Michael J. Garabedian

Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation

The glucocorticoid steroid hormone cortisol is released by the adrenal glands in response to stress and serves as a messenger in circadian rhythms. Transcriptional responses to this hormonal signal are mediated by the glucocorticoid receptor (GR). We determined GR binding throughout the human genome by using chromatin immunoprecipitation followed by next-generation DNA sequencing, and measured related changes in gene expression with mRNA sequencing in response to the glucocorticoid dexamethasone (DEX). We identified 4392 genomic positions occupied by the GR and 234 genes with significant changes in expression in response to DEX. This genomic census revealed striking differences between gene activation and repression by the GR. While genes activated with DEX treatment have GR bound within a median distance of 11 kb from the transcriptional start site (TSS), the nearest GR binding for genes repressed with DEX treatment is a median of 146 kb from the TSS, suggesting that DEX-mediated repression occurs independently of promoter-proximal GR binding. In addition to the dramatic differences in proximity of GR binding, we found differences in the kinetics of gene expression response for induced and repressed genes, with repression occurring substantially after induction. We also found that the GR can respond to different levels of corticosteroids in a gene-specific manner. For example, low doses of DEX selectively induced PER1, a transcription factor involved in regulating circadian rhythms. Overall, the genome-wide determination and analysis of GR:DNA binding and transcriptional response to hormone reveals new insights into the complexities of gene regulatory activities managed by GR.

DIFFERENTIAL REGULATION OF GLUCOCORTICOID RECEPTOR TRANSCRIPTIONAL ACTIVATION VIA AF-1-ASSOCIATED PROTEINS

The hormone‐activated glucocorticoid receptor (GR), through its N‐ and C‐terminal transcriptional activation functions AF‐1 and AF‐2, controls the transcription of target genes presumably through interaction(s) with transcriptional regulatory factors. Utilizing a modified yeast two‐hybrid approach, we have identified the tumor susceptibility gene 101 (TSG101) and the vitamin D receptor‐interacting protein 150 (DRIP150) as proteins that interact specifically with a functional GR AF‐1 surface. In yeast and mammalian cells, TSG101 represses whereas DRIP150 enhances GR AF‐1‐mediated transactivation. Thus, GR AF‐1 is capable of recruiting both positive and negative regulatory factors that differentially regulate GR transcriptional enhancement. In addition, we show that another member of the DRIP complex, DRIP205, interacts with the GR ligand binding domain in a hormone‐dependent manner and facilitates GR transactivation in concert with DRIP150. These results suggest that DRIP150 and DRIP205 functionally link GR AF‐1 and AF‐2, and represent important mediators of GR transcriptional enhancement.

Modulation of Glucocorticoid Receptor Function via Phosphorylation

Abstract: The glucocorticoid receptor (GR) is phosphorylated at multiple serine residues in a hormone‐dependent manner. It has been suggested that GR phosphorylation affects turnover, subcellular trafficking, or the transcriptional regulatory functions of the receptor, yet the contribution of individual GR phosphorylation sites to the modulation of GR activity remains enigmatic. This review critically evaluates the literature on GR phosphorylation and presents more recent work on the mechanism of GR phosphorylation from studies using antibodies that recognize GR only when it is phosphorylated. In addition, we present support for the notion that GR phosphorylation modifies protein‐protein interactions, which can stabilize the hypophosphorylated form of the receptor in the absence of ligand, as well as facilitate transcriptional activation by the hyperphosphorylation of GR via cofactor recruitment upon ligand binding. Finally, we propose that GR phosphorylation also participates in the nongenomic activation of cytoplasmic signaling pathways evoked by GR. Thus, GR phosphorylation is a versatile mechanism for modulating and integrating multiple receptor functions.

Antidepressants increase human hippocampal neurogenesis by activating the glucocorticoid receptor

