Expression of glucocorticoid receptor alpha - and beta -isoforms in human cells and tissues

Laura Pujols1, Joaquim Mullol1,2, Jordi Roca-Ferrer1, Alfons Torrego1,3, Antoni Xaubet1,3, John A. Cidlowski4, and César Picado1,3

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Alternative splicing of the human glucocorticoid receptor (GR) primary transcript generates two protein isoforms: GR-alpha and GR-beta . We investigated the expression of both GR isoforms in healthy human cells and tissues. GR-alpha 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-beta mRNA was much less expressed than GR-alpha 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-beta mRNA was not found in colon. While GR-alpha protein was detected in all cells and tissues, GR-beta was not detected in any specimen. Our results suggest that, in physiological conditions, the default splicing pathway is the one leading to GR-alpha . The alternative splicing event leading to GR-beta is minimally activated.

reverse transcriptase-competitive polymerase chain reaction; Western blotting; healthy human tissues; inflammatory cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha and GR-beta , which originate from the same gene by alternative splicing of the GR primary transcript (16, 23, 33). GR-alpha 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-alpha and GR-beta isoforms. With the development of GR-alpha -specific antibodies, the expression of GR-alpha has been reported in different cell and tissue types (12, 24, 34). In the absence of ligand, GR-alpha resides primarily in the cytoplasm of cells and is held inactive by its binding to heat shock proteins. Upon hormone binding, GR-alpha 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-kappa B (3, 4, 11, 15, 25, 41).

Within the last few years, a number of studies have centered their attention on the GR-beta isoform. GR-beta differs from GR-alpha in its carboxy terminus, where the last 50 amino acids of GR-alpha are replaced by a nonhomologous 15-amino acid sequence. GR-beta does not either bind glucocorticoids or transactivate target genes (22, 23, 33). Transfection studies revealed the ability of GR-beta to act as a dominant negative inhibitor of GR-alpha activity (2, 32, 33) through a mechanism that involves the formation of transcriptionally impaired GR-alpha -GR-beta heterodimers (32). However, other investigators have challenged this concept (5, 14, 22). The expression of GR-beta , both at the mRNA and protein level, seems to be much lower than that of GR-alpha (10, 22, 24, 33, 35). Immunohistochemical studies have reported expression of GR-beta in specific cell types, mostly inflammatory cells (9, 20, 21, 28, 35, 47, 48). Although the physiological significance of GR-beta is still unknown, an overexpression of GR-beta 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-beta in a variety of cells, its relative abundance compared with GR-alpha is still a matter of controversy. Given the scarce information in the literature with regard to the expression of GR-alpha - and GR-beta -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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 at -70°C for RT-PCR and Western blotting analysis. PBMCs were isolated by Ficoll-Hypaque gradient centrifugation, and peripheral blood eosinophils were isolated as reported previously (53). Alveolar macrophages were obtained via fiberoptic bronchoscopy with bronchoalveolar lavage performed as described elsewhere (8). The purity of the cells was always >95%.

A549 cells, an epithelial cell line from human lung carcinoma, BEAS-2B cells, a human bronchial epithelial cell line, and COS-7 cells, a fibroblast-like cell line from kidney simian cells, were cultured as previously reported (31, 40). COS-7 cells were transfected with the GR-beta expression vector pCMVhGRbeta , which contains the GR-beta -specific coding sequences and the GR-beta 3'-untranslated region, using the calcium phosphate coprecipitation technique as previously described (33, 40).

The authorship institutional review board and ethics committee approved the study, and patients gave informed consent.

RT-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-alpha and GR-beta 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-alpha and GR-beta cDNAs were amplified using specific antisense primers that shared the same sense primer, whose sequences were as follows: 5'-GGCAATACCAGGTTTCAGGAACTTACA-3' (GR-alpha /beta sense), 5'-ATTTCACCATCTACTCTCCCATCACTG-3' (GR-alpha antisense), and 5'-ATTATCCAGCACTTCATAGACACAAAT-3' (GR-beta antisense), corresponding to nucleotide start positions 1869, 2692, and 2870, respectively. Twenty-eight PCR cycles were performed for GR-alpha and 38 cycles for GR-beta . 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-alpha and GR-beta 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-alpha -specific antibody (AShGR), and GR-beta -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-alpha and GR-beta proteins, respectively. Both the characterization and specificity of these antibodies have been studied extensively before (34, 35).

