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Endocrinology Vol. 138, No. 11 5028-5038
Copyright © 1997 by The Endocrine Society

ARTICLES

Expression and Subcellular Distribution of the ß-Isoform of the Human Glucocorticoid Receptor1

Robert H. Oakley2, Jeffrey C. Webster, Madhabananda Sar, C. Richard Parker, Jr. and John A. Cidlowski

Laboratory of Signal Transduction (R.H.O., J.C.W., M.S., J.A.C.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; and the Department of Obstetrics and Gynecology, University of Alabama (C.R.P.), Birmingham, Alabama 35233

Address all correspondence and requests for reprints to: Dr. John A. Cidlowski, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, P.O. Box 12233, MD E2–02, Research Triangle Park, North Carolina 27709. E-mail: cidlowski{at}niehs.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two highly homologous protein isoforms, termed hGR{alpha} and hGRß, that differ at their carboxy-termini. In contrast to the well characterized hGR{alpha} isoform, which modulates gene expression in a hormone-dependent fashion, the biological significance of hGRß has only recently begun to emerge. We and others have shown that the hGRß messenger RNA transcript is widely expressed in human tissues and that the hGRß protein functions as a dominant negative inhibitor of hGR{alpha} in transfected cells. Unfortunately, these initial studies did not determine whether the hGRß protein was made in vivo. Such analyses are hindered because available anti-hGR antibodies cannot discriminate between the similarly sized hGR{alpha} and hGRß proteins. Therefore, to investigate the expression of the hGRß protein, we have produced an antipeptide, hGRß-specific antibody termed BShGR. This antibody was made against the unique 15-amino acid peptide at the carboxy-terminus of hGRß and recognizes both the native and denatured conformations of hGRß, but does not cross-react with hGR{alpha}. Using BShGR on Western blots and in immunoprecipitation experiments, we detected the hGRß protein in a variety of human cell lines and tissues. Immunocytochemistry was then performed with BShGR on HeLa S3 and CEM-C7 cells and on tissue sections prepared from lung, thymus, and liver to assess the cellular and subcellular distribution of hGRß. In all immunopositive cells, hGRß was found in the nucleus independent of glucocorticoid treatment. Within tissues, the hGRß protein was expressed most abundantly in the epithelial cells lining the terminal bronchiole of the lung, forming the outer layer of Hassall’s corpuscle in the thymus, and lining the bile duct in the liver. As a potential in vivo inhibitor of hGR{alpha} activity, expression of hGRß may be an important factor regulating target cell responsiveness to glucocorticoids.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GLUCOCORTICOID receptor (GR) belongs to the superfamily of steroid/thyroid/retinoic acid receptor proteins that function as ligand-dependent transcription factors (1, 2, 3). Like other members of this family, the GR is comprised of an amino-terminal trans-activation domain, a central DNA-binding domain, and a carboxy-terminus that contains the hormone-binding domain as well as sequences important for interacting with heat shock protein-90 (hsp90) (4), nuclear translocation (5), receptor dimerization (6), and trans-activation (7). In the absence of ligand, the GR resides in the cytoplasm of cells in a multiprotein complex consisting of the receptor polypeptide, two molecules of hsp90, and several additional proteins (8, 9). The receptor undergoes a change in conformation upon hormone binding, resulting in the dissociation of hsp90 and the other associated proteins. In its new conformation, the activated GR translocates into the nucleus, where it binds to glucocorticoid receptor-responsive elements (GREs) located in the promoter regions of target genes. The receptor then communicates with the basal transcription machinery and either positively or negatively regulates expression of the linked gene depending on the GRE sequence and promoter context. The activated GR can also modulate gene expression apart from DNA binding by physically interacting with other transcription factors, such as activating protein-1 and nuclear factor-{kappa}B (10, 11, 12, 13).

When the human GR (hGR) complementary DNA (cDNA) was cloned in 1985, two cDNA clones were isolated that predicted the existence of two highly homologous receptor isoforms differing only at their carboxy-termini (14). These two isoforms, termed hGR{alpha} and hGRß, are identical through amino acid 727, but then diverge, with hGR{alpha} having an additional 50 amino acids and hGRß having an additional, nonhomologous 15 amino acids. Alternative splicing in exon 9 of the hGR gene was later shown to be responsible for generating the two receptor isoforms (15). Over the last decade, investigators have focused their attention on hGR{alpha}, which, as described above, functions as a ligand-dependent transcription factor. In contrast, the hGRß isoform has been largely ignored because early studies reported that the recombinant hGRß protein did not bind hormone and did not activate glucocorticoid-responsive promoters (14, 16).

Two recent findings have sparked new interest into the role hGRß may play in steroid receptor physiology. First, the hGRß messenger RNA (mRNA) transcript, like hGR{alpha}, has a widespread tissue distribution (15, 17). Second, the recombinant hGRß protein inhibits hGR{alpha}-mediated trans-activation of target genes in a dose-dependent manner (15, 17). The mechanism responsible for this inhibition has not been elucidated, but may involve competition between hGR{alpha} and hGRß for GRE binding, formation of hGR{alpha}/hGRß heterodimers that are transcriptionally inactive, and/or titration of a coactivator needed by hGR{alpha} for full transcriptional activity. The ability of hGRß to antagonize the function of hGR{alpha} suggests that hGRß may be a critical factor regulating target cell responsiveness to glucocorticoids. High levels of hGRß would confer glucocorticoid resistance and low levels of hGRß would confer glucocorticoid hypersensitivity.

