Transcriptional signature of wounded keratinocytes reveals selective roles for ERK1/2, P38 and PI3K signalling pathways
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE6820 | GEO | 2007/12/30
SECONDARY ACCESSION(S): PRJNA99175
REPOSITORIES: GEO
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Project description:Covering denuded dermal surface after injury requires migration, proliferation and differentiation of skin keratinocytes. To clarify the major traits controlling these intermingled biological events, we surveyed the genomic modifications occurring during the course of a scratch closure of cultured human keratinocytes. Using a DNA microarray approach, we report the identification of 161 new markers of epidermal repair. Expression data, combined with functional analysis performed with specific inhibitors of ERK, p38[MAPK] and PI3 kinases, demonstrate that kinase pathways exert very selective functions by precisely controlling the expression of specific genes. Inhibition of the ERK pathway totally blocks the wound closure and inactivates many early transcription factors and EGF-type growth factors. P38[MAPK] inhibition only delays âhealingâ, probably in line with the control of genes involved in the propagation of injury-initiated signalling. In contrast, PI3 kinase inhibition accelerates the scratch closure and potentiates the scratch-dependent stimulation of three genes related to epithelial cell transformation, namely HAS3, HBEGF and Ets1. Our results define in vitro human keratinocyte wound closure as a reparation process resulting from a fine balance between positive signals controlled by ERK and p38[MAPK], and negative ones triggered off by PI3 kinase. The perturbation of any of these pathways might lead to dysfunction in the healing process, as those observed in pathological wounding phenotypes, such as hypertrophic scars or keloids. Keywords: Transcriptome of healing keratinocytes The DNA microarray analysis concerning the identification of the genes altered in response to cell scratching was performed on 3 independent experiments, derived from 3 distinct cell cultures. Results were generated after 2 independent microarray analyses, meaning that each time point was the result of 6 independent analyses. The data relative to the effect of U0126, SB203580 and LY294002 on the transcriptomal healing response were obtained from 2 independent experiments performed using 2 distinct cell cultures. Results were generated after 2 independent microarray analyses. The cDNA microarray contained 4.200 distinct cDNA probes. It has been previously described in Moreilhon et al (11). Gene selection was based on relevance to inflammation, infection, differentiation, ion transport, cell signaling, cell migration, proliferation etc⦠A large fraction of the probes also corresponded to transcripts encoding membrane proteins. The list of the 4.200 probes is available at http://www.microarray.fr/IPMC/cDNA_microarray5k.html, and the microarray is archived in GEO under reference GSE1853. The cDNA probes were PCR amplified from cDNA derived from Universal Human Reference RNA (Stratagene) by reverse transcription. Probes had the following properties: 1) they have a normalized length of 250 +/- 19 bp; 2) they have a normalized GC content of 52 +/- 8%; 3) they were specific for a unique human gene; and 4) they were controlled by DNA sequencing. PCR products were purified by using QIAquick 96 PCR Purification Kit (Qiagen), resuspended in 3SSC with an average spotting concentration of 200 ng/μl. Microarrays were printed with a SDDC-2 (Bio-Rad) on homemade aldehyde-coated glass microscope slides. Data presented into the present manuscript only refer to sequence-verified probes. Each PCR product was spotted four times on each slide (2 independent clusters of 2 spots spatially separated), to reduce positional bias of the fluorescence readout. The CyDye-labelled first-strand cDNAs were generated with the CyScribe First-Strand cDNA Labelling Kit (RPN 2600, Amersham Pharmacia Biotech), as described in Moreilhon et al, using ten micrograms of total RNA as template. Unincorporated CyDye-nucleotides were removed using the Nucleotide Removal Kit (Qiagen). Data Collection and Analysis : Data Collection : Microarrays were scanned either on GenePix4000B or on ScanArray5000 (Perkin Elmer). Both machines provided similar results (not shown). 