Statistical data analysis. Expression of GR-alpha or GR-beta mRNA is expressed as the arithmetic mean ± SE of 106 copies of GR-alpha cDNA or 103 copies of GR-beta 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of GR-alpha and GR-beta mRNAs in human cells and tissues. GR-alpha mRNA was expressed in all analyzed cells and tissues. The abundance of GR-alpha mRNA (×106 GR-alpha 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-alpha 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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Fig. 1.   RT-competitive PCR analysis of glucocorticoid receptor (GR)-alpha mRNA expression in human cells and tissues. After gel electrophoresis of the PCR products, the relative amounts of target cDNA to competitor were compared by densitometric analysis. A: GR-alpha mRNA expression in tissues. Inset: representative gel of the GR-alpha PCR for a tissue expressing high (brain), intermediate (kidney), or low (nasal mucosa) GR-alpha mRNA expression levels. GR-alpha -S and target cDNA refer to amplification of the GR-alpha internal standard (1,091 bp) and GR-alpha target cDNA (824 bp), respectively. *P < 0.05 compared with kidney, liver, heart, nasal mucosa, and colon, dagger P < 0.05 compared with nasal mucosa and colon, and #P < 0.05 compared with colon by Mann-Whitney U-test. B: GR-alpha mRNA expression in inflammatory cells. *P < 0.05 compared with eosinophils, peripheral blood mononuclear cells (PBMCs), and neutrophils by Mann-Whitney U-test.

GR-beta mRNA was detected in all cells and tissues except the colon (n = 4), although in concentrations at least 400 times lower than GR-alpha mRNA. The abundance of GR-beta mRNA (×103 GR-beta cDNA copies/µg total RNA) in tissues (Fig. 2A) was as follows: liver (0.94 ± 0.11; n = 3) >=  skeletal muscle (0.90 ± 0.25; n = 3) > kidney (0.74 ± 0.17; n = 4) > lung (0.44 ± 0.16; n = 5) > brain (0.19 ± 0.07; n = 4) >=  nasal mucosa (0.18 ± 0.04; n = 5) > heart (0.15 ± 0.08; n = 3). GR-beta mRNA abundance in inflammatory cells (Fig. 2B) was as follows: eosinophils (1.55 ± 0.58; n = 3) > PBMCs (1.36 ± 0.44; n = 4) > macrophages (0.67 ± 0.23; n = 3) > neutrophils (0.39 ± 0.11; n = 6).


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Fig. 2.   RT-competitive PCR analysis of GR-beta mRNA expression in human cells and tissues. After gel electrophoresis of the PCR products, the relative amounts of target cDNA to competitor were compared by densitometric analysis. A: GR-beta mRNA expression in tissues. Inset: representative gel of the GR-beta PCR for a tissue expressing high (liver), intermediate (lung), or low (heart) GR-beta mRNA expression levels. GR-beta -S and target cDNA refer to amplification of the GR-beta internal standard (1,118 bp) and GR-beta target cDNA (1,002 bp), respectively. *P < 0.05 compared with lung, brain, nasal mucosa, heart, and colon, dagger P < 0.05 compared with brain, nasal mucosa, heart, and colon, and #P < 0.05 compared with colon by Mann-Whitney U-test. B: GR-beta mRNA expression in inflammatory cells. *P < 0.05 compared with neutrophils by Mann-Whitney U-test.

Expression of GR-alpha and GR-beta proteins in human cells and tissues. In an attempt to quantify the relative abundance of GR-alpha and GR-beta proteins in human tissues, tissue protein extracts were subjected to electrophoresis until achieving separation between GR-alpha and GR-beta 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-beta in GR-beta -transfected COS-7 cells and in a protein mixture of GR-beta -transfected COS-7 cells and BEAS-2B cells (Fig. 3). GR-alpha , which migrated above GR-beta protein, was detected in BEAS-2B cells, a cell line previously reported to contain GR-alpha (40), and in all analyzed tissues. No GR-beta was detected in any of the tissues analyzed, i.e., no protein band comigrated with recombinant GR-beta .