Although the hGRß mRNA transcript has been detected in many tissues, it is still not known whether this message is actually translated into endogenous hGRß protein. Resolving this fundamental issue is critical to further understanding the role hGRß plays in vivo. Investigating hGRß protein expression is problematic because no ligand has been identified for hGRß and all anti-hGR antibodies currently in use recognize both 94-kDa hGR{alpha} and 90-kDa hGRß isoforms. This cross-reactivity is undesirable because of the small difference in size between the two receptor isoforms and the potential for hGR{alpha} to be posttranslationally modified or degraded into a smaller 90-kDa protein. In the present study we describe the production of an hGRß-specific antibody termed BShGR. Using this antibody, we investigate the tissue, cellular, and subcellular distribution of the hGRß protein.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Protease inhibitors were supplied by Boehringer Mannheim (Indianapolis, IN). Dexamethasone was supplied by Steraloids (Wilton, NH). Tran35S-Label (1108 Ci/mmol) was purchased from ICN Radiochemicals (Irvine, CA). The peroxidase-labeled goat antirabbit secondary antibody and chemiluminescent detection reagents were obtained from Amersham (Arlington Heights, IL). Biotinylated goat antirabbit IgG was obtained from Vector Laboratories (Burlingame, CA), and the fluorescein-conjugated goat antirabbit IgG was purchased from Molecular Probes (Eugene, OR). Vinylsulfone-activated Sepharose, protein A-Sepharose CL-4B, and other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell culture and transfections
HeLa S3, CEM-C7, and normal lung epithelial cells (provided by Dr. Paul Nettesheim, NIEHS) were grown as described previously (15, 18). COS-1, HEK-293, JAR, and Hep G2 cells were grown in DMEM supplemented with 2 mM glutamine and 10% (vol/vol) of a mixture (1:1) of heat-inactivated FCS and calf serum. All cultures were maintained in a 5% CO2 humidified atmosphere at 37 C and passaged every 3–4 days. For transfection of COS-1 cells, medium was removed from subconfluent cells (1 x 106) and replaced with fresh DMEM containing 3% serum. Equimolar amounts of pCMVhGR{alpha} (20 µg), pCMVhGRß (18.2 µg), or pCMV5 (10 µg) were prepared as a calcium phosphate precipitate, with the total amount of DNA in each transfection cocktail adjusted to 20 µg with salmon sperm DNA. The precipitates were then added to the cells, and after a 5-h incubation, the medium was removed, and the cells were shocked for 30 s with 15% glycerol and then refed with fully supplemented DMEM. Construction of pCMVhGR{alpha}, pCMVhGRß, and the human cytomegalovirus major intermediate early gene promoter expression vector backbone pCMV5 have been described previously (15).

Production and purification of the hGRß-specific antibody BShGR
A peptide corresponding to amino acids 728–742 at the carboxy-terminus of the hGRß protein was synthesized and purified by the Micro Protein Chemistry Facility at the University of North Carolina (Chapel Hill, NC). An amino-terminal cysteine was added to the peptide as a linker, and an aliquot of the peptide was conjugated to keyhole limpet hemocyanin to enhance antigenicity. A female New Zealand White rabbit was then immunized with the hGRß-specific peptide as a mixture (1:1) of free peptide and keyhole limpet hemocyanin-conjugated peptide in Freund’s complete adjuvant using a combination of intradermal and sc injections. Subsequently, the rabbit was boosted monthly with a mixture (1:1) of the free peptide and conjugated peptide in incomplete Freund’s adjuvant. Serum was collected 2 weeks after each boost and analyzed for hGRß-specific antibodies. Epitope-purified antiserum was obtained by linking the synthetic peptide to vinylsulfone-activated Sepharose and subjecting the serum to affinity chromatography as described previously (19).

Western blotting
Confluent cells (COS-1, HeLa S3, CEM-C7, HEK-293, JAR, Hep G2, and secondary lung epithelial cells) were harvested and resuspended in buffer containing 10 mM Tris-HCl (pH 8.3), 1 mM EDTA, and a mixture of protease inhibitors (0.1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1 µM pepstatin, and 1 µM leupeptin). Cells were then homogenized by four 10-sec bursts on ice using a Tekmar Ultra Turrax homogenizer (Tekmar Co., Cincinnati, OH), and the homogenate was immediately centrifuged at 165,000 x g in a Beckman 50Ti rotor (Palo Alto, CA) for 1 h at 4 C. Supernatants were collected, mixed with an equal volume of Fairbanks solution [2% SDS, 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 10% sucrose, and 20 µg/ml pyronin Y], boiled for 5 min, and stored at -70 C. Proteins (75 µg for COS-1, 150 µg for other cell types) were resolved by electrophoresis through 7.5% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose in Tris-glycine buffer [25 mM Tris-HCl (pH 8.3), 150 mM glycine, and 15% methanol] (20, 21). Membranes were stained with Ponceau S (0.5% in 1% acetic acid) (22) to evaluate loading equivalency and transfer efficiency and blocked overnight at 4 C in Tris-buffered saline [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween-20] containing 10% nonfat dry milk. The blot was then washed in Tris-buffered saline containing 1% nonfat dry milk (TBS-1%) and incubated for 1 h at room temperature with the appropriate dilution of primary antibody in TBS-1% [1:1,000 BShGR for transfected COS-1 cells, 1:500 BShGR for HeLa S3 and CEM-C7 cells, 1:1,000 for antipeptide hGR antibody 59 (23), 1:1,000 for antipeptide hGR antibody 57 (23)]. Subsequently, membranes were washed in TBS-1%, reacted for 1 h at room temperature with a horseradish peroxidase-labeled goat antirabbit secondary antibody (1:15,000 in TBS-1%), washed in TBS-1%, reacted with chemiluminescent reagents, and then processed for autoradiography. The human multiple tissue Western blot (Clontech, Palo Alto, CA) contained 75 µg protein isolated from different human tissues and was processed as described above. Whole cell lysates (40 µg) from various lung, breast, endometrial, bladder, ovary, and melanoma cell lines were kindly provided by Drs. Alicia White and J. Carl Barrett (NIEHS) and analyzed as described above. Blots were stripped by incubating at 55 C for 45 min in 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol.

In vitro transcription and translation
The hGRß cDNA (~3.8 kb) was excised from the expression vector pCMVhGRß at the KpnI-BamHI sites and cloned into the KpnI-BamHI sites in the dual promoter vector pT7/T3–18 (Life Technologies, Gaithersburg, MD) to generate pT7/T3-hGRß. Construction of pT7/T3-hGR{alpha} has been previously described (24). These two plasmids were added to the TNT Coupled Reticulocyte Lysate Translation System (Promega Corp., Madison, WI) for in vitro synthesis of the hGR{alpha} and hGRß proteins. The transcription/translation reactions contained 1.0 µg pT7/T3-hGR{alpha} or pT7/T3-hGRß, 25 µl rabbit reticulocyte lysate, 20 µM methionine-free amino acids, 4 µl Tran35S-Label (11.45 mCi/ml), 1 µl RNasin ribonuclease inhibitor, 1 µl T7 RNA polymerase, and 2 µl of the buffer provided by the supplier in a final volume of 50 µl. After a 90-min incubation at 30 C, the translation reactions were frozen and stored at -70 C until used for immunoprecipitation assays.

