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Raymond E. Boissy - One of the best experts on this subject based on the ideXlab platform.
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A Role for Tyrosinase-Related Protein 1 in 4-tert-Butylphenol-Induced Toxicity in Melanocytes : Implications for Vitiligo
American Journal of Pathology, 2006Co-Authors: Prashiela Manga, Rangaprasad Sarangarajan, David Sheyn, Fan Yang, Raymond E. BoissyAbstract:Vitiligo presents with depigmented cutaneous lesions following localized Melanocyte death. Multiple factors contribute to cell death, including genetically determined susceptibility to trauma, and environmental factors, such as exposure to 4-tert-butylphenol (4-TBP). We demonstrate that 4-TBP induces oxidative stress that is more readily overcome by Melanocytes from normally pigmented individuals than from two individuals with vitiligo. The antioxidant catalase selectively and significantly reduced death of Melanocytes derived from two individuals with vitiligo, indicating a role for oxidative stress in vitiligo pathogenesis. In normal Melanocytes, oxidative stress results in reduced expression of microphthalmia-associated transcription factor (MITF). Melanocyte-stimulating hormone-induced expression of MITF protein caused increased sensitivity to 4-TBP, whereas sensitivity of melanomas correlated with MITF expression. MITF stimulates melanin synthesis by up-regulating expression of melanogenic enzymes such as tyrosinase-related protein-1 (Tyrp1). Although melanin content per se did not affect sensitivity to 4-TBP, expression of Tyrp1 significantly increased sensitivity. Melanocytes and melanomas that express functional Tyrp1 were significantly more sensitive to 4-TBP than Tyrp1-null cells. Thus, normal Melanocytes respond to 4-TBP by reducing expression of MITF and Tyrp1. We hypothesize that Melanocytes in vitiligo demonstrate reduced ability to withstand oxidative stress due, partly, to a disruption in MITF regulation of Tyrp1.
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Melanocyte-Specific Proteins Are Aberrantly Trafficked in Melanocytes of Hermansky-Pudlak Syndrome-Type 3
The American journal of pathology, 2005Co-Authors: Raymond E. Boissy, Yang Zhao, Bonnie L. Richmond, Marjan Huizing, Amanda Helip-wooley, Amy Koshoffer, William A. GahlAbstract:Hermansky-Pudlak Syndrome-type 3 (HPS-3) is a relatively mild subtype of HPS with minimal cutaneous and ocular depigmentation. The HPS-3 gene encodes a novel protein of unknown function with a predicted molecular weight of 114 kd. To assess the role of the HPS3 protein in melanization, cultured Melanocytes developed from HPS-3 patients were evaluated biochemically and histologically for activity and localization of Melanocyte-specific proteins. Endogenous tyrosinase activity of HPS-3 Melanocytes was substantial, but tyrosinase activity and melanin synthesis was suppressed in intact Melanocytes. However, the level of suppression, as well as extent to which up-regulation by isobutylmethylxanthine and cholera toxin was muted, was less that in HPS-1 Melanocytes. Ultrastructurally, HPS-3 Melanocytes contained morphologically normal melanosomes, predominantly of stage I and II with minimal stage III and few stage IV melanosomes. Dihydroxyphenylalanine (DOPA) histochemistry demonstrated an increase in melanization of melanosomes. Unique to HPS-3 Melanocytes were numerous DOPA-positive 50-nm vesicles and tubular elements present throughout the cell body and dendrites. Tyrosinase, tyrosinase-related protein-1 (Tyrp1), dopachrome tautomerase (Dct), and LAMP1 and 3 localization in HPS-3 Melanocytes, as evaluated by immunocytochemistry and confocal microscopy, demonstrated a fine, floccular distribution in contrast to the coarse, granular distribution characteristic of control Melanocytes. The localization profile of other proteins expressed by Melanocytes (ie, Silver/Pmel17, Melan-A/MART-1, LAMP2, Rab 27, transferrin, c-kit, adaptin-3, and the HPS1 protein) appeared normal. These results suggest that a specific subset of Melanocyte proteins are aberrantly trafficked throughout the HPS-3 Melanocyte and may be responsible for the reduction in melanin synthesis.
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Mutant alleles at the brown locus encoding tyrosinase-related protein-1 (TRP-1) affect proliferation of mouse Melanocytes in culture.
Pigment Cell Research, 2000Co-Authors: Rangaprasad Sarangarajan, M. Lynn Lamoreux, Yang Zhao, George F. Babcock, James Cornelius, Raymond E. BoissyAbstract:Tyrosinase related protein-1 (TRP-1) is a Melanocyte-specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP-1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1b (brown) and Tyrp1b-cj (cordovan) compared with wild type Tyrp1B (black) on Melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP-1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan Melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown Melanocytes but not between black and cordovan Melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP-1 in wild type black, brown and cordovan Melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse Melanocyte cultures.
