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Gene regulation in human epidermal keratinocytes by a Rho kinase inhibitor (Y-27632)

ABSTRACT: We previously demonstrated that the lifespan of primary human keratinocytes could be extended indefinitely by culture in the presence of the Rho kinase (ROCK) inhibitor Y-27632. Here, gene expression analysis of primary human epidermal keratinocytes cells grown in the presence of Y-27632 demonstrates that ROCK inhibition primarily inhibits keratinocyte differentiation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE52515 | GEO | 2014/03/31

SECONDARY ACCESSION(S): PRJNA229149

REPOSITORIES: GEO

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Gene regulation in human epidermal keratinocytes by a Rho kinase inhibitor (Y-27632)

Project description:Keratinocytes are a targeted cells for infection of human papillomaviruses (HPV). But, there are very limited information for gene regulation of human karatinocytes by HPV infection. Y-27632, a rho-kinase inhibitor, stimulates proparation of keratinocytes. We identified genes, which are regulated by a drug for karatinocyte proparation (Y-27632). mRNA profiles of primary HEKs regulated by a rho kinase inhibitor (Y-27632, Y-drug) were generated by microarray analysis using Illumina BeadChip.

2014-03-31 | E-GEOD-52515 | biostudies-arrayexpress

Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632

Project description:Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense may be hindered by invasive isolation methods and/or improper culture conditions to support stem cell maintenance and other potential mechanisms contributing to long-term subcultivation in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases.

2018-09-01 | GSE119317 | GEO

Immortalization and characterization of rat lingual keratinocytes in a high-calcium and feeder-free culture system using ROCK inhibitor Y-27632

Project description:The study tried the identify some differentially expressed proteins in rat lingual keratinocytes treated with or without the ROCK inhibitor Y-27632. In brief, rat lingual keratinocytes were treated with or without Y-27632 for 36 h and were subjected to proteomic analysis.

2022-06-23 | PXD025812 | JPOST Repository

Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632

Project description:Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632

| PRJNA488732 | ENA

Irradiated feeder cells and Y-27632

Project description:Expression data from this experiment is part of a larger project aimed at defining the individual effects and synergistic effects of ROCK inhibitor Y-27632 and conditioned media from irradiated J2 cells when applied to epithelial cells. This data set consists of four individual samples, each of which are total RNA collected from human foreskin keratinocyte cells, either grown in F medium (control), treated with Y-27632, grown in conditioned medium (as described in associated publication), or both treatments.

2014-09-15 | GSE61226 | GEO

Expression data from Human Embrionic Stem Cells (hESC) from three different lines cultured on feeders or Matrigel with and without ROCK Inhibitor Y-27632

Project description:Microarray expression data from three different cell lines of human embrionic stem cells (Hs181, Hs293, Hs420), grown under feeder and feeder-free conditions (matrigel) with the addition of Y-27632 (ROCK Inhibitor) We assessed the difference between cell lines and the impact of feeder-free vs matrigel conditions upon the addition of the ROCK Inhibitor Y-27632

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Long-term Y-27632 treatment promotes senescence of primary human dermal fibroblasts by increasing IGFBP-5

Project description:Purpose: Y-27632, a well-characterized Rho-associated kinase (ROCK) inhibitor, has been shown to play an important role in regulation of cell growth in multiple types of cells, the regulation appeared to be cell specific. To further understand its molecular mechanisms, we performed RNA-seq analysis of HDF at 12 hours and 48 hours after Y-27632 treatment. Methods: Total RNAs were isolated from dermal cells with/without Y-27632 treatment for 12 hours or 48 hours respectively. Then mRNAs were purified from above total RNA using poly-T oligo-attached magnetic beads. After sequencing libraries were prepared using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA), the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. Results: We sequenced 12 of 229 differential expressed genes 48 hours after Y-27632 treatment and found genes which by GO analysis are negative regulators of cell growth ). Of these 12 genes, the expression level of 4 genes, IGFBP1, IGFBP-5, HNF4A and NKD1, changed significantly at both 12 hours and 48 hours of Y-27632 treatment. This results, validated by RT-qPCR analysis showed that the expression of IGFBP-5 (Insulin Growth Factor Binding Protein 5) increased by 1.5 fold after 12 hours of treatment, and increased more than 10-fold after 48 hours. Expression of the other 3 genes didn’t change significantly after Y-27632 treatment Conclusions:RNA-seq analysis of gene expression profile in HDFs collected from 12 hours and 48 hours treatment of Y-27632. From these analyses we found the IGFBP-5, a negative regulation of cell proliferation, whose expression increased with the duration of Y-27632 treatment (7-fold increase after the first 24 hours