Immunoprecipitations
HeLa S3, CEM-C7, and transfected COS-1 cells were harvested in PBS and lysed using a Dounce homogenizer (Kontes Co., Vineland, NJ) in TENT buffer [20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 150 mM NaCl, and 0.5% Triton X-100 containing protease inhibitors (0.1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1 µM pepstatin, and 1 µM leupeptin]. After the homogenate was centrifuged at 13,000 x g for 5 min at 4 C, the supernatant was collected, and the protein concentration was determined by the method of Bradford (25) using the Bio-Rad (Richmond, CA) protein assay. Proteins (400 µg for HeLa S3 and CEM-C7 cells, 250 µg for transfected COS-1 cells) in a final volume of 200 µl TENT buffer were precleared with 2.5 µl preimmune serum for 15 min at 4 C and then with 40 µl protein A-Sepharose beads (133 mg/ml TENT buffer) for 1 h at 4 C. After centrifugation, the supernatant was removed and transferred to a new tube. The appropriate antibody at a 1:100 dilution was added, and each tube was incubated for 2.5 h at 4 C with rotation. For binding immune complexes, 80 µl protein A-Sepharose beads were added, and the incubation was continued for an additional 30 min at 4 C with rotation. The protein A-Sepharose-immune complexes were washed three times with 500 µl TENT buffer and then resuspended in a buffer containing 10% glycerol, 2% SDS, 0.2 mg/ml bromophenol blue, 62.5 mM Tris-HCl (pH 6.8), and 5% 2-mercaptoethanol. Immunoprecipitated protein was eluted from the protein A-Sepharose by boiling for 5 min and resolved by the method of Laemmli on 8.5% SDS-polyacrylamide gels (26). After electrophoretically transferring the proteins to nitrocellulose, immunoblotting was carried out as described above using the antipeptide hGR antibody 57. For immunoprecipitation of in vitro translation products, 5.0 µl of each translation reaction were added to 200 µl TENT buffer and processed as described above. Gels were fixed in 30% methanol-10% acetic acid for 15 min and processed for autoradiography using liquid EN3HANCE (DuPont-NEN, Boston, MA).

Immunocytochemistry
HeLa S3 and transfected COS-1 cells were plated in two-chamber glass slides (~1 x 105 cells/chamber slide) and cultured for 24 h in medium containing serum stripped of endogenous glucocorticoids by treatment with dextran-coated charcoal. After a 2-h treatment with or without dexamethasone (1 x 10-7 M, final concentration), slides were washed twice in PBS (pH 7.4), fixed for 10 min at room temperature with 2% paraformaldehyde (pH 7.2), washed again in PBS, and then permeabilized in 0.2% Triton X-100 for 20 min. After an additional wash in PBS, the cells were incubated for 20 min in 0.2% normal goat serum followed by an overnight incubation at 4 C with BShGR diluted in PBS (1:8000 for transfected COS-1 cells, 1:1500 for HeLa S3 cells). Subsequently, the cells were treated for 30 min with biotinylated goat antirabbit IgG at a dilution of 1:200. Immunoreactivity was then visualized by adding avidin-biotin peroxidase (1:200) for 30 min followed by a 10-min reaction with diaminobenzidine-hydrogen peroxide solution.

Surgical sections of adult human tissues, provided by the University of Alabama-Birmingham Tissue Procurement Service, were fixed in buffered formalin and then embedded in paraffin. After deparaffinization, 5-µm tissue sections mounted on Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA) were treated with 3% aqueous hydrogen peroxide to block endogenous peroxidase activity. The sections were treated with 1% swine serum as a blocking reagent and then incubated with a 1:500 dilution of BShGR at 37 C for 30 min. The BioGenix Universal Rabbit kit (BioGenix Laboratories, San Ramon, CA) (biotin/avidin horseradish peroxidase) was used for detection of the primary antibody with diaminobenzidine as chromagen according to the manufacturer’s instructions. Control sections for detecting possible nonspecific immunostaining were incubated as described above, except the primary antiserum was replaced with normal rabbit serum. All sections were lightly counterstained with methyl green.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production and characterization of an hGRß-specific antibody
The 94-kDa hGR{alpha} and 90-kDa hGRß proteins are identical through amino acid 727, but then diverge, with hGR{alpha} having an additional 50 amino acids and hGRß having an additional nonhomologous 15 amino acids (Fig. 1AGo). To produce an antibody specific for the hGRß isoform, we synthesized a peptide corresponding to the unique 15 amino acids at the carboxy-terminus of the hGRß protein (Fig. 1BGo). A rabbit was then immunized with the hGRß-specific peptide, and after multiple immunizations, serum was collected and epitope purified as described in Materials and Methods. To test the specificity of the epitope-purified antibody (referred to as BShGR for ß-specific hGR antibody), we initially transfected receptor-negative COS-1 cells with an hGR{alpha} expression vector (pCMVhGR{alpha}) or an hGRß expression vector (pCMVhGRß). Whole cell extracts were then prepared and analyzed by Western blotting. As shown in Fig. 2AGo, BShGR specifically detected the 90-kDa hGRß protein in the hGRß-expressing cells. This immunoreactivity was not observed with preimmune serum (data not shown) or with BShGR preabsorbed with the peptide antigen. Moreover, BShGR did not cross-react with hGR{alpha}. The antipeptide hGR antibodies 59 and 57, which were made against epitopes common to both receptor isoforms (see Fig. 1AGo) (23), recognize both proteins and demonstrate that hGR{alpha} and hGRß were expressed at similar levels.



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  		Figure 1. Schematic representation of the hGR{alpha} and hGRß protein isoforms (A) and the hGRß-specific peptide antigen (B). A, The hGR{alpha} and hGRß protein isoforms are identical through amino acid 727, but then diverge. The functional domains ({tau}1 and {tau}2, trans-activation domains; DBD, DNA-binding domain; HBD, hormone-binding domain) and putative site of hsp90 interaction are indicated for each isoform. The locations of the peptide epitopes for antipeptide hGR antibodies 59 (amino acids 245–259) and 57 (amino acids 346–367) are also shown (23). B, Sequence of the 15-amino acid peptide (corresponding to amino acids 728–742 at the carboxy-terminus of hGRß) used to produce the hGRß-specific antibody BShGR.

 


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  		Figure 2. Specificity of the BShGR antibody for the hGRß protein. A, Whole cell extracts were prepared from COS-1 cells transfected with equimolar amounts of pCMVhGR{alpha} or pCMVhGRß. Denatured proteins were separated on 7.5% SDS-polyacrylamide gels, transferred to nitrocellulose, and analyzed by Western blotting. Blots processed in parallel were incubated with BShGR, BShGR preabsorbed with the peptide antigen, antibody 59, or antibody 57. B, Reticulocyte lysates programed with pT7/T3-hGRß, pT7/T3-hGR{alpha}, or pT7/T3–18 (mock) were translated as described in Materials and Methods. Radiolabeled proteins were immunoprecipitated with preimmune serum, antibody 59, BShGR, or BShGR preabsorbed with the peptide antigen. Immunoprecipitated proteins were then separated on 8.5% SDS-polyacrylamide gels and processed for autoradiography. Molecular mass standards are indicated in the left margin.