Antidepressants increase adult hippocampal neurogenesis in animal models, but the underlying molecular mechanisms are unknown. In this study, we used human hippocampal progenitor cells to investigate the molecular pathways involved in the antidepressant-induced modulation of neurogenesis. Because our previous studies have shown that antidepressants regulate glucocorticoid receptor (GR) function, we specifically tested whether the GR may be involved in the effects of these drugs on neurogenesis. We found that treatment (for 3–10 days) with the antidepressant, sertraline, increased neuronal differentiation via a GR-dependent mechanism. Specifically, sertraline increased both immature, doublecortin (Dcx)-positive neuroblasts (+16%) and mature, microtubulin-associated protein-2 (MAP2)-positive neurons (+26%). This effect was abolished by the GR-antagonist, RU486. Interestingly, progenitor cell proliferation, as investigated by 5′-bromodeoxyuridine (BrdU) incorporation, was only increased when cells were co-treated with sertraline and the GR-agonist, dexamethasone, (+14%) an effect which was also abolished by RU486. Furthermore, the phosphodiesterase type 4 (PDE4)-inhibitor, rolipram, enhanced the effects of sertraline, whereas the protein kinase A (PKA)-inhibitor, H89, suppressed the effects of sertraline. Indeed, sertraline increased GR transactivation, modified GR phosphorylation and increased expression of the GR-regulated cyclin-dependent kinase-2 (CDK2) inhibitors, p27Kip1 and p57Kip2. In conclusion, our data suggest that the antidepressant, sertraline, increases human hippocampal neurogenesis via a GR-dependent mechanism that requires PKA signaling, GR phosphorylation and activation of a specific set of genes. Our data point toward an important role for the GR in the antidepressant-induced modulation of neurogenesis in humans.

Glucocorticoid Receptor-Mediated Cell Cycle Arrest Is Achieved through Distinct Cell-Specific Transcriptional Regulatory Mechanisms

Glucocorticoids inhibit proliferation of many cell types, but the events leading from the activated glucocorticoid receptor (GR) to growth arrest are not understood. Ectopic expression and activation of GR in human osteosarcoma cell lines U2OS and SAOS2, which lack endogenous receptors, result in a G1 cell cycle arrest. GR activation in U2OS cells represses expression of the cyclin-dependent kinases (CDKs) CDK4 and CDK6 as well as their regulatory partner, cyclin D3, leading to hypophosphorylation of the retinoblastoma protein (Rb). We also demonstrate a ligand-dependent reduction in the expression of E2F-1 and c-Myc, transcription factors involved in the G1-to-S-phase transition. Mitogen-activated protein kinase, CDK2, cyclin E, and the CDK inhibitors (CDIs) p27 and p21 are unaffected by receptor activation in U2OS cells. The receptors N-terminal transcriptional activation domain is not required for growth arrest in U2OS cells. In Rb-deficient SAOS2 cells, however, the expression of p27 and p21 is induced upon receptor activation. Remarkably, in SAOS2 cells that express a GR deletion derivative lacking the N-terminal transcriptional activation domain, induction of CDI expression is abolished and the cells fail to undergo ligand-dependent cell cycle arrest. Similarly, murine S49 lymphoma cells, which, like SAOS2 cells, lack Rb, require the N-terminal activation domain for growth arrest and induce CDI expression upon GR activation. These cell-type-specific differences in receptor domains and cellular targets linking GR activation to cell cycle machinery suggest two distinct regulatory mechanisms of GR-mediated cell cycle arrest: one involving transcriptional repression of G1 cyclins and CDKs and the other involving enhanced transcription of CDIs by the activated receptor.

Target-specific utilization of transcriptional regulatory surfaces by the glucocorticoid receptor

The glucocorticoid receptor (GR) activates or represses transcription depending on the sequence and architecture of the glucocorticoid response elements in target genes and the availability and activity of interacting cofactors. Numerous GR cofactors have been identified, but they alone are insufficient to dictate the specificity of GR action. Furthermore, the role of different functional surfaces on the receptor itself in regulating its targets is unclear, due in part to the paucity of known target genes. Using DNA microarrays and real-time quantitative PCR, we identified genes transcriptionally activated by GR, in a translation-independent manner, in two human cell lines. We then assessed in U2OS osteosarcoma cells the consequences of individually disrupting three GR domains, the N-terminal activation function (AF) 1, the C-terminal AF2, or the dimer interface, on activation of these genes. We found that GR targets differed in their requirements for AF1 or AF2, and that the dimer interface was dispensable for activation of some genes in each class. Thus, in a single cell type, different GR surfaces were used in a gene-specific manner. These findings have strong implications for the nature of gene response element signaling, the composition and structure of regulatory complexes, and the mechanisms of context-specific transcriptional regulation.

