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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 361-367, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4861

Increased Glucocorticoid Receptor ß Expression Converts Mouse Hybridoma Cells to a Corticosteroid-Insensitive Phenotype

Pia J. Hauk, Elena Goleva, Ian Strickland, Alessandra Vottero, George P. Chrousos, Kevin O. Kisich and Donald Y. M. Leung

Department of Pediatrics, National Jewish Medical and Research Center, Denver; Department of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado; and Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Development, National Institutes of Health, Bethesda, Maryland

Address correspondence to: Donald Y. M. Leung, M.D., Ph.D., National Jewish Medical Research Center, 1400 Jackson Street, Room K926i, Denver, CO 80206. E-mail: leungd{at}njc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoid (GC) insensitivity is a challenging clinical problem associated with many chronic inflammatory disorders and life-threatening disease progression. The molecular basis of GC insensitivity, however, is unknown. Alternative splicing of the GC receptor (GCR) pre-mRNA generates a second GCR, termed GCRß, which does not bind GC but antagonizes the transactivating activity of the classic GCR, termed GCR{alpha}. GC-insensitive conditions have been associated with increased GCRß expression. Whether or not increased GCRß expression can contribute to GC insensitivity, however, remains controversial. To more precisely demonstrate the effect of GCRß on steroid responsiveness, we virally transduced GCRß cDNA into mouse DO-11.10 hybridoma cells, as mice are known to be deficient in the GCRß gene. We demonstrate that viral transduction of GCRß cDNA into mouse hybridoma cells to induce stable expression of GCRß results in GC insensitivity of these cells. Furthermore, in such cells GCR{alpha} is complexed with GCRß. Such heterodimer formation may account for the reduced effectiveness of GC action in cells overexpressing GCRß.

Abbreviations: bovine serum albumin, BSA • dexamethasone, DEX • fetal calf serum, FCS • glucocorticoid, GC • glucocorticoid receptor, GCR • green fluorescent protein, GFP • glucocorticoid-responsive element, GRE • human glucocorticoid receptor ß, hGCRß • Iscove's modified Dulbecco's medium, IMDM • mouse mammary tumor virus, MMTV • murine stem cell virus, MSCV • peripheral blood mononuclear cells, PBMC


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoid (GC) insensitivity is increasingly being recognized in the management of chronic inflammatory diseases such as asthma, inflammatory bowel disease, systemic lupus erythematosus, and transplantation rejection (1–5). It poses a major clinical challenge, as corticosteroid therapy is the cornerstone of anti-inflammatory therapy. Such patients are subjected to the unwanted side effects of prolonged high doses of systemic GC therapy. Delineation of the molecular basis for GC insensitivity is critical for the development of new treatment approaches for this group of refractory patients, and may provide new insights into the pathogenesis of chronic inflammation. Previous studies have revealed that GCs are less effective in inhibiting mitogen-induced proliferation of T cells from subjects with GC insensitivity as compared with GC-sensitive individuals (6). Thus, GC insensitivity in these individuals is not absolute, but reflects a shift in the dose-response curve such that higher concentrations of steroids are required to inhibit proliferation of cells from GC-insensitive as compared with GC-sensitive subjects (7).

GC action is mediated through the glucocorticoid receptor (GCR), which is found in the cytosol of most human cells. As the result of alternative splicing of the GCR pre-mRNA, there are two homologous mRNAs and protein isoforms, termed GCR{alpha} and GCRß (8). Both mRNAs contain the same first eight exons of the GCR gene (9). The remainder is derived by alternative splicing of the last exon of the GCR gene, resulting in either inclusion or exclusion of exon 9{alpha}. The two protein isoforms have the same first 727 NH2-terminal amino acids. GCRß differs from GCR{alpha} only in its carboxy terminus with replacement of the last 50 amino acids of GCR{alpha} with a unique 15 amino acid sequence lacking a steroid binding domain. These differences render GCRß unable to bind GCs, reduce its binding affinity for DNA recognition sites, abolish its ability to transactivate GC-sensitive genes, and make it function as a dominant inhibitor of GCR{alpha}.

