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Transcriptomic Network Interactions in Human Skin Treated with Topical Glucocorticoid Clobetasol Propionate.

ABSTRACT: Glucocorticoids are the most frequently used anti-inflammatory drugs in dermatology. However, the molecular signature of glucocorticoids and their receptor in human skin is largely unknown. Our validated bioinformatics analysis of human skin transcriptome induced by topical glucocorticoid clobetasol propionate (CBP) in healthy volunteers identified numerous unreported glucocorticoid-responsive genes, including over a thousand noncoding RNAs. We observed sexual and racial dimorphism in the CBP response including a shift toward IFN-?/IFN-? and IL-6/Jak/Signal transducer and activator of transcription (STAT) 3 signaling in female skin; and a larger response to CBP in African-American skin. Weighted gene coexpression network analysis unveiled a dense skin network of 41 transcription factors including circadian Kruppel-like factor 9 (KLF9), and ?260 of their target genes enriched for functional pathways representative of the entire CBP transcriptome. Using keratinocytes with Kruppel-like factor 9 knockdown, we revealed a feedforward loop in glucocorticoid receptor signaling, previously unreported. Interestingly, many of the CBP-regulated transcription factors were involved in the control of development, metabolism, circadian clock; and 80% of them were associated with skin aging showing similarities between glucocorticoid-treated and aged skin. Overall, these findings indicate that glucocorticoid receptor acts as an important regulator of gene expression in skin-both at the transcriptional and posttranscriptional level-via multiple mechanisms including regulation of noncoding RNAs and multiple core transcription factors.

SUBMITTER: Lili LN

PROVIDER: S-EPMC6814545 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Transcriptomic Network Interactions in Human Skin Treated with Topical Glucocorticoid Clobetasol Propionate.

Lili Loukia N LN Klopot Anna A Readhead Benjamin B Baida Gleb G Dudley Joel T JT Budunova Irina I

The Journal of investigative dermatology 20190625 11

Glucocorticoids are the most frequently used anti-inflammatory drugs in dermatology. However, the molecular signature of glucocorticoids and their receptor in human skin is largely unknown. Our validated bioinformatics analysis of human skin transcriptome induced by topical glucocorticoid clobetasol propionate (CBP) in healthy volunteers identified numerous unreported glucocorticoid-responsive genes, including over a thousand noncoding RNAs. We observed sexual and racial dimorphism in the CBP re ...[more]

PMID: 31247200

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Project description:The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos (Danio rerio) using transcriptomic profiling. Clobetasol propionate, a highly potent synthetic corticosteroid, which is known to have immunosuppressive effects on vertebrates including zebrafish, was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection, the embryos were injected with a mixture of different pathogen-associated molecular patterns (PAMPs) consisting of Pam3CSK4 (synthetic lipopeptide), poly(I:C) (synthetic viral dsRNA) and flagellin (isolated from Bacillus subtilis). Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test (OECD 236), 20 fertilized eggs were exposed to a sub-lethal concentration (250 nM) of clobetasol propionate for 48 hours under semi-static conditions. The test comprised of three treatments with clobetasol propionate and two negative control (NC) groups and was performed in triplicates. At 46 hours post fertilization (hpf), the eggs were manually dechorinated. At 48 hpf, the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection, 8 nL of the mixed PAMPs (0.33 µg/mL Pam3CSK4, 0.33 µg/mL poly(I:C) and 0.033 µg/mL flagellin) were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs, while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. After injection, the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