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Dilated rough endoplasmic reticulum and premature death in Melanocytes cultured from the vitiligo mouse.
The American journal of pathology, 1991Co-Authors: Raymond E. Boissy, K. E. Beato, James J. NordlundAbstract:It has been proposed that the selective Melanocyte destruction in hair bulbs of the murine model for vitiligo (VIT) is instigated by either the local cutaneous environment or an innate Melanocyte defect. To address this problem, the authors have isolated the Melanocyte population from environmental influences by using cell culture technology and have observed reduced proliferation, specific cytologic abnormalities, and premature cell death in cultures of pure VIT Melanocytes established from neonatal skin. Cultured VIT Melanocytes manifest abnormal compartmentalization of melanosomes and some aberrant dihydroxyphenylalanine-positive structures. The most prominent abnormality observed in cultured VIT Melanocytes when compared with the control C57BL/6J cells is a development in dilation of the rough endoplasmic reticulum (RER) that morphologically resembles the in vivo condition. Dilation of the RER can be exaggerated in VIT or induced in C57BL/6J Melanocytes by the addition of Brefeldin A to cultures. Conversely the dilated RER cisternae characteristic of the VIT Melanocyte can be reversed by inhibition of protein synthesis with cyclobeximide. Melanocyte cultures developed from heterozygote neonates, resulting from cross-breedings between the VIT and the C57BL/6J lines, also demonstrated extensive RER dilation along with only slightly reduced proliferation. The results in this report verify that the murine vitiligo Melanocyte expresses an innate defect that affects the structure and presumably the function of the rough endoplasmic reticulum.
Stephen L Johnson - One of the best experts on this subject based on the ideXlab platform.
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Lineage Relationship of Direct-Developing Melanocytes and Melanocyte Stem Cells in the Zebrafish
PloS one, 2011Co-Authors: Robert C. Tryon, Charles W. Higdon, Stephen L JohnsonAbstract:Previous research in zebrafish has demonstrated that embryonic and larval regeneration Melanocytes are derived from separate lineages. The embryonic Melanocytes that establish the larval pigment pattern do not require regulative Melanocyte stem cell (MSC) precursors, and are termed direct-developing Melanocytes. In contrast, the larval regeneration Melanocytes that restore the pigment pattern after ablation develop from MSC precursors. Here, we explore whether embryonic Melanocytes and MSCs share bipotent progenitors. Furthermore, we explore when fate segregation of embryonic Melanocytes and MSCs occurs in zebrafish development. In order to achieve this, we develop and apply a novel lineage tracing method. We first demonstrate that Tol2-mediated genomic integration of reporter constructs from plasmids injected at the 1–2 cell stage occurs most frequently after the midblastula transition but prior to shield stage, between 3 and 6 hours post-fertilization. This previously uncharacterized timing of Tol2-mediated genomic integration establishes Tol2-mediated transposition as a means for conducting lineage tracing in zebrafish. Combining the Tol2-mediated lineage tracing strategy with a Melanocyte regeneration assay previously developed in our lab, we find that embryonic Melanocytes and larval regeneration Melanocytes are derived from progenitors that contribute to both lineages. We estimate 50–60 such bipotent melanogenic progenitors to be present in the shield-stage embryo. Furthermore, our examination of direct-developing and MSC-restricted lineages suggests that these are segregated from bipotent precursors after the shield stage, but prior to the end of convergence and extension. Following this early fate segregation, we estimate approximately 100 embryonic Melanocyte and 90 MSC-restricted lineages are generated to establish or regenerate the zebrafish larval pigment pattern, respectively. Thus, the dual strategies of direct-development and MSC-derived development are established in the early gastrula, via fate segregation of the two lineages.
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Neocuproine ablates Melanocytes in adult zebrafish.
Zebrafish, 2008Co-Authors: Thomas O'reilly-pol, Stephen L JohnsonAbstract:Abstract The simplest regeneration experiments involve the ablation of a single cell type. While methods exist to ablate the Melanocytes of the larval zebrafish,1,2 no convenient method exists to ablate Melanocytes in adult zebrafish. Here, we show that the copper chelator neocuproine (NCP) causes fragmentation and disappearance of melanin in adult zebrafish Melanocytes. Adult Melanocytes expressing eGFP under the control of a Melanocyte-specific promoter also lose eGFP fluorescence in the presence of NCP. We conclude that NCP causes Melanocyte death. This death is independent of p53 and melanin, but can be suppressed by the addition of exogenous copper. NCP is ineffective at ablating larval Melanocytes. This now provides a tool for addressing questions about stem cells and the maintenance of the adult pigment pattern in zebrafish.
