The viral defense gene RNaseL acts as a regeneration repressor
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ABSTRACT: Normal Human Epidermal Keratinocytes treated with either Q-VD-OPh or vehicle,were prepared for protein analysis using proteomics approach.Protein expression changes were discussed.
Project description:Mammalian injury responses are predominantly characterized by fibrosis and scarring rathe than functional regeneration.This limited regenerative capacity in mammals could reflect loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors.
Project description:RNA sequencing of wild-type or Interferon Alpha receptor 1 Knockout MEF cells treated with DMSO or the Broad-spectrum Caspase Inhibitor Q-VD-OPh The mechanism by which cells undergo death determines whether dying cells trigger inflammatory responses or remain immunologically silent. Mitochondria play a central role in the induction of cell death, as well as in immune signaling pathways. Here, we identify of a mechanism by which mitochondria and downstream pro-apoptotic caspases regulate the activation of antiviral immunity. In the absence of active caspases, mitochondrial outer membrane permeabilization by Bax and Bak results in the expression of type I interferons (IFNs). This induction is mediated by mitochondrial DNA-dependent activation of the cGAS/STING pathway and results in the establishment of a potent state of viral resistance. Our results show that mitochondria have the capacity to simultaneously expose a cell-intrinsic inducer of the IFN response, and to inactivate this response in a caspase-dependent manner. This mechanism provides a dual control, which determines whether mitochondria initiate an immunologically silent or a pro-inflammatory type of cell death. To determine whether the pharmacological inhibition of caspases could recapitulate the phenotype caused by genetic deficiencies, we treated WT MEFs with broad-spectrum inhibitors of caspases (Q-VD-OPH). The inhibitor induced an increased expression of ISGs, similar to the effect of caspase or Apaf-1 deficiency. Next, we compared the transcriptional changes induced by caspase inhibition in WT and IFNAR1 KO cells, which lack a critical subunit of the receptor for IFNα/β (Muller et al., 1994). We observed that the absence of the IFNAR receptor abrogated the transcriptional response of the cells to caspase inhibition by Q-VD-OPH, demonstrating the role of type I IFNs in this response. RNA was extracted from duplicate samples and libraries generated for sequencing using the directional RNA-Seq library prep at the Yale Center for Genome Analysis. Libraries were sequenced using a Hiseq2500 sequencer to generate 76bp single-end reads. Duplicate samples were analyzed for each condition.
Project description:Our goal is to find new genes regulated by p21 in human primary cells . To get it we carried out a gene expression profiling in two different models, human myeloid leukemia K562 cells and human keratinocytes both of them with conditional expression of p21. In order to identify genes specifically modulated by p21 we compared with the cell line with overexpression of p27, because p21 and p27 belong to the same gene family and regulated the same genes specially in cell cycle. So, our intention is to identify only genes regulated by p21 and not p27. In order to confirm these results we studied the p21-dependent repression of mitotic genes in a different cellular system. We chose human primary keratinocytes because they are non-tumorigenic, non-immortalized and epithelial cells, in contrast to human myeloid leukemia K562 cells. Human primary keratinocytes were infected with recombinant adenoviruses expressing the full-length p21 protein. A dramatic increase in p21 in infected keratinocytes was demonstrated by RT-qPCR (as we show in the manuscript). As controls, we also infected the keratinocytes with adenovirus carrying the genes for p27 which overexpression was also confirmed by RT-qPCR (as we show in the manuscript). We prepared RNA 24 h after infection and performed large-scale expression assay using the Afftymetrix platform. The clustering analysis revealed that p21 provoked the down-regulation of a number genes involved in cell cycle control not shared by cells expressing p27 (as we show in the manuscript). Our goal, has been getting genes regulated more strongly by p21 and not by p27 in cell cycle and mitosis. Our result are supported because we have found the same genes in two different models and also we have validated (by RT-qPCR) more than 20 cell cycle and mitotic genes, found in our affymetrix arrays. Also we have found the region of p21 that is sufficient for gene regulation and for one gene we have described as p21 bind to the promoter. Finally, we have discussed in our manuscript how p21 can do this regulation by bioinformatic analysis of p21-target genes. The success of this study is to describe a new role of p21 as a transcriptional co-repressor in some systems.