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Fig. 3.   Relative expression of GR-alpha and GR-beta proteins in human tissues. Total cellular proteins from GR-beta -transfected COS-7 cells, BEAS-2B cells, and human tissues were isolated and analyzed by Western blotting with antibody 57, which is directed against an epitope common to both GR-alpha and GR-beta . COS-7 cells (15 µg) and BEAS-2B cells (30 µg) were loaded alone or as a mixture of both protein extracts (BEAS-2B + COS-7).

Because antibody 57 only detected GR-alpha in our tissues (Fig. 3), we sought to analyze the expression of GR-alpha in all of the specimens by immunoblotting with both antibody 57 and AShGR. Antibody 57 revealed expression of GR-alpha in all analyzed samples (Fig. 4A). GR-alpha expression, compared with that of 50 µg total proteins from A549 cells, was high in nasal mucosa, liver, lung, skeletal muscle, brain, kidney, and PBMCs and low in heart, colon, and neutrophils. GR-alpha was detected in neutrophils only after long film exposure, i.e., when GR-alpha signal in A549 cells was fully saturated.


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Fig. 4.   Characterization of GR-alpha protein in cells and tissues with the anti-GR antibody 57 (A) and the GR-alpha -specific antibody (AShGR; B). Total cellular proteins from A549, nasal mucosa (n = 4), kidney (n = 2), liver (n = 3), lung (n = 3), skeletal muscle (n = 2), brain (n = 3), heart (n = 2), colon (n = 2), PBMCs (n = 4), and neutrophils (n = 3) were isolated and analyzed by Western blotting with antibody 57 and AShGR.

The pattern of expression identified with AShGR (Fig. 4B) was similar to that detected with antibody 57 (Fig. 4A). Thus, with the exception of skeletal muscle, those cells and tissues expressing high GR-alpha content with antibody 57 also displayed strong band intensities for GR-alpha with AShGR. As for antibody 57, GR-alpha protein expression was low in heart and was not even detected in colon and neutrophils.

BShGR detected GR-beta in COS-7 cells transfected with pCMVhGRbeta (Fig. 5A). As previously reported (35), no GR-beta was detected in GR-beta -transfected COS-7 cells incubated with BShGR preabsorbed with the peptide antigen. No protein band was found to fully comigrate with recombinant GR-beta in any of the analyzed cells and tissues. BShGR identified one band above and another one below recombinant GR-beta in liver, kidney (Fig. 5B), nasal mucosa, and lung. Immunoreactivity for these two bands was still detected after incubation with BShGR preabsorbed with the peptide antigen, which suggests that these two bands account for nonspecific interactions between the antibody and proteins expressed in these tissues.


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Fig. 5.   Expression of GR-beta protein in cells and tissues. A: specificity of the GR-beta -specific antibody (BShGR) for the GR-beta protein. Total cellular proteins from COS-7 cells transfected with pCMVhGRbeta were isolated and analyzed by Western blotting with BShGR or BShGR preabsorbed with the peptide antigen. B: total cellular proteins from liver and kidney were isolated and analyzed by Western blotting with BShGR or BShGR preabsorbed with the peptide antigen.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha - and GR-beta -specific isoforms. A few years ago, both GR-alpha and GR-beta mRNAs (2, 33), as well as their protein products (12, 35), were detected in human tissues and cell lines. Since then, GR-beta expression has been reported in a number of cell types. However, its relative abundance compared with GR-alpha 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-alpha and GR-beta proteins by Western blotting using isoform-specific antibodies, as well as an antibody that recognizes both receptor isoforms.

GR-alpha mRNA was expressed to varying degrees in all analyzed cells and tissues. GR-alpha protein expression was characterized by immunoblotting with antibody 57, which recognizes both GR-alpha and GR-beta , 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-alpha in this tissue. One explanation for this discrepancy could be that posttranslational modifications of the GR-alpha protein taking place in skeletal muscle might lead to a GR-alpha variant that would not be efficiently recognized by the AShGR antibody.

The expression of GR-alpha protein in most cells and tissues matched up quite well with GR-alpha mRNA levels. One exception to this was found in nasal mucosa, where the relatively high GR-alpha 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-alpha , 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-alpha was found in brain, skeletal muscle, lung, liver, kidney, and PBMCs. The lowest GR-alpha 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-alpha 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-alpha 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-beta 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-alpha 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-alpha mRNA, as has already been pointed out by Oakley and coworkers (33). Thus the alternative splicing event leading to GR-beta 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-beta mRNA than the brain cortex. Conversely, GR-alpha mRNA expression levels in eosinophils and PBMCs were lower than in brain cortex.