Whether GCRß has a functional role in steroid resistance has been a matter of major controversy. Bamberger and coworkers (10) and Oakley and colleagues (11, 12), have both found that transiently transfecting increasing amounts of GCRß expression vectors into various cell types inhibits the trans-activating ability of GCR{alpha}. In contrast, De Lange and associates (13) reported that even in the presence of 10-fold excess of GCRß, GCR{alpha} was still able to activate transcription from the mouse mammary tumor virus (MMTV) promoter at the same rate. Similarly, Hecht and coworkers (14) were unable to demonstrate an inhibitory role for GCRß when transfected into COS7 cells. These discrepant results may be explained by the use of different vector systems, differences in cell or tissue specificity, or insufficient GCRß expression due to the transient transfection methods used. To address these potential confounding technical problems, we virally transduced GCRß cDNA into mouse hybridoma cells, which naturally lack GCRß, and selected for GCRß1 cells by cell sorting to determine whether these cells become GC resistant.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression of GCRß in Murine DO-11.10 Hybridoma Cells
The cDNA encoding the human glucocorticoid receptor ß (hGCRß) isoform (base pairs 23–2296) (9) was subcloned into the replication defective murine stem cell virus (MSCV) retroviral vector as a bicistronic coding unit containing green fluorescent protein (GFP) (15), followed by the encephalomyelitis virus, internal ribosome entry site (IRES), and the GCRß coding region (16). As a control, the vector containing GFP-IRES without the GCRß coding sequence was constructed. Phoenix packaging cells (17) were transiently transfected with the expression vectors for GCRß and GFP, or GFP alone, using calcium phosphate precipitation. At the same time, the plasmid vector pCLeco, which contains cDNA encoding for the viral structural proteins "gag, pol, and env" was transfected into the Phoenix cells to enhance the production of infectious virus (18). In brief, 3 x 105 Phoenix cells in 2 ml of 5% FCS (Gemini, Woodland, CA)/Iscove's modified Dulbecco's medium (IMDM) media (Cellgrow; Mediatech, Herndon, VA) were plated out per well of a six-well culture plate (Nalge Nunc International, Rochester, NY), pretreated with 1 ml of 0.1 mg/ml poly-D-lysine (Sigma, St. Louis, MO) in BSS (Cellgrow, Mediatech), and incubated overnight at 37°C, 7% CO2. The next day, 1 µg of pCLeco plasmid vector, 5 µg of MSCV retroviral vector containing either GFP alone or GFP and GCRß cDNA were mixed in a final volume of 150 µl ddH2O. Fifty microliters of 1 M CaCl2 was added. This mixture was slowly added by drops into a tube containing 200 µl of 2x HBS held on a vortexer. Two hundred microliters of this solution were added per well of Phoenix cells and incubated overnight. Next day the medium was replaced by 2 ml of fresh 5% FCS/IMDM per well, and the cells were cultured overnight again. The transfection efficiency of Phoenix cells was measured as percentage of GFP-positive cells by FACS analysis using a FACSCalibur (Becton Dickinson Biosciences, Sparks, MD). Culture supernatants from transfected Phoenix cells producing recombinant MSCV were used to transduce DO-11.10 hybridoma cells (kindly provided by Dr. P. Marrack, National Jewish Medical and Research Center, Denver, CO). In brief, 1 x 106 DO-11.10 hybridoma cells suspended in 2 ml of 5% FCS/IMDM media were plated out per well of a six-well culture plate. Two milliliters of appropriate culture supernatant were added to the target cells. Hexadimethrine bromide (Polybrene; Sigma) was used at a final concentration of 8 µg/ml to enhance the adherence of infectious viral particles to the target cells. The culture plates were equilibrated at 7% CO2, sealed in plastic bags, and spun at 1,800 rpm for 2 h at room temperature to further enhance attachment of the virus to the target cells (spinfection). After this, DO-11.10 hybridoma cells were washed once and cultured for 4 d in 10% FCS/RPMI media (Cellgrow by Mediatech) at 37°C 5% CO2. DO-11.10 hybridoma cells transduced with human GCRß/GFP, or GFP alone, were sorted for GFP+ cells (Moflo Cytomation, Fort Collins, CO) and further cultured.

