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Physiol Genomics 45: 422–433, 2013. First published April 9, 2013; doi:10.1152/physiolgenomics.00154.2012. Glucocorticoid-induced changes in gene expression in embryonic anterior pituitary cells Sultan A. Jenkins,1 Laura E. Ellestad,1,2 Malini Mukherjee,2 Jyoti Narayana,2 Larry A. Cogburn,3 and Tom E. Porter1,2 1 Department of Animal and Avian Sciences, University of Maryland, College Park, Maryland; 2Molecular and Cell Biology Program, University of Maryland, College Park, Maryland; and 3Department of Animal and Food Sciences, University of Delaware, Newark, Delaware Submitted 19 November 2012; accepted in final form 7 April 2013 pituitary; glucocorticoid; microarray; embryo pleiotropic effects on many tissues, including stimulation of hepatic gluconeogenesis, suppression of immune function, and the generalized stress response. In addition, glucocorticoids induce production of lung surfactant in perinatal mammals. Within the anterior pituitary gland, glucocorticoids are known to decrease adrenocorticotropic hormone secretion in adults and induce growth hormone (GH) production during early development (24). However, glucocorticoid induction of GH gene expression in fetal rat and embryonic chicken pituitary cells requires ongoing synthesis of an unknown protein(s) (2, 20). The chicken embryo provides an excellent model to study effects of glucocorticoids on pituitary cell differentiation, because the ontogenic profile for expression of pituitary genes is similar to that of mammals (11, ADRENAL GLUCOCORTICOIDS HAVE Address for reprint requests and other correspondence: T. E. Porter, Dept. of Animal and Avian Sciences, Univ. of Maryland, College Park, MD 20742 (e-mail: [email protected]). 422 12), the embryo can be manipulated easily without the confounding maternal interactions that occur in mammals, and dozens of embryonic pituitary glands can be collected easily at the same age in a single day for performing primary cell culture experiments. We previously established that somatotroph differentiation occurs around embryonic day (e) 14 of chicken embryonic development (25). Somatotrophs lack the ability to differentiate spontaneously and require an extrapituitary signal (26), and we have shown that one signal capable of prematurely inducing somatotroph differentiation is the adrenal glucocorticoid corticosterone (CORT) (1–3, 9, 10, 14, 17, 19, 24, 27). Treatment of e11 chicken embryos with CORT in vivo increased the number of cells that secreted GH on e13 (3, 9). In cultures of embryonic pituitary cells, CORT increased the abundance of cells expressing GH mRNA (2, 27). Treatment with the protein synthesis inhibitor cycloheximide (CHX) completely blocked CORT induction of GH mRNA, indicating that the response requires the ongoing synthesis of one or more proteins (2). The somatotrophs induced by CORT release GH in response to GH-releasing hormone (27). Taken together, these results implicate involvement of corticosteroids in the terminal steps of somatotroph differentiation and suggest that glucocorticoid induction of GH gene expression is indirect, involving synthesis of another protein(s). Similar findings have been reported previously in rodents. Addition of the synthetic glucocorticoid dexamethasone to the drinking water of pregnant rats prematurely induces GH protein and mRNA in the corresponding fetuses, and treatment of fetal rat pituitary cells with dexamethasone or CORT in vitro induces GH protein and mRNA (20, 21). As in chicken embryonic pituitary cells, induction of GH mRNA by glucocorticoids in rats requires ongoing protein synthesis (20). Thus, it appears that glucocorticoid induction of GH production during development is a common mechanism among vertebrate classes. The purpose of the present study was to identify additional genes that are regulated by glucocorticoids within the embryonic pituitary gland. Chicken embryonic pituitary cells were used because it would be technically difficult to obtain a sufficient number of embryonic pituitary glands from rodents to perform these studies. To identify both direct and indirect glucocorticoid-regulated genes, we used a custom chicken cDNA microarray to characterize changes in transcriptional profiles in embryonic pituitary cells in response to CORT in the absence and presence of a protein synthesis inhibitor. The Del-Mar 14K Integrated Systems microarray contains 14,053 cDNAs assembled from the neuroendocrine system (hypothalamus, pineal, and pituitary gland), the reproductive system, the 1094-8341/13 Copyright © 2013 the American Physiological Society Downloaded from http://physiolgenomics.physiology.org/ by 10.220.32.247 on June 17, 2017 Jenkins SA, Ellestad LE, Mukherjee M, Narayana J, Cogburn LA, Porter TE. Glucocorticoid-induced changes in gene expression in embryonic anterior pituitary cells. Physiol Genomics 45: 422–433, 2013. First published April 9, 2013; doi:10.1152/physiolgenomics.00154.2012.— Within the anterior pituitary gland, glucocorticoids such as corticosterone (CORT) provide negative feedback to inhibit adrenocorticotropic hormone secretion and act to regulate production of other hormones including growth hormone (GH). The ontogeny of GH production during chicken embryonic and rat fetal development is controlled by glucocorticoids. The present study was conducted to characterize effects of glucocorticoids on gene expression within embryonic pituitary cells and to identify genes that are rapidly and directly regulated by glucocorticoids. Chicken embryonic pituitary cells were cultured with CORT for 1.5, 3, 6, 12, and 24 h in the absence and presence of cycloheximide (CHX) to inhibit protein synthesis. RNA was analyzed with custom microarrays containing 14,053 chicken cDNAs, and results for selected genes were confirmed by quantitative reverse transcription real-time PCR (qRT-PCR). Levels of GH mRNA were maximally induced by 6 h of CORT treatment, and this response was blocked by CHX. Expression of 396 genes was affected by CORT, and of these, mRNA levels for 46 genes were induced or repressed within 6 h. Pathway analysis of genes regulated by CORT in the absence of CHX revealed networks of genes associated with endocrine system development and cellular development. Eleven genes that were induced within 6 h in the absence and presence of CHX were identified, and eight were confirmed by qRT-PCR. The expression