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Glucocorticoids are stress hormones that maintain homeostasis through gene regulation mediated by nuclear receptors. We have discovered that other cellular stressors are integrated with glucocorticoid signaling through a new hormone-independent phosphorylation site, Ser134, on the human glucocorticoid receptor (GR). Ser134 phosphorylation is induced by a variety of stress-activating stimuli in a p38 mitogen-activated protein kinase (MAPK)-dependent manner. Cells expressing a mutant glucocorticoid receptor incapable of phosphorylation at Ser134 (S134A-GR) had significantly altered hormone-dependent genome-wide transcriptional responses and associated hormone-mediated cellular functions. The phosphorylation of Ser134 significantly increased the association of the GR with the zeta isoform of the 14-3-3 class of signaling proteins (14-3-3zeta) on chromatin promoter regions, resulting in a blunted hormone-dependent transcriptional response of select genes. These data argue that the phosphorylation of Ser134 acts as a molecular sensor on the GR, monitoring the level of cellular stress to redirect glucocorticoid-regulated signaling through altered 14-3-3zeta cofactor binding and promoter recruitment. This posttranslational modification allows prior cellular stress signals to dictate the transcriptional response to glucocorticoids.
All organisms, from the single cell to the multiorgan human, are in constant communication with their environment and, in doing so, are challenged by external stressors. In order to maintain a homeostatic balance, living multicellular organisms have developed complex physiological adaptive responses to cope with these stressors. Glucocorticoids are a principal mediator of many stressors from fish to humans. When the body perceives stress, the hypothalamic-pituitary-adrenal (HPA) axis is activated, resulting in the release of the primary stress hormones, glucocorticoids, by the adrenal gland. These hormones affect nearly every organ and tissue in the body and are required for life. They influence everything from metabolism, cardiovascular maintenance, immune and inflammatory responses, central nervous system function, reproduction, and cell survival (9).
Glucocorticoids exert their action by binding and activating the glucocorticoid receptor (GR), a ligand-dependent transcription factor. The GR is comprised of a N-terminal transcriptional activation function domain (AF-1), a DNA binding domain, and a C-terminal ligand binding domain. Unliganded GR is sequestered in the cytoplasm within a heat shock protein 90 (hsp90)-chaperone complex. Upon ligand binding, a conformational change within the GR is induced to allow for nuclear translocation. Within the nucleus, the GR binds DNA and recruits transcriptional machinery as well as various cofactors to either positively or negatively regulate the transcription of target genes. The ability of the GR signaling pathway to associate with different cofactors contributes to the transcriptional output of a cell and the subsequent response to glucocorticoid hormones (34).
There are several mechanisms for expanding the plasticity of glucocorticoid signaling, including receptor isoforms and posttranslational modifications, such as phosphorylation, ubiquitination, and SUMOylation (23, 29, 43). All characterized GR posttranslational modifications reported to date require hormone binding to induce a molecular confirmation within the receptor that is susceptible for kinases to modify the GR. The hormone-dependent phosphorylation-mediated events ultimately lead to differential transcriptional responses of the GR. The phosphorylation of the GR also affects the stability of the protein by altering nuclear/cytoplasmic shuttling and targeting the receptor for ubiquitin-mediated proteasomal degradation. Thus, the ligand-dependent phosphorylation of the GR significantly impacts the cellular response to steroids (4, 7, 15–17, 26). In response to glucocorticoids, cyclin-dependent kinases (CDKs), mitogen activated protein kinases (MAPKs), and glycogen synthase kinase 3(GSK-3) gain access to the human GR (hGR) and phosphorylate it at several sites, with serines 203, 211, 226, and 404 being the most thoroughly characterized at the molecular level (15). These data suggest that ligand-dependent GR phosphorylation can act as a convergence point to integrate multiple signaling pathways with steroid signaling subsequent to ligand binding.
Here we report a novel mechanism that cells utilize to integrate cellular stress-mediated signaling pathways with steroid signaling prior to interactions between the nuclear receptor and its ligand. This integration point comes in the form of a novel hormone-independent GR phosphorylation site, serine 134. Serine 134 is hyperphosphorylated under an array of stressful conditions, including glucose starvation, oxidative stress, UV irradiation, and osmotic shock. Interestingly, cells expressing a mutant GR incapable of phosphorylation at Ser134 (Ser134-GR) had a redirection of the transcriptional response to hormone and an altered activation of distinct biological pathways. These alterations in gene expression profiles result from changes in the ability of phosphorylated Ser134-GR (phosphoSer134-GR) to interact with the 14-3-3zeta signaling proteins on different gene promoters within chromatin. Our results suggest that the level of cellular stress prior to hormone stimulation, as measured by changes in the phosphorylation of Ser134, dictates which genes will respond to glucocorticoids.