Project description:The skin serves as a barrier to the environment and is constantly exposed to viral pathogens. Whether viral pathogens contribute to the oncogenesis of rare skin cancers has not been systematically explored. Here, we analyzed 18 skin cancer types by exploiting off-target reads from commonly available next-generation sequencing data. We identified human papillomavirus 42 (HPV42) in all digital papillary adenocarcinoma (DPA) cases but not in any of 11,091 tumors from common cancer types. HPV42 was previously not described as an oncogenic driver, yet we show that HPV42 recapitulates the molecular hallmarks of oncogenic HPVs. Using a machine learning approach, we identified a conserved transcriptomic fingerprint of HPV transformation common to oncogenic HPVs, including HPV42. Collectively, our results establish HPV42 as an oncogenic HPV, which has implications for the diagnosis and treatment for DPA patients.
Project description:Cyclic AMP-induced phosphorylation of the transcription factor CREB elicits expression of genes mediating diverse biological functions. In lymphoid organs, the neurotransmitter norepinephrine stimulates beta(2)-adrenergic receptors on B lymphocytes to promote CREB-dependent expression of genes like the B cell Oct 2 coactivator (OCA-B). Although CREB phosphorylation recruits cofactors such as CBP/p300 to stimulate transcription, bona fide endogenous inhibitors of CREB-coactivator or CREB-DNA interactions have not emerged. Here, we identified RGS13, a member of the Regulator of G protein Signaling (RGS) protein family, as a nuclear factor that suppresses CREB-mediated gene expression. cAMP or Ca(2+) signaling promoted RGS13 accumulation in the nucleus, where it formed a complex with phosphorylated CREB and CBP/p300. RGS13 reduced the apparent affinity of pCREB for both the CRE and CBP. B lymphocytes from Rgs13(-/-) mice had more beta(2)-agonist-induced OCA-B expression. Thus, RGS13 inhibits CREB-dependent transcription of target genes through disruption of complexes formed at the promoter.
Project description:The aim of the study was to describe the function of miR-146a in human skin keratinocytes in relation to chronic skin diseases. miR-146a precursor and the control were transfected into human primary keratinocytes treated with IFN-gamma, TNF-alpha or left untreated. mRNA expression profiles of each conditions were detected.
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
Project description:BackgroundThe transcription factor, Sox2, is central to the behaviour of neural stem cells. It is also one of the key embryonic stem cell factors that, when overexpressed can convert somatic cells into induced pluripotent cells. Although generally studied as a transcriptional activator, recent evidence suggests that it might also repress gene expression.ResultsWe show that in neural stem cells Sox2 represses as many genes as it activates. We found that Sox2 interacts directly with members of the groucho family of corepressors and that repression of several target genes required this interaction. Strikingly, where many of the genes activated by Sox2 encode transcriptional regulators, no such genes were repressed. Finally, we found that a mutant form of Sox2 that was unable to bind groucho was no longer able to inhibit differentiation of neural stem cells to the same extent as the wild type protein.ConclusionsThese data reveal a major new mechanism of action for this key transcription factor. In the context of our understanding of endogenous stem cells, this highlights the need to determine how such a central regulator can distinguish which genes to activate and which to repress.