profiles and canonical pathways defined in this study will be useful for future analyses of glucocorticoid action and regulation of pituitary function. GLUCOCORTICOID-REGULATED GENES IN THE EMBRYONIC PITUITARY liver, adipose tissue, and skeletal muscle (5– 8). We have used these custom cDNA microarrays previously to characterize changes in gene expression during pituitary development in embryonic chickens (11) and differences in hypothalamic gene expression between genetically selected fat and lean chicken lines (4). These microarrays represent more than half of the expressed genes in the chicken genome. We report the identification of genes that are direct and early targets for glucocorticoid effects in the embryonic anterior pituitary gland. MATERIALS AND METHODS National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) data repository (platform accession #GPL1731 http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc⫽GPL1731). The GEO file contains clone identification number, clone name, GenBank accession number, links to the cDNA sequence, contig sequence, BLASTN and BLASTX alignments, and chromosomal location for each cDNA. Samples were hybridized to the microarrays using a reference RNA design (30). An internal reference sample was generated by labeling with Cy5 an aRNA pool made from all aRNA samples from the entire experiment (48 samples total). The experimental samples were labeled with Cy3 and hybridized to an array with an aliquot of the Cy5-labeled pool. This design resulted in the use of 48 microarrays (12 treatment groups per replicate, n ⫽ 4 replicates). Labeling of cDNA with Cy3 and Cy5, microarray hybridization, and image scanning were performed by Microarray Core Facility at the University of Maryland Institute for Bioscience and Biotechnology Research. Cy3- or Cy5-labeled target cDNA was generated in a two-step process from 1 g aRNA using random primers with the Amino Allyl cDNA Labeling Kit (Ambion) followed by coupling of Cy3 or Cy5 mono-reactive NHS esters (Amersham Biosciences, Piscataway, NJ) to the cDNA. Labeled cDNA targets were purified from unincorporated fluorescent dye with the CyScribe GFX Purification Kit (Amersham). Microarrays were hybridized overnight at 42°C with Cy3-labeled experimental samples and an aliquot of the Cy5-labeled reference pool using microarray hybridization buffer (Amersham). The slides were then washed with increasing stringency with salt sodium citrate and scanned with a 418 confocal laser scanner (Affymetrix) at 550 nm for Cy3 and 649 nm for Cy5, generating two TIFF images for each slide. The data obtained from the microarray analysis were processed and normalized using software that is part of the TM4 suite of microarray data analysis applications (29). The two TIFF images for each slide were processed using Spotfinder (version 2.2.4). The raw pixel intensities determined with Spotfinder were exported to the Microarray Data Analysis System (MIDAS, version 2.18) to be normalized. Lowess normalization was carried out on the data from the Cy3 channel without background correction. Next, the data underwent standard deviation regularization first by block then by slide, with Cy5 (the pooled RNA sample) as the reference. Background fluorescence was determined for each slide by taking the mean Cy3 and Cy5 fluorescence values of the eight replicate control spots (salmon testes DNA) on each slide. Any spot whose Cy3 or Cy5 fluorescence intensity levels were below background was deleted from further analysis. Data were then analyzed as log2(Cy3/Cy5), or log2-ratio, for each spot. Primer design and qRT-PCR. Oligonucleotide primers (Sigma Genosys) used for quantitative reverse transcription real-time polymerase chain reactions (qRT-PCR) were designed using Primer Express (Applied Biosystems, Foster City, CA) from the contig or singlet sequence for that cDNA. All cDNA sequences are available from an online searchable database (http://cogburn.dbi.udel.edu/). When possible, primers were selected to span the 3=-most intron of a known gene. The sequences of all PCR primers are listed in Table 1. Reverse transcription (RT) reactions were carried out using SuperScript III (Invitrogen) with random primers (Invitrogen) and 500 ng of aRNA or an oligo dT primer and total RNA as noted. As a negative control for genomic DNA contamination, a pool of all the RNA from a given replicate experiment was made, and the reaction conducted as the others except reverse transcriptase was not added. All reactions were diluted to 100 l (fivefold) prior to PCR analysis. The diluted RT samples (2 l) were then analyzed using SYBR Green PCR Master Mix (Applied Biosystems) and a BioRad iCycler. Cycling parameters were an initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 55°C for 45 s. Dissociation curve analysis and gel electrophoresis were conducted to ensure that a single PCR product of appropriate size was amplified in each reaction. PCR products were sequenced to confirm their identity. Physiol Genomics • doi:10.1152/physiolgenomics.00154.2012 • www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.32.247 on June 17, 2017 Animals and pituitary cell cultures. All animals were Ross broiler strain chicken embryos purchased from Allen’s Hatchery (Seaford, DE). All procedures were approved by the Institutional Animal Care and Use Committee at the University of Maryland. All hormones and other chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise stated. Embryonic day 0 (e0) was defined as the day when the eggs were placed in a humidified incubator (G.Q.F. Manufacturing, Savannah, GA) at 37.5°C. The typical incubation length for chickens is 21 days. On e11, the embryos were removed and their pituitary glands isolated under a dissecting microscope. Approximately 90 embryonic anterior pituitary glands were isolated for each replicate trial. The pituitaries were dispersed into individual cells by trypsin digestion and mechanical agitation as described previously (25). Dispersed pituitary cells were plated (1 ⫻ 106 cells/well) in poly-L-lysine-coated 12-well culture plates in serum-free medium (D-MEM/F-12 nutrient mixture; Invitrogen, Carlsbad, CA) supplemented with 0.1% bovine serum albumin, 5 g/ml bovine insulin, 5 g/ml human transferrin, 100 U/ml penicillin G, and 100 g/ml streptomycin sulfate. Cells were allowed to attach for 1 h in a 37.5°C, 5% CO2 atmosphere. Cells were then either pretreated