16-bits TIF images were quantified with the corresponding softwares (GenePix Pro 5.0 program (Axon Instruments) for the GenePix, and Quantarray for the ScanArray). Intensities (either total or background values) were defined as average intensities for each spot. Negative controls (ânegâ) were spotted on each slide. They corresponded to non mammalian mRNA sequences with no significant identity with any human sequences. Such spots were used as controls. Data was normalized using a dye-swap method as described by Moreilhon et al.. We found that this method, while requiring duplication of experiments, improves the reproducibility of the quantification. Scratched (S) and non-scratched (NS) RNA samples were reverse transcribed in the presence of known amounts of the corresponding non mammalian âcontrolâ RNAs namely DmdNaC and DGNaC added at a respective S/NS ratio of 5, 0.2. Only experiments showing a good correlation between the experimental and true ratios for these controls were kept for analysis. Results are expressed as average ratios of intensities in âscratched cellsâ over intensities in ânon-scratched cellsâ. Data was then analyzed and/or visualized with MEV , or with the stand-alone program GeneANOVA, and the SAMâs Excel⢠plugin . Lists of genes significantly down and up-regulated under the different experimental conditions were selected by analysis of variance or using SAM. For the analysis of variance, signal was modelled as proposed by Kerr and Churchill (17) according to: yijkg = μ + Ai + Dj + Tk + Gg + (TG)kg + (AG)ig + (DG)jg + εijkg (1), where yijkg is the fluorescent intensity of array i and dye j representing treatment k and gene g. μ, Ai, Dj, Tk, and Gg are factors representing average signals among the whole experiment, one array, one dye, one treatment, or one gene, respectively. (TG)kg, (AG)ig, and (DG)jg are two-factor interactions between treatment and gene, array and gene (spot effects), dye and gene, respectively. εijkg represents a residual noise component, modelled as a Gaussian distribution with mean 0 and variance ϲ. (TG)kg denotes differences in expression for particular treatment and gene combinations that are not explained by the average effects on these treatments and genes. They represent the effects of interest, which were modelled using GeneANOVA. SAM analysis was performed as described in Tusher et al. (16). Ontologies attached to each gene were then used to classify altered genes according to main biological themes. Annotation of our probes was also accessible through MEDIANTE, our own microarray database which stores annotations derived from public databases. Additional statistical analyses, including principal component analysis (PCA) were performed with GeneANOVA.
2010-05-27 | E-GEOD-6820 | biostudies-arrayexpress
Project description:BRAF inhibitors are highly effective therapies for patients with BRAF V600 mutated metastatic melanoma. Patients who receive BRAF inhibitors develop a variety of hyper-proliferative skin conditions, whose pathogenic basis is the paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in BRAF wild-type cells. Most of these hyper-proliferative skin changes improve when a MEK inhibitor is co-administered, as a MEK inhibitor blocks paradoxical MAPK activation. We tested whether we could take advantage of the mechanistic understanding of the skin hyper-proliferative side effects of BRAF inhibitors to accelerate skin wound healing by inducing paradoxical MAPK activation. Here we show that the BRAF inhibitor vemurafenib accelerates human keratinocyte proliferation and migration by increasing ERK phosphorylation and cell cycle progression. Topical treatment with vemurafenib in two wound-healing models in mice accelerated cutaneous wound healing and improved the tensile strength of healing wounds through paradoxical MAPK activation; addition of a MEK inhibitor reversed the benefit of vemurafenib-accelerated wound healing. The same dosing regimen of topical BRAF inhibitor did not increase the incidence of cutaneous squamous cell carcinomas in mice even after the application of a carcinogen. Therefore, topical BRAF inhibitors may have clinical applications in accelerating the healing of skin wounds. Full depth incisional wound mice tissues with/without Vemurafenib treatment were sent for RNAseq analysis on day 2, 6 and 14
2016-07-29 | E-GEOD-74558 | biostudies-arrayexpress
Project description:BRAF inhibitors are highly effective therapies for patients with BRAF V600 mutated metastatic melanoma. Patients who receive BRAF inhibitors develop a variety of hyper-proliferative skin conditions, whose pathogenic basis is the paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in BRAF wild-type cells. Most of these hyper-proliferative skin changes improve when a MEK inhibitor is co-administered, as a MEK inhibitor blocks paradoxical MAPK activation. We tested whether we could take advantage of the mechanistic understanding of the skin hyper-proliferative side effects of BRAF inhibitors to accelerate skin wound healing by inducing paradoxical MAPK activation. Here we show that the BRAF inhibitor vemurafenib accelerates human keratinocyte proliferation and migration by increasing ERK phosphorylation and cell cycle progression. Topical treatment with vemurafenib in two wound-healing models in mice accelerated cutaneous wound healing and improved the tensile strength of healing wounds through paradoxical MAPK activation; addition of a MEK inhibitor reversed the benefit of vemurafenib-accelerated wound healing. The same dosing regimen of topical BRAF inhibitor did not increase the incidence of cutaneous squamous cell carcinomas in mice even after the application of a carcinogen. Therefore, topical BRAF inhibitors may have clinical applications in accelerating the healing of skin wounds.
