5-hydroxyeicosatetraenoic acid controls androgen reduction in diverse types of human epithelial cells
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ORGANISM(S): Homo sapiens (human)
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PROVIDER: S-BSST941 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Labelled 5?-dihydrotestosterone (DHT) binding experiments have shown that expression levels of (yet unidentified) membrane androgen receptors (mAR) are elevated in prostate cancer and correlate with a negative prognosis. However, activation of these receptors which mediate a rapid androgen response can counteract several cancer hallmark functions such as unlimited proliferation, enhanced migration, adhesion and invasion and the inability to induce apoptosis. Here, we investigate the downstream signaling pathways of mAR and identify rapid DHT induced activation of store-operated Ca2+ entry (SOCE) in primary cultures of human prostate epithelial cells (hPEC) from non-tumorous tissue. Consequently, down-regulation of Orai1, the main molecular component of Ca2+ release-activated Ca2+ (CRAC) channels results in an almost complete loss of DHT induced SOCE. We demonstrate that this DHT induced Ca2+ influx via Orai1 is important for rapid androgen triggered prostate specific antigen (PSA) release. We furthermore identified alterations of the molecular components of CRAC channels in prostate cancer. Three lines of evidence indicate that prostate cancer cells down-regulate expression of the Orai1 homolog Orai3: First, Orai3 mRNA expression levels are significantly reduced in tumorous tissue when compared to non-tumorous tissue from prostate cancer patients. Second, mRNA expression levels of Orai3 are decreased in prostate cancer cell lines LNCaP and DU145 when compared to hPEC from healthy tissue. Third, the pharmacological profile of CRAC channels in prostate cancer cell lines and hPEC differ and siRNA based knock-down experiments indicate changed Orai3 levels are underlying the altered pharmacological profile. The cancer-specific composition and pharmacology of CRAC channels identifies CRAC channels as putative targets in prostate cancer therapy.
| S-EPMC3875772 | biostudies-literature
Project description:Androgen receptor (AR) signaling is crucial to the development and homeostasis of the prostate gland, and its dysregulation mediates common prostate pathologies. The mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells have been investigated in human and rodent adult prostate. However, the cellular stress response of human prostate epithelial cells is not well understood, though it is central to prostate health and pathology. Here, we report that androgen sensitizes HPr-1AR and RWPE-AR human prostate epithelial cells to cell stress agents and apoptotic cell death. Although 5?-dihydrotestosterone (DHT) treatment alone did not induce cell death, co-treatment of HPr-1AR cells with DHT and an apoptosis inducer, such as staurosporine (STS), TNFt, or hydrogen peroxide, synergistically increased cell death in comparison to treatment with each apoptosis inducer by itself. We found that the synergy between DHT and apoptosis inducer led to activation of the intrinsic/mitochondrial apoptotic pathway, which is supported by robust cleavage activation of caspase-9 and caspase-3. Further, the dramatic depolarization of the mitochondrial membrane potential that we observed upon co-treatment with DHT and STS is consistent with increased mitochondrial outer membrane permeabilization (MOMP) in the pro-apoptotic mechanism. Interestingly, the synergy between DHT and apoptosis inducer was abolished by AR antagonists and inhibitors of transcription and protein synthesis, suggesting that AR mediates pro-apoptotic synergy through transcriptional regulation of MOMP genes. Expression analysis revealed that pro-apoptotic genes (BCL2L11/BIM and AIFM2) were DHT-induced, whereas pro-survival genes (BCL2L1/BCL-XL and MCL1) were DHT-repressed. Hence, we propose that the net effect of these AR-mediated expression changes shifts the balance of BCL2-family proteins, such that androgen signaling sensitizes mitochondria to apoptotic signaling, thus rendering HPr-1AR more vulnerable to cell death signals. Our study offers insight into AR-mediated regulation of prostate epithelial cell death signaling.
| S-EPMC4874596 | biostudies-literature
Project description:The olfactory epithelium constitutes the sole source of regenerating neural cells that can be obtained from a living human. As such, primary cultures derived from human olfactory epithelial biopsies can be utilized to study neurobiological characteristics of individuals under different conditions and disease states. Here, using such human cultures, we report in vitro generation of cells that exhibit a complex neuronal phenotype, encompassing receptors and signaling pathways pertinent to both olfaction and other aspects of CNS function. Using in situ hybridization, we demonstrate for the first time the native expression of olfactory receptors in cultured cells derived from human olfactory epithelial tissue. We further establish the presence and function of olfactory transduction molecules in these cells using immunocytochemistry, calcium imaging and molecular methods. Western blot analysis revealed the expression of neurotransmitter receptors for dopamine (D2R), 5-HT (5HT2C) and NMDA subtypes 1 and 2A/2B. Stimulation with dopamine or 5-HT enhanced receptor G protein activation in a subtype specific manner, based on 35S-guanosine triphosphate incorporation assay. Functional characteristics of the cultured cells are demonstrated through enhanced tyrosine phosphorylation of NMDAR 2A/2B and recruitment of signaling partners in response to NMDA stimulation. The array of neuronal characteristics observed here establishes that proliferating cells derived from the human olfactory epithelium differentiate in vitro to express functional and molecular attributes of mature olfactory neurons. These cultured neural cells exhibit neurotransmitter pathways important in a number of neuropsychiatric disorders. Their ready availability from living humans thus provides a new tool to link functional and molecular features of neural cells with clinical characteristics of individual living patients.
