Tenofovir (TFV) treatment of female reproductive tract (FRT) cells results in differential accumulation of intracellular Tenofovir diphosphate (TFV-DP) in different cell types, with greater concentrations in epithelial cells (100-fold) and fibroblasts (10-fold) than in CD4+ T cells. The possibility that TFV-DP accumulation and retention in epithelial cells and fibroblasts may alter TFV availability and protection of CD4+ T cells against HIV infection, prompted us to evaluate TFV and/or Tenofovir alafenamide (TAF) release from FRT cells. Endometrial, endocervical and ectocervical polarized epithelial cells and fibroblasts were pre-loaded with TFV or TAF, and secretions tested for their ability to inhibit HIV infection of activated blood CD4+ T cells. Epithelial cell basolateral secretions (1, 2 and 3 days post-loading), but not apical secretions, suppressed HIV infection of CD4+ T cells, as did secretions from pre-loaded fibroblasts from each site. Intracellular TFV-DP levels in epithelial cells following preloading with TFV or TAF correlated directly with ARV protection of CD4+ T cells from HIV infection. When added apically to epithelial cells, TFV/TAF was released basolaterally, in part through Multidrug Resistant Protein transporters, taken up by fibroblasts and released into secretions to partially protect CD4+ T cells. These findings demonstrate that epithelial cells and fibroblasts release TFV/TAF for use by CD4+ T cells and suggest that the tissue environment plays a major role in the sustained protection against HIV infection.

HIV prevention research is focused on combining antiretrovirals (ARV) and progestin contraceptives to prevent HIV infection and pregnancy. The possibility that progestins compromise ARV anti-HIV activity prompted us to evaluate the effects of progestins on tenofovir (TFV) and TFV-alafenamide (TAF) on HIV infection and intracellular TFV-diphosphate (TFV-DP) concentrations in blood and genital CD4+T cells. Following incubation of blood CD4+T cells with TFV or TAF, Medroxyprogesterone acetate (MPA), but not Levonorgestrel, Norethisterone or progesterone, suppressed the anti-HIV effect of TFV by reducing intracellular TFV-DP, but had no effect on TAF inhibition of infection or TFV-DP. In contrast, with genital CD4+T cells, MPA suppressed TAF inhibition of HIV infection and lowered of TFV-DP concentrations without affecting TFV protection. These findings demonstrate that MPA selectively compromises TFV and TAF protection in blood and genital CD4+T cells and suggests that MPA may decrease ARV protection in individuals who use ARV intermittently for prevention.

Disruption of the epithelium in the female reproductive tract (FRT) is hypothesized to increase HIV infection risk by interfering with barrier protection and facilitating HIV-target cell recruitment. Here we determined whether Tenofovir (TFV), used vaginally in HIV prevention trials, and Tenofovir alafenamide (TAF), an improved prodrug of TFV, interfere with wound healing in the human FRT. TFV treatment of primary epithelial cells and fibroblasts from the endometrium (EM), endocervix (CX) and ectocervix (ECX) significantly delayed wound closure. Reestablishment of tight junctions was compromised in EM and CX epithelial cells even after wound closure occurred. In contrast, TAF had no inhibitory effect on wound closure or tight junction formation following injury. TAF accumulated inside genital epithelial cells as TFV-DP, the active drug form. At elevated levels of TAF treatment to match TFV intracellular TFV-DP concentrations, both equally impaired barrier function, while wound closure was more sensitive to TFV. Furthermore, TFV but not TAF increased elafin and MIP3a secretion following injury, molecules known to be chemotactic for HIV-target cells. Our results highlight the need of evaluating antiretroviral effects on genital wound healing in future clinical trials. A possible link between delayed wound healing and increased risk of HIV acquisition deserves further investigation.

Arsenic (As) is considered a top environmental chemical of human health because it has been linked to adverse health effects including cancer, diabetes, cardiovascular disease, and reproductive and developmental problems. In several cell culture and animal models, As acts as an endocrine disruptor, which may underlie many of its health effects. Previous work showed that steroid receptor (SR)-driven gene expression is disrupted in cells treated with inorganic As (arsenite, iAs(+3)). In those studies, low iAs(+3) concentrations (0.1-0.7 mu M) stimulated hormone-inducible transcription, whereas somewhat higher but still non-cytotoxic levels (1-3 mu M) inhibited transcription. This investigation focuses on the mechanisms underlying these inhibitory effects and evaluates the role of methylated trivalent As metabolites on SR function. Recent evidence suggests that, compared with iAs, methylated forms may have distinct biochemical effects. Here, fluorescence polarization (FP) experiments utilizing purified, hormone-bound human glucocorticoid (GR) and progesterone receptor (PR) have demonstrated that neither inorganic (iAs(+3)) nor dimethylated (DMA(+3)) species of trivalent As affect receptor interactions with glucocorticoid DNA response elements (GREs). However, monomethylated forms (monomethylarsenite, MMA(+3) and monomethylarsonic diglutathione, MADG) strongly inhibit GR-GRE and PR-GRE binding. Additionally, speciation studies of iAs(+3)-treated H4IIE rat hepatoma cells show that, under treatment conditions that cause inhibition of hormone-inducible gene transcription, the intracellular concentration of MADG is sufficient to inhibit GR-GRE and PR-GRE interactions in vivo. These results indicate that arsenic's inhibitory endocrine disruption effects are probably caused in part by methylated metabolites' disruption of SR ability to bind DNA response elements that are crucial to hormone-driven gene transcription. Copyright (c) 2013 John Wiley & Sons, Ltd.