for 1.5 h with 10 g/ml CHX to inhibit protein synthesis and then subsequently treated with CORT (10⫺9 M) for 0, 1.5, 3, 6, 12, or 24 h or cultured with CORT (10⫺9 M) without CHX pretreatment for 0, 1.5, 3, 6, 12, or 24 h. The concentration of CHX (10 g/ml) was chosen because it was previously shown to block GH mRNA upregulation (2). All cells were maintained in culture for 24 h regardless of treatment, with CORT added to the cultures at the indicated times prior to the end of the 24 h in culture. At the end of the 24 h culture period, cells were detached from the culture plates with trypsin and immediately frozen in liquid nitrogen prior to RNA isolation. RNA isolation, amplification, and in vitro transcription. Total RNA was isolated from the cultured pituitary cells using RNeasy Mini Kits (QIAGEN, Valencia, CA) according to the manufacturer’s protocol and quantified by measuring optical density at 260 nm. To remove any contaminating DNA, we treated each sample with DNase I. Chicken embryonic anterior pituitary glands do not yield sufficient total RNA for microarray analysis. Therefore, a previously detailed (11, 28) modification of the Eberwine procedure (23) was used to amplify mRNA. Briefly, 0.5 g of total RNA was reverse-transcribed with SuperScript II (Invitrogen) and an oligo(dT) primer containing a T7 promoter site (5=-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGT24-3=; Affymetrix, Santa Clara, CA). Following second-strand DNA synthesis, the double-stranded cDNA was phenol-chloroform extracted, purified using a Microcon-30 column (Millipore, Billerica, MA), and used as a template for in vitro transcription with the T7 MEGAscript kit (Ambion, Austin, TX) according to the manufacturer’s protocol. The resulting amplified RNA (aRNA) was phenol-chloroform extracted, purified with a Spin Column-30 (Sigma), and quantified using the RiboGreen RNA Quantitation Kit (Invitrogen). Del-Mar 14K Integrated Systems microarrays, microarray hybridization, and data analysis. An annotated list of clones and their location on the Del-Mar 14K Integrated Systems microarrays is available at the 423 424 GLUCOCORTICOID-REGULATED GENES IN THE EMBRYONIC PITUITARY Table 1. Oligonucleotide primers used for qRT-PCR Forward (5=-3=) Reverse (5=-3=) ACTB ATP1B1 CEPU COX6C DEXRAS1 FKBP5 GH GLIPR2 LHFPL5 LRRN3 LSM7 NDRG1 NOTCH2 PLEKHB1 PMP22a RAS-DVA SEMA7A SENP2 SMARCD1 pgp1c.pk002.f23 TTCTTTTGGCGCTTGACTCA ATGGAAGAACACTTTAACCTT AGAAGGGCATCCTGATGTGT GCATGGAGTGGAAATACGGT GGTCTACCAGCTCGACATCC AGCACTGCATCCTCTACCTG TTCAAGAAGGATCTGCACAAGGT CAGGGACAGGTCATTTCACA GCGGGACATCAGCTTTTGTT AACGTGACCACTAAAGGACTAGATAGG GATGGCGGATAAGGAGAAGA CCTGCAAAGCTTGCGGAA GAGCCAGTTGGAGAAGATGC CTCTGGAGGCAGAGTTCCAT TCTCCTTCCTGGCCTTTGTA AGGAAGCTCTCCATCCAGAA CAAATTCCCTCGTCATCGTT CCACAACCTTGGGAGAAGAG ACTCAGACTCGCCCAGTGAT AAGCCAAACAATGCCAATGTC GCGTTCGCTCCAACATGTT TCAGCTGCTTTTTATGTCAAAT CAGCCCTTTCTGTCCTTCAG GCACATCCGAGAGACACAAG TGAACACGAGGATGAAAACG CTTTGGTGTCCATCTCCCAT CTCAGATGGTGCAGTTGCTCTCT CAGCTGGATCGTACCTAGCC CAGAAGATGTACAAACCCCCAAG TCGTGAGATGCAGCTGTAGAGATAC ACCACTTGCTTCTCTCCCAC AAGGAGGTTACGCTGGAGC CTGCCATGTTACCCTCTGGT GCACGAACCAATTCCTCTTC TCATTCATTGCCCTCCTCTT GGAGGGAATTTGTCCTCCTT AGAGCTCTCCATCCCCTTTC GCCAGTGGAACCCTATCAAA TCCGCTGGGATTCAAATATC GGTTGCTGCAATTTCTCACAAG qRT-PCR, quantitative reverse transcription polymerase chain reactions. Statistical analysis. Statistical analyses were performed using Statistical Analysis System (SAS) version 9.2 (SAS Institute, Cary, NC). Microarray data from cells cultured in the absence and presence of CHX were analyzed separately. Data were first trimmed to eliminate spots with median pixel intensities below background and genes for which fewer than half of the microarrays returned results. Next, genes were eliminated from further analysis if fewer than half of the arrays failed to return data in the absence of CHX (n ⬎ 11 of 24 arrays total). No threshold value for response was used to trim the dataset prior to analysis. For the 11,148 probes remaining, log2 ratios were subjected to one-way analysis of variance (ANOVA) using PROC GLM in SAS to identify spots that were differentially expressed on at least one of the time points. Differences were considered significant at P ⬍ 0.05. No correction for multiple comparisons was performed to minimize the chances of rejecting effects on gene expression that were real (type II error). Of the 11,148 probes analyzed, 396 showed significant differences among the time points. Levels of mRNA reported are the means and standard errors of the relative expression levels described above. Prior to statistical analysis, qRT-PCR data were transformed to correct for heterogeneity of variance among treatment groups by taking the log10 of the relative expression levels. Results were then analyzed by analysis of variance using the PROC MIXED procedure of SAS. Differences between treatments were compared using the PDIFF procedure (SAS), which corrects for multiple comparisons using the least significant difference. Differences were considered significant at P ⬍ 0.05. Ingenuity Pathway Analysis. Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Redwood City, CA) was used to map the differentially expressed genes to gene interaction networks and canonical metabolic/regulatory pathways. The significant gene list of 396 probes was reannotated with GenBank Human Protein ID or UniProt ID and Gene Ontology (GO) terms using the “GeneBase” tool on our website (http://cogburn.dbi.udel.edu/). The reannotation tool provides two output files (UniProt ID list and GORetriever result file) for GO term analysis on the Agbase website (http://www.agbase.msstate. edu/). The UniProt ID list was submitted to GO Retriever tool, while the GORetriever result file was submitted to GO SlimViewer tool on AgBase. RESULTS Time course for CORT induction of GH mRNA. To identify genes regulated by CORT prior to or later than induction of GH mRNA, we first defined the expression of GH mRNA in response to CORT. Chicken e11 pituitary cells were treated with CORT (1 ⫻ 10⫺9 M) for 0, 1.5, 3, 6, 12, and 24 h in the absence and presence of CHX (10 g/ml). GH mRNA levels increased to a maximum at 6 h after CORT treatment and remained elevated compared with the 0 h time point throughout the 24 h culture period (Fig. 1). Microarray analysis. The complete output from our microarray analysis can be found at the GEO website (accession number