Transcriptome Response to Autoantibodies in Human Primary Epidermis Keratinocyte Model for Pemphgus Vulgaris
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ABSTRACT:
PROVIDER: PRJNA1217866 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR32190300_1.fastq.gz | Fastqsanger.gz | |||
| SRR32190300_2.fastq.gz | Fastqsanger.gz | |||
| SRR32190277_1.fastq.gz | Fastqsanger.gz | |||
| SRR32190277_2.fastq.gz | Fastqsanger.gz | |||
| SRR32190278_1.fastq.gz | Fastqsanger.gz |
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Project description:Pemphigus vulgaris (PV) is an autoimmune disease caused by autoantibodies (AAbs) targeting Desmoglein 1 (DSG1) or Desmoglein 3 (DSG3) on keratinocytes, leading to disrupted cell-cell adhesion and epidermal blistering. To investigate the early signaling events triggered by AAb binding, we examined transcriptomic responses in a human primary epidermis keratinocyte (HPEK) model for PV. After incubating the single-chain variable fragment (scFv) PX43, which targets DSG1 and DSG3, and human IgG as control for 5h, 10h and 24h, differentially expressed genes (DEGs) and regulated pathways was analyzed using DESeq2 and pathway enrichment analysis. Few significantly differentially expressed DEGs (under 10) were identified of PX43 compared to hIgG at all three time points (5h, 10h and 24h) with adjusted pvalue below 0.05 and |log2FC| >1. Upregulated inflammatory related pathways including TNFα signaling were identified only at 24h. Correlation analysis of the log2 transformed transcripts per million (TPM) shows the very high positive correlation of PX43 and hIgG at all time points (spearman correlation above 0.9) These findings indicate that AAbs targeting DSG1 and DSG3 do not drive broad transcriptomic or proteomic changes at the HPEK model.
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Project description:To characterize the global transcriptome of human keratinocyte stem cells (KSC) and keratinocyte progenitors (KP), primary basal keratinocyte subpopulations enriched in quiescent stem cells [Itg-α6bright / Trf-Rdim] or in cycling progenitors [Itg-α6bright / Trf-Rbright] were purified by flow cytometry from human skin samples. Sorted keratinocytes were then seeded in culture plates and maintained in culture medium overnight (15 hours), and genome-wide transcriptome analysis of these two subpopulations was directly performed, in order to globally maintain the initial phenotypes. After RNA preparation, gene profiling was performed using oligonucleotide microarrays (26068 probes). LOWESS normalisation was applied. Keywords: epidermis, keratinocyte stem cells, progenitors, transcriptome, stemness
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Project description:To characterize the global transcriptome of human keratinocyte stem cells (KSC) and keratinocyte progenitors (KP), primary basal keratinocyte subpopulations enriched in quiescent stem cells [Itg-?6bright / Trf-Rdim] or in cycling progenitors [Itg-?6bright / Trf-Rbright] were purified by flow cytometry from human skin samples. Sorted keratinocytes were then seeded in culture plates and maintained in culture medium overnight (15 hours), and genome-wide transcriptome analysis of these two subpopulations was directly performed, in order to globally maintain the initial phenotypes. After RNA preparation, gene profiling was performed using oligonucleotide microarrays (26068 probes). LOWESS normalisation was applied. Keywords: epidermis, keratinocyte stem cells, progenitors, transcriptome, stemness Dye-swap hybridization were performed. Slides were scanned with a Genepix 4000 microarray scanner (Axon Instruments, Molecular devices, Sunnyvale, CA). For each hybridized spot, the Cy3 and Cy5 fluorescence values were obtained by using Genepix Pro 4.0 software (Axon Instruments) and were saved as a result file
2015-05-06 | E-GEOD-68583 | biostudies-arrayexpress
Project description:In epidermis, granular keratinocytes are responsible for the synthesis of a great number of proteins of high tissue-specificity, and required for the epidermal barrier establishment and the regulation of the desquamation process. This project aims at characterizing the physiopathological function of genes implicated in these processes, and in the short term, defining their expression level in the course of keratinocyte differentiation. Pangenomic oligonucleotide microarrays are used to compare their expression between two cell populations enriched in basal or granular keratinocytes (T1 and T4 fractions resp.), obtained by successive incubations in trypsin solution of six independent normal human epidermis samples. Real-time quantitative PCR experiments were further performed to ensure the validity of the profiling for each candidate gene identified from microarrays data, and results of this multitechnique approach will be the object of a forthcoming publication. These results will be the basis for a better understanding of dermatological hyperkeratotic pathologies. Within epidermis, keratinocytes achieve a differentiation program culminating in cornification, a programmed cell death required for the establishment of the barrier function. A complex genetic regulatory network underlies the numerous biochemical modifications taking place in the last living cells, the granular keratinocytes (GKs), but very few transcription factors specific from this process were identified. Here we describe a large-scale, multitechnique approach performed on cells purified from normal human epidermis, and mainly focused on identification of these regulators. We performed microarray experiments to compare gene expression in cell fractions enriched in granular or basal keratinocytes. These data, with in silico promoter analysis for 52 differentiation markers, and exploitation of an EST library from GKs, were filtered according to gene products’ known or putative function and ESTs number in UniGene database, and allowed identification of 298 candidates out of 3,662 genes potentially specific from the last differentiation steps. Half were detected for the first time in epidermis and quantified by quantitative real-time PCR. These genes are linked to various aspects of GKs function, including structural components, lipid metabolism and secretory apparatus, cell signaling, and regulation of transcription. Forty-nine new differentiation-associated genes were identified, and 38 transcription factors in 92 were positively or negatively regulated in the course of differentiation, therefore possibly involved in the final differentiation of GKs. These data give new leads to have a global view of the network involved in epidermal barrier function, and allow in the future a better understanding of several pathologies, including genodermatoses of unknown aetiology. Keywords: Cell type comparison Six independent samples of normal human epidermis were used for this study. For three samples, technical replicates (at the labelling step) were performed (ie AQQ003, AQQ005 and AQQ011 slides are dye-swaps of AQQ002, AQQ004 and AQQ010, respectively). Data from each pair of “swap arrays” were merged and considered as coming from three simple arrays (named AQQ002dw, AQQ004dw and AQQ010dw) for statistical analysis. For each slide, T4 is the test sample (total RNA from granular keratinocytes), and T1 the reference sample (total RNA from basal keratinocytes of the same subject), and both come from the same biological sample.
