1 Institut d'Investigacions Biomèdiques August Pi i Sunyer, 2 Servei d'Otorinolaringologia, and 3 Servei de Pneumologia i Al.lèrgia Respiratòria-Institut Clínic de Pneumologia i Cirurgia Toràcica, Hospital Clínic, Departament de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain; and 4 Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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Alternative splicing of the human
glucocorticoid receptor (GR) primary transcript generates two protein
isoforms: GR- and GR-
. We investigated the expression of both GR
isoforms in healthy human cells and tissues. GR-
mRNA abundance
(×106 cDNA copies/µg total RNA) was as follows: brain
(3.83 ± 0.80) > skeletal muscle > macrophages > lung > kidney > liver > heart > eosinophils > peripheral blood mononuclear cells (PBMCs) > nasal mucosa > neutrophils > colon (0.33 ± 0.04).
GR-
mRNA was much less expressed than GR-
mRNA. Its abundance
(×103 cDNA copies/µg total RNA) was as follows:
eosinophils (1.55 ± 0.58) > PBMCs > liver
skeletal muscle > kidney > macrophages > lung > neutrophils > brain
nasal mucosa > heart (0.15 ± 0.08). GR-
mRNA was not found in colon. While GR-
protein was
detected in all cells and tissues, GR-
was not detected in any
specimen. Our results suggest that, in physiological conditions, the
default splicing pathway is the one leading to GR-
. The alternative
splicing event leading to GR-
is minimally activated.
reverse transcriptase-competitive polymerase chain reaction; Western blotting; healthy human tissues; inflammatory cells
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INTRODUCTION |
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GLUCOCORTICOIDS
MODULATE a large number of metabolic, cardiovascular, immune, and
behavioral functions. The biological action of glucocorticoids is
mediated through the activation of intracellular glucocorticoid
receptors (GR). The GR belongs to the superfamily of
steroid/thyroid/retinoid acid receptor proteins that function as
ligand-dependent transcription factors (3, 4, 17, 25). Two
human isoforms of GR have been identified, termed GR- and GR-
,
which originate from the same gene by alternative splicing of the GR
primary transcript (16, 23, 33). GR-
is the predominant isoform of the receptor and the one that shows steroid binding activity
(23). However, most of the studies analyzing GR expression did not distinguish between GR-
and GR-
isoforms. With the
development of GR-
-specific antibodies, the expression of GR-
has
been reported in different cell and tissue types (12, 24,
34). In the absence of ligand, GR-
resides primarily in the
cytoplasm of cells and is held inactive by its binding to heat shock
proteins. Upon hormone binding, GR-
is phosphorylated, dissociated
from heat shock proteins, and subsequently translocated to the cell nucleus, where it mediates either transactivation or transrepression of
target genes. The mechanisms for gene activation are mediated through
binding of a GR homodimer to specific glucocorticoid response elements
on the promoter region of target genes. The mechanisms for gene
repression, which account for most of the immunosuppressive and
anti-inflammatory responses of glucocorticoids, mostly involve protein-protein interactions between the GR and transcription factors,
such as activator protein-1 and nuclear factor-
B (3, 4, 11,
15, 25, 41).
Within the last few years, a number of studies have centered their
attention on the GR- isoform. GR-
differs from GR-
in its
carboxy terminus, where the last 50 amino acids of GR-
are replaced
by a nonhomologous 15-amino acid sequence. GR-
does not either bind
glucocorticoids or transactivate target genes (22, 23,
33). Transfection studies revealed the ability of GR-
to act
as a dominant negative inhibitor of GR-
activity (2, 32,
33) through a mechanism that involves the formation of
transcriptionally impaired GR-
-GR-
heterodimers
(32). However, other investigators have challenged this
concept (5, 14, 22). The expression of GR-
, both at the
mRNA and protein level, seems to be much lower than that of GR-
(10, 22, 24, 33, 35). Immunohistochemical studies have
reported expression of GR-
in specific cell types, mostly
inflammatory cells (9, 20, 21, 28, 35, 47, 48). Although
the physiological significance of GR-
is still unknown, an
overexpression of GR-
has been reported in glucocorticoid-resistant
diseases, such as asthma (20, 28, 47), ulcerative colitis
(24), chronic lymphocytic leukemia (45), and
nasal polyposis (21).
All of the factors triggering the glucocorticoid-induced cascade of events leading to the modulation of gene transcription influence the sensitivity to glucocorticoids. For instance, the GR binding affinity to either the hormone or the DNA, the interaction with cofactors and transcription factors, and the GR expression levels influence the sensitivity of the tissue to glucocorticoids (3). With reference to the latter, it is well established that GR cellular levels parallel the GR-mediated response (19) and that glucocorticoids themselves downregulate the expression of their own receptor through transcriptional, posttranscriptional, and posttranslational mechanisms (6, 7, 36, 40).
Although a number of studies report the expression of GR- in a
variety of cells, its relative abundance compared with GR-
is still
a matter of controversy. Given the scarce information in the literature
with regard to the expression of GR-
- and GR-
-specific isoforms
in humans tissues, we sought to determine the pattern of mRNA and
protein expression of both receptor isoforms in a variety of healthy
human inflammatory cells and tissues.
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MATERIALS AND METHODS |
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Materials.
Random hexanucleotide primers, SuperScript II RNase H
reverse transcriptase, and the RT-PCR buffers were obtained from Life Technologies (Barcelona, Spain). Dextran and Ficoll-Hypaque were purchased from Amersham Pharmacia (Barcelona, Spain), the PCR primers
and the protease inhibitor cocktail tablet were from Boehringer Mannheim (Barcelona, Spain), TRI-Reagent was from MRC (Cincinnati, OH),
and the RNAqueous-4PCR kit was from Ambion (Austin, TX).
Human cells and tissues. Human tissues from brain cortex, heart, lung parenchyma, kidney cortex, skeletal muscle, colonic and nasal mucosa, and liver from healthy individuals were kindly provided by different physicians and surgeons from our Institution (see acknowledgments) and used for RT-PCR and Western blotting studies. Total RNAs from brain, kidney, and liver were purchased from Ambion and ClonTech (Palo Alto, CA), and used for RT-PCR studies.
Peripheral blood mononuclear cells (PBMCs), neutrophils, eosinophils, and alveolar macrophages were obtained from healthy individuals. Peripheral blood neutrophils were isolated using a Ficoll-Hypaque gradient. Briefly, 40 ml of 2% (wt/vol) dextran were added to 40 ml of heparinized venous blood. The blood suspension was left for 45 min at room temperature to allow sedimentation of red blood cells. The supernatant was then layered over Ficoll-Hypaque and centrifuged at 400 g for 30 min. The resulting neutrophil pellet was washed with PBS, centrifuged, and stored atRT-competitive PCR.
Total RNA from the tissue specimens was isolated using a rapid
extraction method (TRI-Reagent), as described elsewhere
(40). Total RNA from inflammatory cells was isolated using
the RNAqueous-4PCR kit according to the manufacturer's instructions.
Total RNA (2-4 µg) was reverse transcribed to cDNA using random
hexanucleotide primers and SuperScript II RNase H reverse
transcriptase, following the manufacturer's recommendations. GR-
and GR-
mRNAs were measured by competitive PCR, in which known
amounts of an exogenous DNA (competitor or internal standard) were
coamplified in competition with the target cDNA in the same test tube
(40). GR-
and GR-
cDNAs were amplified using
specific antisense primers that shared the same sense primer, whose
sequences were as follows: 5'-GGCAATACCAGGTTTCAGGAACTTACA-3'
(GR-
/
sense), 5'-ATTTCACCATCTACTCTCCCATCACTG-3' (GR-
antisense), and 5'-ATTATCCAGCACTTCATAGACACAAAT-3' (GR-
antisense),
corresponding to nucleotide start positions 1869, 2692, and 2870, respectively. Twenty-eight PCR cycles were performed for GR-
and 38 cycles for GR-
. The RT-PCR reaction conditions have been described
extensively elsewhere (40). To ensure that the RNA was
effectively reverse transcribed to cDNA, the PCR for the housekeeping
gene glyceraldehyde-3-phosphate dehydrogenase was routinely performed
for each sample.
Western blotting.
Human tissues from brain cortex, heart, lung parenchyma, kidney
cortex, skeletal muscle, colonic mucosa, nasal mucosa, and liver, as
well as human neutrophils, PBMCs, A549, BEAS-2B, and COS-7 cells were
resuspended in lysate buffer containing one protease inhibitor cocktail
tablet (Complete), 50 mM HEPES buffer, 0.05% Triton X-100, 0.62 mM
phenylmethylsulfonyl fluoride, and 20 mM sodium molybdate. Total
proteins from tissues and cells were isolated as described elsewhere
(40) and were resolved by electrophoresis through 8%
SDS-polyacrylamide Tris-glycine gels. Protein electrophoresis (100 µg) was routinely performed on a Bio-Rad Mini-Protean II cell
(Hercules, CA). To achieve optimal separation between GR- and GR-
isoforms, proteins (200 µg) were electrophoresed on a Hoefer SE600
unit (San Francisco, CA). After electrophoresis, proteins were
transferred to nitrocellulose. To check for equal loading and transfer
efficiency, membranes were stained with Ponceau S (0.5% in 1% acetic
acid). The immunostaining was performed as described elsewhere
(40). Antibody 57, GR-
-specific antibody (AShGR), and
GR-
-specific antibody (BShGR) were used as polyclonal primary
antibodies. Antibody 57 is raised against epitopes common to both
receptor isoforms. AShGR and BShGR antibodies are raised against a
peptide corresponding to the 15 nonhomologous amino acids of the
carboxy-terminus of human GR-
and GR-
proteins, respectively.
