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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
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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.
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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
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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).
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Gene
GLUCOCORTICOID-REGULATED GENES IN THE EMBRYONIC PITUITARY
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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).
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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
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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,
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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
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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-
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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.
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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
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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
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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
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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.
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