Dexamethasone (Dex) (1,4-pregnadien-9-fluoro-16-methyl-11β,17,21-triol-3,20-dione) was purchased from Steraloids (Newport, RI). Doxycycline, hydrogen peroxide, and lipopolysaccharide (LPS) were purchased from Sigma (St. Louis, MO). Tumor necrosis factor alpha (TNF-α) and transforming growth factor β (TGF-β) were purchased from R&D Systems (Minneapolis, MN). The kinase inhibitors SB203580, LY294002, KT5720, SP600125, and compound C were purchased from Calbiochem (San Diego, CA). Rabbit anti-phosphoSer134-GR antibodies were produced by using peptides made by AnaSpec (San Jose, CA), and the antisera were produced by Covance (Denver, PA). pTRE-hGRα-S134A and pTRE-hGRα-S134D were generated by the site-directed mutagenesis of pTRE-hGRα (30) using a QuikChange kit (Stratagene, La Jolla, CA). N-terminally Flag-tagged GRα (pcDNA-hGRα-Flag) was described previously (16). The knockdown of p38α was accomplished with Mission short hairpin RNA (shRNA) lentiviral transduction particles (Sigma), and 14-3-3zeta knockdowns were accomplished with small interfering RNA (siRNA) SMARTpools purchased from Dharmacon (Lafayette, CO). An expression vector containing pCMV 14-3-3zeta was purchased from Open Biosystems (Huntsville, AL), and pCMV DN 14-3-3zeta was created by PCR as previously described (45). All cloning and mutagenesis products were verified by DNA sequencing at the DNA Sequencing Core at the National Institute of Environmental Health Sciences (NIEHS).
U2-OS, A549, H9C2, hepatocytoma (HTC), and HeLa cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium (DMEM)-F-12 medium supplemented with 10% fetal calf serum. Geneticin (500 μg/ml; Invitrogen)-hygromycin (200 μg/ml; Invitrogen) was used to establish U2-OS cell lines stably expressing a polyclonal mixed population of wild-type hGRα (WT-hGRα), S134A-hGRα, and S134D-hGRα similar to that described previously (16). Receptor levels were compared by Western blot analysis, and cell populations expressing comparable receptor levels were used. Results were validated with the use of three independent mixed populations for each mutant of the GR. Some cell treatments were done in glucose-free DMEM (Invitrogen) supplemented with 5 g/liter glucose and/or 10% fetal bovine serum (FBS) or 250 mM mannitol. Primary thymocytes from adrenalectomized male Sprague-Dawley rats were isolated by Lindsay Smith as previously described (40). All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the NIEHS, National Institutes of Health.
Following the indicated treatments, cells were washed in phosphate-buffered saline (PBS) and lysed on ice in Triton X-100 lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 [pH 7.4]) supplemented with phosphatase inhibitor cocktail set II (Calbiochem) and protease inhibitor cocktail tablets (Roche). For immunoprecipitations, cells were collected in HEM buffer (10 mM HEPES [pH 7.4], 1 mM EDTA, 20 mM sodium molybdate) supplemented with phosphatase and protease inhibitor cocktails. The resulting whole-cell extracts were sonicated for 10 s, clarified by microcentrifugation, and quantitated by using the BCA protein quantitation kit (Pierce, Rockford, IL). Immunoprecipitations were carried out by the addition of anti-Flag (Sigma), anti-14-3-3zeta (Millipore, San Diego, CA), or anti-p38 MAPK (Cell Signaling, Beverly, MA) antibody and protein A-Sepharose beads to whole-cell extracts and incubation for 16 h at 4°C with slow rotation. The resulting immunocomplexes were collected by microcentrifugation and washed. All immunocomplexes and cell extracts were resolved on 4 to 20% ReadyGels Tris-Gly gels (Bio-Rad, Hercules, CA). All proteins were electrophoretically immobilized onto nitrocellulose membranes, which were subsequently probed with anti-57-GR antibodies (1:1,000) (10), anti-phosphoSer134-GR antibodies (1:1,000), anti-phosphoSer211-GR antibodies (1:2,000; Cell Signaling), anti-β-actin antibodies (1:2,500; Chemicon, Billerica, MA), anti-p38 MAPK antibodies (1:1,000; Cell Signaling), anti-phospho-p38 MAPK antibodies (1:500; Cell Signaling), anti-phosphoSer404 GR antibodies (1:1,000) (16), anti-pan-14-3-3 antibodies (1:1,000; Millipore), anti-14-3-3zeta antibodies (1:1,000; Millipore), or anti-Hsp90 antibodies.
The identification of phosphorylated GR residues by mass spectrometry was described previously (16). Briefly, immunoprecipitated Flag-tagged human GRα was digested with trypsin (Promega, Madison, WI) and analyzed for phosphorylated peptides by nano-liquid chromatography electrospray ionization tandem mass spectrometry (nanoLC-ESI-MS/MS) both with and without the use of immobilized metal ion affinity chromatography. The extent of phosphorylation was estimated by stable isotope-free relative and absolute quantitations determined by comparing the areas under the peaks for each eluted peptide.
