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Jennifer B. Collins,2
Sherry F. Grissom,2 and
John A. Cidlowski1*
Molecular Endocrinology Group, Laboratory of Signal Transduction,1 Microarray Center, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 277092
Received 12 February 2007/ Returned for modification 16 April 2007/ Accepted 25 July 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An unresolved question in the field is how glucocorticoids selectively kill bone cells and protect other cells such as hepatocytes from death. Although tissue-selective ligand availability and cofactor recruitment can partially explain the cell type-selective effects of glucocorticoids (1, 9, 11, 12), recent evidence suggests that tissue-selective expression of glucocorticoid receptor (GR) isoforms may also play a critical role in the tissue-selective responsiveness of glucocorticoids. GR isoforms include GR
and GRß, which are generated via alternative splicing, with GR being expressed at relatively higher levels in the majority of the tissues examined (17). In addition, each GR transcript generates additional isoforms via alternative translation initiation mechanisms (18). The GR-A isoform, one of the eight translational isoforms, is the full-length receptor, and the other GR isoforms have smaller N termini (18). We previously demonstrated that translationally generated GR isoforms regulate both common and distinct sets of genes in the same cell (18). Here, we expressed wild-type human GR and individual GR isoforms in U-2 OS cells, a human osteoblastic sarcoma cell line that lacks endogenous GR, and show that these translationally generated GR isoforms selectively regulated the genome. Importantly, the GR isoforms had distinct capabilities to activate the cell death program despite having identical DNA binding and ligand binding domains. The molecular basis for this functional difference among GR isoforms appeared to involve selective coactivator recruitment and chromatin modification on proapoptotic genes. Interestingly, the ability to repress NF-
B activity was comparable among GR isoforms.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-fluoro-16-methyl-11ß,17,21-triol-3,20-dione) (DEX) was purchased from Steraloids (Newport, RI). Rabbit anti-GR antibody 57 was previously described (6). All other primary antibodies were obtained from Upstate (Charlottesville, VA), except rabbit anti-granzyme A (GZMA) (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-poly(ADP-ribose) polymerase (PARP) (Pharmingen, San Diego, CA). Goat anti-rabbit antibodies conjugated with horseradish peroxidase were obtained from Jackson Immunoresearch (West Grove, PA). All other reagents were obtained from Sigma (St. Louis, MO) unless otherwise specified. Animals. The studies were approved by the institutional animal use committee at Northwestern University. After terminal anesthesia, spleens were harvested from 6-week-old male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME), and calvaria were harvested from 1- to 2-day-old C57BL/6 pups. Primary spleen cells were subjected to red blood cell lysing and gradient centrifugation purification. Calvaria were trimmed and subjected to 15 min of collagenase (20 U/ml; MP Biomedicals, Solon, OH) digestion threetimes. Cells from the last two digestions were harvested and cultured until Western blot analysis.
Cell lines.
COS-1 cells, U-2 OS parental cells, and U-2 OS cells expressing each of the GR isoforms were described previously (18). U-2 OS cell subclones expressing two GR isoforms were produced as described previously (18). The expression of one GR isoform in these subclones can be down-regulated by doxycycline, and the expression of the second GR isoform, the GR-A isoform, derived from pcDNA-human GR containing an optimal Kozak context, is constitutive. Cells were treated with vehicle, DEX (100 nM), and/or lipopolysaccharide (LPS) (1 µg/ml).
Stably expressing siRNAs in U-2 OS cells. U-2 OS cells expressing the GR-C isoform were plated onto a 100-mm dish at 80% confluence and transfected with 15 µg of plasmid DNA expressing each small interfering RNA (siRNA). Sequences of siRNAs against GZMA were CACCTCAACTGGATAATTA, ACGCGAAGGTGACCTTAAA, and TAACCTACAGTACGTATCA (scrambled); and sequences of siRNAs against caspase-6 (CASP6) were ACATTAACTGGCTTGTTCA, GATGCAGCCTCCGTTTACA, and CTATAATTACCAAGTGCTC (scrambled). Each siRNA was cloned into pSUPER.retro.puro (Oligoengine, Seattle, WA) and transfected into U-2 OS GR-C cells using Transit LT1 reagent (Mirus Corp., Madison, WI) at 5 µl per 1 µg DNA according to the manufacturer's protocol. One day after transfection, cells were passaged onto three 150-mm dishes, and selection was initiated 24 h later by supplementing the growth medium with 5 µg/ml of puromycin (Invitrogen, Carlsbad, CA). Selection lasted for 4 weeks, and the medium was replenished every 5 days. Healthy colonies were then transferred onto 12-well plates and propagated in growth medium containing 1 µg/ml of puromycin. Positive clones were screened using Western blot analysis.