HDL promotes rapid atherosclerosis regression in mice and alters inflammatory properties of plaque monocyte-derived cells

HDL cholesterol (HDL-C) plasma levels are inversely related to cardiovascular disease risk. Previous studies have shown in animals and humans that HDL promotes regression of atherosclerosis. We hypothesized that this was related to an ability to promote the loss of monocyte-derived cells (CD68+, primarily macrophages and macrophage foam cells) from plaques. To test this hypothesis, we used an established model of atherosclerosis regression in which plaque-bearing aortic arches from apolipoprotein E-deficient (apoE−/−) mice (low HDL-C, high non–HDL-C) were transplanted into recipient mice with differing levels of HDL-C and non–HDL-C: C57BL6 mice (normal HDL-C, low non–HDL-C), apoAI−/− mice (low HDL-C, low non–HDL-C), or apoE−/− mice transgenic for human apoAI (hAI/apoE−/−; normal HDL-C, high non–HDL-C). Remarkably, despite persistent elevated non–HDL-C in hAI/apoE−/− recipients, plaque CD68+ cell content decreased by >50% by 1 wk after transplantation, whereas there was little change in apoAI−/− recipient mice despite hypolipidemia. The decreased content of plaque CD68+ cells after HDL-C normalization was associated with their emigration and induction of their chemokine receptor CCR7. Furthermore, in CD68+ cells laser-captured from the plaques, normalization of HDL-C led to decreased expression of inflammatory factors and enrichment of markers of the M2 (tissue repair) macrophage state. Again, none of these beneficial changes were observed in the apoAI−/− recipients, suggesting a major requirement for reverse cholesterol transport for the beneficial effects of HDL. Overall, these results establish HDL as a regulator in vivo of the migratory and inflammatory properties of monocyte-derived cells in mouse atherosclerotic plaques, and highlight the phenotypic plasticity of these cells.

Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

Synergistic Transcriptional Activation of the Tissue Inhibitor of Metalloproteinases-1 Promoter via Functional Interaction of AP-1 and Ets-1 Transcription Factors

The tissue inhibitor of metalloproteinases-1 (TIMP-1) is an inhibitor of the extracellular matrix-degrading metalloproteinases. We characterized response elements that control TIMP-1 gene expression. One contains a binding site that selectively binds c-Fos and c-Jun in vitro and confers a response to multiple AP-1 family members in vivo. Adjacent to this is a binding site for Ets domain proteins. Although c-Ets-1 alone did not activate transcription from this element, it enhanced transcription synergistically with AP-1 either in the context of the natural promoter or when the sequence was linked upstream of a heterologous promoter. Furthermore, a complex of c-Jun and c-Fos interacted with c-Ets-1 in vitro. These results suggest that AP-1 tethers c-Ets-1 to the TIMP-1 promoter via protein-protein interaction to achieve Ets-dependent transcriptional regulation. Collectively, our results indicate that TIMP-1 expression is controlled by several DNA response elements that respond to variations in the level and activity of AP-1 and Ets transcriptional regulatory proteins.

POTENTIATION OF HUMAN ESTROGEN RECEPTOR ALPHA TRANSCRIPTIONAL ACTIVATION THROUGH PHOSPHORYLATION OF SERINES 104 AND 106 BY THE CYCLIN A-CDK2 COMPLEX

Both estradiol binding and phosphorylation regulate transcriptional activation by the human estrogen receptor α (ER). We have previously shown that activation of the cyclin A-CDK2 complex by overexpression of cyclin A leads to enhanced ER-dependent transcriptional activation and that the cyclin A-CDK2 complex phosphorylates the ER N-terminal activation function-1 (AF-1) between residues 82 and 121. Within ER AF-1, serines 104, 106, and 118 represent potential CDK phosphorylation sites, and in this current study, we ascertain their importance in mediating cyclin A-CDK2-dependent enhancement of ER transcriptional activity. Cyclin A overexpression does not enhance transcriptional activation by an ER derivative bearing serine-to-alanine changes at residues 104, 106, and 118. Likewise, the cyclin A-CDK2 complex does not phosphorylate this triple-mutated derivative in vitro. Individual serine-to-alanine mutations at residues 104 and 106, but not 118, decrease ER-dependent transcriptional enhancement in response to cyclin A. The same relationship holds for ER phosphorylation by cyclin A-CDK2 in vitro. Finally, enhancement of ER transcriptional activation by cyclin A is evident in the absence and presence of estradiol, as well as in the presence of tamoxifen, suggesting that the effect of the cyclin A-CDK2 on ER transcriptional activation is AF-2-independent. These results indicate that the enhancement of ER transcriptional activation by the cyclin A-CDK2 complex is mediated via the AF-1 domain by phosphorylation of serines 104 and 106. We propose that these residues control ER AF-1 activity in response to signals that affect cyclin A-CDK2 function.