U2-OS cells stably expressing WT-, S134A-, or S134D-GR or HTC cells were plated onto 35-mm dishes with glass-bottom inserts. The cells were treated as indicted, fixed in 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. The cells were then blocked with goat serum and incubated with anti-57-GR (1:500), anti-14-3-3zeta (1:2,000), or anti-BuGR2 GR (1:1,000) antibodies followed by Alexa Fluor 488 or 593 staining (1:1,000; Invitrogen). Confocal images of U2-OS cells were taken on a Zeiss LSM510-NLO Meta confocal microscope using a C-Apochromat 40×/1.2-W Corr differential interference contrast (DIC) objective. Confocal images of HTC cells were taken on a Zeiss 710 confocal microscope equipped with a 63× Plan Apo 1.4-numerical-aperture (NA) objective and a 1-μm pinhole. Colocalization images and coefficients were obtained by using Zen2010 software. Thresholds were set to average intensity values for each channel. The coefficients were calculated as the relative number of colocalizing pixels in the GR channel compared to the total number of pixels above the threshold value, with a value range of 0 to 1 (with 0 being no colocalization and 1 being that all pixels colocalized). Quantitation was performed with three independent images from each treatment to obtain colocalization coefficient values.
U2-OS cells stably expressing WT- or S134A-GR were treated with either vehicle (H2O) or 100 nM Dex for 6 h. The total RNAs from six biological replicates of WT-GR-expressing cells and three biological replicates of S134A-GR-expressing cells were harvested (Qiagen, Valencia, CA), and gene expression analysis was conducted by using Agilent Human Whole Genome 4-by-44 multiplex format oligonucleotide arrays (catalog number 014850; Agilent Technologies, Santa Clara, CA) according to the Agilent one-color microarray-based gene expression analysis protocol, as previously described (16). In order to identify differentially expressed probes, analysis of variance (ANOVA) was used to determine if there was a statistical difference between the means of groups. In addition, a Benjamini-Hochberg multiple-test correction with a P value of <0.01, to reduce the number of false-positive results, was performed by using the Rosetta Resolver system (version 7.0; Rosetta Biosoftware, Kirkland, WA). Finally, these statistically significant genes, regardless of fold change, were loaded into Ingenuity Pathways Analysis software (Ingenuity Systems) for functional analysis. Area-proportional Venn diagrams were drawn by using free software available from Bioinforx.
U2-OS cells stably expressing WT- or S134A-GR were treated with 100 nM Dex, 100 μM H2O2, or vehicle (H2O) for 6 h, and total RNA was isolated by using the 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 primer/probe set of the unregulated housekeeping gene beta-actin (Applied Biosystems). Each primer/probe set was analyzed with three different biological replicates of RNA.
Chromatin immunoprecipitation (ChIP) assays were performed by using the Magna ChIP A chromatin immunoprecipitation kit (Millipore) according to the manufacturer's protocol. Briefly, cells were plated onto 150-mm dishes and cultured for 24 h in DMEM-F-12 medium supplemented with 10% charcoal-dextran-treated (stripped) FBS. Cells were then treated with 100 μM H2O2 for 30 min, followed by 100 nM Dex for 90 min. The cells were fixed in 1% formaldehyde and harvested in lysis buffer containing protease inhibitors. The nuclear contents were then sonicated by using a Branson Sonifier 150 at setting 4 with 10-s pulses, four times on ice. Sheared chromatin was then immunoprecipitated overnight with 10 μl of 57-GR or 5 μl of 14-3-3zeta antibodies. After the elution of protein-DNA complexes and DNA purification, real-time PCR analysis was performed by use of the following primers: forward primer 5′-CCAGCGGTTTGCGTAG-3′, probe 5-TGAACACTCAGCTCCTAGCGTGC-3, and reverse primer 5′-GCCACTTGCACCAGGA-3′ for insulin-like growth factor binding protein 1 (IGFBP1) (positions −150 to −43) and forward primer 5′-AGGCTTGATGACCAGAGAGGTTTG-3′, probe 5′-ATTCAGCTGCAAGTCTGGCATGGGAA-3′, and reverse primer 5′-GCTCAGAGACTTTGTGGCTATTTGG-3′ for glucocorticoid-induced leucine zipper (GILZ) (positions −1919 to −1794). These promoter regions were shown previously to bind GR (8).