Western blot analysis. Procedures for preparing cell lysates and animal tissues for Western blot analysis were previously described (20). Lysates containing 10 to 50 µg of proteins were resolved on 4 to 12% NuPage Bis-Tris gels (Invitrogen), and titers for antibodies were 1:1,000 to 1:2,500 for anti-GR 57 antibodies, 1:150 for anti-GZMA, 1:500 for anti-CASP6, 1:2,000 for anti-PARP, and 1:10,000 to 1:40,000 for anti-ß-actin. Densitometry analysis was performed using NIH Image software.
[3H]DEX binding assay. Cytosolic GR was prepared by resuspending cells in binding buffer (10 nM HEPES, 20 nM NaMoO4, 1 nM EDTA, and 1 nM dithiothreitol containing protease inhibitor cocktails [Roche, Indianapolis, IN] [pH 7.35] at 4°C) and sonication for 10 s at setting 4 on a Branson 150 sonifier (Branson Ultrasonics Co., Danbury, CT), followed by centrifuging at 55,000 rpm for 45 min at 4°C. Eight concentrations of [3H]DEX (Perkin-Elmer, Wellesley, MA) ranging from 0.5 to 2,500 nM were used for binding reactions. After incubation for 16 h at 4°C, unbound [3H]DEX was absorbed and stripped using activated charcoal. The amount of [3H]DEX bound to GR was counted on a 1450 Microbeta Wallac Jet (Perkin-Elmer). Nonspecific binding was defined by the addition of a 100-fold excess of unlabeled DEX. Saturable binding was normalized to protein content. The one-site binding subroutine in Prism (GraphPad, San Diego, CA) was used to obtain the total binding (Bmax) and apparent Kd (dissociation constant) values.
Microarray analysis.
Gene expression analysis was conducted using Agilent Whole Human arrays (Agilent Technologies, Palo Alto, CA). Three separate biological replicates of cytoplasmic RNA samples were harvested and purified from each of the U-2 OS cell lines stably expressing human GR (hGR), -A, -B, -C3, or -D3 treated with 100 nM DEX for 0 or 6 h using RNeasy Midi kits (Invitrogen). Total RNA was amplified using the Agilent Low RNA Input fluorescent linear amplification lit protocol. Starting with 500 ng of total RNA, Cy3-labeled cRNA was produced according to the manufacturer's protocol. For each sample, 1.5 µg of Cy3-labeled cRNAs was fragmented using the Agilent In Situ Hybridization kit protocol. Hybridizations were performed for 17 h in a rotating hybridization oven at 65°C at 4 rpm. Slides were washed with a solution containing 6x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7]) plus 0.005% N-lauroyl sarcosine for 1 min and then washed in a solution containing 0.06x SSPE plus 0.005% N-lauroyl sarcosine for 1 min at 37°C. The slides were dried by slowly removing from second wash solution and then scanned with an Agilent G2565 scanner (10 µm and with extended dynamic range) and processed with Agilent Feature Extraction v9.1. Consequently, three separate chips were used for each of the isoforms at each treatment condition, yielding 30 chips in total: five isoforms by three biological replicates by two time points. The resulting files were imported into the Rosetta Resolver system (version 6.0; Rosetta Biosoftware, Kirkland, WA). This system performs data preprocessing and error modeling (33). The ratios (treated to controls) were analyzed at the Entrez gene level in Resolver and were considered to be differentially expressed if the P value was less than 0.0001. Clustering analysis was performed using the Rosetta Resolver analysis software. The list of genes used in the clustering came from GeneSpring's Gene Ontology SLIMS, Cell Death GO 0008219 (2).
