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Expression data from retinoic acid-sufficient and retinoic acid-deficient mouse airway smooth muscle [mouse ASMs]

ABSTRACT: Observational studies in human suggest involvement of vitamin A/retinoic acid (RA) signaling in the regulation of airway smooth muscle (ASM) function, but the precise mechanisms by which RA impacts ASM phenotype is not clear. Here, we generated trascriptional profiles from two different models of RA-sufficient and RA-deficient mouse ASM in order to determine the molecular targets of RA in ASM (VAS/VAD, CTR/BMS) Mouse ASMs were isolated from Acta2-hrGFP;Cspg4-DsRED adult mice using flow cytometry. VAS mice were given normal, vitamin A-sufficient diet (15 IU vitamin/g) while the VAD mice were given 3 days of vitamin A-deficient diet (< 0.1 IU vitamin/g). CTR/BMS mouse ASMs were also isolated from Acta2-hrGFP;Cspg4-DsRED adult mice using flow cytometry. CTR mice were given 3 days of powdered, vitamin A sufficient diet mixed with vehicles (corn oil) while the BMS diet were given 3 days of powdered, vitamin A sufficient diet mixed with a potent pan-RA-receptor antagonist (BMS, 5 mcg/g).

ORGANISM(S): Mus musculus

PROVIDER: GSE116846 | GEO | 2018/12/25

REPOSITORIES: GEO

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Project description:Vitamin A (retinol) is an essential precursor for the production of retinoic acid (RA), which in turn is a major regulator of gene expression, affecting cell differentiation throughout the body. Understanding how vitamin A nutritional status, as well as therapeutic retinoid treatment, regulates the expression of retinoid homeostatic genes is important for improving dietary recommendations and therapeutic strategies using retinoids. This study investigated genes central to processes of retinoid uptake and storage, release to plasma, and oxidation in the liver of rats under steady-state conditions after different exposures to dietary vitamin A (deficient, marginal, adequate and supplemented), and acutely after administration of a therapeutic dose of all-trans-RA. Over a very wide range of dietary vitamin A, lecithin:retinol acyltransferase (LRAT) as well as multiple cytochrome P450s (CYP26A1, CYP26B1, and CYP2C22) differed by diet and were highly correlated with one another and with vitamin A status assessed by liver retinol concentration (all correlations, P<0.05). After acute treatment with RA, the same genes were rapidly and concomitantly induced, preceding RARß, a classical direct target of RA. CYP26A1 mRNA exhibited the greatest dynamic range (change of log26 in 3 h). Moreover, CYP26A1 increased more rapidly in the liver of RA-primed rats than naïve rats. By in situ hybridization, CYP26A1 mRNA was strongly regulated within hepatocytes, closely resembling RBP4 in location. Overall, whether RA is produced endogenously from retinol or administered exogenously, changes in retinoid homeostatic gene expression simultaneously favor both retinol esterification and RA oxidation, with CYP26A1 exhibiting the greatest dynamic change. All rats were housed in a room maintained at 22°C with a 12-h dark:light cycle, and food and water were freely available. For the Steady-State Vitamin A Study (experiment 1) and the Retinoic Acid 16-hour Kinetic Study (experiment 2), lactating female Sprague-Dawley rats with 12 female pups (purchased from Charles River Laboratories, Willmington, MA) were fed a vitamin A-deficient purified diet [AIN-93G diet, prepared by Research Diets, New Brunswick, NJ] to reduce the transfer of vitamin A in milk from mother to pups prior to the start of the study. For experiment 1, from weaning, the offspring were fed the same diet modified to contain vitamin A at one of four levels: 0 (vitamin A deficient), 0.4 mg retinol/kg diet (vitamin A marginal), 4 mg retinol/kg diet (vitamin A adequate control), or 100 mg retinol/kg diet (vitamin A supplemented). All rats were studied at 8 weeks of age. Rats were euthanized by carbon dioxide asphyxiation and blood and liver were collected rapidly and frozen in liquid nitrogen for storage at -80°C before analysis. In experiment 2, at 8 weeks of age female vitamin A-deficient rats were treated with ~100 ug of All-trans-Retinoic acid (at-RA) for 0 (vehicle only), 3, 6, 10 or 16 h (n=3-4/group). Tissues were collected and RNA was prepared in the same manner as in experiment 1. In the 90-minute "first pass" kinetic study (experiment 3), female rats were purchased at 6 weeks of age and fed a stock rodent diet. When the rats were 8 weeks old they were assigned to a control (naïve) group. Rats in the naive group received an equal amount of vehicle only (vegetable oil/5% ethanol). Food was removed immediately. Sixteen hours after priming, each rat was lightly anesthetized by isoflurane-oxygen inhalation and treated with ~25 ug all-trans-RA bound to albumin [10 ug RA per 100 g bo dy weight], injected into the exposed left common iliac vein. The incision was closed with a surgical staple. The rats were allowed to recover from the anesthesia. Rats were killed at 0 minute (vehicle injection), and 30, 60, and 90 min (n = 3/group) after injection of the RA test dose. Rats were euthanized by carbon dioxide asphyxiation and blood and liver were collected rapidly and frozen in liquid nitrogen for storage at -80°C before analysis. For experiment 1, five biological repeats were Vitamin A deficient, seven biological repeats were Vitamin A marginal, six biological repeats were Vitamin A adequate, and two biological repeats were Vitamin A supplemented. In experiment 2, three biological repeats were performed for each of the following treatments: untreated, at-RA for 3 hours, at-RA for 6 hours and at-RA for 16 hours. Four biological repeats were treated with at-RA for 10 hours. For experiment 3, two biological repeats were untreated and three biological repeats were performed for each of the following at-RA treatment times: 30, 60 and 90 minutes. A total of 47 samples were analyzed.

