Dataset Information

Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress.

ABSTRACT: Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy, and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus, whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes.

SUBMITTER: Mosenson JA

PROVIDER: S-EPMC3947476 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress.

Mosenson Jeffrey A JA Flood Kelsey K Klarquist Jared J Eby Jonathan M JM Koshoffer Amy A Boissy Raymond E RE Overbeck Andreas A Tung Rebecca C RC Le Poole I Caroline IC

Pigment cell & melanoma research 20140113 2

Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured ...[more]

PMID: 24354861

Similar Datasets

Project description:BackgroundOxidative stress has a vital role in the early stages of vitiligo. Autoantigens released from apoptotic melanocytes (MC) under oxidative stress are involved in the presentation and recognition of antigens. However, the transport of autoantigens to the cell surface and their release to the extracellular environment are still unclear. Apoptotic bodies (ABs) have always been considered as a key source of immunomodulators and autoantigens. Yet, the role of ABs in the immune mechanism of vitiligo is still unknown.PurposeTo explore whether MC's autoantigens translocate into ABs during oxidative stress-induced apoptosis and study the molecular mechanisms underlying autoantigen migration and AB formation.MethodsPIG3V (an immortalized human vitiligo melanocyte cell line) were treated with H2O2, and ABs were separated. Transmission electron microscopy, flow cytometry, Western blot, mass spectrometry, and other methods were used to determine the relocation of specific antigens in PIG3V cells to ABs. After pretreatment with specific inhibitors (Rho kinase (Y-27632), myosin light chain kinase (MLCK, ML-9), pan-caspase (zVAD-FMK), and JNK (SP600125)), the pathway of autoantigen translocation into ABs and the formation of apoptotic bodies were determined.ResultsWhen treated with 0.8 mM H2O2, ABs were released from these cells. Autoantigens such as tyrosinase-related protein 1 (TYRP-1) and cleavage nuclear membrane antigen Lamin A/C (Asp230) were concentrated in ABs. The expression of autoantigens and the formation of ABs increased in a time- and dose-dependent manner after treatment with H2O2, while the application of specific inhibitors inhibited the formation of apoptotic bodies, i.e., the expression of antigens.ConclusionVitiligo autoantigens translocate into ABs in the process of apoptosis induced by oxidative stress. The cytoskeletal protein activation pathway and the JNK-related apoptosis pathway are involved in the transport of autoantigens and the formation of ABs. ABs may be the key bridge between MC cell apoptosis and cellular immunity.

Project description:Purpose: In order to explore the mechanism of hydroxychloroquine (HCQ) in treating vitiligo. We report the application of transcriptome profiling (RNA-seq) in human vitiligo melanocytes cell line PIG3V after HCQ treatment. Methods: The PIG3V cells were divided randomly in two groups: HCQ treatment (1 μg/mL, 24 hours) and the cells without treatment (only medium). RNA samples extracted from PIG3V cells were quantified by NanoDrop and Bioanalyzer. After 2 rounds of purification Poly A RNA was extracted then from total RNA by using Dynabeads Oligo. Small pieces of Poly A RNA were returned through Magnesium RNA Fragmentation Module under the condition of 94℃ for 5 minutes. Subsequently, SuperScript Reverse Transcriptase was used to reverse-transcribe the cleaved RNA pieces into cDNA. Then, U-labeled second-stranded DNAs was synthesized. After adding of A-base to each strand, preparing for indexed adapters, and heat-labile UDG enzyme treatment, PCR amplification was performed under (1) 95℃, 3 minutes; (2) 98℃, 15 seconds for 8 cycles; (3) 60℃, 15 seconds, 72℃, 5 minutes. Ultimately, RNA sequencing was performed according to protocol of Illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China). After calculation, differential expressed genes were screened by fold change (FC)＞2 or FC＜0.5 and p value＜0.05. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample and identified 60612 genes and 229420 transcripts. Approximately 0.3% of the genes showed differential expression between the HCQ treated PIG3V cells and the cells without HCQ, with a fold change＞2 or＜0.5 , p value＜0.05. Conclusions: Our research showed the first detailed transcriptome analysis of human vitiligo melanocytes PIG3V, with or without HCQ treatment. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic mechanisms of HCQ in treating vitiligo.

Dataset Information

Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress.

Publications

Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets