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 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 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
). 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.