Project description:Although evidence has shown that very small electric currents produce a beneficial therapeutic result for wounds, non-invasive EMF therapy has consisted mostly of anecdotal clinical reports with very few well controlled laboratory mechanistic studies. In this study, we evaluated the effects and potential mechanisms of a non-invasive EMF device on skin wound repair. In vitro analyses with human skin keratinocyte cultures demonstrated that the non-invasive EMF has a very strong effect on accelerating keratinocyte migration and a relatively weaker effect on promoting keratinocyte proliferation. The positive effects of the non-invasive EMF on cell migration and proliferation seem keratinocyte specific without such effects seen on dermal fibroblasts. cDNA microarray and RT-PCR performed revealed increased expression of CRK7 and HOXC8 genes in treated keratinocytes. This study suggests that a non-invasive electric magnetic field accelerates wound reepithelialization through a mechanism of promoting keratinocyte migration and proliferation, possibly due to upregulation of CRK7 and HOXC8 genes. Experiment Overall Design: Non-invasive EMF: Experiment Overall Design: In this study, we used the Field Therapy Accelerator (FTA; Advatech, Miami, Florida) device to produce a magnetic field which in turn induces a non-invasive electric current in the targetâs surroundings. All human and animal studies have been approved by the authors' Institutional Review Board. The FTA device generates a continuous series of direct current (DC)-like pulses of voltage followed for a very short time interval by a negative voltage spike. The induced EMF comprises of a series of concentric circles centered on the axis of the coil. The EMF vectors are at each point perpendicular to the surface of the circle and in the plane of that circle, that plane being parallel to the face of the coil. Experiment Overall Design: In vitro Studies with Cell Cultures: Experiment Overall Design: Cells and Cell Cultures: Normal human keratinocytes were maintained in growth medium of EpiLife (Cascade Biologics, Portland, Oregon) with human keratinocyte growth supplement at concentration of 0.2% v/v of bovine pituitary extract, 5 ug/ml bovine insulin, 0.18 ug/ml hydrocortisone, 5 ug/ml bovine transferrin, 0.2 ng/ml human epidermal growth factor (Cascade Biologics, Portland, Oregon), plus antibiotics of 100 U/ml penicillin G and 100 ug/ml streptomycin at 37ºC and 5% CO2. Media were changed every 24 hours. Experiment Overall Design: cDNA Microarray and RT-PCR Analysis: Experiment Overall Design: Cell cultures and migration assay: Normal human epidermal keratinocytes were maintained in 35-mm cell culture dishes and in EpiLife at 37ºC and 5% CO2. Keratinocytes were maintained in growth media and grown to confluency. At time 0, a cross-shaped wound gap or cell-free zone was made among confluent monolayer cells in the center of the culture dish, and detached cells were washed off with PBS and then replaced with growth medium. Non-invasive EMF treatment: Group 1 was treated for 1 hour immediately after wounding, at a frequency of 2080 cycles/second, electric field strength of 20mV/cm, and duty cycle of 90%. Group 2 was the control group, and received no treatment but was placed in the same hood at temperatures of 31±2ºC for the same period of time as their treatment counterparts. Each group was performed in triplicates. Experiment Overall Design: cDNA Microarray: RNA was isolated from cells using an RNeasy Mini RNA Isolation Kit (Qiagen Sciences, Germantown, Maryland), and converted to double stranded cDNA using a cDNA RT Kit (Applied Biosystems, Foster City, California). An in vitro transcription reaction was subsequently performed to produce biotin-labeled complementary RNA (cRNA) from the cDNA. Gene expression profiles were assessed using the Affymetrix Human Genome HU133A 2.0 GeneChip array, containing 22,277 sequenced human genes. GeneChip arrays were scanned using a GeneArray Scanner (Hewlett-Packard, Santa Clara, Calif). Hybridization data were analyzed using MAS 5.
Project description:Mice with deficient expression of RASSF9 exhibit intriguing phenotypes of skin-related pathology, including abnormal thickening of the epidermis, dysregulated proliferation of keratinocytes, and alopecia. To delineate the underlying mechanism, we profiled gene expression in keratinocytes of RASSF9-mutant mice to identify targets whose expressions were affected by RASSF9 gene deletion. Primary keratinocytes from neonatal ICR mice of wildtype control (WT) or homozygous RASSF9 deletion (RASSF9-/-) were harvested for RNA extraction and hybridization on Affymetrix microarrays. For WT, a single microarray hybridization was performed. For RASSF9-/-, one hybridization was performed for each of two independent samples.