To estimate the titer of infectious virus particles, 105 NIH 3T3 cells (kindly provided by Dr. P. Marrack) in 2 ml of 5% FCS/IMDM media had been plated out the day before the spinfection. In parallel to DO-11.10 hybridoma cells, these cells were transduced with 1 µl and 10 µl of viral supernatant. After the spinfection, the media was replaced by 2 ml of 5% FCS/IMDM, and the cells were cultured overnight. The transduction frequency of NIH 3T3 cells was measured as percentage of GFP-positive cells by FACS analysis. The formula % of GFP+ NIH 3T3 cells/100 x 2 x 105 x dilution factor (100 for 10 µl or 1,000 for 1 µl of virus supernatant) was used to roughly estimate the titer of infectious virus particles. All transduced hybridoma cells which had been sorted for GFP+ cells were subjected to retransduction with the appropriate expression vector, either for GCRß/GFP, or GFP alone, to further enhance gene expression.

For further experiments, the resulting GFP cell populations were sorted for GFPdim and GFPbright expressing cells. After gating on live DO-11.10 hybridoma cells according to forward and side scatter and doublet exclusion, GFPdim cells were defined as the 3–4% of gated live cells expressing GFP at the lowest fluorescence intensity. Accordingly, GFPbright cells were defined as the 3–4% of gated live cells expressing GFP at the highest fluorescence intensity. The sorted cells were then cultured in 10% FCS/RPMI medium.

Detection of Human GCRß in Murine DO-11.10 Hybridoma Cells by Intracellular Immunofluorescence Staining
Transduced DO-11.10 hybridoma cells were washed once and resuspended in PBS. A quantity of 1 to 2 x 106 cells were used per staining condition. Staining was performed at room temperature in 96-well round-bottom culture plates (Nalge Nunc International, Rochester, NY) as previously described (19). After fixing in PBS containing 3% paraformaldehyde and 3% sucrose, cells were washed and permeabilized in PBS containing 0.2% Triton-X-100 and 0.02% NaN3. Nonspecific binding was blocked by incubation in IMDM-media containing 5% FCS and 0.02% NaN3 (blocking solution). Cells were then stained for 1 h in blocking solution with the GCRß-specific polyclonal rabbit-anti-human GCRß-antibody (Affinity Bioreagents, Golden, CO). In negative controls, the specific antibodies were replaced by non-specific rabbit immunoglobulin (Southern Biotechnology Associates INC, Birmingham, AL). In a second set of controls, the cells were preincubated for 1 h at 37°C in the presence of a 20-fold excess of the GCRß-peptide used to raise the antibody (NVMWLKPESTSHTLI) (National Jewish Molecular Resource Center, Denver, CO) before staining. After washing, cells were incubated for 1 h in blocking solution containing a Cyanine (Cy) 3-conjugated donkey-anti-rabbit Fab2 fragment (Jackson ImmunoResearch Laboratories, West Grove, PA) as secondary antibody. Stained cells were suspended in PBS containing 5% FCS and 0.02% NaN3, and were analyzed with a Becton Dickinson FACScalibur for GFP+/GCRß1 cells, using Cellquest software (Becton Dickinson Biosciences). Intracellular staining was verified by fluorescence microscopy (Nikon, Garden City, NY), using image analysis software (IPLab Spectrum; Signal Analytics Corporation, Fairfax, VA).