Previous studies by our laboratory have demonstrated an important role of GR serine phosphorylation in regulating glucocorticoid signaling within cells (16, 44). The human GR has four well-characterized phosphorylation sites (serines 203, 211, 226, and 404), and their phosphorylation status influences the transcriptional response to glucocorticoids (15). Since the phosphorylation of the GR has a profound effect on glucocorticoid signaling, we utilized mass spectrometry to search for additional phosphorylated residues within the human GR sequence, resulting in the discovery of several novel phosphorylated residues. Interestingly, one of these residues, serine 134, was highly phosphorylated in a hormone-independent manner (Fig. 1A and B). When mass spectrometry experiments were performed without phosphopeptide enrichment, the nonphosphorylated and phosphorylated forms of the peptide corresponding to residues 132 to 154 were readily observed. Relative quantitation suggests that a large fraction (approximately 50%) of the wild-type GR is phosphorylated at Ser134, as estimated by a comparison of the areas under the curves of unphosphorylated and Ser134-phosphorylated peptides (see Fig. S1 in the supplemental material). Additionally, a mass spectrometer-based screen of phosphoproteins during mitosis also revealed a phosphoSer134-GR peptide (12), confirming the presence of an endogenous Ser134-phosphorylated GR. Therefore, due to the abundance and unique hormone-independent nature of this phosphorylation, we further characterized the phosphorylation status of serine 134 of GR. Antiphosphopeptide antibodies directed toward phosphorylated Ser134 validated the mass spectrometry results showing that Ser134 phosphorylation was not altered in response to the synthetic glucocorticoid hormone dexamethasone (Dex) in U2-OS cells stably expressing WT-GR (Fig. 1C). We also created a phospho-deficient mutant of the GR (S134A) to demonstrate the specificity of our antibody (Fig. 1C). Blots were reprobed for a known hormone-dependent phosphorylation site, serine 211, confirming the responsiveness to Dex (Fig. 1C). The status of Ser134 (or homologous Ser154 in rat) phosphorylation on the endogenous GR was assayed with rat liver hepatocytoma (HTC), human lung carcinoma A549, rat cardiomyocyte H9C2, and human cervical carcinoma HeLa cells. Figure 1D shows that Ser134 was constitutively phosphorylated, whereas Ser211 was phosphorylated in a hormone-dependent manner. Together, these data demonstrate an abundant and novel hormone-independent phosphorylation site of the GR, Ser134 (rat Ser154 [rSer154]), which is conserved in human and rat cell lines, suggesting that it may be important for GR function in cells.
In order to identify the mechanism responsible for this GR phosphorylation event, we removed serum and glucose to potentially block the phosphorylation of the GR. Surprisingly, glucose starvation enhanced phosphorylation at Ser134 (Fig. 2A). Energy starvation in cells induces stress, activating several metabolic signaling pathways, including 5′-AMP-activated protein kinase (AMPK), protein kinase A (PKA), and Akt (14), as well as the stress-activated MAPKs p38 and Jun N-terminal protein kinase (JNK) (24). To determine which, if any, of these pathways contribute to the phosphorylation of the GR on Ser134, cells were starved of glucose before treatment with specific inhibitors of each pathway. Our results show that the p38 MAPK inhibitor SB203580 was able to block the starvation-induced hyperphosphorylation of Ser134 of the GR, while the other inhibitors were ineffective (Fig. 2B). In order to determine what other cellular stressors could induce Ser134 hyperphosphorylation, we stimulated U2-OS cells stably expressing WT-GR with several mediators of cellular stress pathways (28). Our results reveal that the hyperphosphorylation of Ser134 was induced by glucose starvation, UVC irradiation, and oxidative stress (H2O2). Importantly, Ser134 phosphorylation correlated with the activation of p38 MAPK, as determined by the phosphorylation of Thr180/Tyr182 (Fig. 2C). To evaluate if hyperphosphorylation also occurs in cells containing the endogenous GR, rat HTC cells were stimulated with multiple mediators of cellular stress. Osmotic shock (OSMO) and UVC irradiation induced both GR Ser154 phosphorylation and p38 MAPK activity in HTC cells (Fig. 2D). Based on these data, we conclude that hSer134/rSer154 of the GR becomes hyperphosphorylated in response to multiple cellular stress signals that activate the p38 MAPK pathway.
To access the role of p38 MAPK during stress-induced Ser134 hyperphosphorylation, we next preformed immunoprecipitation assays to determine if p38 MAPK and the GR associate. The GR and p38 MAPK formed a weak association in both the absence and presence of Dex in U2-OS cells stably expressing WT-GR (Fig. 3A). Interestingly, this association was enhanced significantly in cells expressing S134A-GR, which suggests that p38 MAPK could not catalyze the phosphorylation of Ser134 and therefore had a prolonged interaction with the GR. Our results also demonstrate a decreased association of p38 MAPK with the GR in the presence of hormone, suggesting that GR nuclear translocation decreases p38 MAPK-GR complex formation, or ligand binding results in a conformational change preventing p38 MAPK from binding the GR. The ability of p38 MAPK to phosphorylate the GR in a ligand-dependent manner at Ser211 was reported previously; however, recent studies challenge this notion (17, 22, 32). To determine if p38 MAPK activity is necessary for the phosphorylation of serines 134 and 211 of the GR, we pretreated U2-OS cells expressing WT-GR with the p38 MAPK inhibitor SB203580 before stimulation with Dex and/or irradiation with UVC. The hormone-dependent phosphorylation of the GR at Ser211 was not affected by p38 MAPK inhibition in our studies. Conversely, Ser134 was phosphorylated in an UVC-dependent manner and was blocked by p38 MAPK inhibition (Fig. 3B). Since Ser211 was reported to be a substrate of p38 MAPK, we assayed for the sensitivity of this phosphorylation to our p38 MAPK inhibitors. We pretreated U2-OS cells with increasing concentrations of SB203580 (0 to 20 μM) and then treated them with UVC and Dex simultaneously. As expected, the GR was phosphorylated on serines 134 and 211 in response to cotreatment with UVC and Dex. Importantly, the phosphorylation of Ser134 was inhibited by as little as 1 μM SB203580, while the inhibition of Ser211 phosphorylation required 20 μM inhibitor. These results establish the Ser134 residue as a much stronger substrate of p38 MAPK (Fig. 3C). To further support the involvement of p38 MAPK in the phosphorylation of Ser134 of the GR, we transduced U2-OS cells expressing WT-GR with a lentivirus encoding either nonspecific (shScram) or p38α MAPK-specific short hairpin RNAs (sh p38α) and assayed for Ser134 and Ser211 phosphorylation after Dex, OSMO, H2O2, or UVC treatment. As shown in Fig. 3D, the knockdown of p38α MAPK led to a signification decrease in OSMO-, H2O2-, and UVC-mediated Ser134 phosphorylation, further validating the important role of p38 MAPK in the phosphorylation of the GR at Ser134. However, the knockdown of p38α MAPK did not prevent the Dex-mediated phosphorylation of Ser211. The role of p38 MAPK in the phosphorylation of Ser154 was then assayed in rat cells with endogenous GR expression. As shown in Fig. 3E, the H2O2-induced phosphorylation of Ser154 was sensitive to the inhibition of p38 MAPK. Similarly, in primary thymocytes isolated from adrenalectomized rats, UVC irradiation induced the phosphorylation of Ser134, and the inhibition of p38 MAPK blocked this increase (Fig. 3F). Together, our results clearly demonstrate the role of p38 MAPK in mediating the phosphorylation of the GR on hSer134/rSer154 downstream of several different stress-activated pathways.