GSE5067, http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc⫽GSE5067). Of the 14,053 cDNAs contained on the microarrays, a total of 396 genes showed a significant difference (P ⬍ 0.05) between any two time points in response to CORT in the absence of CHX with at least half of the Fig. 1. Time course of growth hormone (GH) mRNA after corticosterone (CORT) administration. Chicken embryonic day (e) 11 pituitary cells were either pretreated for 1.5 h with 10 g/ml cycloheximide (CHX) and then subsequently cultured in the presence of CORT (10⫺9 M) for 0, 1.5, 3, 6, 12, or 24 h (open bars) or cultured with CORT (10⫺9 M) with no CHX pretreatment (black bars). Cells were then harvested, and total cellular RNA extracted and amplified to produce amplified (a)RNA. Levels of -actin (ACTB) and GH mRNA were determined by qRT-PCR performed on aRNA. Levels of GH mRNA were normalized to levels of ACTB mRNA, and results are expressed relative to 0 h time point. Results presented are the means and SE of the relative expression levels for 4 replicate experiments. Means without a common superscript differ (P ⬍ 0.05) within the no CHX pretreatment group. *Significantly different from CHX pretreated group at the same time point (P ⬍ 0.05). Physiol Genomics • doi:10.1152/physiolgenomics.00154.2012 • www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.32.247 on June 17, 2017 Gene GLUCOCORTICOID-REGULATED GENES IN THE EMBRYONIC PITUITARY 425 Physiol Genomics • doi:10.1152/physiolgenomics.00154.2012 • www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.32.247 on June 17, 2017 Fig. 2. Comparison of the microarray analysis (open bars) of anterior pituitary expression levels for 5 categories of gene expression with results from qRT-PCR analysis (black bars) for the same genes. Genes from the microarray analysis were put into 1 of 5 categories: no effect, repressed early, repressed late, induced late, and induced early. Levels of the analyzed genes were normalized to levels of ACTB mRNA, and results are expressed relative to the highest expression level for each technique. Results are presented for cytochrome c oxidase subunit VIc (COX6C); SWI/ SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 1 (SMARCD1); LSM7 homolog, U6 small nuclear RNA associated (LSM7); pleckstrin homology domain containing, family B member 1 (PLEKHB1); CEPU-Se alpha 2 isoform (CEPU); GLI pathogenesis-related 2 (GLIPR2); peripheral myelin protein 22 a (PMP22a); SUMO1/sentrin/SMT3-specific peptidase 2 (SENP2); semaphorin 7A, GPI membrane anchor (SEMA7A); and notch 2 (NOTCH2). The qRT-PCR analysis for all genes was conducted on aRNA. Relative levels of mRNA were determined by subtracting the cycle threshold (Ct) of ACTB from that of the gene of interest (⌬Ct ⫽ CtGene ⫺ CtACTB). Then the ⌬Ct of the 0 h, no CHX group was subtracted from the ⌬Ct of the other time points (⌬⌬Ct ⫽ ⌬1.5, 3, 6, 12, or 24 h ⫺ ⌬0 h). The fold difference relative to basal (0 h) was then calculated as 2⫺⌬⌬Ct. *Values within a given technique are significantly different from control (0 h) (P ⬍ 0.05, n ⫽ 4). 426 GLUCOCORTICOID-REGULATED GENES IN THE EMBRYONIC PITUITARY possible observations (n ⱖ 12). Based on the results for GH mRNA levels, we categorized our microarray data by grouping genes into one of five possible categories: 1) genes that were induced early (i.e., at least twofold within 3 h), 2) genes that were repressed early, 3) genes that were induced late (i.e., at least twofold between 6 and 24 h), 4) genes that were repressed late, and 5) genes that displayed no significant effect at any time point compared with basal (0 h). A total of 46 transcripts Downloaded from http://physiolgenomics.physiology.org/ by 10.220.32.247 on June 17, 2017 Fig. 3. Gene interaction networks predicted by Ingenuity Pathway Analysis (IPA) of 335 genes whose mRNA levels were affected by CORT treatment. A: 26 pituitary genes assigned by IPA to cellular compromise, inflammatory response, and cellular movement functions. B: another gene interaction network of 24 genes involved in drug metabolism, endocrine system development and function, and lipid metabolism functions. Genes upregulated by CORT are shown in red, while those downregulated by CORT are shown in green. Color intensity reflects magnitude of change. Genes without color were not affected by CORT treatment. Direct relationships are shown by the solid lines, and indirect ones are shown as dashed lines. Physiol Genomics • doi:10.1152/physiolgenomics.00154.2012 • www.physiolgenomics.org GLUCOCORTICOID-REGULATED GENES IN THE EMBRYONIC PITUITARY increased early, 48 decreased early, 20 increased late, and 13 decreased late after CORT treatment (Supplemental Table S1).1 To verify the data obtained from the microarray, qRTPCR analysis was performed on genes from each of these gene 1 The online version of this article contains supplemental material. 427 expression categories (Fig. 2). The genes that were verified for the no significant effect category were cytochrome c oxidase subunit VIc (COX6C) and SWI/SNF related, matrix associated, actin-dependent regulator of chromatin, subfamily d, member 1 (SMARCD1). LSM7 homolog, U6 small nuclear RNA associated (LSM7) and pleckstrin homology domain containing, Downloaded from http://physiolgenomics.physiology.org/ by 10.220.32.247 on June 17, 2017 Fig. 4. Gene interaction networks in the chicken pituitary responding to CORT treatment. A: 28 differentially expressed genes assigned by IPA to cancer and the cell cycle functions. B: a gene interaction network of 16 genes involved in cell death, hematological system development and function, and cellular development. Physiol Genomics • doi:10.1152/physiolgenomics.00154.2012 • www.physiolgenomics.org 428 GLUCOCORTICOID-REGULATED GENES IN THE EMBRYONIC PITUITARY Table 2. Summary of IPA and assignment of biological functions and canonical pathways to differentially expressed genes in pituitary glands treated with CORT P Value Genes, n 1.34E-05 4.07E-04 4.40E-04 4.74E-04 5.08E-04 110 50 55 7 20 2.46E-05 7.30E-05 1.40E-04 2.11E-04 2.52E-04 27 77 27 70 53 3.40E-06 3.40E-06 3.40E-06 5.31E-04 5.31E-04 18 45 37 21 30 1.06E-04 4.98E-05 6.33E-04 6.60E-05 1.96E-03 14 12 10 9 6 Top biological functions Diseases and Disorders Cancer Reproductive system disease Neurological disease Inflammatory response Renal and urological disease Molecular and Cellular Functions Cellular compromise Cell death Carbohydrate