2016-07-29 | GSE74558 | GEO
Project description:Skin wound healing is one of the major prevalent medical problems in the worldwide. Wound healing involves multi-process synergy and re-epithelialization is an essential part of wound healing. Histone H3K36 tri-methylase Setd2 has been extensively studied in different biological processes and diseases. However, the function of Setd2 in the wound healing remains unclear. To elucidate the biological role of Setd2 in the skin wound healing, conditional gene targeting was employed to establish epidermis-specific Setd2-deficient mice. We found that Setd2 deficiency resulted in accelerated re-epithelialization during cutaneous wound healing by promoting keratinocytes proliferation and migration. Furthermore, we demonstrated that loss of Setd2 activated the AKT/mTOR pathway, and pharmacological inhibitions of AKT and mTOR with MK2206 and rapamycin delayed wound closure, respectively. In conclusion, our results reveal the essential role of Setd2 in skin wound healing that is Setd2 loss promotes cutaneous wound healing via activation of AKT/mTOR signaling.
2020-12-25 | GSE163860 | GEO
Project description:Skin wound healing is one of the major prevalent medical problems in the worldwide. Wound healing involves multi-process synergy and re-epithelialization is an essential part of wound healing. Histone H3K36 tri-methylase Setd2 has been extensively studied in different biological processes and diseases. However, the function of Setd2 in the wound healing remains unclear. To elucidate the biological role of Setd2 in the skin wound healing, conditional gene targeting was employed to establish epidermis-specific Setd2-deficient mice. We found that Setd2 deficiency resulted in accelerated re-epithelialization during cutaneous wound healing by promoting keratinocytes proliferation and migration. Furthermore, we demonstrated that loss of Setd2 activated the AKT/mTOR pathway, and pharmacological inhibitions of AKT and mTOR with MK2206 and rapamycin delayed wound closure, respectively. In conclusion, our results reveal the essential role of Setd2 in skin wound healing that is Setd2 loss promotes cutaneous wound healing via activation of AKT/mTOR signaling.
2020-12-25 | GSE163858 | GEO
Project description:Epidermal keratinocytes respond to extracellular influences by activating cytoplasmic signal transduction pathways that change the transcriptional profiles of affected cells. To define responses to two such pathways, p38 and ERK, we used SB203580 and PD98059 as specific inhibitors, and identified the regulated genes after 1, 4, 24 and 48 hrs, using Affymetrix’ Hu133Av2 microarrays. Additionally, we compared genes specifically regulated by p38 and ERKs with those regulated by JNK and by all three pathways simultaneously. We find that the p38 pathway induces the expression of extracellular matrix and proliferation-associated genes, while suppressing microtubule-associated genes; the ERK pathway induces the expression of nuclear envelope and mRNA splicing proteins, while suppressing steroid synthesis and mitochondrial energy production enzymes. Both pathways promote epidermal differentiation and induce feedback inactivation of MAPK signaling. c-FOS, SRY and N-Myc appear to be the principal targets of the p38 pathway, Elk-1 SAP1 and HLH2 of ERK, while FREAC-4, ARNT and USF are common to both. The results for the first time comprehensively define the genes regulated by the p38 and ERK pathways in epidermal keratinocytes and suggest a list of targets potentially useful in therapeutic interventions. Human epidermal keratinocytes are grown in Keratinocyte Serum-Free Medium (Gibco) supplemented with 0.05 mg/ml bovine pituitary extract, 2.5 ng/ml epidermal growth factor, 0.09 mM CalCl2 and 1% penicillin/streptomycin (KGM). They are switched to Keratinocyte Serum Free-Media (Gibco) supplemented only with 1% penicillin/streptomycin (KBM) 24 h prior to commencing experiments. A set is left as controls, others treated with 5 uM JNK inhibitor SP600125, 15 uM p38 inhibitor SB203580, or 50 um ERK inhibitor PD98059. Timecourse of treated and parellel control samples over a 48 hr period was performed.