To examine if serine 134 regulates GR signaling within cells, we assessed the cellular localization of the GR in U2-OS cells in the absence or presence of Dex. The phosphorylation status of the GR on Ser134 did not alter the hormone-dependent translocation of the GR into the nucleus (see Fig. S2A in the supplemental material). Additionally, the hyperphosphorylation of Ser134 induced by UVC irradiation or oxidative stress in WT-GR-expressing cells did not alter GR nuclear translocation (Fig. S2B). As shown for WT- and S134A-GR-expressing cells, the half-life of GR proteins in both the presence and absence of hormone was unaffected by the phosphorylation status of Ser134 (Fig. S2C). U2-OS cells expressing the phospho-deficient GR (S134A) still maintained hormone-dependent phosphorylation patterns at serines 211 and 404 comparable to those of WT-GR cells (Fig. 4A). Therefore, the phosphorylation of Ser134 did not alter the hormone-dependent nuclear translocation, protein stability, or phosphorylation pattern of the GR. Since the basic properties and mechanisms of the GR were not modified, the physiological significance of Ser134 phosphorylation was determined by examining gene transcriptional events. To accomplish this goal, total cellular RNAs isolated from at least three biological replicates of U2-OS cells stably expressing WT- or S134A-GR were subjected to whole-human-genome microarray analysis. Figure 4B demonstrates the level of GR Ser134 phosphorylation in the microarray analysis samples. We observed that only 20 out of nearly 44,000 genes were uniquely regulated in the absence of hormone, suggesting that the status of Ser134 phosphorylation did not significantly alter gene regulation in resting cells. Conversely, we show that gene expression was significantly altered in the presence of hormone (Fig. 4C). The WT receptor significantly regulated 1,388 genes after a 6-h stimulation with Dex, whereas the phosphorylation-deficient S134A mutant significantly regulated 2,434 genes. Of these genes, 889 required Ser134 phosphorylation, and 1,935 occurred only with the lack of phosphorylation, leaving only 499 genes to be commonly regulated in both WT-GR and S134A-GR. These data suggest that the phosphorylation status of Ser134 is critical for over 85% of the hormone-regulated genes in U2-OS cells (Fig. 4C). When hormone-regulated genes were divided into induced and repressed genes, our data demonstrated that Ser134 phosphorylation did not alter the proportion of induced versus repressed genes in response to hormone but rather the overall number of genes regulated (Fig. 4D). To analyze the biological pathways involved in Ser134-dependent signaling, the 889 genes regulated only in phosphorylated WT-GR-expressing U2-OS cells and the 1,935 genes regulated only in the phospho-deficient cells were examined by Ingenuity Pathways Analysis software. Glucocorticoid signaling is important for numerous cellular processes, including growth, death, development, and the cell cycle, through its ability to regulate gene expression (19, 20, 33). Therefore, we compared the WT-GR-regulated and S134A-GR-regulated gene lists for their abilities to regulate genes in these classical hormone-dependent pathways. The cells lacking Ser134 phosphorylation regulated 2- to 5-fold more genes in these pathways than did cells expressing the phosphorylated receptor. Aberrant glucocorticoid action is also strongly associated with metabolic and endocrine disorders as well as inflammatory diseases (3, 36, 37, 41). Since such diseases are also associated with high levels of p38 MAPK activity (11), we accessed the involvement of Ser134 phosphorylation in the regulation of these pathways. Interestingly, although WT-GR-expressing cells regulated fewer genes than did S134A-GR-expressing cells, genes associated with endocrine disorders and the inflammatory response were highly regulated in WT-GR-expressing cells, with no significant regulation in the phospho-deficient cells (Fig. 4E). We utilized Ingenuity Pathways Analysis software to further explore the role of glucocorticoid receptor Ser134 phosphorylation in regulating genes involved in the inflammatory response. We show that WT-GR- and S134A-GR-expressing cells commonly regulated 54 genes within this pathway in response to hormone. Interestingly, WT-GR-expressing cells regulated an additional 137 genes within this pathway, while S134A-GR-expressing cells regulated only an additional 8 genes (see Fig. S3 in the supplemental material). We validated the microarray and Ingenuity Pathway Analysis results with the use of real-time PCR (Fig. 4F). Two well-characterized hormone-responsive genes, SGK (serum- and glucocorticoid-induced protein kinase) and GILZ (glucocorticoid-induced leucine zipper), were both found to be equally induced by hormone regardless of the Ser134 phosphorylation status. Since Ingenuity Pathways Analysis showed differential biological pathway activation based on the Ser134 phosphorylation status, we validated genes within those specific pathways. HDAC1 and p53 have a role in the regulation of cell growth and death (5, 21), and interestingly, HDAC1 and p53 were significantly repressed only in cells lacking Ser134 phosphorylation. Foxo3 is associated with both metabolic and endocrine disorders (31) and was regulated by hormone only in cells expressing WT-GR. Another gene associated with endocrine disorders, Bcl6 (48), was selectively regulated by hormone in WT-GR-expressing cells. When genes involved in the inflammatory response were evaluated, CEBPB was induced in WT cells and repressed in S134A cells in response to hormone (6, 21). Our results indicate that although the GR nuclear translocation, protein stability, and phosphorylation status of other serines remain unaltered, the absence of Ser134 phosphorylation enhanced the hormone-mediated transcriptional response and significantly altered the repertoire of GR-regulated genes. These findings suggest a role for stress in mediating the selective activation of specific hormone-regulated pathways.