Project description:Overall hypothesis: RNAseq can be used to examine and compare the vitamin A (VA) effect, Infection effect caused by Citrobacter rodentium (C. rodentium), and the Interaction effect (VA status × C. rodentium infection) in mouse small intestine (SI) and colon, on the scale of global transcriptome. Methods: Vitamin A deficient (VAD) mice were generated by feeding VAD diet to the pregnant C57/BL6 dams and their post-weaning offspring. Vitamin A sufficient (VAS) mice were fed with purified diet containing normal amount of VA. At weaning, mice were maintained on their respective diets and assigned to one of the four treatment groups (non-infected VAD, infected VAD, non-infected VAS and infected VAS) until the end of the experiments. For the infected groups, age-matched VAD or VAS mice were orally inoculated with C. rodentium. Mice during early (SI study) or peak (colon study) C. rodentium infection were euthanized in two separate studies. The SI study focused on SI and analyses were performed on samples collected 5 days post-infection. The colon study analyzed samples on post-infection day 10, the peak of C. rodentium infection. Total mRNA extracted from SI and colon were sequenced using Illumina HiSeq 2500 platform. Three to six biological replicates were sequenced in each group. Differentially Expressed Gene (DEG), Gene Ontology (GO) enrichment, and Weighted Gene Co-expression Network Analysis (WGCNA) were performed to characterize expression patterns and co-expression patterns. Results: a) differentially expression genes (DEGs) corresponding to VA effect were present in both the SI and colon. In SI, DEGs altered by VA were mainly involved in the retinoid metabolic pathway and immunity-related pathways. Novel target genes (e.g. Mbl2, Mmp9, Cxcl14, and Nr0b2) and cell types (based on Tuft cell and mast cell markers) under the regulation of VA were suggested by differential expression analysis coupled with the co-expression network analysis. In regard to the colon, VA demonstrated an overall suppressive effect on the cell division pathway. Finally, the comparison of co-expression modules between SI and colon indicated distinct regulatory networks in these two organs. b) In the SI, no Infection effect was detected during early infection, whereas in the colon, during peak of C. rodentium infection: 1) It has been shown that infection upregulated innate and adaptive immune functions, epithelial proliferation, apoptosis, endoplasmic reticulum stress, and reactive oxygen species production, as well as downregulated ion and water absorption. This helped to confirm the findings in previous studies, consolidating the knowledge-base about the host response to C. rodentium, 2) Pathways (e.g. neuron activity, smooth muscle contraction, vascular constriction, and developmental regulation) and additional ion transporters previously not known to be influenced by C. rodentium were suggested as potential mechanisms responsible for the diarrhea pathology and host response. 3) Retinoic acid receptor (RAR) and vitamin D receptor (VDR) signaling pathways appeared to be downregulated by infection, indicating that the malabsorption of micronutrients might be a danger signal for the host to switch from homeostasis to an anti-infection mode. c) In the SI, no Interaction (VA status × C. rodentium infection) effect was detected during early infection. In colon, 1329 genes corresponded to the Interaction effect. During the peak of C. rodentium infection, the sufficient stimulation of cell division, protein catabolism, apoptosis, transcription regulation, MHC class I, interferons, and cytokine signaling might be of key importance for the resistance of VAS mice. In addition, the proper maintenance of the proliferation, differentiation, migration, and activation of T cells and leukocytes during peak infection may also confer the VAS hosts with higher survival and clearance rate. On the other hand, our data suggested that the higher rate of lethal dehydration observed in infected VAD mice, may be attributed to the disrupted of HCO3- metabolism and an improperly high level of Cl- secretion. Conclusions: VA alters the gene expression profile in both SI and colon, whereas the Infection effect and the Interaction effect were only observed in colon during peak of C. rodentium infection, but not in the SI during early infection. Our studies suggest novel target genes and cell types under the regulation of VA in the SI, and indicate that the resistance of the VAS hosts may be attributed to the concordant control over ion/water absorption, immune functions, as well as the balance between epithelial hyperplasia and apoptosis.

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Expression data from retinoic acid-sufficient and retinoic acid-deficient mouse airway smooth muscle [mouse ASMs]

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