metabolism Gene expression Lipid metabolism Physiological System Development and Function Skeletal and muscular system Tissue development Tissue morphology Embryonic development Organism development Top canonical pathways Glucocorticoid Receptor Signaling ERK/MAPK signaling NRF2-mediated oxidative stress response PTEN signaling Ceramide signaling P values were determined by the Ingenuity Pathway Analysis (IPA) software. CORT, corticosterone. erythroid 2-related factor (NRF2)-mediated oxidative stress response, phosphatase and tensin homolog (PTEN) signaling, and ceramide signaling (Table 2, see Table 3 for list of genes). Fourteen genes are involved in glucocorticoid receptor signaling, which included three genes upregulated by CORT [FKBP5, TAF2, and menage a trois homolog 1 (MNAT1)] and 11 downregulated genes (i.e., POMC, HRAS, DUSP1, STAT3, PIK3R1, and SGK1). Twelve pituitary genes are involved in ERK-MAPK signaling, with two upregulated genes [protein phosphatase 2A activator, regulatory subunit 4 (PPP2R4), and kinase suppressor of ras 1 (KSR1)] and 10 downregulated genes, including integrin, alpha 4 (ITGA4), HRAS, and a member of RAS oncogene family (RAP1A). Ten CORT-responsive genes are members of the NRF2-mediated oxidative stress response, including three upregulated genes (FKBP5, PRDX1, and TXNRD1) and seven downregulated genes (i.e., GSTA1, AKR1A1, and SOD1). The PTEN signaling pathway involved one upregulated gene, the apoptosis facilitator (BCL2L11), and eight downregulated genes, including FGFR2, IGF2R, and BMPR2. The six (two upregulated and four downregulated) genes implicated in ceramide signaling were also assigned to the other signaling pathways indicated above. IPA identified 26 different transcription factors known to regulate target genes in our dataset (Table 4). Accordingly, the top five transcription factors associated with the differentially expressed genes were the glucocorticoid receptor [nuclear receptor subfamily 3, group C, member 1(NR3C1)], v-myc myelocytomatosis viral oncogene (MYC), tumor protein p53 (TP53), hepatocyte nuclear factor 4-alpha (HNF4A), and hun- Physiol Genomics • doi:10.1152/physiolgenomics.00154.2012 • www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.32.247 on June 17, 2017 family B member 1 (PLEKHB1) were verified for our repressed early category. Genes that were repressed late included CEPU-Se alpha 2 isoform (CEPU) and GLI pathogenesisrelated 2 (GLIPR2). The induced late category was validated with peripheral myelin protein 22 a (PMP22a) and SUMO1/ sentrin/SMT3-specific peptidase 2 (SENP2). The category of genes that were induced early (within 3 h) was validated with semaphorin 7A, GPI membrane anchor (SEMA7A), and notch 2 (NOTCH2). As shown in Fig. 2, effects of CORT on mRNA levels for each of these genes were comparable when determined by microarray or qRT-PCR, confirming the validity of our gene expression categories. IPA. The 396 genes whose mRNA levels were affected by CORT treatment in the absence of CHX were submitted to IPA software, which identified 335 chicken pituitary transcripts as ready for pathway analysis. Sixty-one transcripts were not annotated in the IPA database. IPA placed the 335 genes into gene interaction networks and canonical metabolic and regulatory pathways. Two of the top five gene interaction networks showed genes involved in cellular compromise, inflammatory response, and cellular movement (Fig. 3A) and those involved in drug metabolism, endocrine system development and function, and lipid metabolism (Fig. 3B). Each network contained one or more genes that were either upregulated or downregulated by CORT treatment. Upregulated genes included known targets of glucocorticoids, FKBP5 and RASD1, and one identified in Fig. 3B as prefoldin subunit 5 (PFDN5). It should be noted, however, that the percent identity between the predicted open reading frame of the cDNA clone (pgp1c.pk002.f23) printed on the microarray and the PFDN5 nucleotide sequence is marginal. Two additional gene interaction networks (Fig. 4) show 28 CORT-responsive genes (i.e., ERBB2, HRAS, FAT1, and QKI; Fig. 4A), which are involved in cancer, cell cycle and connective tissue development and function, and 16 genes that are involved in cell death, hematological development and function, and cellular development (i.e., APBA1, LPAR2, POMC, RASD2, and STAT3; Fig. 4B). The majority of pituitary genes shown in these two networks are downregulated by CORT. The major biological functions and canonical pathways involved in the transcriptional responses of the embryonic pituitary cells to CORT treatment are summarized in Table 2. The largest number of genes in the diseases and disorders category was assigned to cancer (100 genes, including 107 genes involved in tumorigenesis), reproductive system disease (50 genes), and neurological disease (55 genes) functions. In the molecular and cellular function category, a large number of CORT-responsive genes were related to cell death (77 genes), gene expression (70 genes), and lipid metabolism (53 genes, which included 25 genes involved in lipid synthesis). Tissue development, tissue morphology, and organismal development were highly represented in the physiological systems development and function category in the Ingenuity Knowledge Base. Our differentially expressed pituitary gene list contains 66 genes that are associated with proliferation of cells. These observations support extensive involvement of CORT in differentiation and proliferation of cells in the chicken embryo pituitary gland. The top five canonical pathways involved in the pituitary response to CORT treatment include glucocorticoid receptor (NR3C1) signaling, ERK-MAPK signaling, nuclear factor- 429 GLUCOCORTICOID-REGULATED GENES IN THE EMBRYONIC PITUITARY Table 3. Genes assigned by IPA to top five canonical pathways Symbol Gene Name PPP2R4 KSR1 PPP1CA PIK3R3 PPP2R5A YWHAH PIK3R1 STAT3 RAP1A DUSP1 HRAS ITGA4 protein phosphatase 2A activator, regulatory subunit 4 kinase suppressor of ras 1 protein phosphatase 1, catalytic subunit, alpha isozyme phosphoinositide-3-kinase, regulatory subunit 3 (gamma) protein phosphatase 2, regulatory subunit B, alpha tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide phosphoinositide-3-kinase, regulatory subunit 1 (alpha) signal transducer and activator of transcription 3 (acute-phase response factor) RAP1A, member of RAS oncogene family dual specificity