 
To demonstrate further the specificity of the BShGR antibody, the hGRß and hGR{alpha} proteins were translated in vitro in the presence of radiolabeled methionine. Immunoprecipitations were then performed on the labeled proteins using preimmune serum, antibody 59, BShGR, or BShGR preabsorbed with the peptide antigen (Fig. 2BGo). BShGR only immunoprecipitated the hGRß protein (lane 3); it did not immunoprecipitate hGR{alpha} (lane 7). In contrast, antibody 59 immunoprecipitated both receptor isoforms (lanes 2 and 6). Thus, the Western blot and immunoprecipitation experiments clearly show that BShGR is specific for the hGRß protein. These experiments also reveal the major problem associated with using an antibody that recognizes both receptor isoforms for studying expression of the endogenous hGRß protein. In COS-1 cells and reticulocyte lysates expressing only the 94-kDa hGR{alpha} protein, antibodies 59 and 57 also detected a low abundant 90-kDa protein (Fig. 2Go, A and B). This protein appears to be an hGR{alpha} degradation product but comigrates with hGRß. Therefore, in cells expressing both hGR{alpha} and hGRß, nonspecific hGR antibodies (such as 59 and 57) cannot distinguish the 90-kDa hGRß protein from a 90-kDa hGR{alpha} degradation product.

Expression of the endogenous hGRß protein
The hGRß mRNA transcript is expressed in many tissues (15, 17); however, it is not known whether this message is actually translated into endogenous hGRß protein. With the hGRß-specific antibody BShGR, we now had the reagent to definitively answer this question of fundamental importance. Initially, we performed Western blots on proteins isolated from HeLa S3 cells (a human cervical carcinoma cell line) and CEM-C7 cells (a human leukemia cell line). These cell lines were chosen because both express the hGRß mRNA transcript (15). Blots processed in parallel were probed with BShGR, BShGR preabsorbed with the peptide antigen, or antibody 57 (Fig. 3Go, A and B). In both cell lines, the hGRß-specific antibody detected a 90-kDa protein that was not observed with preimmune serum (data not shown) or with BShGR preabsorbed with the peptide antigen. The size of the protein and the specificity of the BShGR interaction suggested that this protein was hGRß.



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  		Figure 3. Expression of the hGRß protein in HeLa S3 and CEM-C7 cells. Whole cell extracts were prepared from confluent cells, and denatured proteins were separated on 6.0% SDS-polyacrylamide gels, transferred to nitrocellulose, and analyzed by Western blotting. Blots processed in parallel were incubated with BShGR, BShGR preabsorbed with the peptide antigen, or antibody 57. Molecular mass standards are indicated in the left margin.

 
Antibody 57 detected not only the 94-kDa hGR{alpha} protein, but also a 90-kDa protein that comigrated with the protein recognized by BShGR (Fig. 3Go, A and B). Whether this protein is hGRß or a hGR{alpha} degradation product cannot be determined because antibody 57 cross-reacts with both receptor isoforms (Figs. 1AGo and 2AGo). Regardless of its identity, this 90-kDa protein is less abundant than hGR{alpha}. If one assumes that antibody 57 has the same affinity for the denatured hGR{alpha} and hGRß proteins, this result suggests that hGR{alpha} is more abundant than hGRß in HeLa S3 and CEM-C7 cells. Consistent with these results, we have shown previously that the hGR{alpha} mRNA transcripts are more abundant than the hGRß message in these two cell lines (15).

Although the BShGR antibody specifically detected a 90-kDa protein on the Western blot in Fig. 3Go, we sought further proof that hGRß was expressed in these cells. Our strategy was to analyze proteins immunoprecipitated by BShGR on Western blots using antibody 57. If hGRß is present, antibody 57 should detect a 90-kDa protein in the BShGR immunoprecipitates. In this way, hGRß can be double probed with two different antibodies recognizing epitopes in different regions of the protein (see Fig. 1AGo). The feasibility of this approach was first tested in COS-1 cells transfected with pCMVhGR{alpha}, pCMVhGRß, or pCMV5 (mock). Immunoprecipitations were performed on whole cell extracts with preimmune serum, antibody 59, BShGR, or BShGR preincubated with the peptide antigen (Fig. 4AGo). Antibody 57 successfully detected the 90-kDa hGRß protein in the BShGR immunoprecipitates from the hGRß-transfected cells (compare lanes 3, 7, and 9). This reactivity was not observed with preimmune serum (lane 1) or with BShGR preincubated with the peptide antigen (lane 4).



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  		Figure 4. Immunoprecipitation of the recombinant (A) and endogenous (B) hGRß protein. A, Whole cell extracts were prepared from COS-1 cells transfected with equimolar amounts of pCMVhGR{alpha}, pCMVhGRß, or pCMV5 (mock). Proteins were immunoprecipitated with preimmune serum (lanes 1 and 5), antibody 59 (lanes 2, 6, and 8), BShGR (lanes 3, 7, and 9), or BShGR preincubated with the peptide antigen (lane 4). B, Whole cell extracts were prepared from HeLa S3 and CEM-C7 cells. Proteins were immunoprecipitated with antibody 59 (lane 1), nonimmune serum (lane 2), BShGR (lane 3), or BShGR preincubated with the peptide antigen (lane 4). Epitope-purified anticyclophilin A antiserum prepared in our laboratory served as the nonimmune control. In both experiments, equal aliquots of immunoprecipitated proteins were separated on 8.5% SDS-polyacrylamide gels, transferred to nitrocellulose, and analyzed by Western blotting using antibody 57. Molecular mass standards are indicated in the left margin.

 
When this strategy was applied to protein extracts prepared from HeLa S3 and CEM-C7 cells, antibody 57 detected a 90-kDa protein in the BShGR immunoprecipitates (Fig. 4BGo, lane 3, both panels). This protein was not immunoprecipitated by nonimmune serum or by BShGR preincubated with the peptide antigen (lanes 2 and 4, both panels), suggesting that this 90-kDa protein is hGRß. In CEM-C7 cells a higher molecular mass protein (~100 kDa) was also immunoprecipitated by BShGR (lane 3, right panel). However, the interaction of BShGR with this protein appears to be nonspecific, as this protein was also immunoprecipitated by BShGR preabsorbed with the peptide antigen (lane 4, right panel). As expected, the 94-kDa hGR{alpha} protein was detected in the antibody 59 immunoprecipitates prepared from transfected COS-1 cells (Fig. 4AGo, lane 6) and from HeLa S3 and CEM-C7 cells (Fig. 4BGo, lane 1, both panels).