Real-time PCR. RNA samples were extracted from cells using an Absolutely RNA RT-PCR Miniprep kit (Stratagene, La Jolla, CA) and treated with DNase according to the manufacturer's protocol. The level of individual mRNA in each sample was measured using a one-step reverse transcription-PCR procedure on a Prism 7900HT thermocycler (Applied Biosystems, Foster City, CA). Each reaction mixture (25-µl total volume) contained 12.5 µl of 2x Universal PCR Master Mix, No AmpErase (Applied Biosystems), 1.25 µl of the predeveloped gene expression system (Applied Biosystems), 250 ng of total RNA, 2 U/µl of RNase inhibitor (Roche Diagnostics), and 0.4 U/µl murine leukemia virus reverse transcriptase (Roche). Each experiment was performed in duplicates at least twice. Quantification was achieved using the Sequence Detection software 2.0 absolute level subroutine (Applied Biosystems). Human cyclophilin B mRNA levels were measured as described above by using primers 5'-AGATGGCAAGCATGTGGTGTT and 5'-TACTCCTTGGCGATGGCAA and probe 5'-ATCATCGCAGACTGCGGCAAGATCGA labeled with a 6-carboxyfluorescein (FAM) reporter at the 5' end and a 6-carboxytetramethylrhodamine quencher at the 3' end. Human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) levels were measured as described above by using primers 5'-GAAGGTGAAGGTCGGAGTC and 5'-GAAGATGGTGATGGGATTT and probe 5'-CAAGCTTCCCGTTCTCAGCC labeled with FAM reporter at the 5' end and a 6-carboxytetramethylrhodamine quencher at the 3' end. Cyclophilin B mRNA or GAPDH levels were used to normalize the levels of genes of interests.
Flow cytometry analysis.
Labeling with annexin V and propidium iodide (PI) was processed using the TACS annexin V-fluorescein isothiocyanate apoptosis detection kit (Trevigen, Inc., Gaithersburg, MD) and 10 µg/ml PI according to the manufacturer's instructions. Caspase activity was determined using CaspaTag In Situ Assay kits (Chemicon International, Temecula, CA) for the fluorescence detection of caspase-3 and caspase-7 activities according to the manufacturer's instructions. A total of 1.5 x 105 control or treated cells were processed, and 1 x 104 cells were analyzed using a BD (Franklin Lakes, NJ) FACSort flow cytometer with an excitation wavelength of 488 nm and emission wavelength of 530 nm (for annexin V-fluorescein isothiocyanate and CaspaTag) or 585 nm (for PI). The DNA content of each sample was determined by fixing cells via the slow addition of cold 70% ethanol to a volume of 
1.5 ml, which was then adjusted to 5 ml with cold 70% ethanol. The samples were stored at 4°C overnight before analysis. For flow cytometric analysis, the fixed cells were pelleted, washed once in phosphate-buffered saline, and stained in 1 ml of 20 µg/ml of PI containing 1 mg/ml RNase in phosphate-buffered saline for 20 min. Cells (7,500) were analyzed by flow cytometry by gating onto a PI area-versus-width dot plot to exclude cell debris and cell aggregates. The percentage of degraded DNA was determined by dividing the number of cells with subdiploid DNA by the total number of cells examined under each experimental condition.
NF-B reporter assays.
COS-1 cells were plated at approximately 80% confluence on 12-well plates. Twenty nanograms of pTRE-GR, -A, -B, -C, or -D; 200 ng pCMVp65 (27); 150 ng pGL2-3XMHCLuc (27); and 20 ng of pGL3-hRL (18) were transfected into COS-1 cells using Transit LT1 reagent at 3 µl per 1 µg DNA according to the manufacturer's protocol. Drug or vehicle treatment started 4 h after transfection and lasted for 20 h in growth medium supplemented with 5% charcoal-dextran-stripped fetal calf serum. Luciferase activity was measured as previously described (19). In each experiment, the firefly luciferase activity normalized to the Renilla luciferase activity was measured in triplicate and averaged. Each experiment was repeated three times.
ChIP assay.