Our microarray data reveal a profound effect of the status of Ser134 phosphorylation on gene regulation, which led us to hypothesize that the activation of cellular stress pathways, and the hyperphosphorylation of Ser134, would also have a substantial impact on the profiles of gene expression within cells. Studies from other laboratories have demonstrated that the phosphorylation status of Ser211 and Ser226 on the GR largely impacts the hormone-dependent expression levels of LAD1 (ladinin 1), IGFBP1 (insulin-like growth factor binding protein 1), and GILZ (7). Our microarray data revealed no significant differences in hormone-mediated LAD1, IGFBP1, and GILZ expression in basally phosphorylated WT-GR cells compared to S134A-GR cells. To determine if Ser134 hyperphosphorylation would impact these glucocorticoid target genes, we induced hyperphosphorylation with oxidative stress by treating WT- or S134A-GR-expressing U2-OS cells with H2O2. We then stimulated the cells with Dex for 6 h, harvested the cells for total RNA, and preformed quantitative PCR (Fig. 5). The induction of oxidative stress significantly blunted the hormone-mediated upregulation of LAD1 and IGFBP1 but had no effect on GILZ in WT-GR cells (Fig. 5A, C, and E). Cells expressing the phospho-deficient GR (S134A-GR) were unaltered by the induction of oxidative stress (Fig. 5B, D, and F). Notably, when p38 MAPK activity was inhibited in WT-GR-expressing cells, the hormone-dependent regulation of LAD1 and IGFBP1 was partially restored following oxidative stress (Fig. 5A and C). Conversely, this was not observed for cells expressing S134A-GR (Fig. 5B, D, and F). Thus, our data support the hypothesis that stress inhibits GR transcriptional activity through the p38 MAPK-mediated phosphorylation of Ser134 in a gene-dependent manner. Additionally, our results also suggest that the inhibition of stress-activated pathways can restore hormone responsiveness within cells.
The phosphorylation status of the GR was reported previously to alter cofactor recruitment, which in turn alters target gene responses (7). To test the hypothesis that altered GR signaling due to the Ser134 phosphorylation status was a result of differential cofactor recruitment, we utilized motif scanning of the GR sequence to identify new potential cofactor binding sites. Sequence scanning revealed that serine 134 was contained within a potential consensus site for 14-3-3 proteins (Fig. 6A). There have been no reports of 14-3-3 proteins interacting with the N terminus of the GR; however, the ligand binding domain of the GR was previously shown to interact with the eta and sigma isoforms of 14-3-3 (25, 27). 14-3-3zeta is associated with oxidative stress (47), and its binding pocket closely matched the GR consensus site; thus, we chose to explore its role in GR signaling. To determine whether phosphorylation alters the ability of the GR to form a complex with 14-3-3zeta, we performed coimmunoprecipitation experiments with cells expressing WT or phospho-deficient GR. WT-GR had strong complex formation with 14-3-3zeta, and cells lacking Ser134 phosphorylation had a decreased association of 14-3-3zeta with the GR both before and after Dex stimulation (Fig. 6B). Based on these results, we wanted to determine if the hyperphosphorylation of Ser134 could alter the interaction between the GR and 14-3-3zeta. Treatment with H2O2 enhances the basal interaction of the GR with 14-3-3zeta, and the association was not affected by hormone (Fig. 6C). WT-GR and phospho-deficient GR (S134A) had similar interactions with Hsp90 (Fig. 6C), another GR cofactor, suggesting that the phosphorylation of the GR at Ser134 selectively alters the association with 14-3-3 proteins. As shown in Fig. 6D, the endogenous GR and 14-3-3zeta form a strong complex in A549 cells, which was further enhanced by the H2O2-induced phosphorylation of Ser134 of the GR. Finally, to determine the cellular location of GR-14-3-3zeta interactions, we treated HTC cells with Dex and H2O2 before staining for the endogenous GR and 14-3-3. Confocal microscopy images and analysis show that 14-3-3zeta proteins were located in both the cytoplasm and nucleus of HTC cells and that H2O2 treatment promoted the redistribution of 14-3-3 into the nucleus (24% ± 3.1% to 32.2% ± 1.6%). Data also demonstrated that the GR translocated from the cytoplasm to the nucleus upon hormone binding. Image analysis revealed that the GR and 14-3-3zeta proteins colocalized in both the cytoplasm and nucleus of HTC cells and that their colocalization was further enhanced by the H2O2-induced phosphorylation of Ser134 of the GR (Fig. 6E).
