U-2 OS, MG-63, Hep-G2, and HeLa cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco modified Eagle medium-F-12 medium supplemented with 10% fetal calf serum. Wild-type (WT) and GSK-3β-null mouse embryonic fibroblasts (MEFs) were generously provided by Jim Woodgett (Mount Sinai Hospital-SLRI) and cultured as previously described (21). Geneticin (500 μg/ml; Invitrogen) and hygromyocin (200 μg/ml; Invitrogen) were used to establish U-2 OS cell lines stably expressing WT-, S404A-, and S404D-hGRα, similar to that described previously (29). Briefly, U-2 OS cells were transfected with pTET-OFF and clonally selected with Geneticin to produce the parental U-2 OS cell line (null). Parental U-2 OS cells were then transfected with pTRE2 vector containing WT-, S404A-, or S404D-GR and clonally selected with Geneticin and hygromycin. Receptor levels were compared by Western blot analysis, and clones expressing comparable receptor levels were used. All results were validated with the use of several independent clones for each mutant of the GR. FuGENE reagent (Roche, Indianapolis, IN) was used at 3 μl per 1 μg of DNA for transfection and transient expression of hGRα-Flag, GSK-3β, and shGSK. All Dex treatments were performed in growth medium supplemented with 10% charcoal-dextran-stripped fetal calf serum.

Following treatments as indicated, the cells were washed in ice-cold phosphate-buffered saline (PBS) and lysed for 60 min on ice in Triton X-100 lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EGTA, 1% Triton X-100 [pH 7.4]) supplemented with phosphatase inhibitor cocktail set II (Calbiochem) and protease inhibitor cocktail tablets (Roche). The resulting detergent-solubilized whole-cell extracts were clarified by microcentrifugation and quantitated using a BCA protein quantitation kit (Pierce, Rockford, IL). Immunoprecipitations were carried out by adding anti-Flag (Sigma), anti-GR 57 (11), or anti-GR (BD Biosciences, San Jose, CA) antibodies to 500 μg of whole-cell extracts, followed by incubation for 16 h at 4°C with slow rotation. The resulting immunocomplexes were collected by microcentrifugation and washed several times with PBS. For in vitro kinase assays, GR immunocomplexes were incubated with active GSK-3β (3 ng; Chemicon), 20 mM MOPS (morpholinepropanesulfonic acid), 10 mM magnesium acetate, 100 μM ATP, and 1 μCi of γ-³²P for 10 min at 30°C. Kinase reactions were stopped with the addition of 3× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer and boiled for 5 min. All immunocomplexes were then resolved on 4 to 20% ReadyGel Tris-Gly gels (Bio-Rad, Hercules, CA). For Western blot analysis, 20 to 50 μg of protein from whole-cell extracts was similarly resolved. Nuclear and/or cytoplasmic fractionation was performed by using a Biovision kit (Biovision, Mountain View, CA) according to the manufacturer's directions. All proteins were electrophoretically immobilized to nitrocellulose membranes, which were subsequently probed with anti-GR antibodies (1:1,000; BD Biosciences), anti-GR 57 antibodies (1:500), anti-pSer404 GR antibodies (1:1,000), anti-pSer211 GR antibodies (1:2,000), anti-β-actin antibodies (1:2,500; Chemicon, Billerica, MA), anti-Bcl-xL antibodies (1:1,000; Chemicon), anti-GSK-3β antibodies (1:500; Cell Signaling, Danvers, MA), anti-GSK-3α antibodies (1:500; Chemicon), anti-p65 (RelA) antibodies (1:500; Cell Signaling), anti-CBP/p300 antibodies (1:500; Santa Cruz Biotechnologies, Santa Cruz, CA), anti-GRIP1 antibodies (1:500; Chemicon), anti-cIAP antibodies (1:1,000; Cell Signaling), anti-GAPDH antibodies (1:500; Cell Signaling), anti-nucleolin antibodies (1:1,000; Santa Cruz Biotechnologies), or anti-survivin antibodies (1:1,000; Stressgen, Ann Arbor, MI). Densitometry and quantitation of three independent experiments was performed by using NIH ImageJ software and normalized to β-actin expression.