Testing for Steroid Sensitivity of Murine DO-11.10 Hybridoma Cells after Transduction with Human GCRß
After co-transduction with the expression vector for GFP and human GCRß or transduction with GFP alone, GFP+ murine DO-11.10 hybridoma cells were further divided into GFPdim and GFPbright cells by cell sorting (Moflo Cytomation). All GFP+ DO-11.10 hybridoma cells were cultured for 48 h in 96-well flat bottom culture plates (Nalge Nunc International) at 1 x 104 cells/well in 10% FCS/RPMI media in the absence and presence of 10-8 to 10-4 M hydrocortisone (HC) (Sigma). As controls, untransduced DO-11.10 hybridoma cells were tested. Six hours before harvest, the cells were pulsed with 1 µCi/well of 3H-thymidine (ICN Biomedicals, Costa Mesa, CA). At the end of the culture, cells were harvested onto glass fiber filters (Harvester 96; Tomtec, Orange, CT), placed into a liquid scintillation cocktail (Wallac, Milton Keynes, UK), and incorporated tritium was counted in a liquid scintillation counter (1,450 Microbeta Plus Counter; Wallac Oy, Turku, Finland). Steroid sensitivity was assessed by expressing the results of experimental conditions with HC as percentage proliferation of control cultures without HC (= 100%). The results shown are the mean ± SEM of six independent experiments. For all proliferation assays, the experimental values reported represent the mean of triplicate determinations.

Quantitative Western Blot Analysis
Quantitative Western blot analysis was performed using specific anti-GCR{alpha} and -GCRß polyclonal antibodies as previously described (8). The GCR{alpha}-directed antibody has been extensively employed in detailed analyses of GCR{alpha} proteolytic cleavage (20). It has also been shown to recognize murine GCR{alpha}. DO-11.10 hybridoma cells were lysed at 4°C, and 100 µg protein was applied to 8% precast polyacrylamide-SDS gel (Novex, San Diego, CA) and electrophoresed in parallel with prestained markers (SeeBlue; Novex) to estimate molecular weights; increasing concentrations of peptide-bovine serum albumin (BSA) conjugate standard (5–25 ng, corresponding to 70–360 fmol) were also used for quantitation. Proteins were transferred to nitrocellulose membranes and blocked in 5% nonfat milk for 1 h. Immunoblotting was performed at 4°C overnight, using purified GCR{alpha} and GCRß specific polyclonal antibodies at 10 µg/ml. After washing, the blots were incubated at room temperature with peroxidase-conjugated anti-rabbit immunoglobulin antibody (Dako, Carpinteria, CA) at a dilution of 1:4,000. Blots were washed and exposed to chemiluminescence solution for 1 min (ECL kit; Amersham LifeScience, Piscataway, NJ), followed by exposure to X-OMAT AR films (Eastman Kodak Co, Rochester, NY). Band densities of the standards and the samples developed on the film were scanned and analyzed by densitometry using the NIH image system 1.61 program.