| S-EPMC2951119 | biostudies-literature
Project description:Study questionWhat is the transcriptional network governed by the androgen receptor (AR) in human epididymis epithelial (HEE) cells from the caput region and if the network is tissue-specific, how is this achieved?Summary answerAbout 200 genes are differentially expressed in the caput HEE cells after AR activation; the AR transcriptional network is tissue-specific and may be mediated in part by distinct AR co-factors including CAAT-enhancer binding protein beta (CEBPB) and runt-related transcription factor 1 (RUNX1).What is known alreadyLittle is known about the AR transcriptional program genome wide in HEE cells, nor its co-factors in those cells. AR has been best studied in the prostate gland epithelium and prostate cancer cell lines, due to the important role of this factor in prostate cancer. However AR-associated differentially expressed genes (DEGs) and AR co-factors have not yet been compared between human epididymis and prostate epithelial cells.Study design, size, durationCaput HEE cells from two donors were exposed to the synthetic androgen R1881 at 1 nM for 12-16 h after 72 h of hormone starvation.Participants/materials, setting, methodsChromatin was prepared from R1881-treated and vehicle control HEE cells. AR-associated chromatin was purified by chromatin immunoprecipitation (ChIP) and AR occupancy genome wide was revealed by deep sequencing (ChIP-seq). Two independent biological replicates were performed. Total RNA was prepared from R1881 and control-treated HEE cells and gene expression profiles were documented by RNA-seq. The interaction of the potential novel AR co-factors CEBPB and RUNX1, identified through in-silico motif analysis of AR ChIP-seq data, was examined by ChIP-qPCR after siRNA-mediated depletion of each co-factor individually or simultaneously.Main results and the role of chanceThe results identify about 200 genes that are differentially expressed (DEGs) in HEE cells after AR activation. Some of these DEGs show occupancy of AR at their promoters or cis-regulatory elements suggesting direct regulation. However, there is little overlap in AR-associated DEGs between HEE and prostate epithelial cells. Inspection of over-represented motifs in AR ChIP-seq peaks identified CEBPB and RUNX1 as potential co-factors, with no evidence for FOXA1, which is an important co-factor in the prostate epithelium. CEBPB and RUNX1 ChIP-seq in HEE cells showed that both these factors often occupied AR-binding sites, though rarely simultaneously. Further analysis at a single AR-regulated locus (FK506-binding protein 5, FKPB5) suggests that CEBPB may be a co-activator. These data suggest a novel AR transcriptional network governs differentiated functions of the human epididymis epithelium.Large scale dataAR ChIP-seq and RNA-seq data are deposited at GEO: GSE109063.Limitations, reasons for cautionThere is substantial donor-to-donor variation in primary HEE cells cultures. We applied stringent statistical tests with a false discovery rate (FDR) of 0.1% for ChIP-seq and standard pipelines for RNA-seq so it is possible that we have missed some AR-regulated genes that are important in caput epididymis function.Wider implications of the findingsOur data suggest that a novel AR transcriptional network governs differentiated functions of the human epididymis epithelium. Since this cell layer has a critical role in normal sperm maturation, the results are of broader significance in understanding the mechanisms underlying the maintenance of fertility in men.Study funding/competing interestsThis work was funded by the National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Development: R01 HD068901 (PI: Harris). The authors have no competing interests to declare.
| S-EPMC6454485 | biostudies-literature
Project description:PurposeAndrogens exert a significant influence on the structure, function and/or pathophysiology of the meibomian gland and conjunctiva. We sought to determine whether this hormone action involves the regulation of epithelial cell gene expression in these tissues.MethodsImmortalized human meibomian gland and conjunctival epithelial cells were treated with placebo or dihydrotestosterone (DHT) and processed for molecular biologic procedures. Gene expression was evaluated with BeadChips and data were analyzed with bioinformatic and statistical software.ResultsAndrogen treatment significantly influenced the expression of approximately 3,000 genes in immortalized human meibomian gland and conjunctival epithelial cells. The nature of DHT action on gene activity was predominantly cell-specific. Similarly, DHT exerted a significant, but primarily cell-specific, influence on many gene ontologies and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. These included groups of genes related, for example, to lipid dynamics, innate immunity, cell cycle, Janus kinase (JAK)-signal transducer and activator of transcription (stat) cascades, oxidative phosphorylation, the proteasome, and mammalian target of rapamycin (mTOR), Wnt, and peroxisome proliferator-activated receptor (PPAR) signaling.ConclusionsOur findings support our hypothesis that androgens regulate gene expression in human meibomian gland and conjunctival epithelial cells. Our ongoing studies are designed to determine whether many of these genes are translated and play a role in the health and well being of the eye.