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mutations in gfpt1 and skiv2l2 cause distinct stage specific defects in larval Melanocyte regeneration in zebrafish
PLOS Genetics, 2005Co-Authors: Chaotsung Yang, Anna Hindes, Keith A Hultman, Stephen L JohnsonAbstract:The establishment of a single cell type regeneration paradigm in the zebrafish provides an opportunity to investigate the genetic mechanisms specific to regeneration processes. We previously demonstrated that regeneration Melanocytes arise from cell division of the otherwise quiescent Melanocyte precursors following larval Melanocyte ablation with a small molecule, MoTP. The ease of ablating Melanocytes by MoTP allows us to conduct a forward genetic screen for mechanisms specific to regeneration from such precursors or stem cells. Here, we reported the identification of two mutants, eartha j23e1 and julie j24e1 from a Melanocyte ablation screen. Both mutants develop normal larval Melanocytes, but upon Melanocyte ablation, each mutation results in a distinct stage-specific defect in Melanocyte regeneration. Positional cloning reveals that the eartha j23e1 mutation is a nonsense mutation in gfpt1 (glutamine:fructose-6-phosphate aminotransferase 1), the rate-limiting enzyme in glucosamine-6-phosphate biosynthesis. Our analyses reveal that a mutation in gfpt1 specifically affects Melanocyte differentiation (marked by melanin production) at a late stage during regeneration and that gfpt1 acts cell autonomously in Melanocytes to promote ontogenetic Melanocyte darkening. We identified that the julie j24e1 mutation is a splice-site mutation in skiv2l2 (superkiller viralicidic activity 2-like 2), a predicted DEAD-box RNA helicase. Our in situ analysis reveals that the mutation in skiv2l2 causes defects in cell proliferation, suggesting that skiv2l2 plays a role in regulating melanoblast proliferation during early stages of Melanocyte regeneration. This finding is consistent with previously described role for cell division during larval Melanocyte regeneration. The analyses of these mutants reveal their stage-specific roles in Melanocyte regeneration. Interestingly, these mutants identify regeneration-specific functions not only in early stages of the regeneration process, but also in late stages of differentiation of the regenerating Melanocyte. We suggest that mechanisms of regeneration identified in this mutant screen may reveal fundamental differences between the mechanisms that establish differentiated cells during embryogenesis, and those involved in larval or adult growth. Citation: Yang CT, Hindes AE, Hultman KA, Johnson SL (2007) Mutations in gfpt1 and skiv2l2 cause distinct stage-specific defects in larval Melanocyte regeneration in
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A requirement for kit in embryonic zebrafish Melanocyte differentiation is revealed by melanoblast delay
Development Genes and Evolution, 2004Co-Authors: Eve M. Mellgren, Stephen L JohnsonAbstract:Exploring differences in gene requirements between species can allow us to delineate basic developmental mechanisms, provide insight into patterns of evolution, and explain heterochronic differences in developmental processes. One example of differences in gene requirements between zebrafish and mammals is the requirement of the kit receptor tyrosine kinase in Melanocyte development. kit is required for migration, survival and differentiation of all neural crest-derived Melanocytes in mammals. In contrast, zebrafish kit is not required for differentiation of embryonic Melanocytes during normal development. When melanoblast development in zebrafish embryos is delayed by injecting morpholinos targeted to the mitfa gene, we show that these delayed melanoblasts fail to differentiate in kit mutants. Thus, we show that there is a kit requirement for Melanocyte differentiation in zebrafish when melanoblast development is delayed. Furthermore, we show that kit is not involved in maintaining Melanocyte precursors through the developmental delay, but instead is required for differentiation of Melanocytes after the block on their development is removed. Finally, we suggest there is a heterochronic shift in the onset of Melanocyte differentiation between fish and mouse, and developmental delay of melanoblast development in zebrafish removes this heterochronic difference.
Fabian V Filipp - One of the best experts on this subject based on the ideXlab platform.