Flag-tagged human GRα was transiently expressed in U-2 OS cells for 48 h and then immunoprecipitated with anti-Flag antibodies conjugated to agarose beads (Sigma). Immunoprecipitates were diluted in 3× SDS-PAGE sample buffer and electrophoresed on 4 to 20% Tris-glycine gels. Gel bands were stained with Simply Blue Stain (Invitrogen), excised, and digested with trypsin (Promega, Madison, WI) for 8 h (9). Afterward, the samples were analyzed for phosphorylated peptides by nanoLC-ESI-MS/MS both with and without the use of immobilized metal ion affinity chromatography essentially as previously described (7, 9). The extent of phosphorylation was estimated by stable isotope-free relative and absolute quantitation essentially as described by Steen et al. (43).

U-2 OS cells stably expressing WT-, S404A-, or S404D-GR were plated onto 35-mm dishes with glass bottom inserts. The cells were treated as indicated, washed in PBS, fixed in 4% paraformaldehyde, and permeabilized by Triton X-100 (0.2% in PBS). The cells were then blocked in PBS supplemented with 1.5% normal goat serum, followed by incubation with anti-GR (1:500 dilution; BD Biosciences) or anti-pSer404-GR antibodies, followed by Alexa Fluor 488 (1:1,000; Invitrogen) or Alexa Fluor 594 antibodies (1:1,000; Invitrogen). Cells were then mounted in Vectashield containing DAPI (4′,6′-diamidino-2-phenylindole; Vecta Laboratories, Burlingame, CA). Confocal images were taken on a Zeiss LSM510-NLO meta using a C-Apochromat ×40/1.2 W Corr DIC objective lens.

U-2 OS cells stably expressing WT- or S404A-GR were incubated in growth media supplemented with 10% charcoal-dextran-stripped fetal calf serum for 48 h and then treated, in triplicate, with either vehicle (H₂O) or 100 nM Dex for 6 h. The total RNA was harvested (Qiagen, Valencia, CA), and gene expression analysis was conducted by using Agilent whole-genome 4 × 44 multiplex format oligonucleotide arrays (catalog no. 014850; Agilent Technologies, Santa Clara, CA) according to the Agilent 1-color microarray-based gene expression analysis protocol. Starting with 500 ng of total RNA, Cy3-labeled cRNA was generated according to the manufacturer's protocol. For each sample, 1.65 μg of Cy3-labeled cRNAs was fragmented and hybridized for 17 h in a rotating hybridization oven. Slides were washed and then scanned with an Agilent scanner. The data were obtained using Agilent Feature Extraction software (v9.5), with the one-color defaults for all parameters. Agilent Feature Extraction software performed error modeling, adjusting for additive and multiplicative noise. In order to identify differentially expressed genes (P < 0.01), an error-weighted analysis of variance (ANOVA) with the Bonferroni correction was performed using the Rosetta Resolver system (version 7.0; Rosetta Biosoftware, Kirkland, WA). The Bonferroni test correction was used to reduce the number of false positives. Finally, the ANOVA results were subjected to Ingenuity Pathways (Ingenuity Systems, Redwood City, CA) analysis for functional analysis.

U-2 OS cells stably expressing WT- or S404A-GR were treated with 100 nM Dex or vehicle (H₂O) for 6 h, and total RNA was isolated by using a Qiagen RNeasy minikit. Real-time PCR was performed by using the 7900HT sequence detection system and predesigned primer/probe sets from Applied Biosystems (Foster City, CA) according to the manufacturer's instructions. The signal obtained from each gene primer-probe set was normalized to that of the unregulated housekeeping gene β-actin primer-probe set (also available from Applied Biosystems). Each primer-probe set was analyzed with at least three different sets of RNA.

U-2 OS cells were plated into 24-well plates (35,000 per well). pGRE₂-luc and pGL2-3XMHC-luc reporter constructs were used for GR transactivation and transrepression assays, respectively. Cells were treated with Dex or vehicle (20 h) 24 h after reporter transfection. The luciferase activity was measured as previously described (29). In each experiment, the firefly luciferase activity normalized to the Renilla luciferase activity was measured in duplicate and averaged. Each experiment was repeated three times.