2019-08-19 | GSE135934 | GEO

Transcriptional profiling of the cellular transformation induced by Rho subfamily GTPases

Project description:We have used microarray technology to identify the transcriptional targets of Rho subfamily GTPases. This analysis indicated that murine fibroblasts transformed by these proteins show similar transcriptomal profiles. Functional annotation of the regulated genes indicate that Rho subfamily GTPases target a wide spectrum of biological functions, although loci encoding proteins linked to proliferation and DNA synthesis/transcription are up-regulated preferentially. Rho proteins promote four main networks of interacting proteins nucleated around E2F, c-Jun, c-Myc, and p53. Of those, E2F, c-Jun and c-Myc are essential for the maintenance of cell transformation. Inhibition of Rock, one of the main Rho GTPase targets, leads to small changes in the transcriptome of Rho-transformed cells. Rock inhibition decreases c-myc gene expression without affecting the E2F and c-Jun pathways. Loss-of-function studies demonstrate that c-Myc is important for the blockage of cell-contact inhibition rather than for promoting the proliferation of Rho-transformed cells. However, c-Myc overexpression does not bypass the inhibition of cell transformation induced by Rock blockage, indicating that c-Myc is essential, but not sufficient, for Rock-dependent transformation. These results reveal the complexity of the genetic program orchestrated by the Rho subfamily and pinpoint protein networks that mediate different aspects of the malignant phenotype of Rho-transformed cells Keywords: Rho/Rac GTPases, microarray, oncogenesis, proliferation, gene expression, transcription, Rock, c-Myc

2007-01-03 | GSE5913 | GEO

Comparative transcriptome analysis of DFAT cells after the treatment with Y-27632 and the transfection of Mkl1 siRNA

Project description:Cellular differentiation is regulated through activation and repression of defined transcription factors. A hallmark of differentiation is a pronounced change in cell shape, which is determined by dynamics of the actin cytoskeleton. In de-differentiated fat (DFAT) cells and 3T3-L1 cells, we showed that treatment with the ROCK inhibitor Y-27632, by inducing remodeling of the actin cytoskelton, causes adipocyte differentiation. In addition, we found that depletion of MKL1, an actin binding transcriptional coactivator, elicits adipogenesis. To investigate whether regulation of MKL1 by actin cytoskeleton dynamics drives adipocyte differentiation, we compared the gene expression changes resulting from DFAT cells after treatment with Y-27632 and transfection of Mkl1 siRNA. Using Affymetrix mouse genome array, we compared global gene expression profiles in DFAT cells after treatment with Y-27632 and transfection with Mkl1 siRNA to search for particular biological functions in genes of which expression intensities were increased and decreased.

2014-03-28 | E-GEOD-52334 | biostudies-arrayexpress

mTORC1 Loss Impairs Epidermal Adhesion via Rho Kinase Activation

Project description:In this study, we use the epidermis as a model system to elucidate the cellular effects and signaling feedback sequelae of mTORC1 loss-of-function in epithelial tissue. In mice with conditional epidermal loss of mTORC1 components Rheb or Rptor, we find that mTORC1 loss-of-function unexpectedly results in a profound skin barrier defect with epidermal abrasions, blistering and early postnatal lethality, due to a thinned epidermis with decreased desmosome expression and incomplete biochemical differentiation. Impaired cell-cell adhesion in the context of mTORC1 loss-of-function is caused by constitutive activation of Rho kinase (ROCK) signaling with increased cytoskeletal tension, and inhibition or silencing of ROCK1 is sufficient to rescue keratinocyte adhesion and biochemical differentiation. mTORC1 loss-of-function results in marked feedback up-regulation of upstream TGF-β signaling, triggering ROCK activity and its downstream effects on desmosomal gene expression. These findings elucidate a novel role for mTORC1 in the regulation of epithelial barrier formation, cytoskeletal tension and cell adhesion and underscore the complexity of signaling feedback after mTORC1 inhibition

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