GCs are currently used to treat a wide variety of pathological conditions from an isolated skin rash to the chronic condition of rheumatoid arthritis. Therefore, the sensitivity of tissues to GCs plays a key role in their ability to treat pathological conditions. Recent work has shown that several factors contribute to the hormonal actions of GR signaling, and alterations in these factors lead to GC resistance or hypersensitivity. The major contributing factors in GR signaling are ligand availability, receptor isoform expression, promoter association, and GR stability (25
). In addition, several recent studies suggest that covalent additions such as acetylation, ubiquitylation, and phosphorylation affect steroid receptor stability and activity (17
). Therefore, the presence or absence of posttranslational modifications within different tissues would provide a potential mechanism for cell- or gene-specific regulation of GR function.
The work presented here describes a novel convergence point between GSK-3β and the GR pathway. Specifically, we found that upon hormone stimulation, GSK-3β can directly phosphorylate GR on Ser404 (Fig. and ). Furthermore, we found that this phosphorylation event alters the ability of GR to function as a transcription factor (Fig. , , and ) and ultimately effects how U-2 OS osteosarcoma cells respond to GCs (Fig. ). Therefore, the phosphorylation status of Ser404 has an important role in dictating how the GR will respond to GC stimulation. One could also speculate that cells with aberrant GSK-3β activity would have an altered response to GCs.
FIG. 9. The binding of hormone to the GR allows for DNA binding and the subsequent alteration of gene transcription. The phosphorylation of the GR on Ser404 by GSK-3β significantly alters the GR-mediated transcriptional response. Shown is a cluster analysis (more ...)
GSK-3α/β kinase was originally identified as a regulator of glycogen synthesis (49
) and has since been shown to regulate, both positively and negatively, a broad range of substrates by phosphorylation (3
). The majority of GSK-3α/β substrates are formed via prior phosphorylation by an additional kinase at position P+4
(pS/TXXXpS/T), also referred to as the priming site. However, several substrates, such as β-catenin, tau, and axin, do not require a priming site prior to GSK-3α/β phosphorylation (18
). We did not discover a priming site for GSK-3β within the GR; however, further experiments will determine whether such a site exists and the requirement of a priming event.
GSK-3α/β is active in resting cells and regulates glucose metabolism by phosphorylating and inactivating glycogen synthase. An insulin stimulus to normal cells inhibits GSK-3α/β kinase activity and thus relieves the inhibitory control of glucose metabolism. However, cells that have become resistant to insulin fail to regulate glucose and display elevated GSK-3α/β activity (20
). Due to the ability of chronic GC treatment to induce insulin resistance, it would be interesting to explore the role of GSK-3β-mediated GR phosphorylation in pathological conditions such as metabolic syndrome and obesity. Our lab is currently exploring such possibilities.
Recently, several groups have demonstrated a role for GSK-3β kinase in NF-κB activation and cell survival in a variety of cell types (13
). Their results show that loss of GSK-3β function in cells leads to excessive tumor necrosis factor alpha toxicity, resulting in enhanced cellular death. These observations are consistent with our present findings that decreased GSK-3β activity leads to enhanced cellular death in osteoblasts. Similarly, we observed that GSK-3β activity can affect NF-κB-mediated gene regulation. Our work here opens the possibility that GSK-3β activity may control NF-κB transcriptional responses by Ser404 phosphorylation of the GR and altered binding to p65 (Fig. and ). Therefore, it would be of interest to explore the possibility that aberrant NF-κB activation, as in cancer and autoimmunity, may be partially due to an alteration in the phosphorylation status of the GR.
Activation of the GR by hormone resulted in a transcription alteration of nearly 20% of the human genome (Fig. ). Therefore, pathways that modify the GR response will have a global impact on the function and properties of the cell. Here, we illustrate that a single posttranslational modification to the GR can significantly redirect the transcriptional output of the cell in response to hormone. As shown in Fig. , hormone-activated WT and phospho-deficient GRs are capable of activating the same pathways, though with significantly different gene regulation patterns. More surprisingly, the phospho-deficient GR mutant can selectively regulate additional pathways in excess of the WT receptor (Fig. ). Therefore, we show a global importance of GSK-3β-mediated phosphorylation of the GR to significantly redirect the transcriptional output of cells and thus alter their response to hormone. Finally, our results are not without precedence. While the present study was in review, Chen et al. published a study showing that GR phosphorylation at Ser211 and Ser226 alters the expression profile of several GC-responsive genes (8
Finally, chronic GC treatments often lead to tissue- and cell-type-specific resistance. Mechanisms resulting in GC resistance are rarely attributed to mutation of the GR but often attributed to the convergence of additional activated cell signaling components with the GR pathway. Such pathways include mitogen-activated kinases, protein kinase A, CDKs, and receptor tyrosine kinase (2
). We show here the convergence of another pathway, the GSK-3β signaling pathway, which also exerts a form of cellular resistance to GC administration. Specifically, we find that when Ser404 of GR is mutated to aspartic acid (S404D), a cellular mutation to mimic GSK-3 phosphorylation of GR, osteoblasts are protected against apoptosis (Fig. ). These results suggest that Ser404 phosphorylation of GR is a possible mechanism to explain the ability of some cells to become resistant to GC-induced apoptosis. Taken together, our results demonstrate the important and novel role of GSK-3β-mediated phosphorylation of Ser404 of the GR in regulating GC signaling.