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bromodomain and extra terminal domain bet proteins regulate Melanocyte differentiation
Epigenetics & Chromatin, 2020Co-Authors: Archit Trivedi, Aanchal Mehrotra, Caitlin E Baum, Brandon Lewis, Tupa Basuroy, Thomas M Blomquist, Robert J Trumbly, Fabian V FilippAbstract:Pharmacologic inhibition of bromodomain and extra-terminal (BET) proteins is currently being explored as a new therapeutic approach in cancer. Some studies have also implicated BET proteins as regulators of cell identity and differentiation through their interactions with lineage-specific factors. However, the role of BET proteins has not yet been investigated in Melanocyte differentiation. Melanocyte inducing transcription factor (MITF) is the master regulator of Melanocyte differentiation, essential for pigmentation and Melanocyte survival. In this study, we tested the hypothesis that BET proteins regulate Melanocyte differentiation through interactions with MITF. Here we show that chemical inhibition of BET proteins prevents differentiation of unpigmented melanoblasts into pigmented Melanocytes and results in de-pigmentation of differentiated Melanocytes. BET inhibition also slowed cell growth, without causing cell death, increasing the number of cells in G1. Transcriptional profiling revealed that BET inhibition resulted in decreased expression of pigment-specific genes, including many MITF targets. The expression of pigment-specific genes was also down-regulated in melanoma cells, but to a lesser extent. We found that RNAi depletion of the BET family members, bromodomain-containing protein 4 (BRD4) and bromodomain-containing protein 2 (BRD2) inhibited expression of two melanin synthesis enzymes, TYR and TYRP1. Both BRD4 and BRD2 were detected on Melanocyte promoters surrounding MITF-binding sites, were associated with open chromatin structure, and promoted MITF binding to these sites. Furthermore, BRD4 and BRD2 physically interacted with MITF. These findings indicate a requirement for BET proteins in the regulation of pigmentation and Melanocyte differentiation. We identified changes in pigmentation specific gene expression that occur upon BET inhibition in melanoblasts, Melanocytes, and melanoma cells.
Dustin Anderson - One of the best experts on this subject based on the ideXlab platform.
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yy1 regulates Melanocyte development and function by cooperating with mitf
PLOS Genetics, 2012Co-Authors: Jun S. Song, Robert J. A. Bell, Thanh-nga T. Tran, Rizwan Haq, Huifei Liu, Kevin T. Love, Robert Langer, Dustin AndersonAbstract:Studies of coat color mutants have greatly contributed to the discovery of genes that regulate Melanocyte development and function. Here, we generated Yy1 conditional knockout mice in the Melanocyte-lineage and observed profound Melanocyte deficiency and premature gray hair, similar to the loss of Melanocytes in human piebaldism and Waardenburg syndrome. Although YY1 is a ubiquitous transcription factor, YY1 interacts with M-MITF, the Waardenburg Syndrome IIA gene and a master transcriptional regulator of Melanocytes. YY1 cooperates with M-MITF in regulating the expression of piebaldism gene KIT and multiple additional pigmentation genes. Moreover, ChIP–seq identified genome-wide YY1 targets in the Melanocyte lineage. These studies mechanistically link genes implicated in human conditions of Melanocyte deficiency and reveal how a ubiquitous factor (YY1) gains lineage-specific functions by co-regulating gene expression with a lineage-restricted factor (M-MITF)—a general mechanism which may confer tissue-specific gene expression in multiple lineages.
Tupa Basuroy - One of the best experts on this subject based on the ideXlab platform.
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bromodomain and extra terminal domain bet proteins regulate Melanocyte differentiation
Epigenetics & Chromatin, 2020Co-Authors: Archit Trivedi, Aanchal Mehrotra, Caitlin E Baum, Brandon Lewis, Tupa Basuroy, Thomas M Blomquist, Robert J Trumbly, Fabian V FilippAbstract:Pharmacologic inhibition of bromodomain and extra-terminal (BET) proteins is currently being explored as a new therapeutic approach in cancer. Some studies have also implicated BET proteins as regulators of cell identity and differentiation through their interactions with lineage-specific factors. However, the role of BET proteins has not yet been investigated in Melanocyte differentiation. Melanocyte inducing transcription factor (MITF) is the master regulator of Melanocyte differentiation, essential for pigmentation and Melanocyte survival. In this study, we tested the hypothesis that BET proteins regulate Melanocyte differentiation through interactions with MITF. Here we show that chemical inhibition of BET proteins prevents differentiation of unpigmented melanoblasts into pigmented Melanocytes and results in de-pigmentation of differentiated Melanocytes. BET inhibition also slowed cell growth, without causing cell death, increasing the number of cells in G1. Transcriptional profiling revealed that BET inhibition resulted in decreased expression of pigment-specific genes, including many MITF targets. The expression of pigment-specific genes was also down-regulated in melanoma cells, but to a lesser extent. We found that RNAi depletion of the BET family members, bromodomain-containing protein 4 (BRD4) and bromodomain-containing protein 2 (BRD2) inhibited expression of two melanin synthesis enzymes, TYR and TYRP1. Both BRD4 and BRD2 were detected on Melanocyte promoters surrounding MITF-binding sites, were associated with open chromatin structure, and promoted MITF binding to these sites. Furthermore, BRD4 and BRD2 physically interacted with MITF. These findings indicate a requirement for BET proteins in the regulation of pigmentation and Melanocyte differentiation. We identified changes in pigmentation specific gene expression that occur upon BET inhibition in melanoblasts, Melanocytes, and melanoma cells.