Having assured ourselves that the BShGR antibody was reacting specifically with the endogenous hGRß protein, we performed Western blots on whole cell lysates isolated from a variety of human cell lines (Table 1Go). With the exception of melanoma, SK-OV-3 (ovarian adenocarcinoma), and Hep G2 (hepatocellular carcinoma) cells, the 90-kDa hGRß protein was detected by the BShGR antibody in each cell type. Antibody 57 reacted with the 94-kDa hGR{alpha} protein in all cell lines and with a 90-kDa protein in many of the cells. In general, the 94-kDa hGR{alpha} protein was much more abundant than this 90-kDa protein. However, in a few cell lines (NCI-H520, NCI-H661, MDA-MB-435S, Ishikawa, and HEC59), the 94- and 90-kDa proteins were made at comparable levels. Whether this 90-kDa protein detected by antibody 57 is hGRß or an hGR{alpha} degradation product cannot be determined at this time, but it raises the possibility that the hGR{alpha} and hGRß proteins are made at similar levels in certain cells.


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  		Table 1. Expression of hGRß and hGR{alpha} proteins in various human cell lines

 
We next investigated the expression of the hGRß protein in human tissues. For this experiment, a Western blot containing proteins isolated from brain, heart, kidney, lung, and skeletal muscle was probed with BShGR. As shown in the left panel of Fig. 5Go, a protein consistent in size with hGRß is recognized by BShGR in extracts prepared from brain and lung. This protein was weakly detected in heart, but was not observed in kidney and skeletal muscle. As the hGRß mRNA transcript has been detected in kidney and skeletal muscle (15, 17), the hGRß protein may be expressed at very low levels in these cells. The blot was then stripped and reprobed with antibody 57, revealing, as expected, expression of hGR{alpha} in each tissue (Fig. 5Go, right panel). With the exception of heart, antibody 57 did not detect a 90-kDa protein in any of the tissues. Assuming that antibody 57 has the same affinity for the denatured hGR{alpha} and hGRß proteins, this result indicates that hGRß is less abundant than hGR{alpha} at the whole tissue level, consistent with the relative levels of the hGR{alpha} and hGRß mRNA transcripts (15). Interestingly, both BShGR and antibody 57 recognized what appears to be the same 100-kDa protein in heart and skeletal muscle. This muscle-specific protein may be another form of hGRß resulting from alterations in the hGRß mRNA transcript and/or posttranslational modifications of the hGRß protein. Alternatively, this signal may reflect a nonspecific interaction between the antibodies and a protein expressed in the two tissues.



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  		Figure 5. Expression of the hGRß protein in human tissues. A multiple tissue Western blot containing protein isolated from various human tissues (brain, heart, kidney, lung, and skeletal muscle) was probed with BShGR (left panel). The blot was then stripped and reprobed with antibody 57 (right panel). Shown are a 4-h exposure for BShGR (used at a 1:500 dilution) and a 2-h exposure for antibody 57 (used at a 1:1000 dilution). Molecular mass standards are indicated in the left margin.

 
Subcellular distribution of the endogenous hGRß protein
We have shown previously using antibody 57 that the hGRß protein, in contrast to hGR{alpha}, resides primarily in the nucleus of transfected COS-1 cells independent of glucocorticoid treatment (15). Unfortunately, these immunocytochemical studies could not be extended to include the endogenous hGRß protein because of the cross-reactivity of antibody 57 with hGR{alpha}. Therefore, we tested the ability of BShGR to recognize the native conformation of the hGRß protein. For these experiments, COS-1 cells were transfected with pCMVhGRß, and immunocytochemistry was performed with preimmune serum, BShGR, or BShGR preabsorbed with the peptide antigen (Fig. 6Go). BShGR stained the cells that acquired the expression vector, but showed no staining of the untransfected cells (middle panel). Consistent with our earlier observations using antibody 57 (15), hGRß was localized preferentially to the nucleus, and its subcellular distribution was unaffected by the administration of glucocorticoids. Significant cytoplasmic staining was observed in some of the cells (left side of middle panel), perhaps reflecting overexpression of the hGRß protein. Immunoreactivity was not observed with preimmune serum (upper panel) or with BShGR preabsorbed with the peptide antigen (lower panel), confirming the specificity of the staining. Moreover, BShGR did not stain cells expressing the hGR{alpha} protein (data not shown).



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  		Figure 6. Immunocytochemical localization of the hGRß protein in transfected COS-1 cells. COS-1 cells were transfected with pCMVhGRß and cultured in the absence (CON) or presence (DEX) of 1 x 10-7 M dexamethasone. Immunocytochemistry was then performed with preimmune serum (top panel), BShGR (middle panel), or BShGR preabsorbed with the peptide antigen (lower panel). Immunoreactivity was visualized by staining with avidin-biotin-peroxidase.

 
Because BShGR specifically recognized the native conformation of hGRß, we used this antibody to investigate the cellular and subcellular distribution of the endogenous hGRß protein. Immunocytochemistry was conducted initially on HeLa S3 cells. As shown in Fig. 7Go, BShGR stained the nucleus of these cells, and this staining pattern was not altered by glucocorticoids. No significant staining was observed with preimmune serum or with BShGR preabsorbed with the peptide antigen. Similar results were observed in CEM-C7 cells (data not shown). We next performed immunohistochemistry on human tissue sections prepared from lung, thymus, and liver with the BShGR antibody. Heavy immunostaining was observed in the epithelial cells lining the terminal bronchiole of the lung (Fig. 8AGo), in the epithelial cells forming the outer layer of Hassall’s corpuscle in the thymus (Fig. 8BGo), and in the epithelial cells lining the bile duct in the liver (Fig. 8CGo). Thymic lymphocytes showed very little staining and hepatocytes showed moderate staining (Fig. 8Go, B and D). In all cases, immunostaining was restricted to the nucleus. Thus, in agreement with our findings for the recombinant hGRß protein, the endogenous hGRß protein resides in the nucleus of cells. Tissue sections incubated with preimmune serum did not display any immunostaining (data not shown).



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  		Figure 7. Immunocytochemical localization of the hGRß protein in HeLa S3 cells. After a 2-h incubation in the absence (CON) or presence (DEX) of 1 x 10-7 M dexamethasone, HeLa S3 cells were fixed and processed for immunocytochemistry, as described in Materials and Methods, with preimmune serum, BShGR, or BShGR preincubated with the peptide antigen. Immunoreactivity was visualized by staining with avidin-biotin-peroxidase.