| S-EPMC3351406 | biostudies-literature
Project description:Human neurons engineered from induced pluripotent stem cells (iPSCs) through neurogenin 2 (NGN2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here, we used single-cell transcriptomics to dissect the cell states that emerge during NGN2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to NGN2 expression level and the duration of NGN2-forced expression. Our data reveal that NGN2 dosage can regulate neuron fate acquisition, and that NGN2-iN heterogeneity can confound results that are sensitive to neuron type.
| S-EPMC8452516 | biostudies-literature
Project description:Prevalence of asthma is higher in women than in men, but the mechanisms underlying this sex bias are unknown. Group 2 innate lymphoid cells (ILC2s) are key regulators of type 2 inflammatory responses. Here, we show that ILC2 development is greatly influenced by male sex hormones. Male mice have reduced numbers of ILC2 progenitors (ILC2Ps) and mature ILC2s in peripheral tissues compared with females. In consequence, males exhibit reduced susceptibility to allergic airway inflammation in response to environmental allergens and less severe IL-33-driven lung inflammation, correlating with an impaired expansion of lung ILC2s. Importantly, orchiectomy, but not ovariectomy, abolishes the sex differences in ILC2 development and restores IL-33-mediated lung inflammation. ILC2Ps express the androgen receptor (AR), and AR signaling inhibits their differentiation into mature ILC2s. Finally, we show that hematopoietic AR expression limits IL-33-driven lung inflammation through a cell-intrinsic inhibition of ILC2 expansion. Thus, androgens play a crucial protective role in type 2 airway inflammation by negatively regulating ILC2 homeostasis, thereby limiting their capacity to expand locally in response to IL-33.
| S-EPMC5461006 | biostudies-literature
Project description:Gut microbiota play an important role in regulating the development of the host immune system, metabolic rate, and at times, disease pathogenesis. The factors and mechanisms that mediate interactions between microbiota and the intestinal epithelium are not fully understood. We provide novel evidence that microbiota may control intestinal epithelial stem cell (IESC) proliferation in part through microRNAs (miRNAs). We demonstrate that miRNA profiles differ dramatically across functionally distinct cell types of the mouse jejunal intestinal epithelium and that miRNAs respond to microbiota in a highly cell type-specific manner. Importantly, we also show that miRNAs in IESCs are more prominently regulated by microbiota compared with miRNAs in any other intestinal epithelial cell subtype. We identify miR-375 as one miRNA that is significantly suppressed by the presence of microbiota in IESCs. Using a novel method to knockdown gene and miRNA expression ex vivo enteroids, we demonstrate that we can knock down gene expression in Lgr5+ IESCs. Furthermore, when we knock down miR-375 in IESCs, we observe significantly increased proliferative capacity. Understanding the mechanisms by which microbiota regulate miRNA expression in IESCs and other intestinal epithelial cell subtypes will elucidate a critical molecular network that controls intestinal homeostasis and, given the heightened interest in miRNA-based therapies, may offer novel therapeutic strategies in the treatment of gastrointestinal diseases associated with altered IESC function.
| S-EPMC5314158 | biostudies-literature
Project description:The progression of prostate cancer to the castration-recurrent phenotype remains a major problem medically. The present study examined the transcriptomics of de novo androgen synthesis as a potential mechanism to escape from dependence on circulating androgen. VCaP, LNCaP and LAPC4 cells were acclimated to 1 nM testosterone for five generations before subjecting them to a reduced level of 0.03 nM testosterone. Changes in gene expression were quantified using qRT-PCR. Analyses of the cholesterol biosynthesis pathway and the ?4, ?5 and backdoor steroidogenic pathways were carried out. VCaP cells showed no change in the transcriptome of cholesterol biosynthesis. However, several receptors for cholesterol transport were upregulated. The ?4 and ?5 steroidogenic pathways, but not the backdoor pathway, were stimulated. Additionally, androgen receptor (AR) expression was increased. Taken together, the above changes might allow recovery of AR activity to a near normal level. In contrast, LNCaP cells showed only minimal adjustment in the transcriptome of steroidogenesis. LAPC4 cells were equally unresponsive to boosting the machinery of androgen production. In brief, our results suggest that the VCaP model is an appropriate model for further investigation of targeting the androgen-AR axis to block the emergence of castration-resistant prostate cancer.
| S-EPMC2939298 | biostudies-literature
Project description:AimReactive oxygen species (ROS) produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine, due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut, and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut.ResultsNoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS induced colon cancer, NoxO1 has a protective role and may influence the population of natural killer cells.ConclusionNoxO1 affects colon epithelium homeostasis and prevents inflammation.
| S-EPMC5951971 | biostudies-literature