Expression data from primary keratinocytes obtained from WT and keratinocyte specific Glut1-deficient mice
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ABSTRACT:
PROVIDER: PRJNA399529 | ENA |
REPOSITORIES: ENA
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Project description:Glut1 is highly expressed in basal cells of keratinocytes, but the regulation of Glut1 and expression of additional glucose transporters in the skin has not been explored, here we specifically ablate Glut1 in epidermal keratinocytes to elucidate the role of glucose transport in the keratinocytes. we performed microarray analysis in WT and Glut1 deficient primary keratinocytes to determine the pathways might contribute to the impaired proliferation in Glut1 deficient keratinocytes.
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Project description:Glut1 is highly expressed in basal cells of keratinocytes, but the functions and regulation of Glut1 has not been explored, here we specifically ablate Glut1 in epidermal keratinocytes to elucidate the role of glucose transport in the skin. We performed microarray analysis in WT and Glut1 deficient primary keratinocytes to determine the pathways might contribute to the impaired proliferation in Glut1 deficient keratinocytes,
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Project description:MED1 (Mediator complex subunit 1) is expressed by human epidermal keratinocytes and functions as a coactivator of several transcription factors. To elucidate the role of MED1 in keratinocytes, we established keratinocyte-specific MED1-null (MED1epi-/-) mice using the K5Cre-LoxP system. To elucidate the mechanism(s) underlying abnormalities of keratinocytes derived from MED1epi-/- mice, we compared the gene expression patterns of MED1epi-/--derived keratinocytes with their wild type counterparts by microarray analysis.
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Briefly, Human Primary Keratinocytes were isolated and treated with the cytokine IL-17A (50ng/ml) in incomplete media devoid of any growth factors. Tryptic digested and desalted peptide samples were injected in Thermoscientific Q-Exactive Plus instruments through EasyNLC HPLC autosampler. The instruments were set to MS1 resolution of 70000 and MS2 resolution of 17500. The acquisition experiments were optimized to run on 120 min gradients.
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This dataset helped to simulate the IL-17A-driven inflammation in keratinocytes and uncovered many putative druggable targets in the context of psoriasis.
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Project description:We conducted RNA sequencing analysis on skin tissues obtained from spontaneous inflammatory skin lesions in keratinocyte-specific Mettl3 knockout (K14CreERT2;Mettl3fl/fl) mice and their controls (Mettl3fl/fl). In brief, we observed spontaneous skin inflammation in the tail of K14CreERT2;Mettl3fl/fl mice 2-3 weeks following tamoxifen injection. The objective of this study was to investigate the impact and underlying mechanism of Mettl3 in epidermal keratinocytes regarding skin inflammation
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Project description:MED1 (Mediator complex subunit 1) is expressed by human epidermal keratinocytes and functions as a coactivator of several transcription factors. To elucidate the role of MED1 in keratinocytes, we established keratinocyte-specific MED1-null (MED1epi-/-) mice using the K5Cre-LoxP system. To elucidate the mechanism(s) underlying abnormalities of keratinocytes derived from MED1epi-/- mice, we compared the gene expression patterns of MED1epi-/--derived keratinocytes with their wild type counterparts by microarray analysis. Generation of the MED1 conditional null mutation in epidermal keratinocytes and cell culture: MED1flox/flox mice (Jia et al, J Biol Chem 279:24427. 2004) were mated with K5Cre mice (Tarutani et al, Proc Natl Acad Sci U S A 94:7400. 1997) to generate F1 K5Cre+;MED1flox/+ mice. The K5Cre+;MED1flox/+ mice were then mated with MED1flox/flox mice to generate K5Cre+;MED1flox/flox mice (MED1epi-/-). Mice were screened for presence of the transgene by PCR using specific primers for the floxed MED1 gene and the human K5 gene according to the previous reports. Skin of newborn mice was excised after the mice were sacrificed with excessive anesthetic and were treated with dispase followed by trypsin to separate the epidermis from the dermis. Keratinocytes were seeded on type I collagen coated dishes, and were cultured in CnT07 conditioned culture medium (CELLnTEC, Bern, Switzerland). Keratinocytes were used for the experiment as a primary culture. All animal studies were conducted according to protocols approved by the Institutional Animal Care and Use Committee at Osaka University. Microarray analysis: For comprehensive comparison of gene expression patterns between keratinocytes derived from MED1epi-/- and from WT mice, microarray analysis was used. Total RNAs were extracted using an RNeasy kit (Qiagen, San Diego, CA). Then, 2μg total RNA was reverse transcribed to cDNA with T7 oligo d(T) primer (Affymetrix, Santa Clara, CA). The cDNA synthesis products were used for in vitro transcription reactions containing T7 RNA polymerase and biotinylated nucleotide analogue (pheudouridine base) cRNAs. The labeled cRNA products were then fragmented and loaded onto GeneChip(R) Mouse Genome 430 2.0 arrays (Affymetrix), and were hybridized according to the manufacturer’s protocol. Streptavidin-Phycoerythrin (Molecular Probes) was used as the fluorescent conjugate to detect hybridized target sequences. Raw intensity data from the GeneChip array were analyzed by GeneChip Operating Software (Affymetrix).
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Project description:The genetic expression profile of a Wnt signal agonist, BIO, was evaluated in human primary keratinocytes. Accelerating scaling up of primary keratinocytes benefits skin autografts for severely burned patients. Wnt signal, a conserved pathway controlling cell cycle and morphogenesis of embryo, has been postulated to promote the cell proliferation and tumorigenesis in adult. Here, the effects of Wnt signal on the growth of interfollicular keratinocytes were investigated. We demonstrated that recombinant Wnt3a significantly promoted the primary keratinocyte growth at a low cell density. A well-characterized GSK-3beta inhibitor, BIO, activated the Wnt signals and also enhanced the colony formation of keratinocytes dose-dependently. Gene expression profile of the BIO-treated keratinocytes revealed the linkage of the BIO with the cell mitosis and indicated that epithelial cell adhesion molecule (EpCAM), a Wnt target gene, was upregulated. Comparing to the EpCAM- keratinocytes, the EpCAM+ cells showed higher proliferation rate and efficacy of the colony formation. Especially, inhibiting the EpCAM expression by shRNA attenuated the proliferation effect of BIO and the growth advantage of the EpCAM+ keratinocytes. These evidences emphasize the positive role of canonical Wnt and EpCAM on the regulation of cell growth and self-renewal for human keratinocytes.
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Project description:Our aim was to investigate the interaction between epidermal differentiation and VZV infection. By means of a calcium-induced keratinocyte differentiation model and RNA-seq we show VZV infection has a profound effect on differentiating keratinocytes and hijacks the normal process of epidermal gene expression to generate a signature resembling patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Analysis of the viral transcriptome provides evidence that VZV replication in skin is tightly linked to differentiation and critically, that late viral gene expression is associated with cellular differentiation. The experiment was performed on human primary keratinocytes under four conditions: undifferentiated/uninfected, uninfected/differentiated, VZV-infected/undifferentiated and VZV-infected/differentiated.
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