dopamine, and L-3,4-Dihydroxyphenylalanine Substrates Facilitate the Proliferation of Melanocytes and Embryonal Carcinoma Cells NTERA-2.

This study presents the effects of treating polystyrene (PS) cell culture plastic with oxidoreductase enzyme laccase and the catechol substrates caffeic acid (CA), L-DOPA, and dopamine on the culturing of normal human epidermal melanocytes (NHEMs) and human embryonal carcinoma cells (NTERA-2). The laccase-substrate treatment improved PS hydrophilicity and roughness, increasing NHEM and NTERA-2 adherence, proliferation, and NHEM melanogenesis to a level comparable with conventional plasma treatment. Cell adherence dynamics and proliferation were evaluated. The NHEM endpoint function was quantified by measuring melanin content. PS surfaces treated with laccase and its substrates demonstrated the forming of polymer-like structures. The surface texture roughness gradient and the peak curvature were higher on PS treated with a combination of laccase and substrates than laccase alone. The number of adherent NHEM and NTERA-2 was significantly higher than on the untreated surface. The proliferation of NHEM and NTERA-2 correspondingly increased on treated surfaces. NHEM melanin content was enhanced 6-10-fold on treated surfaces. In summary, laccase- and laccase-substrate-modified PS possess improved PS surface chemistry/hydrophilicity and altered roughness compared to untreated and plasma-treated surfaces, facilitating cellular adherence, subsequent proliferation, and exertion of the melanotic phenotype. The presented technology is easy to apply and creates a promising custom-made, substrate-based, cell-type-specific platform for both 2D and 3D cell culture.

Petanin Potentiated JNK Phosphorylation to Negatively Regulate the ERK/CREB/MITF Signaling Pathway for Anti-Melanogenesis in Zebrafish.

Petanin, an acylated anthocyanin from the Solanaceae family, shows potential in tyrosinase inhibitory activity and anti-melanogenic effects; however, its mechanism remains unclear. Therefore, to investigate the underlying mechanism of petanin's anti-melanogenic effects, the enzyme activity, protein expression and mRNA transcription of melanogenic and related signaling pathways in zebrafish using network pharmacology, molecular docking and molecular dynamics simulation were combined for analysis. The results showed that petanin could inhibit tyrosinase activity and melanogenesis, change the distribution and arrangement of melanocytes and the structure of melanosomes, reduce the activities of catalase (CAT) and peroxidase (POD) and enhance the activity of glutathione reductase (GR). It also up-regulated JNK phosphorylation, inhibited ERK/RSK phosphorylation and down-regulated CREB/MITF-related protein expression and mRNA transcription. These results were consistent with the predictions provided through network pharmacology and molecular docking. Thus, petanin could inhibit the activity of tyrosinase and the expression of tyrosinase by inhibiting and negatively regulating the tyrosinase-related signaling pathway ERK/CREB/MITF through p-JNK. In conclusion, petanin is a good tyrosinase inhibitor and anti-melanin natural compound with significant market prospects in melanogenesis-related diseases and skin whitening cosmetics.

Paeoniflorin regulates RhoA/ROCK1 and Nrf2 pathways in PDLIM1-dependent or independent manners in oxidative stressed melanocytes.

BACKGROUND: The adhesive properties of vitiligo melanocytes have decreased under oxidative stress., cytoskeleton proteins can control cell adhesion. Paeoniflorin (PF) was proved to resist hydrogen peroxide (H2O2)-induced oxidative stress in melanocytes via nuclear factorE2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. OBJECTIVES: This study was to investigate whether PF exerts anti-oxidative effect through influencing cytoskeleton markers or potential signaling pathway. METHODS: Human Oxidative Stress Plus array was used to identify the differentially expressed genes between H2O2 + PF group and H2O2 only group, in PIG1 and PIG3V melanocyte cell lines respectively. Western blotting was used to verify the PCR array results and to test the protein expression levels of cytoskeleton markers including Ras homolog family member A (RhoA), Rho-associated kinase 1 (ROCK1) and antioxidative marker Nrf2. Small interfering RNA was used to knock down PDZ and LIM domain 1 (PDLIM1). RESULTS: PF increased the expressions of PDLIM1, RhoA and ROCK1 in H2O2-induced PIG1, in contrast, decreased the expressions of PDLIM1 and ROCK1 in H2O2-induced PIG3V. Knockdown of PDLIM1 increased the expressions of RhoA and Nrf2 in PF-pretreated H2O2-induced PIG1, and ROCK1 and Nrf2 in PF-pretreated H2O2-induced PIG3V. CONCLUSIONS: PF regulates RhoA/ROCK1 and Nrf2 pathways in PDLIM1-dependent or independent manners in H2O2-induced melanocytes. In PIG1, PF promotes PDLIM1 to inhibit RhoA/ROCK1 pathway or activates Nrf2/HO-1 pathway, separately. In PIG3V, PF directly downregulates ROCK1 in PDLIM1-independent manner or upregulates Nrf2 dependent of PDLIM1.