Chromatin immunoprecipitation (ChIP) assays were performed using a ChIP kit from Upstate according to the manufacturer's protocol. Briefly, 5 x 106 cells grown on 150-mm dishes supplemented with charcoal-stripped fetal bovine serum for 3 days were treated with vehicle or DEX (100 nM) for 6 h. Cells were then harvested in lysis buffer in the presence of protease inhibitors sonicated on a Branson Sonifier 150 at setting 4 with 10-s pulses four times on ice. The amount of total input DNA for each immunoprecipitation reaction was 35 µg. Sources of antibodies are listed above. The amounts of antibodies per immunoprecipitation reaction were 4 µl for anti-GR, 2 µl for anti-CBP, 5 µl for anti-p300, 5 µl for anti-acetylated H4, and 2 µl for anti-phosphorylated polymerase II. The level of precipitated DNA containing the GZMA promoter region (146 bp) was quantified using real-time PCR analysis. The primer sequences were 5'-GCACTGTGCCCTATTCAAGAAACC and 5'-ACACAAGGCAAACCATACATGCAG, and the probe (FAM-labeled) sequence was 5'-ATCCAAGAACATCTGGTGCAGGAGGT. All measurements were normalized to the levels of input DNA. In addition, precipitated DNA containing other promoter regions was subjected to PCR analysis, and the primer sequences used were 5'-GCCACATGCTGTCCTTGTAGTGAA and 5'-AGGGAAGGCTTTGGAGGGTTGTAA for H+/K+-exchanging ATPase (ATP4A), 5'-ATC TGT GGG ATC AGG CTG AG and 5'-CTC TGT GGG ACA GAG TTG TC for vitamin D receptor (VDR), 5'-AAC GGA AAG GAC CGG CAG TTG and 5'-CTG GAA AGT CCT TCC GAC CA for inhibitor of nuclear factor-B (I-B), and 5'-CTT AAC TCA GGA ACC ATC CTC TCT G and 5'-TGC CCT CCT CTT CCT GAA G for CASP6.
Statistical analysis. One-way analysis of variance (ANOVA) was performed, followed by a Tukey post hoc comparison using Prism software (GraphPad, San Diego, CA) to compare the differences among treated cells and controls. A P value of <0.05 was considered to be significant.
Microarray data accession number. The microarray data in this publication were deposited in NCBI's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) (7) and are accessible through Gene Expression Omnibus series accession number GSE6711.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in U-2 OS cells.
To determine whether glucocorticoids can directly induce programmed cell death via GR in bone cells, we stably expressed wild-type hGR in U-2 OS cells, a human osteosarcoma cell line lacking endogenous GR (Fig. 1). When treated with the synthetic glucocorticoid DEX (100 nM), a striking contrast was observed between the parental cells and cells expressing GR: cells expressing GR were killed upon exposure to DEX, while parental cells lacking GR were not killed. To determine whether DEX-induced cell death in cells expressing GR is occurring by apoptotic mechanisms, we examined a series of morphological and biochemical parameters that are well-recognized integrative components of the programmed cell death process. Apoptotic characteristics displayed by U-2 OS cells expressing GR included distinct chromatin condensation (Fig. 1B), a loss of cell membrane integrity (Fig. 1C), externalization of phosphatidyl serine (Fig. 1D), PARP cleavage (Fig. 1E), DNA degradation (Fig. 1F), and caspase activity (Fig. 1G). In stark contrast, U-2 OS cells lacking GR did not exhibit any of these apoptotic phenotypes following glucocorticoid treatment. These findings support the notion that U-2 OS cells are fully capable of undergoing apoptosis in response to glucocorticoids and that GR expression is required for the proapoptotic actions of glucocorticoids in these cells.
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message produces multiple GR isoforms (18). However, the physiologic consequences of the expression of these multiple isoforms of GR are not fully understood. To determine whether the GR isoforms differ in their ability to induce apoptosis in bone cells, we stably expressed GR-A, -B, -C (C3 as a representative of three GR-C isoforms, C1, C2, and C3), and -D (D3 as a representative of three GR-D isoforms, D1, D2, and D3) isoforms individually in U-2 OS cells (18). GR protein levels were comparable among individual isoforms as determined by both Western blot analysis and [3H]DEX ligand binding assays (Fig. 2A). Western blot analysis showed that individual isoforms had similar expression levels in the absence or presence of DEX (100 nM for 2 h) and that the clone expressing wild-type GR contained the combination of all GR isoforms. The Bmax and apparent Kd values for cytosolic receptors obtained from cells expressing GR, -A, -B, -C, and -D isoforms were similar (Fig. 2A). In addition, the half-life of each of the GR isoforms was measured using Western blot analysis as well. There was no difference among the half-lives of the GR isoforms. The half-lives of the GR isoforms in the absence of ligand were 12 to 15 h, and exposure to DEX (100 nM) reduced the half-live of the GR isoforms to 6 h (Fig. 2B), indicating that all GR isoforms are fully capable of undergoing ligand-dependent downregulation (31). Previous studies indicate that all GR isoforms produced in these U-2 OS cells are functional, as all GR isoforms regulate transiently expressed reporter genes and endogenous U-2 OS genes (18).