U-2 OS cells stably expressing WT- or S404A-GR were treated with 100 nM Dex for 1 h. Then, anti-GR 57 antibodies were used to immunoprecipitate GR from cellular extracts overnight at 4°C. The immunocomplexes were then washed two times in cold PBS before incubation with 1 μM Dex for 10 min at 25°C. The GR was then incubated with 50 ng of sequence-grade trypsin (Promega) for 15 min at 25°C. A 3× SDS sample loading dye was then added, and the samples were boiled 10 min before being resolved on 4 to 20% Tris-glycine gels and probed with anti-GR antibodies (1:2,000; BD Biosciences).

Cell death was measured by propidium iodide (PI) staining. After treatment, adherent and suspended cells were collected, centrifuged, and resuspended in a PBS solution containing 10 μg/ml of PI (Invitrogen). A total of 2 × 10⁵ control or treated cells were processed, and 1 × 10⁴ cells were immediately analyzed by using a BD FACSort flow cytometer (Franklin Lakes, NJ) with an excitation wavelength of 488 nm and an emission wavelength of 585 nm. For GSK-3 knockdown experiments, pShuttle-GFP-shScramble and pShuttle-GFP-shGSK-3 constructs were expressed for 3 days, after which flow and Western analyses were performed in order to select for the green fluorescent protein-positive cell populations and confirm GSK-3 knockdown, respectively. Each experiment was conducted in triplicate.

Since we found that the GR was phosphorylated at Ser404 in an agonist-dependent manner, we next investigated whether the phosphorylation status of Ser404 affected GR signaling. GR is a nuclear receptor and functions primarily to enhance and/or repress gene transcription (19); therefore, we first studied the effects of Ser404 phosphorylation on nuclear localization of the GR. In U-2 OS cells expressing WT-GR, GR was predominantly cytoplasmic, and stimulation with Dex for 1 h drove its nuclear accumulation (Fig. ​(Fig.3A).3A). In addition, U-2 OS cells expressing the unphosphorylatable GR (S404A) or a Ser404 to aspartic acid mutant of GR (S404D-GR), a mutation that mimics both the size and the negative charge of a phosphorylated serine residue, were assayed for GR localization. We found that cells expressing S404A-GR or S404D-GR had complete nuclear localization of the GR after 1 h of Dex stimulation (Fig. ​(Fig.3A).3A). These results suggest that Ser404 phosphorylation does not affect the ability of the receptor to translocate from the cytoplasm to the nucleus upon hormone stimulation. Since Dex stimulation enhanced Ser404 phosphorylation and GSK-3β is primarily a nuclear kinase, we hypothesized that phosphorylation of the GR occurs in the nucleus. Therefore, we determined the cellular location of pSer404-GR. Immunofluorescent staining of U-2 OS cells expressing WT-GR demonstrated that the GR was cytoplasmic and contained a basal amount of pSer404 phosphorylation. Upon Dex treatment, GR translocated to the nucleus and Ser404 phosphorylation of GR increased. Interestingly, pSer404-GR was mainly localized to the perinuclear region (Fig. ​(Fig.3B),3B), suggesting that once phosphorylated in the nucleus, the GR maybe exported from the nucleus. To address this question, we inhibited Ser404-GR phosphorylation with the GSK-3α/β inhibitor BIO and found enhanced nuclear localization of GR and a prevention of the accumulation of pSer404 in the perinuclear space, further supporting the hypothesis of receptor phosphorylation leading to nuclear export. To confirm these results via an alternative approach, WT-GR-expressing U-2 OS cells were stimulated with 100 nM Dex for 1 h and separated into cytoplasmic and nuclear fractions. Figure ​Figure3C3C shows that GR was primarily in the cytoplasm in the absence of hormone and, upon Dex treatment, GR accumulated in the nucleus. Hormone treatment also increased GR phosphorylation on Ser211 and Ser404. However, while pSer211-GR was both cytoplasmic and nuclear (47), pSer404-GR was largely cytoplasmic (Fig. ​(Fig.3C).3C). Together, these results suggest that Ser404-GR phosphorylation does not affect the import of the GR into the nucleus but may affect its export into the cytoplasm.