Immunoprecipitation of Heterodimers of Murine GCR and hGCRß
GFP+/GCRß1 and GFP+ DO11.10 hybridoma cells were harvested in PBS and lysed 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 a 30-min incubation on ice, cell lysates were centrifuged at 13,000 x g for 10 min at 4°C, the supernatant was collected, and the protein concentration was determined by the method of Bradford using the Bio-Rad (Hercules, CA) protein assay. Proteins (400 µg for GFP+/GCRß1 and GFP+ DO11.10 hybridoma cells) were incubated with appropriate precipitating antibody: 2 µg of anti-GCR total (M-20) (Santa Cruz Biotechnologies), 2 µg of anti-GCR{alpha} (P-20) antibody (Santa Cruz Biotechnologies), or 2 µg of anti-GCRß (PA3–514) antibody (Affinity Bioreagents, Golden, CO) for 1 h at 4°C with rotation. For binding immune complexes, 20 µl protein A-agarose (Santa Cruz Biotechnologies) beads were added, and the incubation was continued for overnight at 4°C with rotation. The protein A-agarose immune complexes were washed four times with 200 µl PBS and then resuspended in loading buffer (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-agarose by boiling for 3 min. Proteins were resolved on 10% Tris-HCl gels (Bio-Rad) and electrophoretically transferred to nitrocellulose in Tris/CAPS buffer (Bio-Rad) containing 15% methanol. Membranes were stained with Poinceau S (0.5% in 1% acetic acid) to evaluate loading equivalency and transfer efficiency and blocked for 30 min at room temperature in PBS (5 mM Na2HPO4, 0.15 M NaCl, and 0.05% Tween-20) containing 5% nonfat dry milk (wt/vol). The blot was then incubated overnight with the appropriate primary antibody in phosphate-buffered saline with 0.25% BSA, 1:500 Tween-20, and 1:500 anti-GCR{alpha} (P-20) antibody or 1:500 anti-GCRß (PA3–514) antibody. Subsequently, membranes were washed in PBS with 0.05% Tween-20, incubated for 1 h at room temperature with a horseradish peroxidase–labeled protein A (Amersham Pharmacia Biotech, Piscataway, NJ) (1:10,000 in PBS with 0.05% Tween-20, 0.25% BSA, and 5% dry milk), washed in PBS with 0.05% Tween-20, incubated with chemiluminescent reagents (Western Blot Chemiluminescence Reagent Plus; Perkin Elmer Life Sciences, Boston, MA), and then processed for autoradiography.

Statistical Analysis
Cell proliferation in the absence or presence of increasing doses of HC were compared by the Tukey-Kramer HSD multicomparisons procedure. The overall {alpha} level for the set of comparisons was fixed at 0.05 for statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The Expression of GFP Correlates with the Expression of hGCRß in Murine DO-11.10 Hybridoma Cells
Murine DO-11.10 hybridoma cells that had been transduced with the MSCV-GFP, or MSCV-GCRß/GFP, were sorted into two populations based on the intensity of GFP fluorescence (Figure 1A) . GCRß/GFPdim and GCRß/GFPbright cells were then evaluated for the expression of hGCRß. hGCRß was expressed in 93.5% of all GCRß/GFPdim and 92.1% of GCRß/GFPbright cells (Figures 1C and 1D). However, GCRß/GFPdim hybridoma cells expressed hGCRß with a lower intensity than GCRß/GFPbright cells, showing a direct correlation between the level of GFP and the degree of hGCRß expression. The mean fluorescence intensity (MFI) for GFP was seven times higher in GFPbright than in GFPdim cells, and the MFI for hGCRß was three times higher in GCRßbright than in GCRßdim cells (Figure 1B).



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  		Figure 1. Transduction of DO-11.10 hybridoma cells with retrovirus modified to express GCRß results in GCRß expression. (A) The GFP+ population was sorted into the dimmest and brightest 4%. The GFPbright population (solid line) is 7-fold brighter than the GFPdim population (broken line). (B) GCRß expression by GFPbright (solid line) is 3-fold greater than by GFPdim DO-11.10 hybridoma cells (dotted line), both of which are clearly distinguishable from cells expressing only GFP (negative control line). (C) Dot plot of GFP versus GCRß positivity for GFPdim cells. (D) Dot plot of GFP versus GCRß positivity for GFPbright cells.

 
Quantitation of GCR Isoforms Expressed in DO-11.10 Hybridoma Cells
To precisely determine the amounts of murine GCR and hGCRß present in the various cell populations, we performed quantitative Western analysis on their cell lysates (Figure 2) . Table 1 shows the results of densitometry of the bands shown. Naive DO-11.10 cells contained 8.2 fmol of murine GCR per µg of total cellular protein. Neither naive cells nor cells transduced with GFP alone expressed any detectable hGCRß. The GFP/hGCRßdim population expressed 6.2 fmol hGCRß/µg protein, whereas the GFP/hGCRßbright population expressed 10.2 fmol hGCRß/µg protein. The ratios of hGCRß to murine GCR were 1.1:1 and 2.5:1 in the GFP/hGCRßdim and GFP/hGCRßbright populations, respectively.