To determine if the association of the GR with 14-3-3zeta affects hormone-dependent transcription, we examined changes in gene expression following the knockdown of 14-3-3zeta. We achieved a 75% knockdown of 14-3-3zeta protein expression in both WT- and S134A-GR-expressing cells (Fig. 7A). Control and 14-3-3zeta knockdown cells were then used to assay for the Dex-mediated regulation of LAD1, IGFBP1, and GILZ mRNA expression levels. The knockdown of 14-3-3zeta partially restored the hormone-dependent transcription of LAD1 and IGFBP1 following oxidative stress (Fig. 7A). We then mutated the two hydrophobic residues lying within the phosphoserine binding pocket of 14-3-3zeta (R56A and R60A) to create a dominant negative (DN) form (45) and assayed for the hormone-dependent transcription of LAD1 and IGFBP1 following oxidative stress. The expression of DN 14-3-3zeta in H2O2-treated WT-GR-expressing U2-OS cells restored the induction of LAD1 and IGFBP1 by hormone (Fig. 7B), indicating that the presence of Ser134 phosphorylation on the GR affects 14-3-3zeta binding. Since the GR and 14-3-3 interact within the nucleus and affect hormone-dependent transcription, we next determined if GR-14-3-3zeta interactions occur on gene promoter regions of chromatin. Our chromatin immunoprecipitation assays show that hormone-stimulated cells were able to recruit the GR to the GILZ promoter regardless of the Ser134 phosphorylation status. Conversely, the oxidative-stress-induced phosphorylation of the GR decreased its recruitment to the IGFBP1 promoter (Fig. 7C). Interestingly, 14-3-3zeta proteins were recruited to the GILZ promoter when the GR was phosphorylated on Ser134. In contrast, increased GR Ser134 phosphorylation led to the decreased recruitment of 14-3-3zeta to the IGFBP1 promoter (Fig. 7D). These results indicate that the GR can bind GILZ promoters independently of the Ser134 phosphorylation status, whereas the IGFBP1 promoter preferentially binds the unphosphorylated GR. Together, our findings suggest that the serine 134 phosphorylation of the GR has global effects on hormone-dependent transcriptional events due to the differential binding and recruitment of 14-3-3zeta on different gene promoters, as modeled in Fig. 8.
The novel idea that cellular stress pathways converge on glucocorticoid pathways to determine how cells respond to circulating stress hormones is intriguing and insightful in an understanding of the complete stress response in humans. We described here a new ligand-independent posttranslational modification on the GR, serine 134, that was phosphorylated in response to several diverse cellular stress signals but not in response to hormone. We further elucidated that the phosphorylation of Ser134 was mediated by p38 MAPK. The activation of the glucocorticoid receptor by hormone resulted in the transcriptional alteration of over 3,300 genes, but more importantly, the regulation of 2,800 of those genes was dependent on the Ser134 phosphorylation status. Therefore, the level of molecular stress, as measured by Ser134-GR phosphorylation, has a global impact on the function and properties of the cell. Biological pathway analysis using Ingenuity Pathways Analysis software highlights an important role of Ser134 phosphorylation in the regulation of genes associated with endocrine and immunological diseases. Specifically, the GR lacking Ser134 phosphorylation preferentially regulated classical glucocorticoid-mediated pathways such as the cell cycle, growth, and development. Conversely, while the phosphorylated receptor can still activate these classical glucocorticoid-mediated pathways, we observed a significant induction of disease-associated genes that are specific for the phosphorylated receptor. Mechanistically, our data support the notion that this altered gene regulation is a result of 14-3-3 proteins binding to the phosphorylated GR at Ser134 on gene promoter regions of chromatin and altering the transcription of target genes. Ser134-GR phosphorylation affects the transcription of LAD1 and IGFBP1 but not GILZ, thus suggesting that 14-3-3zeta cofactor binding alters transcription in a gene-specific manner. Thus, the phosphorylation status of serine 134 has an important role in determining which genes will be regulated by hormone, which will ultimately affect cellular responses to hormone. Interestingly, the 14-3-3zeta binding site in the amino terminus of the glucocorticoid receptor is conserved in human, mouse, and rat, suggesting that 14-3-3zeta-GR interactions are important for mammalian stress responses. Furthermore, this 14-3-3zeta binding site is also found in a small subset of nuclear receptors, including RXR and PXR, suggesting a potential role of 14-3-3zeta in mediating other nuclear receptor pathways.