2013-09-05 | E-GEOD-50591 | biostudies-arrayexpress
Project description:In skin homeostasis, dermal fibroblasts are responsible for coordinating the migration and differentiation of overlying epithelial keratinocytes. As hairy skin heals faster than non-hairy skin, we hypothesised that follicular fibroblasts would accelerate skin re-epithelialisation after injury faster than interfollicular fibroblasts. We found that hair follicle dermal papilla fibroblast conditioned media (DPFi CM) could significantly accelerate wound closure compared to controls partly due to the presence of sAXL in this media. We used microarrays to identify upregulated and downregulated genes in human epidermal keratinocytes incubated with sAXL, DPFi CM and Epilife (keratinocyte growth media;control) after scratch wounds in vitro.
2019-11-01 | GSE131615 | GEO
Project description:Bis-2-chloroethyl sulfide (sulfur mustard, SM) is a potent alkylating agent and vesicant. Exposure to SM results in activation of numerous signaling cascades, including mitogen-activated protein kinase (MAPK) signaling pathways. These pathways include the Erk, p38, and JNK pathways, which are involved in cell growth, inflammation, and stress signaling. However, the precise roles of these pathways in SM toxicity have not been fully elucidated. We used Western blotting and microarray analysis to examine the activation and role of each pathway following SM exposure in primary human epidermal keratinocytes. Western blotting revealed increased phosphorylation of p38 and JNK following SM exposure; however, phosphorylation of Erk was equivocal, suggesting that growth conditions may impact activation of Erk by SM. We used pharmacologic inhibitors to target each MAPK and then compared the gene expression profiles to identify SM-induced gene networks regulated by each MAPK. Cells were pretreated with 10 µM SB 203580 (p38 inhibitor), PD 98059 (Erk inhibitor), or SP 600125 (JNK inhibitor) 60 minutes before exposure to 200 µM SM. Cells were harvested at 1h, 4h, and 8h post-exposure, and RNA was extracted for synthesis of microarray probes. Probes were hybridized to Affymetrix U133 Plus 2.0 arrays for gene expression profiling. Analysis of variance was performed to identify genes significantly modulated due to pharmacologic inhibition in SM-exposed cells. Pathway mapping confirmed alterations in SM-induced Erk, JNK, and p38 MAPK signaling due to pharmacologic inhibition. SM-induced expression of IL-8, IL-6, and TNF-alpha was decreased by p38 MAPK inhibition, but not by inhibition of other MAPKs. Based on the number of significant pathways mapped to each MAPK in the presence and absence of inhibitors, the p38 MAPK pathway appeared to be the MAPK pathway most responsive to SM exposure. Interestingly, pathway mapping of the microarray data identified potential cross-talk between MAPK signaling pathways and other pathways involved in SM-induced signaling. Mining of these results will increase our understanding of the role of MAPK pathways in SM-induced signal transduction and may identify potential therapeutic targets for medical countermeasure development.
2018-06-15 | GSE29587 | GEO
Project description:The aim of this study was to provide proof of principle as to whether probiotic bacteria or their extracts, could stimulate cutaneous wound healing. To this end, we have used a keratinocyte monolayer scratch assay which assesses one important aspect of wound healing, re-epithelialization. Primary human keratinocyte monolayers were scratched and then exposed to lysates of Lactobacillus rhamnosus GG
2016-03-23 | E-MTAB-3485 | biostudies-arrayexpress
Project description:MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples.
2013-11-09 | E-GEOD-52233 | biostudies-arrayexpress