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  		Figure 2. Quantitation of GCR isoforms expressed in transduced DO-11.10 hybridoma cells. (A) Quantitative Western blot probed with specific antibody for GCR{alpha}, which also recognizes murine GCR. (B) Quantitative Western blot probed with antibody specific for human GCRß. Results of densitometry readings from these films are presented in Table 1.

 

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  		TABLE 1 Quantitation of GCR isoforms in naive and transduced DO-11.10 hybridoma cells

 
Murine DO-11.10 Hybridoma Cells Become Steroid-Insensitive after Transduction with hGCRß
To assess whether murine DO-11.10 hybridoma cells that had been transduced with human GCRß were more steroid-insensitive than control cells not expressing GCRß, we performed proliferation assays in the presence and absence of different concentrations of HC. To investigate if the expression of hGCRß was associated with a higher cell proliferation in the presence of GCs, the assays were set up in the presence and absence of 10-8 to 10-4M HC. DO-11.10 hybridoma cells expressing both GFP and GCRß proliferated more rapidly than hybridoma cells expressing only GFP, even in the absence of additional HC. This may be due to cortisol normally present in fetal bovine serum, which was included in the basal growth medium. However, a dose-dependent trend was observed for GFP/GCRß1 cells to proliferate at higher levels than control cells when cultured in the presence of all concentrations of HC tested, suggesting a shift of these cells toward steroid insensitivity (Figures 3A and 3B) .



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  		Figure 3. GCRß expression induces steroid resistance in DO-11.10 cells. [3H]thymidine incorporation was used to measure lymphocyte proliferation by DO-11.10 cells transduced with either GFP, or GFP + GCRß and sorted into high and low expression populations as described for Figure 1. HC concentrations from 10-8 M to 10-4 M are shown on the x axis. (A) [3H]thymidine incorporation by GFP/GCRß expressing DO-11.10 cells relative to cells expressing GFP only, or naive cells. (B) [3H]thymidine incorporation by DO-11.10 cells with high expression (GFP/GCRßbright), or low expression (GFP/GCRßdim) of GCRß. (C) Proliferation of GFP/GCRß expressing DO-11.10 cells relative to cells expressing GFP only, or naive cells. (D) Proliferation of DO-11.10 cells with high expression (GFP/GCRßbright), or low expression (GFP/GCRßdim) of GCRß. Data are normalized in C and D, and are presented as percentage of baseline proliferation on the y axis. {ddagger}P < 0.001 relative to GFP/GCRßdim cells. The results shown are the mean ± SEM of six independent experiments. *P < 0.001 relative to naive cells. {dagger}P < 0.001 relative to GFP expressing cells by t test.

 
To directly compare the sensitivity of murine DO-11.10 hybridoma cells expressing human GCRß versus controls to GCs, we also expressed the data as percentage of proliferation reached in the presence of different concentrations of HC, normalized to their baseline control proliferation without HC (Figures 3C and 3D). Importantly, the proliferation of GFP/GCRßdim cells was significantly greater than GFPdim cells in the presence of 10-7 M or more HC (P < 0.05). The induction of steroid resistance in cells expressing GCRß was dependent on the level of GCRß expressed, as the GFP/GCRßbright population was significantly more (P < 0.05) steroid resistant than the GFP/GCRßdim population. Similar results were obtained when using dexamethasone (DEX) as the inhibitory steroid.