Both the characterization and specificity of these antibodies have been
studied extensively before (34, 35).
Statistical data analysis.
Expression of GR- or GR-
mRNA is expressed as the arithmetic
mean ± SE of 106 copies of GR-
cDNA or
103 copies of GR-
cDNA per microgram of total RNA.
Statistical comparisons were performed using the nonparametric
Mann-Whitney U-test. P < 0.05 was regarded
as statistically significant.
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RESULTS |
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Expression of GR- and GR-
mRNAs in human cells and tissues.
GR-
mRNA was expressed in all analyzed cells and tissues. The
abundance of GR-
mRNA (×106 GR-
cDNA copies/µg
total RNA) in tissues (Fig.
1A) was as follows: brain
(3.83 ± 0.80; n = 4) > skeletal muscle
(3.11 ± 0.07; n = 3) > lung (2.16 ± 0.98; n = 5) > kidney (1.35 ± 0.32;
n = 4) > liver (0.99 ± 0.31;
n = 3) > heart (0.89 ± 0.51;
n = 3) > nasal mucosa (0.59 ± 0.15;
n = 5) > colon (0.33 ± 0.04;
n = 4). GR-
mRNA abundance in inflammatory cells
(Fig. 1B) was as follows: macrophages (2.29 ± 0.12;
n = 3) > eosinophils (0.83 ± 0.25;
n = 3) > PBMCs (0.74 ± 0.13;
n = 4) > neutrophils (0.53 ± 0.10; n = 6).
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Expression of GR- and GR-
proteins in human cells and
tissues.
In an attempt to quantify the relative abundance of GR-
and GR-
proteins in human tissues, tissue protein extracts were subjected to
electrophoresis until achieving separation between GR-
and GR-
bands. Proteins were then immunoblotted with the anti-GR antibody 57, which is raised against epitopes common to both receptor isoforms. This
antibody efficiently detected GR-
in GR-
-transfected COS-7 cells
and in a protein mixture of GR-
-transfected COS-7 cells and BEAS-2B
cells (Fig. 3). GR-
, which migrated
above GR-
protein, was detected in BEAS-2B cells, a cell line
previously reported to contain GR-
(40), and in all
analyzed tissues. No GR-
was detected in any of the tissues
analyzed, i.e., no protein band comigrated with recombinant GR-
.
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DISCUSSION |
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The multiple actions of glucocorticoids are mediated through
activation of a unique receptor. A number of studies have analyzed GR
expression in individual human tissues, using different approaches. However, the variations in the expression levels of the receptor in
different tissues has barely been investigated. In addition, the
earliest reports on the analysis of GR expression did not distinguish
between GR-- and GR-
-specific isoforms. A few years ago, both
GR-
and GR-
mRNAs (2, 33), as well as their protein products (12, 35), were detected in human tissues and cell lines. Since then, GR-
expression has been reported in a number of
cell types. However, its relative abundance compared with GR-
is as
yet unknown and is still a matter of controversy. In the present study,
we report the mRNA expression of both receptor isoforms in a variety of
human inflammatory cells and tissues and further quantify their mRNA
expression levels by means of RT-competitive PCR. In addition, we have
analyzed the expression of GR-
and GR-
proteins by Western
blotting using isoform-specific antibodies, as well as an antibody that
recognizes both receptor isoforms.
GR- mRNA was expressed to varying degrees in all analyzed cells and
tissues. GR-
protein expression was characterized by immunoblotting
with antibody 57, which recognizes both GR-
and GR-
, and AShGR.
It is of significant interest that, although AShGR and antibody 57 are
raised against different epitopes of the GR protein, both antibodies
displayed similar patterns of expression. The main discrepancy was
found in skeletal muscle. Thus, while antibody 57 detected relatively
high GR levels, AShGR antibody did not appear to detect much GR-
in
this tissue. One explanation for this discrepancy could be that
posttranslational modifications of the GR-
protein taking place in
skeletal muscle might lead to a GR-
variant that would not be
efficiently recognized by the AShGR antibody.