 


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  		Figure 8. Immunohistochemical localization of the hGRß protein in human tissues. Human tissue sections were fixed and processed for immunohistochemistry using the BShGR antibody as described in Materials and Methods. A, Lung from an 84-yr-old man. B, Thymus from a 24-yr-old woman. C and D, Liver from a 28-yr-old woman.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have investigated the expression and subcellular distribution of the hGRß protein. To distinguish hGRß from the wild-type hGR{alpha} isoform, we have produced, characterized, and used an hGRß-specific antibody (BShGR). This antibody binds both the native and denatured conformations of the hGRß protein and does not cross-react with hGR{alpha} under any circumstances. In addition, the interaction of BShGR with hGRß is specific, as demonstrated by the inability of the antibody to react with hGRß when preincubated with the peptide antigen. Using BShGR on Western blots and in immunoprecipitation experiments, we show that the hGRß protein is expressed in a variety of cell lines and tissues. Immunohistochemical analysis of human tissue sections revealed that hGRß is expressed at high levels only in certain epithelial cells and that, within cells, hGRß is located in the nucleus. Expression of the hGRß protein in vivo and its unique cellular and subcellular distribution suggest that hGRß has an important physiological function.

In transfected cells, hGRß has been shown to function as a dominant negative inhibitor of hGR{alpha} (15, 17). Therefore, the relative ratio of the hGR{alpha} and hGRß proteins in vivo may be a critical factor regulating target cell responsiveness to glucocorticoids. Our Western blot data using antibody 57 (which presumably has the same affinity for the denatured hGR{alpha} and hGRß proteins) suggest that hGR{alpha} is much more abundant than hGRß in most cells, consistent with the reported levels of the hGR{alpha} and hGRß mRNA transcripts (15). However, our immunohistochemical analysis with BShGR indicates that within tissues, hGRß is expressed at high levels in a cell type-specific manner. Epithelial cells lining the terminal bronchiole of the lung, forming the outer layer of Hassall’s corpuscle of the thymus, and lining the bile duct of the liver showed strong immunostaining. In contrast, thymic lymphocytes and other epithelial cells in these tissues showed very little immunoreactivity. Moderate immunoreactivity was observed in hepatocytes. Therefore, the relative levels of hGR{alpha} and hGRß may vary considerably among different cells and not at all reflect the ratio determined for the whole tissue. The physiological consequence of cell type-specific differences in the hGR{alpha} to hGRß protein ratio may be cell type-specific differences in glucocorticoid responsiveness.

Exogenous glucocorticoids play an essential role in the acute and chronic therapy of many inflammatory and immune diseases because of their immunosuppressive and antiinflammatory actions. Unfortunately, resistance to the therapeutic effects of glucocorticoids develops in a small proportion of patients being treated for asthma (27, 28), rheumatoid arthritis (29), degenerative osteoarthritis (30), ulcerative colitis (31), transplantation rejection (32), eosinophilic gastritis (33), and complications from acquired immunodeficiency syndrome (34). Glucocorticoids have also been used successfully as chemotherapeutic agents in the treatment of many hematological cancers, including Hodgkin’s lymphoma, acute lymphoblastic leukemia, and multiple myeloma. Again, the beneficial effect of this therapy is sometimes limited by the occurrence of glucocorticoid-resistant tumors (35, 36). The molecular mechanisms underlying these tissue-specific glucocorticoid resistant states are largely unknown. However, it is known for many of these glucocorticoid-resistant patients that changes in the number and/or ligand-binding properties of hGR{alpha} cannot account for the observed resistance (28). Perhaps overexpression of the hGRß protein contributes to the glucocorticoid insensitivity. By preventing hGR{alpha} from binding GREs, forming transcriptionally inactive hGR{alpha}/hGRß heterodimers, and/or titrating a coactivator needed by hGR{alpha} for full transcriptional activity, increased levels of hGRß would prevent hGR{alpha} from activating target genes. Interestingly, a marked reduction in the ability of hGR{alpha} to bind a GRE was recently demonstrated in patients with glucocorticoid-resistant asthma (37).

Glucocorticoid insensitivity is observed not only in disease states, but also during normal physiological processes. For example, we and others have shown that genes positively regulated by hGR{alpha} are unresponsive to glucocorticoids during the G2 phase of the cell cycle (38, 39). During development, the chicken retina is resistant to circulating glucocorticoids before embryonic day 6, but thereafter becomes progressively more sensitive even though the level of wild-type GR does not change significantly over this time period (40). In each case, cell cycle or developmentally regulated induction of hGRß might account for the temporary resistance. Indeed, alternative splicing is often regulated in a cell type-specific fashion, in a developmental stage-specific fashion, or in response to specific cellular signals.

Our immunocytochemical analysis of a variety of cells using the BShGR antibody demonstrated that the hGRß protein is located predominantly in the nucleus of cells independent of glucocorticoid treatment. The nuclear localization suggests that hGRß may regulate gene expression independently of its proposed antagonism of ligand-bound hGR{alpha}. For example, hGRß may bind GREs and repress expression of glucocorticoid-responsive genes in the absence of glucocorticoids. In the presence of glucocorticoids, ligand-bound hGR{alpha} would then displace hGRß from the GRE and subsequently activate gene expression. Although hGRß can bind a GRE (17), evidence supporting a role for hGRß as a constitutive repressor has yet to be found. For example, hGRß did not repress the basal activity of the glucocorticoid-responsive mouse mammary tumor virus promoter in transfected COS-1 cells (15).

In contrast to hGRß’s constitutive nuclear localization, hGR{alpha} is generally thought to reside in the cytoplasm of cells in the absence of ligand, but to translocate to the nucleus after hormone treatment (8, 41). In support of this two-step translocation model, we have shown previously in HeLa S3 cells that hGR{alpha} translocates from the cytoplasm to the nucleus in a hormone-dependent manner (23). However, in this and other immunocytochemical studies performed on hGR{alpha}, significant nuclear staining was observed even in the absence of glucocorticoids (42, 43, 44). Because the antibodies employed in these experiments recognize both hGR{alpha} and hGRß proteins, the observed nuclear staining may, in fact, reflect expression of hGRß, and this might account for some of the controversy surrounding the subcellular distribution of hGR{alpha} (45, 46). An hGR{alpha}-specific antibody is needed for the most accurate and definitive determination of hGR{alpha} compartmentalization.