The fact that we did not detect GR-beta 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-beta protein (18, 40) in various human cell types and tissues. In agreement with us, Gagliardo and coworkers (18) did not detect GR-beta in PBMCs, and Honda and coworkers (24) only detected GR-beta in PBMCs from certain patients with ulcerative colitis. In contrast, as revealed by immunohistochemistry, GR-beta has been reported in specific cell types, mostly inflammatory cells (9, 20, 21, 28, 35, 47, 48). However, we have not found GR-beta protein in inflammatory cells claimed to contain GR-beta , such as PBMCs (20, 28) and neutrophils (48).

Although positive immunoreactivity for GR-beta has been reported in a variety of cells, there is still a lot of controversy concerning the relative abundance of GR-beta compared with GR-alpha protein. For instance, Strickland and coworkers (48) have recently reported high constitutive expression of GR-beta in neutrophils from healthy individuals. The authors also reported higher expression of GR-beta than GR-alpha in these cells. Our findings do not agree with the results of Strickland et al. Thus, although we report low expression of GR-alpha mRNA and protein in neutrophils, the expression of the GR-beta transcript was still much lower than that of GR-alpha , and GR-beta 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-beta than GR-alpha in these cells, whereas two different groups (22, 52), using an antibody that recognized both GR isoforms, demonstrated that GR-alpha was more abundant than GR-beta . 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-alpha -to-GR-beta ratio of the cell with accuracy because the antibodies may have different affinities to the epitopes. In addition, absolute quantification of GR-alpha and GR-beta 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-alpha and GR-beta 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-alpha and GR-beta proteins in our tissues by immunoblotting with antibody 57. Only GR-alpha was detected in all samples. As with BShGR antibody, no GR-beta 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-beta protein, our results demonstrate that GR-alpha is clearly predominant over GR-beta in all the cells and tissues analyzed so far.

The possible physiological role of GR-beta is currently a matter of debate. In cotransfection studies, it has been shown that, when GR-beta is more abundant than GR-alpha , GR-beta acts as a dominant negative inhibitor of GR-alpha activity (2, 33) through a mechanism that mostly involves the formation of transcriptionally impaired GR-alpha -GR-beta heterodimers (32). However, other investigators (5, 14, 22) found no evidence for a specific dominant negative effect of GR-beta on GR-alpha activity. It has been argued that the ability of GR-beta to regulate GR-alpha activity in vivo would depend on its expression level relative to that of GR-alpha and the strength of its association with heat shock protein (hsp) 90 (32). With reference to the latter, GR-beta , as well as GR-alpha , binds to hsp90, but GR-beta -hsp90 complexes are less stable than those of GR-alpha -hsp90 (32). An overexpression of GR-beta in pathological conditions, together with a GR-beta -hsp90 unstable binding, might increase the dimerization of GR-beta with GR-alpha and therefore inhibit GR-alpha activity. Increased expression of GR-beta 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-beta , compared with GR-alpha , reported herein suggest that, at least in physiological conditions, GR-beta is not expressed at levels sufficient to inhibit GR-alpha function. Nevertheless, further studies analyzing the relative amounts of GR-alpha and GR-beta proteins, particularly in those cell types claimed to contain high levels of GR-beta (9, 20, 35, 47, 48), are needed.

In summary, we report the mRNA and protein expression of GR-alpha - and GR-beta -specific isoforms in a variety of human inflammatory cells and tissues. The expression of GR-alpha mRNA was at least 400 times in excess over GR-beta mRNA expression. Characterization of GR-alpha and GR-beta proteins by using isoform-specific antibodies and an antibody that recognizes both receptor isoforms revealed that GR-alpha was expressed to varying degrees in all cells and tissues, whereas GR-beta protein was not detected in any specimen. Our results suggest that the alternative splicing event leading to GR-beta is minimally activated in most cells and tissues. Because of the low expression of GR-beta , compared with GR-alpha , GR-beta is unlikely to have any inhibitory effect on GR-alpha function.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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