The expression of GR- protein in most cells and tissues matched up
quite well with GR-
mRNA levels. One exception to this was found in
nasal mucosa, where the relatively high GR-
protein levels, as
revealed by both AShGR and antibody 57, contrasted with the low
expression of its transcript. The actual mechanisms accounting for this
discrepancy are unknown. Compared with other tissues, the nasal mucosa
might have a differential regulation of GR gene expression, such as an
increased translational efficiency or a decreased protein degradation.
The fact that GR-, both message and protein, was detected in all
cells and tissues is consistent with the numerous and widespread physiological effects of glucocorticoids in humans (42).
Corticosteroids have effects in the brain on memory, the aging process,
the stress response, and the maintenance of homeostasis.
Glucocorticoids also influence the normal function of skeletal muscle,
stimulate liver gluconeogenesis, control the renal fluid and
electrolyte balance, affect the cardiovascular system, regulate lung
maturation, and have profound anti-inflammatory and immunosuppressive
effects. In keeping with their relevant physiological functions,
significant expression of GR-
was found in brain, skeletal muscle,
lung, liver, kidney, and PBMCs. The lowest GR-
expression was found in heart, colonic mucosa, and neutrophils. Several studies have reported high expression levels of non-isoform-specific GR in rat and
human brain (13, 38, 50, 51) as well as in human liver and
kidney (50, 51). Total GR has also been found in skeletal
muscle (29, 49), heart (26), nasal mucosa
(27), lung (1), colon (44),
neutrophils (30), PBMCs (18, 30), alveolar
macrophages (37), and eosinophils (39), but,
because of the use of different techniques, data obtained from these
studies are not always comparable. It is important to point out that
the low GR-
levels we have found in neutrophils concur with the
results published by Miller and coworkers (30) in which
the authors detected low expression of GR-
protein by both Western
blotting with antibody 57 and radioligand binding techniques. Both our findings and those of Miller are consistent with the low sensitivity of
neutrophils to glucocorticoids (43).
GR- mRNA was detected in all inflammatory cells, i.e., PBMCs,
eosinophils, macrophages, neutrophils, and tissues, except the colonic
mucosa. However, its concentration was at least 400 times lower than
the GR-
message. Our results are in line with RT-PCR and Northern
blot analysis performed on whole human tissues and cell lines
(10, 18, 24, 33, 40, 52) and suggest that the default
splicing pathway is the one leading to GR-
mRNA, as has already been
pointed out by Oakley and coworkers (33). Thus the
alternative splicing event leading to GR-
mRNA would be a minor
pathway. Alternative splicing is tightly regulated in a cell-type- or
developmental-stage-specific manner (46). We report
significant tissue-specific differences in the primary GR mRNA pattern
of splicing. For instance, eosinophils and PBMCs expressed higher
levels of GR-
mRNA than the brain cortex. Conversely, GR-
mRNA
expression levels in eosinophils and PBMCs were lower than in brain cortex.
The fact that we did not detect GR- protein, either with BShGR or
with antibody 57, in any of the examined cells and tissues is in line
with the low expression of its transcript. Similarly, using Western
blots, several researchers have detected little (22, 24,
35) or no GR-
protein (18, 40) in various human
cell types and tissues. In agreement with us, Gagliardo and coworkers
(18) did not detect GR-
in PBMCs, and Honda and coworkers (24) only detected GR-
in PBMCs from certain
patients with ulcerative colitis. In contrast, as revealed by
immunohistochemistry, GR-
has been reported in specific cell types,
mostly inflammatory cells (9, 20, 21, 28, 35, 47, 48).
However, we have not found GR-
protein in inflammatory cells claimed
to contain GR-
, such as PBMCs (20, 28) and neutrophils
(48).
Although positive immunoreactivity for GR- has been reported in a
variety of cells, there is still a lot of controversy concerning the
relative abundance of GR-
compared with GR-
protein. For instance, Strickland and coworkers (48) have recently
reported high constitutive expression of GR-
in neutrophils from
healthy individuals. The authors also reported higher expression of
GR-
than GR-
in these cells. Our findings do not agree with the
results of Strickland et al. Thus, although we report low expression of GR-
mRNA and protein in neutrophils, the expression of the GR-
transcript was still much lower than that of GR-
, and GR-
protein was not detected. A similar discrepancy has been reported in HeLa cells
as follows: de Castro and coworkers (12) reported five times more GR-
than GR-
in these cells, whereas two different groups (22, 52), using an antibody that recognized both GR isoforms, demonstrated that GR-
was more abundant than GR-
. In
our opinion, conflicting results can mainly be explained by the
different methodological approaches used. First, immunohistochemical studies based on the use of different antibodies for the detection of
each GR isoform do not reflect the GR-
-to-GR-
ratio of the cell
with accuracy because the antibodies may have different affinities to
the epitopes. In addition, absolute quantification of GR-
and GR-
proteins by Western blotting (12, 48) may not be technically accurate enough to determine the actual proportion of each
receptor isoform. Because of this, various investigators have pointed
out that the best way to compare the relative levels of GR-
and
GR-
proteins would be by using a single antibody that recognized a
common epitope in both isoforms of the receptor (22, 52).