Transient receptor potential mucolipin 1 circumvents oxidative stress in primary human melanocytes.

BACKGROUND: Transient Receptor Potential Mucolipin 1 (TRPML1) serves as a pivotal reactive oxygen species (ROS) sensor in cells, which is implicated in the regulation of autophagy. However, its function in melanocyte autophagy under oxidative stress remains elusive. METHODS: The expression and ion channel function of TRPML1 were investigated using immunofluorescence and calcium imaging in primary human melanocytes (MCs). After activating TRPML1 with MLSA1 (TRPML1 agonist), autophagy-related molecules were investigated via western blot. ROS level, apoptosis- and autophagy-related molecules were investigated after pretreatment with MLSA1. After interference with TRPML1 expression, mitochondrial structures were visualized by electron microscopy with hydrogen peroxide (H2O2）treatment. RESULTS: TRPML1 was expressed and functionally active in primary human MCs, and its activation promotes elevated expression of LC3-II and reduced apoptosis and ROS levels under oxidative stress. TRPML1 downregulation caused mitochondrial swelling and disruption of cristae structures under oxidative stress in primary human MCs. CONCLUSIONS: TRPML1 might mediate lysosomal autophagy in primary human MCs under oxidative stress, participating in mechanisms that maintain the oxidative and antioxidant systems in balance.

Identification of a third myosin-5a-melanophilin interaction that mediates the association of myosin-5a with melanosomes.

Transport and localization of melanosome at the periphery region of melanocyte are depended on myosin-5a (Myo5a), which associates with melanosome by interacting with its adaptor protein melanophilin (Mlph). Mlph contains four functional regions, including Rab27a-binding domain, Myo5a GTD-binding motif (GTBM), Myo5a exon F-binding domain (EFBD), and actin-binding domain (ABD). The association of Myo5a with Mlph is known to be mediated by two specific interactions: the interaction between the exon-F-encoded region of Myo5a and Mlph-EFBD and that between Myo5a-GTD and Mlph-GTBM. Here, we identify a third interaction between Myo5a and Mlph, that is, the interaction between the exon-G-encoded region of Myo5a and Mlph-ABD. The exon-G/ABD interaction is independent from the exon-F/EFBD interaction and is required for the association of Myo5a with melanosome. Moreover, we demonstrate that Mlph-ABD interacts with either the exon-G or actin filament, but cannot interact with both of them simultaneously. Based on above findings, we propose a new model for the Mlph-mediated Myo5a transportation of melanosomes.

Capsaicin combined with stem cells improved mitochondrial dysfunction in PIG3V cells, an immortalized human vitiligo melanocyte cell line, by inhibiting the HSP70/TLR4/mTOR/FAK signaling axis.

BACKGROUND: Vitiligo is a common autoimmune skin disease. Capsaicin has been found to exert a positive effect on vitiligo treatment, and mesenchymal stem cells (MSCs) are also confirmed to be an ideal cell type. This study aimed to explore the influence of capsaicin combined with stem cells on the treatment of vitiligo and to confirm the molecular mechanism of capsaicin combined with stem cells in treating vitiligo. METHODS AND RESULTS: PIG3V cell proliferation and apoptosis were detected using CCK-8 and TUNEL assays, MitoSOX Red fluorescence staining was used to measure the mitochondrial ROS level, and JC-1 staining was used to detect the mitochondrial membrane potential. The expression of related genes and proteins was detected using RT‒qPCR and Western blotting. Coimmunoprecipitation was used to analyze the protein interactions between HSP70 and TLR4 or between TLR4 and mTOR. The results showed higher expression of HSP70 in PIG3V cells than in PIG1 cells. The overexpression of HSP70 reduced the proliferation of PIG3V cells, promoted apoptosis, and aggravated mitochondrial dysfunction and autophagy abnormalities. The expression of HSP70 could be inhibited by capsaicin combined with MSCs, which increased the levels of Tyr, Tyrp1 and DCT, promoted the proliferation of PIG3V cells, inhibited apoptosis, activated autophagy, and improved mitochondrial dysfunction. In addition, capsaicin combined with MSCs regulated the expression of TLR4 through HSP70 and subsequently affected the mTOR/FAK signaling pathway CONCLUSIONS: Capsaicin combined with MSCs inhibits TLR4 through HSP70, and the mTOR/FAK signaling pathway is inhibited to alleviate mitochondrial dysfunction and autophagy abnormalities in PIG3V cells.