phosphatase 1 v-Ha-ras Harvey rat sarcoma viral oncogene homolog integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) FKBP5 PRDX1 TXNRD1 DNAJC16 PIK3R3 SOD1 AKR1A1 PIK3R1 HRAS GSTA1 FK506 binding protein 5 peroxiredoxin 1 thioredoxin reductase 1 DnaJ (Hsp40) homolog, subfamily C, member 16 phosphoinositide-3-kinase, regulatory subunit 3 (gamma) superoxide dismutase 1, soluble aldo-keto reductase family 1, member A1 (aldehyde reductase) phosphoinositide-3-kinase, regulatory subunit 1 (alpha) v-Ha-ras Harvey rat sarcoma viral oncogene homolog glutathione S-transferase alpha 1 BCL2L11 BMPR2 PIK3R3 IGF2R YWHAH PIK3R1 HRAS ITGA4 FGFR2 BCL2-like 11 (apoptosis facilitator) bone morphogenetic protein receptor, type II (serine/threonine kinase) phosphoinositide-3-kinase, regulatory subunit 3 (gamma) insulin-like growth factor 2 receptor tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide phosphoinositide-3-kinase, regulatory subunit 1 (alpha) v-Ha-ras Harvey rat sarcoma viral oncogene homolog integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) fibroblast growth factor receptor 2 PPP2R4 KSR1 PIK3R3 PPP2R5A PIK3R1 HRAS protein phosphatase 2A activator, regulatory subunit 4 kinase suppressor of ras 1 phosphoinositide-3-kinase, regulatory subunit 3 (gamma) protein phosphatase 2, regulatory subunit B=, alpha phosphoinositide-3-kinase, regulatory subunit 1 (alpha) v-Ha-ras Harvey rat sarcoma viral oncogene homolog Log Ratio Entrez Gene ID 0.308 0.283 ⫺0.134 ⫺0.171 ⫺0.221 ⫺0.273 ⫺0.305 ⫺0.328 ⫺0.330 ⫺0.333 ⫺0.365 ⫺0.415 5524 8844 5499 8503 5525 7533 5295 6774 5906 1843 3265 3676 1.623 0.529 0.248 ⫺0.096 ⫺0.171 ⫺0.204 ⫺0.228 ⫺0.305 ⫺0.365 ⫺0.391 2289 5052 7296 23341 8503 6647 10327 5295 3265 2938 0.645 ⫺0.145 ⫺0.171 ⫺0.218 ⫺0.273 ⫺0.305 ⫺0.365 ⫺0.415 ⫺0.419 10018 659 8503 3482 7533 5295 3265 3676 2263 0.308 0.283 ⫺0.171 ⫺0.221 ⫺0.305 ⫺0.365 5524 8844 8503 5525 5295 3265 ERK-MAPK signaling PTEN signaling Ceramide signaling tingtin (HTT). It is of particular interest that the glucocorticoid receptor (NR3C1) affects 26 target genes in our dataset, including POMC, FKBP4, FKBP5, DUSP1, PIK3R1, and PIK3R3 (see Table 4). In contrast, the mineralocorticoid receptor (MR or NR3C2) had only six target genes among the 335 differentially expressed pituitary genes. We have reported that pituitary cells on e11 express both GR and MR (1). The other major transcription regulators and their targets (in parentheses) in this dataset were: MYC (25 genes), HTT (23 genes), TP53 (31 genes), and hepatocyte nuclear factor 4, alpha (HNF4A), which directly affects 45 genes. Many of the target genes of these transcription factors are also shown in the four gene interaction networks (Figs. 3 and 4) and top canonical pathways (Table 3). Identification of genes induced in the absence of protein synthesis. As stated above, our hypothesis is that ongoing synthesis of one or more proteins is required for CORT induction of GH mRNA. The results from Fig. 1 show that GH mRNA was maximally induced within 6 h of CORT treatment and that this response was blocked by CHX. Table 5 provides a list of 46 transcripts that were regulated within 6 h in response to CORT in the absence of CHX. Of these, 11 were induced in the presence of CHX, indicating that they are likely direct targets of glucocorticoids. These direct early-induced transcripts are shown in boldface in Table 5. The identity of one of these transcripts is presently unknown. Effects of CORT in the absence and presence of CHX on mRNA levels for eight genes were confirmed by qRT-PCR (Fig. 5). Genes chosen for confirmation by qRT-PCR included those with the greatest responses to CORT: DEXRAS1, dexamethasone-induced ras-related 1; RASDVA, ras-dorsal ventral anterior; FKBP5, FK506-binding protein 5; LHFPL5, lipoma high mobility group protein I-C (HMGIC) fusion partner-like 5; LRRN3, leucine-rich repeat neuronal 3; ATP1B1, ATPase, Na⫹/K⫹ transporting, beta 1 polypeptide; NDRG1, N-myc downstream regulated gene 1; and pgp1c.pk002.f23, a cDNA with no known identity. CORT increased mRNA levels for all eight genes within 6 h in the presence of CHX. Physiol Genomics • doi:10.1152/physiolgenomics.00154.2012 • www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.32.247 on June 17, 2017 NRF2-mediated oxidative stress 430 GLUCOCORTICOID-REGULATED GENES IN THE EMBRYONIC PITUITARY Table 4. Transcription factors associated with target genes regulated by CORT, as determined by IPA Transcription Factor Regulation z-Score P Value of Overlap Target Genes, n 1.669 1.586 1.455 1.423 1.413 1.154 2.04E-02 3.12E-02 1.70E-02 3.80E-02 9.13E-03 6.34E-05 6 5 4 13 12 25 NR3C2 AR TP73 JUN HOXA10 NR3C1 0.742 0.631 0.475 0.442 0.429 0.405 8.61E-04 2.03E-03 1.82E-02 2.72E-02 2.34E-02 1.08E-06 6 10 7 10 7 26 HTT 0.219 3.33E-04 23 SKIL E2F1 SMARCB1 CTNNB1 MYCN STAT4 ESRRA CEBPB SATB1 TP53 0.100 0.100 0.069 0.054 ⫺0.059 ⫺0.193 ⫺0.471 ⫺0.519 ⫺0.615 ⫺0.745 2.23E-03 3.10E-02 1.93E-04 3.53E-02 1.54E-02 1.76E-02 2.62E-02 2.67E-03 2.26E-02 1.18E-04 10 4 10 13 8 8 5 12 5 31 HNF4A ⫺0.780 6.45E-03 45 XBP1 NOTCH1 ⫺1.415 ⫺1.753 1.61E-02 1.87E-01 7 4 BTG1,ERBB2,FKBP5,LTF,PDLIM1,STAT3 CYB5A,FABP5,KIF2C,PHF20,PLS3 COL4A1,COL6A2,DKK3,SFTPA1 BCL2L11,FN1,FSHB,GJA1,HPGDS,LTBP1,PCDHGC3,POMC,PRDX1,QKI,SELENBP1,SMG1,SNN AKR1A1,ESD,FKBP5,FN1,GSTA1,NRSN1,PRDX1,PREP,PSMB5,SOD1,TTR,TXNRD1 ACAT1,COL4A1,COX7A2L,CSRP2,DUSP1,ERBB2,FABP5,FN1,GJA1,GLYR1,HMOX2,HSPE1, MAN2A1,MBP,PFKFB1,PLS3,PREP,RB1,RPL35,SERBP1,SGK1,SNRPN,VLDLR,WRN,YBX1 FKBP4,FKBP5,FN1,HMOX2,RGS2,VLDLR C4B,CDT1,ERBB2,FABP5,FKBP5,GJA1,GLCCI1,PLS3,POMC,SULT1E1 AEN,BCL2L11,LTBP1,PPL,RB1,YBX1,ZMAT3 BCL2L11,BTG1,DUSP1,FSHB,GJA1,LTBP1,PRDX1,PTPN6,SGK1,XIAP BCL2L11,FN1,GJA1,LTF,P4HB,PIK3R1,ST6GAL1 ACAT1,BCKDHA,BCL2L11,BTG1,C4B,DUSP1,ELMOD3,FKBP4,FKBP5,FN1,GLCCI1,GRIN2C, IRF8,MTCH2,PHLDA2,PIK3R1,PIK3R3,POMC,PSMG2,RB1,RGS2,SAP30BP,SFTPA1,SGK1, STAT3,YWHAH B3GNT2,BCL2L11,CD74,COL4A1,DKK3,FAM173A,FKBP4,FN1,GRIN2C,HRAS,HSPE1,LTF,POR, RASD2,RGS4,SGK1,SLMAP,SNN,SOD1,SPTAN1,SYVN1,XIAP,YBX1 COL4A1,FSHB,KPNA1,SLC44A1 BCL2L11,CYB5A,DUSP1,FGFR2,HSPE1,PIK3R1,PRPSAP1,QKI,RACGAP1,RB1 ACAT1,ACTR2,BCL2L11,BTG1,C4B,CDT1,ERBB2,GJA1,PFKFB1,SLC37A4 COL4A1,CYB5A,DPEP1,FN1,GJA1,HNRNPUL1,HSPE1,HTRA1,IRF8,LMO2,PHLDA2,PLS3,SGK1 CDK9,COL4A1,DKK3,FGFR2,FN1,LRRN3,NACA,RPL35 BCL2L11,GJA1,PGP,PYGL,RNF128,RRAGD,SELENBP1,VLDLR CSRP2,CYB5A,LTF,MTCH2,TRA2B CDT1,CTSC,CYB5A,GLIPR2,GSTA1,HSPE1,MBP,NDRG4,POMC,RGS4,SGK1,VLDLR HLA-DMB,IRF8,LRRN3,PIK3IP1,SGK1 