In keeping with this, we attempted to quantify the relative expression
of GR-
and GR-
proteins in our tissues by immunoblotting with
antibody 57. Only GR-
was detected in all samples. As with BShGR
antibody, no GR-
protein was detected in any specimen. Although we
cannot ultimately rule out the possibility that our Western blotting
conditions were not sensitive enough to detect small amounts of GR-
protein, our results demonstrate that GR-
is clearly predominant
over GR-
in all the cells and tissues analyzed so far.
The possible physiological role of GR- is currently a matter of
debate. In cotransfection studies, it has been shown that, when GR-
is more abundant than GR-
, GR-
acts as a dominant negative
inhibitor of GR-
activity (2, 33) through a mechanism that mostly involves the formation of transcriptionally impaired GR-
-GR-
heterodimers (32). However, other
investigators (5, 14, 22) found no evidence for a specific
dominant negative effect of GR-
on GR-
activity. It has been
argued that the ability of GR-
to regulate GR-
activity in vivo
would depend on its expression level relative to that of GR-
and the
strength of its association with heat shock protein (hsp) 90 (32). With reference to the latter, GR-
, as well as
GR-
, binds to hsp90, but GR-
-hsp90 complexes are less stable than
those of GR-
-hsp90 (32). An overexpression of GR-
in
pathological conditions, together with a GR-
-hsp90 unstable binding,
might increase the dimerization of GR-
with GR-
and therefore
inhibit GR-
activity. Increased expression of GR-
has been
reported in patients with glucocorticoid-insensitive asthma (20,
28, 47), ulcerative colitis (24), chronic lymphocytic leukemia (45), and nasal polyposis
(21). The low levels of GR-
, compared with GR-
,
reported herein suggest that, at least in physiological conditions,
GR-
is not expressed at levels sufficient to inhibit GR-
function. Nevertheless, further studies analyzing the relative amounts
of GR-
and GR-
proteins, particularly in those cell types claimed
to contain high levels of GR-
(9, 20, 35, 47, 48), are needed.
In summary, we report the mRNA and protein expression of GR-- and
GR-
-specific isoforms in a variety of human inflammatory cells and
tissues. The expression of GR-
mRNA was at least 400 times in excess
over GR-
mRNA expression. Characterization of GR-
and GR-
proteins by using isoform-specific antibodies and an antibody that
recognizes both receptor isoforms revealed that GR-
was expressed to
varying degrees in all cells and tissues, whereas GR-
protein was
not detected in any specimen. Our results suggest that the alternative
splicing event leading to GR-
is minimally activated in most cells
and tissues. Because of the low expression of GR-
, compared with
GR-
, GR-
is unlikely to have any inhibitory effect on GR-
function.
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ACKNOWLEDGEMENTS |
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We thank all the people and doctors from our Institution for providing the following human tissues: brain cortex (Banc de Teixits Neurològics, Serveis Científico-Tècnics, Universitat de Barcelona), heart and skeletal muscle (Dr. J. M. Grau, Department of Internal Medicine), kidney (Drs. A. Alcaraz and R. Álvarez, Department of Urology), liver (Dr. J. Caballería, Department of Hepathology), and colon (Dr. J. Panés, Department of Gastroenterology).
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FOOTNOTES |
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This study was supported in part by Fondo de Investigaciones Sanitarias Grant 99-0133, a Sociedad Española de Neumología y Cirugía Torácica-Fundacíon Española de Patología Respiratoria grant, and Departament d'Universitats, Recerca i Societat de la Informacío Grant 2001SGR-384.
Address for reprint requests and other correspondence: C. Picado, Servei de Pneumologia. Hospital Clínic. Villarroel 170, 08036 Barcelona, Catalonia, Spain (E-mail: cpicado{at}medicina.ub.es).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 20, 2002;10.1152/ajpcell.00363.2001
Received 31 July 2001; accepted in final form 11 June 2002.
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