Although it was once thought that ligand-dependent Ser211 phosphorylation on the glucocorticoid receptor was also mediated through the p38 MAPK pathway (32), recent work suggests that this may not be correct. Indeed, recent work by Chen et al. suggests that Ser211 is not a primary target for p38 MAPK activity (7). Our data shown in Fig. 3 suggest that p38 MAPK primarily mediates the phosphorylation of the GR on the ligand-independent Ser134 phosphorylation site.
Glucocorticoids are currently some of the most prescribed therapeutics in the world due to their effects on cells of the immune system. Oral and inhaled glucocorticoids are used to suppress inflammatory, autoimmune, and allergic disorders as well as for the treatment of asthma (18). Therefore, the sensitivity and responsiveness of tissues to glucocorticoids play a key role in their ability to treat these pathological conditions. Here we show that the activation of the p38 MAPK signaling pathway directly modifies GR signaling through the phosphorylation of Ser134, resulting in the alternation of global gene expression profiles. Therefore, the phosphorylation status of the GR is vital for determining the cellular response of cells to glucocorticoids. Interestingly, p38 MAPK is activated under some of the same pathological conditions as those for which glucocorticoids are used (39). Extensive work has shown that p38 MAPK becomes active in response to various proinflammatory cytokines, such as interleukins and tumor necrosis factor alpha (46). Patients needing therapeutic glucocorticoid administration may also have active p38 MAPK signaling pathways due to high levels of inflammatory cytokines present, and thus, cells will have a Ser134-hyperphosphorylated GR. This raises the question of whether pathological inflammation would cause the GR to signal differently in diseased compared to healthy tissues. Our results strongly suggest that glucocorticoid-mediated gene expression profiles shift from normal pathway regulation to genes associated with diseases as a function of GR Ser134 phosphorylation, showing that the level of cellular stress will ultimately determine the response of tissues to glucocorticoids. These data also suggests that chronic stress, i.e., constitutive p38 MAPK activation, will lead to cells with stable populations of Ser134-hyperphosphorylated GR and, thus, cells with resistant and/or significantly altered glucocorticoid responses.
For example, inflammation was shown previously to induce oxidative stress and be an important factor in inducing glucocorticoid resistance in chronic obstructive pulmonary disease (1). We found that the oxidative-stress-induced hyperphosphorylation of Ser134 blunted the hormone-dependent induction of LAD1 and IGFBP1 but not that of GILZ. Thus, oxidative stress led to enhanced Ser134-GR phosphorylation and may explain the altered response to glucocorticoid therapies. Previously reported data also suggest that p38 MAPK inhibitors have potential in reversing glucocorticoid insensitivity and restoring the beneficial effects of glucocorticoids in patients with severe asthma (22). Since we demonstrate that p38 MAPK inhibitors prevented the Ser134 phosphorylation of the GR, and other groups have shown that p38 inhibitors reestablish normal glucocorticoid function, it would be interesting to determine if this glucocorticoid insensitivity was caused by elevated GR Ser134 phosphorylation levels.
The Ser134 phosphorylation status of the GR may also have a metabolic role in the regulation of insulin resistance. We demonstrated in Fig. 2 that the GR becomes hyperphosphorylated in response to glucose starvation, therefore allowing Ser134 to act as a metabolic sensor within cells. There is an extensive body of evidence showing that stress combined with chronic and excessive glucocorticoid exposure in tissues leads to insulin resistance (2, 38). Additionally, there is an important role for p38 MAPK activity in the induction of glucocorticoid-mediated insulin resistance through reduced glucose transporter expression levels and increased leptin production (42). Finally, 14-3-3zeta proteins have a critical role in insulin sensitivity by binding to insulin receptor substrate 1 (IRS1) to block its association with the insulin receptor (35). These findings suggest that stress factors (including prolonged glucocorticoid exposure, increased body fat, and inflammation) typically seen in patients with insulin resistance would lead to p38 MAPK activation and subsequent Ser134 phosphorylation. Therefore, stress-induced Ser134 phosphorylation may have a critical role in the ability of the GR to regulate genes involved in insulin signaling, thereby potentially facilitating insulin resistance.
We thank Katina Johnson for assistance with mass spectrometry analysis. We thank Kevin Gerrish and Laura Wharey of the NIEHS Microarray Core for their help with the microarray data and analysis. We also thank Jeff Tucker, Agnes Janoshazi, and the Confocal Core Facility at the NIEHS. We are also grateful to Lindsay Smith for the isolation of primary rat thymocytes. Finally, we thank members of our laboratory for their critical reading of the manuscript.
This research was supported by the Intramural Research Program of the NIH National Institute of Environmental Health Sciences (Z01E5090057-12). We declare no conflicts of interest.
†Supplemental material for this article may be found at http://mcb.asm.org/.
Published ahead of print on 19 September 2011.