In the absence of hormone, hGR{alpha} has been shown to associate with two molecules of hsp90 (9). The hsp90 molecules are thought to sequester hGR{alpha} in the cytoplasm of cells by maintaining the receptor in a conformation that masks or inactivates its nuclear localization signals (NLS). Upon hormone binding, the hsp90 molecules dissociate from the receptor, resulting in the unmasking or activation of the NLS, allowing hGR{alpha} to translocate into the nucleus. The hGR{alpha} sequences proposed to interact with hsp90 are intact in the hGRß protein (4), but whether hGRß interacts with hsp90 is not known. The nuclear distribution of hGRß makes this an interesting question. The unique carboxy-terminus of hGRß may make the site for hsp90 binding inaccessible, resulting in constitutively active NLS and nuclear residence of hGRß. In support of this hypothesis, hGR{alpha} mutants incapable of associating with hsp90 localize in the nucleus of cells independent of glucocorticoid treatment (41, 47). Alternatively, the conformation of hGRß may be altered in such a way that the NLS are exposed or activated even when hsp90 is associated with the protein. Cadepond et al. have shown recently that sequences at the carboxy-terminus of hGR{alpha} exert an inhibitory influence on the NLS (47). Deletion and/or modification of this sequence, as would occur in hGRß, might inactivate this repression and allow hGRß to translocate into the nucleus independent of hsp90 association.

In summary, we have produced an hGRß-specific antibody termed BShGR, and have used this antiserum to show that the hGRß protein is expressed in human cells. In agreement with our findings, the Chrousos laboratory recently reported that the hGRß protein is expressed in many different tissues, as revealed by Western blot analysis (48). It is important to note, however, that the hGRß antibodies used by the two independent studies were prepared against different immunogens, and therefore, it is difficult to directly compare the results. We also have demonstrated, for the first time, that the endogenous hGRß protein resides in the nucleus of cells and that, within tissues, expression of hGRß is most prominent in a certain epithelial cells. These results should provide important clues toward elucidating the physiological function of this hGR splice variant. The immediate goal of future studies will be not only to identify the type of epithelial cell expressing hGRß at high levels, but also to investigate epithelial cells in other tissues, such as kidney and digestive tract, for hGRß expression. Moreover, it will be important to measure changes in the expression of hGRß in physiological and pathophysiological resistant states. With its unique cellular and subcellular distribution, the hGRß protein may profoundly alter GR physiology.


   Acknowledgments
 
We thank Dr. Paul Nettesheim for the normal human lung epithelial cells and Drs. Alicia White and J. Carl Barrett for providing the lung, breast, endometrial, bladder, ovary, and melanoma cell lysates. We thank Dr. Darryl C. Zeldin, Dr. Wayne P. Bocchinfuso, and Ms. Daphne Bofetiado for critical comments on the manuscript.


   Footnotes
 
1 The immunohistochemistry studies conducted on the human tissue sections were supported by a grant from the Office of Naval Research (N00014–96-I-0255; to C.R.P.). Back