AEN,BCL2L11,BTG1,CARHSP1,CCDC80,CDT1,COL4A1,COL6A2,DUSP1,ERBB2,FAT1,FKBP5, FN1,HRAS,KSR1,LMO3,LTBP1,P4HB,PDLIM1,PIK3R1,PPIC,PPP1CA,PRNP,PRODH,PTPN6, RACGAP1,RB1,SGK1,TSPAN6,WSB2,ZMAT3 ACAT1,ACOT13,BCKDHA,BCS1L,BTG1,C21orf33,C4B,CCT8,CHCHD2,COX7A2L,COX7C,CROT, ELMOD3,FAM46A,FARSB,GMDS,GPX7,HEXA,HMOX2,HSPE1,KIAA1704,MRPL15,MRPL51, NDUFV1,NME7,OGDH,PGM1,PIK3R3,PPIP5K2,PPP1CA,PSMB5,PYGL,SAP30BP,SEMA7A, SGK1,SIX2,SLC37A4,SLC44A1,SUCLG1,THYN1,TTC26,TTR,TXNRD1,VPS29,YBX1 ATF6,GOLPH3,PHLDA2,SDF2L1,SOD1,SYVN1,TTR C21orf33,ERBB2,LYVE1,RB1 DISCUSSION Glucocorticoids have a wide array of effects in many tissues. We characterized effects of CORT on gene expression in chicken embryonic pituitary cells. Expression of 396 genes was affected by CORT treatment. Overall, mRNA levels for genes were increased and decreased in response to CORT in approximately equal numbers. This finding suggests a diverse response to glucocorticoids in embryonic pituitary cells. Approximately three times as many genes were induced or repressed within 3 h of CORT treatment as those that were first affected later. This is consistent with rapid effects of steroid hormones on gene expression. Furthermore, it could be taken to indicate that these early effects were due to a direct action of CORT. However, effects of CORT on expression levels for the majority of these genes were blocked by inhibition of protein synthesis with CHX. This would indicate that ongoing protein synthesis is required for glucocorticoid regulation of these genes and that the effects may, therefore, be indirect through another gene product. Consistent with this, we noted that induction or repression of the majority of genes that occurred at later time points was blocked by inclusion of CHX, indicating that these later effects of glucocorticoid treatment are secondary to an earlier effect. The results of our study are archived in the NCBI GEO database (accession number GSE5067, http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc⫽GSE5067) and provide a resource for investigators interested in the actions of glucocorticoids or in the regulation of pituitary function. GO and pathway analysis of 335 genes differentially expressed in response to CORT revealed both expected and unanticipated results. The top biological functions identified included cancer and cell death, indicating that glucocorticoids may affect pituitary cell abundance by regulating cell division and apoptosis, possibly within different cell populations. Other top biological functions identified included tissue development and morphology and embryonic and organism development, supporting a role for glucocorticoids in regulating pituitary development and cellular differentiation. Not surprisingly, the top canonical pathway affected by CORT treatment was glucocorticoid signaling. More surprising was the identification of ERK/MAPK signaling as a top canonical pathway affected by CORT. This might indicate that some of the actions of glucocorticoids within the embryonic pituitary gland are indirect and require ERK/MAPK signaling. Consistent with this possibility was the identification of a number of genes associated with ERK/MAPK signaling at key regulatory nodes in the gene networks and pathways identified by IPA. Among these were Ras, HRAS, RASD1, RASD2, and ERBB2. We reported previously that the Ras inhibitor manumycin blocked CORT induction of GH mRNA in embryonic pituitary cells (2). Recent results from our laboratory indicate involvement of ERK1/2 Physiol Genomics • doi:10.1152/physiolgenomics.00154.2012 • www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.32.247 on June 17, 2017 PGR KDM5B SPDEF FOS NFE2L2 MYC Target Molecules in Dataset GLUCOCORTICOID-REGULATED GENES IN THE EMBRYONIC PITUITARY 431 Table 5. Chicken pituitary transcripts regulated by CORT within 6 h of in vitro treatment cDNA Clone ID Gene Description Fold Increase BM492047 BM490725 BM490724 BI390179 BI393856 BM490189 BG712159 BI066724 BI393669 BM491452 BM426104 BI066929 BM491138 BM427377 AW355238 BM427001 BI393340 BM491321 BG712199 BG712150 BI067361 BM491267 BM427102 BM488046 pgp2n.pk003.j19 pgp2n.pk004.e24 pgp2n.pk004.e23 pgp1c.pk002.f23 pgp1n.pk012.k19 pgp2n.pk002.k5 pgl1n.pk011.j8 pgf1n.pk009.b16 pgp1n.pk012.a14 pgp2n.pk006.h10 pgf2n.pk001.l12 pgf1n.pk009.p5 pgp2n.pk005.i11 pgf2n.pk006.j13 pnfb.pk0003.b12 pgf2n.pk005.g16 pgp1n.pk010.o21 pgp2n.pk006.a23 pgl1n.pk011.g10 pgl1n.pk011.c4 pgf1n.pk011.n22 pgp2n.pk005.o1 pgf2n.pk005.l13 pgm2n.pk006.g13 pnl1 s.pk002.f9 pgm2n.pk007.k19 pgf1n.pk007.m14 pgf1n.pk010.k2 pgr1n.pk005.p23 pgl1n.pk014.n2 pgm2n.pk007.l19 pgf1n.pk011.b14 pgm2n.pk004.k19 pgf2n.pk002.j19 pgf1n.pk005.p1 pgr1n.pk001.i5 pgf1n.pk007.n4 pgr1n.pk002.j2 pgl1n.pk014.i14 pgl1n.pk014.j16 pgf1n.pk006.g21 pgm2n.pk008.j23 pgf1n.pk001.n7 pgf1n.pk007.o5 pgf1n.pk008.b20 pgf1n.pk007.m18 RAP2B, member of RAS oncogene family (RAS-DVA) scavenger receptor cysteine-rich type 1 protein FKBP5, FK506-binding protein 5; no hits found OGDHL, oxoglutarate dehydrogenase-like USP25,ubiquitin-specific peptidase 25 TXNDC16, thioredoxin domain containing 16 CCDC80, coiled-coil domain containing 80 dexamethasone-induced Ras-related protein 1 X-linked retinopathy protein protein AARS, alanyl-tRNA synthetase LHFPL5, lipoma HMGIC fusion partner-like 5 DDIT4 DNA-damage-inducible transcript 4 no hits found cytochrome c oxidase II epoxide hydrolase 3-like LRRN3, leucine-rich repeats neuronal protein 3 GMPPA, GDP-mannose pyrophosphorylase A complement C4 NDRG1, N-myc downstream regulated gene 1 ATPase, Naⴙ/Kⴙ transporting, beta 1 polypeptide DAT1 neuronal specific transcription factor branched chain keto acid dehydrogenase E1, alpha SMG1, phosphatidylinositol 3-kinase-related kinase no hits found PTCHD1, patched domain containing 1 FAM89A, family with sequence similarity 89, A no hits found no hits found PYGB, phosphorylase, glycogen; brain no hits found no hits found no hits found HISPPD, histidine acid phosphatase domain containing 1 no hits found no hits found no hits found no hits found MAF, v-maf musculoaponeurotic fibrosarcoma oncogene THBS3, thrombospondin 3 no hits found UBE2F, ubiquitin-conjugating enzyme E2F CTRC, chymotrypsin C no hits found MAK16, MAK16 homolog no hits found 2.37 2.11 1.62 1.60 1.28 1.26 1.20 1.17 1.15 1.04 1.02 1.00 0.97 0.95 0.93 0.92 0.89 0.85 0.85 0.84 0.77 0.74 0.74 0.69 0.55 0.27 0.21 0.15 0.14 0.10 0.05 0.05 0.02 0.01 0.00 ⫺0.01 ⫺0.01 ⫺0.02 ⫺0.03 ⫺0.04 ⫺0.05 ⫺0.06 ⫺0.07 ⫺0.09 ⫺0.17 ⫺0.20 BM488446 BI066326 BI067103 CD217894 BG712991 BM488465 BI067208 BM487474 BM426405 BI065808 BM439889 BM440251 BG712919 BG712932 BI065928 BM488716 BI064585 BI066375 BI066422 BI066329 GenBank accession number, cDNA clone name, gene name as assigned by the highest BLASTX score, and log2 fold increase in response to CORT (maximum difference relative to basal) in the absence of cycloheximide (CHX) are provided. Expression of genes in boldface was significantly affected by CORT in the presence of CHX (P ⬍ 0.05). signaling in glucocorticoid induction of GH (L. E. Ellestad and T. E. Porter, unpublished observations). One aim of the present study was to identify candidate genes that are rapidly induced by CORT treatment of chicken embryonic pituitary cells in the absence of ongoing protein synthesis. Inhibition of protein synthesis blocks glucocorticoid induction of GH mRNA in both embryonic chickens (2) and fetal rats (20), indicating that an intermediary protein may be required for glucocorticoid induction of GH expression. The microarray analysis performed in the present study measured mRNA levels for 14,053 genes. Of those, 396 showed a significant difference in expression levels between any two time points following CORT treatment. Of the 396 genes that we categorized, 46 were induced within 6 h, and of those 11 were induced in the presence of CHX. Two of the transcripts characterized are members of the Ras superfamily of signal transduction proteins. DEXRAS1 was originally identified in a murine corticotroph cell line (AtT-20) as a gene that was rapidly induced in response to glucocorticoid treatment (16). Dexras1 has been shown to have a variety of possible functions, such as suppression of G protein signaling downstream of ligand binding (13, 31). The other ras protein identified in our analysis was Ras-dva, which has been shown to be an essential component of the signaling network that patterns the early anterior neural plate and the adjacent ectoderm in Xenopus laevis embryos (32). DEXRAS1 and RASDVA are particularly intriguing as potential candidate genes for involvement in CORT regulation of GH mRNA, because we have previously shown that the ras inhibitor manumycin blocks CORT induction of GH mRNA (2). Another gene induced in the presence Physiol Genomics • doi:10.1152/physiolgenomics.00154.2012 • www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.32.247 on June 17, 2017 GenBank Acc. No. 432 GLUCOCORTICOID-REGULATED GENES IN THE EMBRYONIC PITUITARY of CHX was FKBP5. FKBP5 is an immunophilin that binds FK506 and has been shown to be involved in nuclear translocation of the glucocorticoid receptor (34) and to stimulate GH release from rat somatotrophs (22). Pituitary levels for FKBP5, RASDVA, and DEXRAS1 were also found to increase from e10 to e17 of chicken embryonic development (11), the period during which somatotrophs normally differentiate (25). This finding is also consistent with the rise in serum glucocorticoids that occurs in the chick embryo on e14 (15). Potential involvement of the other candidate genes is less clear. Mutations in LHFPL5 in mice cause deafness and vestibular dysfunction (spontaneous hurry-scurry phenotype) (18). LRRN3 encodes for a predicted protein with no known function. ATP1B1 encodes for the noncatalytic component of the Na/K ATPase that exchanges Na⫹ and K⫹ ions across the plasma membrane. NDRG1 encodes for N-myc downstream-regulated gene 1, which may function in growth arrest and cell differentiation (33). The other candidate cDNA that was induced within 6 h in the presence of CHX was pgp1c.pk002.f23, whose identity and function are unknown. A putative function for LHFPL5, LRRN3, ATP1B1, NDRG1, and pgp1c.pk002.f23 in mediating CORT induction of GH mRNA cannot be predicted. Future work will be required to determine the role if any for DEXRAS1, RASDVA, FKBP5, or the other candidate genes in CORT induction of GH production by embryonic pituitary cells. Potential approaches could involve the use of siRNA to knock down candidate genes and assess the effects on GH production. In summary, we have characterized effects of CORT on gene expression in chicken embryonic pituitary cells. Levels of mRNA for 396 genes were affected within 24 h by CORT treatment, and 11 of these were induced within 6 h in the presence of CHX. These 11 genes are of particular interest as candidate genes that may function to mediate effects of glucocorticoids on embryonic GH production. GRANTS This project was supported by National Research Initiative Competitive Grants 2003-035206-12836, 2006-35206-16617, and 2009-35206-05189 from the USDA National Institute of Food and Agriculture to T. E. Porter. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: S.A.J. and T.E.P. conception and design of research; S.A.J., L.E.E., M.M., J.N., and T.E.P. performed experiments; S.A.J., L.A.C., and T.E.P. analyzed data; S.A.J., L.A.C., and T.E.P. interpreted results of experiments; S.A.J., L.A.C., and T.E.P. prepared figures; S.A.J. and T.E.P. drafted manuscript; S.A.J., L.E.E., M.M., J.N., L.A.C., and T.E.P. approved Physiol Genomics • doi:10.1152/physiolgenomics.00154.2012 • www.physiolgenomics.org Downloaded from http://physiolgenomics.physiology.org/ by 10.220.32.247 on June 17, 2017 Fig. 5. Effects of CORT in the absence and presence of CHX on relative mRNA levels for selected genes induced within 6 h, in the presence and absence of CHX. Total RNA was analyzed by qRT-PCR, normalized to levels of ACTB mRNA, and are presented relative to levels under basal conditions in the absence and presence of CHX. *Means and SE are significantly different (P ⬍ 0.05, n ⫽ 4) from basal for that condition (absence or presence of CHX). DEXRAS1, dexamethasone-induced rasrelated 1; RASDVA, ras-dorsal ventral anterior; FKBP5, FK506-binding protein 5; LHFPL5, lipoma HMGIC fusion partner-like 5, tetraspan membrane protein of hair cell stereocilia homolog; LRRN3, leucine-rich repeat neuronal 3; ATP1B1, ATPase, Na⫹/K⫹ transporting, beta 1 polypeptide; NDRG1, N-myc downstream regulated gene 1; and pgp1c.pk002.f23 (no known identity). GLUCOCORTICOID-REGULATED GENES IN THE EMBRYONIC PITUITARY final version of manuscript; L.E.E., M.M., J.N., L.A.C., and T.E.P. edited and revised manuscript. REFERENCES 18. Longo-Guess CM, Gagnon LH, Cook SA, Wu J, Zheng QY, Johnson KR. 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