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, -A, or -B isoform underwent apoptosis (Fig. 3), whereas more than 50% of cells expressing the GR-C isoform were killed by the same treatment (P < 0.05). Cells expressing the GR-D isoform had significantly less cell death than did cells expressing any of the other GR isoforms. These experiments indicate that GR-C isoform-expressing bone cells were killed by DEX earlier (by 12 h) and to a greater extent than other GR isoforms, whereas the GR-D isoform-expressing cells were relatively resistant to the apoptosis-inducing actions of glucocorticoid. This observation was confirmed in a second set of U-2 OS cell clones expressing each GR isoform (Fig. 4). These results suggest the possibility that GR isoform-specific apoptosis may occur in U-2 OS cells. Indeed, apoptotic landmarks including the externalization of phosphatidyl serine (Fig. 3C), PARP cleavage (Fig. 3D), DNA degradation (Fig. 3E), and caspase activity (Fig. 3F) all indicated GR isoform-selective regulation. For all apoptotic markers examined, the GR, -A, and -B isoforms had similar efficacies, and the GR-C isoform was twice as efficacious. For several markers, the GR-D isoform had significantly less activity than the GR, -A, and -B isoforms (Fig. 3).
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), 3,451 (GR-A), 2,945 (GR-B), 3,252 (GR-C), and 1,761 (GR-D). Collectively, the total number of genes regulated by at least one GR isoform reached 6,501. Consistent with a previous report that analyzed a limited number of genes (18), we show here that the majority of the GR target genes were selectively regulated by the GR isoforms. The number of genes commonly regulated by all GR isoforms was 468 (Fig. 5B). Each GR isoform regulated a unique set of genes in addition to the commonly regulated genes.
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, -A, -B, -C, and -D isoforms, respectively. Only 25 genes in this list were commonly regulated by all GR isoforms (Table 1). Many of the induced genes are proapoptotic. These included CASP6, caspase-7, receptor-interacting serine/threonine kinase 1, tumor necrosis factor (TNF) receptor superfamily members 1b and 10b, GZMA, DNase I-like 3, and caspase recruitment domain family members 12 and 14. In addition, there were repressed antiapoptotic genes. These included B-cell CLL/lymphoma 2 (BCL2)-associated athanogene 2, myeloid cell leukemia sequence 1 (MCL1), and Bcl-xL (BCL2L1) genes.
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B, CASP6, and GZMA (Fig. 7A). This observation is consistent with the fact that all the GR isoforms have identical DNA binding domains. Interestingly, in the absence of DEX, the GR-D isoform, but not any of the other GR isoforms, was found to bind to certain promoters. The mechanism for ligand-independent DNA binding by this GR isoform is not known, and it may be related to its unique nuclear localization pattern (18). The GR-D isoform is in the nucleus regardless of ligand activation; all the other GR isoforms reside in the cytoplasm in the absence of ligand and translocate to the nucleus upon ligand exposure. Additional ChIP assays were also performed using selective antibodies against cyclic AMP response element binding (CREB) binding protein (CBP) and p300, both of which are GR coregulators with histone acetylase activity (Fig. 7B). Significantly higher amounts of CBP and p300 were recruited to the GZMA promoter region by the GR-C isoform, whereas a significantly lower amount of each was recruited by the GR-D isoform than by the other GR isoforms. A similar relationship was found for acetylated histone H4 and phosphorylated RNA polymerase II on the same segment of the GZMA promoter (Fig. 7B). In contrast, the amount of GR protein recruited to the same GZMA promoter region was the same for different GR isoforms, as shown in Fig. 7A. Together, these data indicate that different GR isoforms have different abilities to recruit cofactors, to modify chromatin, and to recruit active RNA polymerase. Quantitative analysis using real-time reverse transcription-PCR confirming the preferential recruitment of cofactors by the GR-C isoform is shown in Fig. 7B. The preferential recruitment of cofactors by the GR-C isoform is gene specific, since coregulator recruitment by GR isoforms onto the promoter region of another gene, I-B, was similar among GR isoforms (data not shown).