2 Member of the Department of Physiology, University of North Carolina (Chapel Hill, NC). Back

Received March 19, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–895[Abstract/Free Full Text]
  2. Carson-Jurica MA, Schrader WT, O’Malley BW 1990 Steroid receptor family: structure and functions. Endocr Rev 11:201–220[Abstract]
  3. Yamamoto KR 1985 Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet 19:209–252[CrossRef][Medline]
  4. Dalman FC, Scherrer LC, Taylor LP, Akil H, Pratt WB 1991 Localization of the 90-kDa heat shock protein-binding site within the hormone-binding domain of the glucocorticoid receptor by peptide competition. J Biol Chem 266:3482–3490[Abstract/Free Full Text]
  5. Picard D, Yamamoto KR 1987 Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J 6:3333–3340[Medline]
  6. Dahlman-Wright K, Wright APH, Gustafsson J-A 1992 Determinants of high-affinity DNA binding by the glucocorticoid receptor: evaluation of receptor domains outside the DNA-binding domain. Biochemistry 31:9040–9044[CrossRef][Medline]
  7. Hollenberg SM, Evans RM 1988 Multiple and cooperative trans-activation domains of the human glucocorticoid receptor. Cell 55:899–906
  8. Webster JC, Jewell CM, Sar M, Cidlowski JA 1994 The glucocorticoid receptor: maybe not all steroid receptors are nuclear. Endocrine 2:967–969
  9. Pratt WB 1993 The role of the heat shock proteins in regulating the function, folding, and trafficking of the glucocorticoid receptor. J Biol Chem 268:21455–21458[Free Full Text]
  10. Jonat C, Rahmsdorf HJ, Park KK, Cato AC, Gebel S, Ponta H, Herrlich P 1990 Antitumor promotion and anti-inflammation: down-modulation of AP-1 (fos/jun) activity by glucocorticoid hormone. Cell 62:1189–1204[CrossRef][Medline]
  11. Yang Yen HF, Chambard JC, Sun YL, Smeal T, Schmidt TJ, Drouin J, Karin M 1990 Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62:1205–1215[CrossRef][Medline]
  12. Schule R, Rangarajan P, Kliewer S, Ransone LJ, Bolado J, Yang N, Verma IM, Evans RM 1990 Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor. Cell 62:1217–1226[CrossRef][Medline]
  13. Scheinman RI, Gualberto A, Jewell CM, Cidlowski JA, Baldwin AS 1995 Characterization of mechanisms involved in transrepression of NF-{kappa}B by activated glucocorticoid receptors. Mol Cell Biol 15:943–953[Abstract]
  14. Hollenberg SM, Weinberger C, Ong ES, Cerelli G, Oro A, Lebo R, Thompson EB, Rosenfeld MG, Evans RM 1985 Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature 318:635–641[CrossRef][Medline]
  15. Oakley RH, Sar M, Cidlowski JA 1996 The human glucocorticoid receptor isoform: expression, biochemical properties, and putative function. J Biol Chem 271:9550–9559[Abstract/Free Full Text]
  16. Giguere V, Hollenberg SM, Rosenfeld MG, Evans RM 1986 Functional domains of the human glucocorticoid receptor. Cell 46:645–652[CrossRef][Medline]
  17. Bamberger CM, Bamberger A-M, de Castro M, Chrousos GP 1995 Glucocorticoid receptor ß, a potential endogenous inhibitor of glucocorticoid action in humans. J Clin Invest 95:2435–2441
  18. Gray T, Guzman K, Davis C, Abdullah L, Nettesheim P 1996 Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 14:104–112[Abstract]
  19. Weigel NL, Schrader WT, O’Malley BW 1989 Antibodies to the chicken progesterone receptor peptide 523–536 recognize a site exposed in receptor-deoxyribonucleic acid complexes but not in receptor-heat shock protein-90 complexes. Endocrinology 125:2494–2501[Abstract]
  20. Fairbanks G, Steck TL, Wallach DFH 1971 Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606–2617[CrossRef][Medline]
  21. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354[Abstract/Free Full Text]
  22. Salinovich D, Montelaro RC 1986 Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 156:341–347[CrossRef][Medline]
  23. Cidlowski JA, Bellingham DL, Powell-Oliver FE, Lubahn DB, Sar M 1990 Novel antipeptide antibodies to the human glucocorticoid receptor: recognition of multiple receptor forms in vitro and distinct localization of cytoplasmic and nuclear receptors. Mol Endocrinol 4:1427–1437[Abstract]
  24. Burnstein KL, Jewell CM, Cidlowski JA 1991 Evaluation of the role of ligand and thermal activation on specific DNA binding by in vitro synthesized human glucocorticoid receptor. Mol Endocrinol 5:1013–1022[Abstract]
  25. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  26. Laemmli UK 1979 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
  27. Corrigan CJ, Brown PH, Barnes NC, Szefler SJ, Tsai J-J, Frew AJ, Kay AB 1991 Glucocorticoid pharmacokinetics, glucocorticoid receptor characteristics, and inhibition of peripheral blood T cell proliferation by glucocorticoids in vitro. Am Rev Respir Dis 144:1016–1025[Medline]
  28. Barnes PJ, Greening AP, Crompton GK 1995 Glucocorticoid resistance in asthma. Am J Respir Crit Care Med 152:S125–S142
  29. Kirkham BW, Corkill MM, Davison SC, Panayi GS 1991 Response to glucocorticoid treatment in rheumatoid arthritis: in vitro cell mediated immune assay predicts in vivo responses. J Rheumatol 18:821–825
  30. DiBattista JA, Martel-Pelletier J, Antakly T, Tardif G, Cloutier J-M, Pelletier J-P 1993 Reduced expression of glucocorticoid receptor levels in human osteoarthritic chondrocytes. role in the suppression of metalloprotease synthesis. J Clin Endocrinol Metab 76:1128–1134[Abstract]
  31. Lichtiger S, Present DH, Kornbluth A, Gelernt I, Bauer J, Galler G, Michelassi F, Hanauer S 1994 Cyclosporine in severe ulcerative colitis refractory to steroid therapy. N Engl J Med 330:1842–1845
  32. Langhoff E, Ladefoged J, Jakobsen BK, Platz P, Ryder LP, Svejgaard A, Thaysen JH 1986 Recipient lymphocyte sensitivity to methylprednisolone affects cadaver kidney graft survival. Lancet 2:1296–1297
  33. Quan SF, Sedgwick JB, Nelson MV, Busse AW 1993 Corticosteroid resistance in eosinophilia gastritis: relation to in vitro eosinophil survival and interleukin 5. Ann Allergy 70:256–260[Medline]
  34. Norbiato G, Bevilacqua M, Vago T, Baldi G, Chebat E, Bertora P, Moroni M, Galli M, Oldenburg N 1992 Cortisol resistance in acquired immunodeficiency syndrome. J Clin Endocrinol Metab 74:608–613[Abstract]
  35. Moalli PA, Pillay S, Krett NL, Rosen ST 1993 Alternatively spliced glucocorticoid receptor messenger RNAs in glucocorticoid-resistant human multiple myeloma cells. Cancer Res 53:3877–3879[Abstract/Free Full Text]
  36. Strasser-Wozak EMC, Hattmannstorfer R, Hala M, Hartmann BL, Fiegl M, Geley S, Kofler R 1995 Splice site mutation in the glucocorticoid receptor gene causes resistance to glucocorticoid-induced apoptosis in a human acute leukemic cell line. Cancer Res 55:348–353[Abstract/Free Full Text]
  37. Adcock IM, Lane SJ, Brown CR, Peters MJ, Lee TH, Barnes PJ 1995 Differences in binding of glucocorticoid receptor to DNA in steroid-resistant asthma. J Immunol 154:3500–3505[Abstract]
  38. Fanger BO, Currie RA, Cidlowski JA 1986 Regulation of epidermal growth factor receptors by glucocorticoids during the cell cycle in HeLa S3 cells. Arch Biochem Biophys 249:116–125[CrossRef][Medline]
  39. Hsu S, DeFranco DB 1995 Selectivity of cell cycle regulation of glucocorticoid receptor function. J Biol Chem 270:3359–3364[Abstract/Free Full Text]
  40. Gorovits R, Ben-Dror I, Fox LE, Westphal HM, Vardimon L 1994 Developmental changes in the expression and compartmentalization of the glucocorticoid receptor in embryonic retina. Proc Natl Acad Sci USA 91:4786–4790
  41. Jewell CM, Webster JC, Burnstein KL, Sar M, Bodwell JE, Cidlowski JA 1995 Immunocytochemical analysis of hormone mediated nuclear translocation of wild type and mutant glucocorticoid receptors. J Steroid Biochem Mol Biol 55:135–146[CrossRef][Medline]
  42. Norman J, Franz M, Schiro R, Nicosia S, Docs J, Fabri PJ, Gower WR 1994 Functional glucocorticoid receptor modulates pancreatic carcinoma growth through an antocrine loop. J Surg Res 57:33–38
  43. Akner G, Mossberg K, Wikstrom A-C, Sundqvist K-G, Gustafsson J-A 1991 Evidence for colocalization of glucocorticoid receptor with cytoplasmic microtubules in human gingival fibroblasts, using two different monoclonal anti-GR antibodies, confocal laser scanning microscopy and image analysis. J Steroid Biochem Mol Biol 39:419–432[CrossRef][Medline]
  44. DiBattista JA, Martel-Pelletier J, Wosu LO, Sandor T, Antakly T, Pelletier J-P 1991 Glucocorticoid receptor mediated inhibition of interleukin-1 stimulated neutral metalloprotease synthesis in normal human chondrocytes. J Clin Endocrinol Metab 72:316–326[Abstract]
  45. Gorski J, Malayer JR, Gregg DW, Lundeen SG 1994 Just where are the steroid receptors anyway? Endocrine 2:99–100
  46. Welshons WV, Judy BM 1995 Nuclear vs translocating steroid receptor models and the excluded middle. Endocrine 3:1–4
  47. Cadepond F, Gasc J-M, Delahaye F, Jibard N, Schweizer-Groyer G, Segard-Maurel I, Evans R, Baulieu E-E 1992 Hormonal regulation of the nuclear localization signals of the human glucocorticosteroid receptor. Exp Cell Res 201:99–108[CrossRef][Medline]
  48. de Castro M, Elliot S, Kino T, Bamberger C, Karl M, Webster E, Chrousos GP 1996 The non-ligand binding ß-isoform of the human glucocorticoid receptor (hGRß): tissue levels, mechanism of action, and potential physiologic role. Mol Med 2:597–607[Medline]



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