Project description:Background:The clobetasol propionate cream formulations (CLB Cr ) belong to the "strongest" group, and are used widely. In addition, those formulations are often used as a mixture with moisturizer. Recently, we evaluated pharmaceutical properties of the CLB Cr using near infrared (NIR) spectroscopy, and characteristic NIR spectra depending on the formulation were observed. In the present study, we attempted to evaluate the more diverse pharmaceutical properties of CLB Cr , including the stability of mixture of CLB Cr and moisturizer. Method:Pharmaceutical properties of CLB Cr were evaluated using from rheological characteristics, microscopic observation, dye permeability observations, electrical conductivity method, thermogravimetry-differential thermal analysis (TG-DTA) and near infrared (NIR) spectroscopy. Stability of mixtures of CLB Cr and moisturizer were evaluated using from dye method and NIR spectroscopy. Results:The hardness of Dermovate® (DRM), Glydil® (GDL), and Myalone® (MYA) was greater than that of CLB Cr . High concentrations of white beeswax were considered the reason for the hardness of DRM and GDL. On the other hand, the hardness of MYA may be due to the presence of macrogol 6000. After storage of the cream formulations discharged from the tube at room temperature, mass reduction and attenuation of the peak reflecting water of NIR spectroscopy occurred in a time-dependent manner, except for GDL and MYA. Only GDL was shown to be a w/o-type formulation by dye and electric conductivity measurements, which suggested that this was the reason for the lack of changes in the mass or NIR spectrum of samples after storage. In the NIR spectrum of MYA, the peak reflecting water slightly increased in a time-dependent manner, suggesting the water absorption of macrogol 6000. TG-DTA provided curves indicating the presence of water in each formulation, except for MYA, which was consistent with water quantification previously reported. Finally, when mixing the CLB Cr with a moisturizer, in any CLB Cr , the stability of the mixture with w/o-type moisturizer varies greatly depending on the each CLB Cr . Conclusion:Thus, even for cream formulations with the same active pharmaceutical ingredient, pharmaceutical properties and stability of mixture with moisturizer may different significantly.

Project description:BackgroundToxic epidermal necrolysis (TEN) is a rare systemic allergic drug eruption with high patient mortality. Currently, no established treatments have been shown to be effective for TEN beyond supportive care. Prior studies of systemic corticosteroids have yielded conflicting data, with some showing a possible benefit and others reporting in increased mortality. However, topical steroids have shown promise for treatment of ocular sequelae of TEN, such as scarring and vision loss. We have designed a randomized controlled trial to evaluate topical clobetasol for treatment of the epidermal manifestations of TEN. In addition, we propose genetic studies to characterize the TEN transcriptome and alterations in cutaneous gene expression that might occur following topical steroid treatment.Methods/designThis split-body randomized, double-blind, placebo-controlled Phase IIa proof-of-concept trial will evaluate the safety and efficacy of once-daily topical clobetasol applied to the skin of patients with TEN. This multicenter trial will recruit a total of 15 patients between the ages of 12 and 85 from the University of California Davis Medical Center and Shriners Hospital for Children inpatient burn units. Designated treatment areas on opposite sides of the body will be treated with blinded clobetasol 0.05% ointment or control petrolatum ointment daily for 14 days. On day 3 of therapy, a biopsy will be taken from the treated area for genetic studies. The primary study aims will be to establish the safety of topical clobetasol treatment and determine the time to cessation of skin detachment for the control and clobetasol-treated areas. Secondary endpoints will evaluate efficacy using parameters such as time to 90% re-epithelialization and percentage of affected skin at 0, 3, 6, 9, 12 and 15 days. Genomic DNA and RNA will be obtained from biopsy samples, to characterize the TEN transcriptome and identify changes in gene expression after topical steroid treatment.DiscussionTopical steroids have shown promise for treating ocular complications of TEN, but to date have not been evaluated for cutaneous manifestations of the disease. This trial will investigate clinical and molecular outcomes of topical clobetasol application and hopefully provide insight into the disease pathophysiology.Trial registrationClinicalTrials.gov NCT02319616. https://clinicaltrials.gov/ct2/show/NCT02351037.

Dataset Information

Transcriptomic Network Interactions in Human Skin Treated with Topical Glucocorticoid Clobetasol Propionate.

Publications

Transcriptomic Network Interactions in Human Skin Treated with Topical Glucocorticoid Clobetasol Propionate.

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