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B reporter gene approximately 50% (Fig. 10A), equivalent to the degree of NF-B repression obtained by the other GR isoforms (Fig. 10A). As an index of NF-B activity, we determined the level of proinflammatory cytokines induced by LPS in the absence and presence of DEX in U-2 OS cells (Fig. 10B). TNF, interleukin-8 (IL-8), and granulocyte-monocyte colony-stimulating factors are all significantly induced by LPS in cells expressing different GR isoforms. Upon the addition of DEX, these LPS-induced genes were suppressed down to the baseline level by each of the GR isoforms, including the GR-D isoform. In summary, the GR-D isoform inhibits NF-B activity without stimulating apoptosis in bone cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GR is a transcription factor that impacts the genome on a large scale. Microarray analysis in particular revealed that each GR isoform had a distinct set of target genes. Collectively, the number gene targets of GR isoforms was over 6,000. Whole human genome surveys and unique contributions from individual GR isoforms result in such a large number compared to those obtained by microarray analyses performed previously by other groups (15, 26). Our results are consistent with the widespread physiological functions of glucocorticoids that span from metabolism, immunity, growth, and differentiation. The unique gene targets of GR isoforms may be a basis for the accurate timing and fine-tuning of each of the physiologic processes governed by glucocorticoids.
Our experiments also provide important evidence that translationally generated GR isoforms have distinct functions. The GR isoforms displayed distinct apoptosis-inducing activities, consistent with the observation that GR isoforms selectively regulated genes involved in apoptosis. The GR-C isoform was significantly more active in doing so than any other GR isoform, and the ability of the GR-D isoform to regulate these apoptosis-related genes was significantly reduced. The enhanced activity of the GR-C isoform was evident by the earlier onset of cell death and by the higher percentage of cell death in the GR-C isoform-expressing cells than in the cells expressing other GR isoforms. We also demonstrated that selective cofactor recruitment was likely the mechanism underlying the functional differences among GR isoforms. GR isoforms differ structurally at the length of the N-terminal domain. The N-terminal domain contains the main transactivation activity of the receptor and the residues pivotal in ligand-dependent coregulator recruitment (16). Consistent with those reports, we found that GR proteins recruited coregulators to the promoter region of the GZMA gene in an isoform-specific manner. It will be interesting to determine whether the N-terminal domains of GR isoforms have different molecular structures presenting different coregulator-interacting surfaces. While this notion is still speculative, recent evidence supports the hypothesis that after ligand and DNA binding, the GR N-terminal domain folds into an organized structure that promotes both intra- and intermolecular interactions and cofactor recruitment (14).
Patients taking glucocorticoids frequently encounter serious side effects. Osteoporosis, for instance, is one of the side effects that can be life-threatening (22). Therefore, much research has focused on developing new approaches to reduce the untoward effects of glucocorticoids, although few of these approaches have been successful. We showed that the GR-D isoform reduced U-2 OS cell-killing capability and that it maintained the NF-B-repressing activity. These data may provide a basis for the development of improved glucocorticoid regimens with reduced bone cell-killing side effects if the GR-D isoform maintains anti-inflammatory actions of glucocorticoids, such as inhibiting NF-B activity in immune cells. Further studies are needed to determine whether the GR-D isoform maintains the anti-inflammatory benefits of glucocorticoids and alleviates osteoporosis.
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ACKNOWLEDGMENTS |
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This research was supported by the Intramural Research Program of the NIH National Institute of Environmental Health Sciences and a divisional fund from the Division of Allergy-Immunology, Department of Medicine, Northwestern University.
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FOOTNOTES |
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Published ahead of print on 6 August 2007.
Present address: Division of Allergy-Immunology, Department of Medicine, and Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL 60611.
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