GCR{alpha} and GCRß Are Physically Associated in Steroid-Insensitive Cells
Because DO-11.10 hybridoma cells that express GCRß are less sensitive to the effects of HC, we examined whether there was evidence of direct physical interaction between the two receptor isoforms. Whole cell lysates were prepared, and murine GCR was immunoprecipitated with specific anti-GCR{alpha} antibody; GCRß was precipitated with specific anti-GCRß antibody; and total GCR proteins were precipitated with anti-GCR antibody that can recognize GCR{alpha} and GCRß as well. The samples were then separated via SDS-PAGE, and transferred to membranes. Western blot analysis was performed using a specific anti-GCRß antibody to determine if immunoprecipitation of murine GCR also captured GCRß, demonstrating a physical interaction between the two GCR isoforms.

Figure 4 illustrates that GCRß is clearly present in GCRß/GFP samples after immunoprecipitation of murine GCR (B). The complex between murine GCR and hGCRß was also evident when using anti-GCRß antibody to immunoprecipitate, and then probing the Western blot with anti-GCR{alpha} antibody (A). In transduced cells, immunoprecipitation with total glucocorticoid receptor antibody resulted in clear detectable bands for GCR{alpha} (A, lane 1) or GCRß (B, lane 1). Treatment of the cells with DEX did not change overall levels of GCR{alpha} (A, lane 2) or GCRß (B, lane 2) detectable in the cells. DEX treatment did not affect the amount of glucocorticoid receptor in DO-11.10 hybridoma cells expressing GFP either (A, lanes 3 and 4). Interestingly, heterodimer formation was not strictly dependent on the addition of DEX, as the amount of heterodimers detected in the cells was not enhanced by DEX treatment (A and B, lanes 5 and 6). Bands for GCRß are only present in cells expressing GCRß (A and B, lanes 5 and 6), and are absent in cells expressing only GFP (A and B, lanes 7 and 8).



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  		Figure 4. GCR{alpha}-GCRß heterodimer formation in DO-11.10 hybridoma cell lines. Total cell lysates from DO-11.10 GFP/GCRß1 and DO-11.10 GFP+ hybridoma cells before (Lanes 1 and 5, and 3 and 7, respectively) and after DEX stimulation (Lanes 2 and 6, and 4 and 8, respectively) were precipitated with anti-GCR total (A, lanes 1–4; and B, lanes 1–4), anti-GCRß (A, lanes 5–8), or anti-GCR{alpha} (B, lanes 5–8) antibodies. Immunoprecipitates were blotted for GCR{alpha} (A) or GCRß (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The sensitivity of cells to the actions of GCs, including endogenous molecules such as cortisol and pharmaceuticals such as DEX, is an important mechanism by which the degree and duration of inflammatory responses are controlled (21, 22). Suboptimal responses to GCs often lead to prolonged treatment with high-dose GC therapy accompanied by serious adverse effects despite persistent tissue inflammation. An understanding of the mechanisms that lead to GC insensitivity or resistance is important for the development of new approaches for the treatment and control of inflammation.

There have been two postulated mechanisms, both of which are likely to occur concurrently in the setting of inflammation and therefore likely synergistically inhibit GCR action. The first is based on in vitro observations that cytokines induce the activation of transcription factors and that overexpression of transcription factors such as AP-1 or NF-{kappa}B interferes with GCR binding and function (23). Indeed, increased AP-1 expression has been reported in the peripheral blood mononuclear cells of patients with steroid-resistant asthma, suggesting that GCR/AP-1 interactions might contribute to GC insensitivity in asthma (24).

The second candidate mechanism for dampening sensitivity to GCs is via induction of GCRß (10). Oakley and coworkers have shown that hGCRß overexpression in HeLa and COS-1 cells inhibits the ability of GCR{alpha} to bind to glucocorticoid-responsive element (GRE) containing promoters, and is a dominant-negative repressor of the trans-activating activity of ligand-bound GCR{alpha} in several cell types (11), but the role in steroid-induced cell death of T cells has not been examined. Previous studies have demonstrated elevated levels of GCRß in several diseases associated with GC insensitivity or resistance. These include GC-insensitive asthma, ulcerative colitis, chronic sinusitis, and fatal asthma unresponsive to intravenous doses of GCs (1–3, 25, 26). Interestingly, Sousa and coworkers (27) reported increased levels of GCRß expression in the T cells of their cohort of GC-resistant subjects with asthma previously described to have elevated c-fos expression in their mononuclear cells. However, direct evidence of a role for GCRß in resistance or insensitivity of cells to steroid action, independent of AP-1 activation, has not yet been demonstrated.