2010-08-31 | E-GEOD-13263 | biostudies-arrayexpress
Project description:SAGE libraries from cultured, differentiated keratinocytes and human epidermis, both normal and affected by actinic keratosis Keywords = Keratinocyte, Epidermis, Homo sapiens, Actinic Keratosis, TNF alpha
2002-02-21 | E-GEOD-31 | biostudies-arrayexpress
Project description:Pemphigus vulgaris (PV) is an autoimmune disease caused by autoantibodies (AAbs) targeting Desmoglein 1 (DSG1) or Desmoglein 3 (DSG3) on keratinocytes, leading to disrupted cell-cell adhesion and epidermal blistering. To investigate the early signaling events triggered by AAb binding, we examined transcriptomic and proteomic responses in a human skin organ culture (HSOC) model for PV. After injecting the single-chain variable fragment (scFv) PX43, which targets DSG1 and DSG3, or the mouse-derived DSG3 AAb AK23, we quantified transcriptome changes at 0, 5, 10, and 24 hours and proteome responses at 0 and 24 hours, using human or murine IgG as controls. Differential pathway regulation was analyzed over time using gene set variation and enrichment analysis, with pathway responses compared to in vitro and in situ responses of normal human epithelial keratinocytes (NHEKs) under conditions such as stretching, differentiation, migration, detachment, and acute wounding. Only PX43 induced split formation in the HSOC model, which was associated with significant transcriptomic and proteomic changes, while AK23 did not alter gene expression or protein levels. Enriched pathways in response to split formation included TNFα, Interferon α/γ, IL2-STAT5, and IL5-STAT3 signaling across all time points. The transcriptomic response closely resembled that of acute skin wounding and keratinocyte detachment, with an inverse correlation to cell stretching and differentiation and no correlation to keratinocyte migration. These findings indicate that AAbs targeting DSG1 and/or DSG3 alone do not drive broad transcriptomic or proteomic changes; rather, observed changes result from split formation, resembling the response to wounding and inflammation.
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Project description:SAGE libraries from cultured, differentiated keratinocytes and human epidermis, both normal and affected by actinic keratosis Keywords = Keratinocyte, Epidermis, Homo sapiens, Actinic Keratosis, TNF alpha
2002-02-22 | GSE31 | GEO
Project description:Transcription profiling of Lgr6+ keratinocyte populations in the epidermis and hair follicle
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Project description:In epidermis, granular keratinocytes are responsible for the synthesis of a great number of proteins of high tissue-specificity, and required for the epidermal barrier establishment and the regulation of the desquamation process. This project aims at characterizing the physiopathological function of genes implicated in these processes, and in the short term, defining their expression level in the course of keratinocyte differentiation. Pangenomic oligonucleotide microarrays are used to compare their expression between two cell populations enriched in basal or granular keratinocytes (T1 and T4 fractions resp.), obtained by successive incubations in trypsin solution of six independent normal human epidermis samples. Real-time quantitative PCR experiments were further performed to ensure the validity of the profiling for each candidate gene identified from microarrays data, and results of this multitechnique approach will be the object of a forthcoming publication. These results will be the basis for a better understanding of dermatological hyperkeratotic pathologies. Within epidermis, keratinocytes achieve a differentiation program culminating in cornification, a programmed cell death required for the establishment of the barrier function. A complex genetic regulatory network underlies the numerous biochemical modifications taking place in the last living cells, the granular keratinocytes (GKs), but very few transcription factors specific from this process were identified. Here we describe a large-scale, multitechnique approach performed on cells purified from normal human epidermis, and mainly focused on identification of these regulators. We performed microarray experiments to compare gene expression in cell fractions enriched in granular or basal keratinocytes. These data, with in silico promoter analysis for 52 differentiation markers, and exploitation of an EST library from GKs, were filtered according to gene products’ known or putative function and ESTs number in UniGene database, and allowed identification of 298 candidates out of 3,662 genes potentially specific from the last differentiation steps. Half were detected for the first time in epidermis and quantified by quantitative real-time PCR. These genes are linked to various aspects of GKs function, including structural components, lipid metabolism and secretory apparatus, cell signaling, and regulation of transcription. Forty-nine new differentiation-associated genes were identified, and 38 transcription factors in 92 were positively or negatively regulated in the course of differentiation, therefore possibly involved in the final differentiation of GKs. These data give new leads to have a global view of the network involved in epidermal barrier function, and allow in the future a better understanding of several pathologies, including genodermatoses of unknown aetiology. Keywords: Cell type comparison
2010-08-31 | GSE13263 | GEO