Our results above raise the possibility that Ser404 phosphorylation may affect GR export from the nucleus. Once GR exits the nucleus, it can be targeted by the proteasome for degradation, which limits the cellular response to GCs (27). The GR protein has been shown to have a half-life of 15 to 20 h, and this half-life decreases to 7 to 9 h upon agonist stimulation (30). Previous work by our lab demonstrated that the phosphorylation status of mouse GR affected receptor half-life (48). Therefore, we wanted to determine whether Ser404 phosphorylation of the human GR also alters the stability of the protein in a ligand-dependent or -independent manner. In accordance with previous reports, we found that WT-GR had a half-life of 21.9 h, and this half-life was decreased to 9.8 h upon exposure to Dex (Fig. ​(Fig.4A).4A). Interestingly, the half-life of unphosphorylatable GR (S404A) had an increased half-life of 32.6 h that was decreased to 13.1 h when exposed to Dex (Fig. ​(Fig.4B).4B). In contrast, the phospho-mimic mutant of GR (S404D) had a decreased half-life of 18.1 h, which was further decreased to only 2.5 h upon Dex exposure (Fig. ​(Fig.4C).4C). These data suggest that the phosphorylation of Ser404 has an important role in GR protein stability. Furthermore, our results raise the possibility that cells with aberrant GSK-3β activity resulting in enhanced Ser404 phosphorylation may preferentially downregulate GR protein, leading to states of GC resistance.

Our results thus far identified Ser404 as a potentially important residue in regulating GR signaling. Therefore, we wanted to determine whether there was physiological significance of Ser404 phosphorylation. To accomplish this goal, we first examined the ability of WT-GR and phosphorylation-deficient GR (S404A) to regulate gene transcription in U-2 OS cells. For these experiments, WT- and S404A-GR-expressing U-2 OS cells were treated with vehicle or 100 nM Dex for 6 h, and the total cellular RNA was collected for whole human genomic microarray analysis. It is important to note that the level of GR expression after Dex treatment was comparable in both WT- and S404A-GR samples (Fig. ​(Fig.5A).5A). We found that the WT receptor regulated 7,772 genes, while the phosphorylation-deficient mutant S404A regulated 9,617 genes. Of these genes, 4,993 were common to both WT- and S404A-GR, 2,779 were unique to WT-GR, and 4,624 were unique to S404A-GR (Fig. ​(Fig.5A).5A). To help elucidate the mechanism of enhanced gene regulation by S404A-GR, we separated the Dex-induced genes from the Dex-repressed genes and found that S404A-GR had an increased number of genes repressed compared to the WT receptor (Fig. ​(Fig.5B).5B). The receptor phosphorylation-specific regulation of endogenous genes was also confirmed by real-time PCR (Fig. ​(Fig.5C).5C). Some genes such as FOXO4 (induced) and Bcl 3 (repressed) were commonly regulated by WT- and S404A-GR. However, S404A-GR regulated additional genes such as the genes encoding Kip2 (induced) and interleukin-1β (repressed). Finally, in order to understand the repertoire of gene regulation by WT and phospho-deficient GR, data were loaded into Ingenuity Pathways analysis software and compared for biological pathway regulation. Genes involved in cell growth and proliferation, hematological system development and function, and immune response were highly regulated in the WT-GR-expressing cells (Fig. ​(Fig.6A).6A). Interestingly, the regulation of these same genes was significantly different in cells expressing S404A-GR. Of greater interest, we found that S404A-GR-expressing cells had the ability to regulate genes involved in lipid metabolism, small molecule biochemistry, and cancer, whereas the WT-GR-expressing cells did not significantly regulate this same pathway (Fig. ​(Fig.6B).6B). Real-time PCR analysis was also performed to verify differentially regulated genes involved in these two pathways and is shown in the right panels of Fig. ​Fig.5C.5C. Indeed, SLC16A5, STAT4, GPR126, and interleukin-4 were differentially regulated by Dex in WT-GR and S404A-GR-expressing cells. These data indicate that the lack of Ser404 phosphorylation not only enhances GR-mediated gene regulation but also significantly alters the repertoire of GR-regulated genes. These results also suggest that the GSK-3β activity in cells is an important determinant in the GR transcriptional response to GC hormones.