In the current studies, we have asked whether stable transduction of a murine T cell hybridoma would result in conversion to a steroid-insensitive phenotype, and whether such insensitivity was a result of direct interaction of GCRß with GCR{alpha}. Consistent with the report by Otto and colleagues (28) that the mouse genome was missing the GCR 9 ß exon, we have demonstrated at the protein level by immunohistochemical staining that mouse DO-11.10 hybridoma cells do not have the GCRß isoform. More importantly, we have found that expression of GCRß by DO-11.10 hybridoma cells does indeed result in steroid insensitivity, which correlates well with its expression level.

The likely mechanisms for GC insensitivity in GCRß overexpressed DO-11.10 hybridoma cells is via the generation of GCR{alpha}:GCRß heterodimers. We demonstrated direct interactions between GCRß and murine GCR by immunoprecipitation of GCRß, followed by Western blot analysis for GCR{alpha}. It has been found that heterodimers between GCR{alpha} and GCRß are able to bind to a consensus GRE, but that they are unable to stimulate transcription as efficiently as GCR{alpha} homodimers (12). Consistent with this concept, a truncated form of GCRß (hGR728T) which lacks the unique 15 amino acids at the COOH terminus does not repress the transcriptional activity of GCR{alpha}. Of note, this final portion of the GCRß molecule is also important in heterodimerization with GCR{alpha}.

Finally, it has been reported that even in the presence of 10-fold excess of GCRß, GCR{alpha} was still able to activate transcription from the MMTV promoter at the same rate (13). However, these studies used an inefficient transient transfection system in which only a minority of cells likely co-expressed both GCRß and GCR{alpha} in the same cell. Therefore, the relative amounts of GCRß needed to interfere with GCR{alpha} are not known. In the current study, we demonstrate that as small as a 2.5-fold increase of GCRß relative to GCR{alpha} (Figure 2) leads to a functional shift in corticosteroid sensitivity. These are levels that should be physiologically achievable, because Webster and coworkers (29) have demonstrated that following treatment of HeLa cells with TNF-{alpha}, relative levels of GCR{alpha}:GCRß rose from a ratio of 4:1 to 1:2. Importantly, such changes were associated with the development of corticosteroid resistance to hormone-mediated transactivation (29). Furthermore, we have recently found that the ratio of GCRß: GCR{alpha} in neutrophils, known to be a naturally steroid-resistant cell type, is 73-fold higher than steroid-sensitive T cells (19).

In summary, the current studies support the concept that increased GCRß expression can contribute to corticosteroid insensitivity independent of the activation of other transcription factors. Although the molecular basis for GCRß-induced corticosteroid insensitivity remains to be determined, it is likely that it involves the formation of GCR{alpha}:GCRß heterodimers. Finally, the cellular levels of GCRß needed to interfere with GCR{alpha} function are likely to be achievable in vivo under conditions of tissue inflammation.


   Acknowledgments
 
The authors would like to thank Anne Dunlap and Johnny Gutierrez for their technical help in the laboratory. They greatly appreciate the assistance of Hannah and Abraham Kupfer in developing the intracellular staining protocol for GCRß, and they would like to thank Thomas Mitchell for his most helpful advice in retroviral transfections. They also thank Maureen Sandoval for her patient assistance in preparation of this manuscript. This study was supported in part by NIH grants HL36577, AR41256, HL37260, and HL34303; by General Clinical and Research Center grant MO1 RR00051 from the Division of Research Resources; by an American Lung Association Asthma Research Center Grant; and by the University of Colorado Cancer Center.

Received in original form March 25, 2002

Received in final form May 7, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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