The conflicting results of recent pre-exposure prophylaxis (PrEP) trials utilizing tenofovir (TFV) to prevent HIV infection in women led us to evaluate the accumulation of intracellular TFV-diphosphate (TFV-DP) in cells from the female reproductive tract (FRT) and whether sex hormones influence the presence of TFV-DP in these cells. Following incubation with TFV, isolated epithelial cells, fibroblasts, CD4+ T cells and CD14+ cells from the FRT as well as blood CD4+ T cells and monocyte-derived macrophages convert TFV to TFV-DP. Unexpectedly, we found that TFV-DP concentrations (fmol/million cells) vary significantly with the cell type analyzed and the site within the FRT. Epithelial cells had 5-fold higher TFV-DP concentrations than fibroblasts; endometrial epithelial cells had higher TFV-DP concentrations than cells from the ectocervix. Epithelial cells had 125-fold higher TFV-DP concentrations than FRT CD4+ T cells, which were comparable to that measured in peripheral blood CD4+ T cells. These findings suggest the existence of a TFV-DP gradient in the FRT where epithelial cells > fibroblasts > CD4+ T cells and macrophages. In other studies, estradiol increased TFV-DP concentrations in endometrial and endocervical/ectocervical epithelial cells, but had no effect on fibroblasts or CD4+ T cells from FRT tissues. In contrast, progesterone alone and in combination with estradiol decreased TFV-DP concentrations in FRT CD4+ T cells. Our results suggest that epithelial cells and fibroblasts are a repository of TFV-DP that is under hormonal control. These cells might act either as a sink to decrease TFV availability to CD4+ T cells and macrophages in the FRT, or upon conversion of TFV-DP to TFV increase TFV availability to HIV-target cells. In summary, these results indicate that intracellular TFV-DP varies with cell type and location in the FRT and demonstrate that estradiol and/or progesterone regulate the intracellular concentrations of TFV-DP in FRT epithelial cells and CD4+ T cells.

ABSTRACT Tenofovir (TFV) is a reverse transcriptase inhibitor used in microbicide preexposure prophylaxis trials to prevent HIV infection. Recognizing that changes in cytokine/chemokine secretion and nucleotidase biological activity can influence female reproductive tract (FRT) immune protection against HIV infection, we tested the hypothesis that TFV regulates immune protection in the FRT. Epithelial cells, fibroblasts, CD4+ T cells, and CD14+ cells were isolated from the endometrium (Em), endocervix (Cx), and ectocervix (Ecx) following hysterectomy. The levels of proinflammatory cytokines (macrophage inflammatory protein 3α [MIP-3α], interleukin 8 [IL-8], and tumor necrosis factor alpha [TNF-α]), the expression levels of specific nucleotidases, and nucleotidase biological activities were analyzed in the presence or absence of TFV. TFV influenced mRNA and/or protein cytokines and nucleotidases in a cell- and site-specific manner. TFV significantly enhanced IL-8 and TNF-α secretion by epithelial cells from the Em and Ecx but not from the Cx. In contrast, in response to TFV, IL-8 secretion was significantly decreased in Em and Cx fibroblasts but increased with fibroblasts from the Ecx. When incubated with CD4+ T cells from the FRT, TFV increased IL-8 (Em and Ecx) and TNF-α (Cx and Ecx) secretion levels. Moreover, when incubated with Em CD14+ cells, TFV significantly increased MIP-3α, IL-8, and TNF-α secretion levels relative to those of the controls. In contrast, nucleotidase biological activities were significantly decreased by TFV in epithelial (Cx) and CD4+ T cells (Em) but increased in fibroblasts (Em). Our findings indicate that TFV modulates proinflammatory cytokines, nucleotidase gene expression, and nucleotidase biological activity in epithelial cells, fibroblasts, CD4+ T cells, and CD14+ cells at distinct sites within the FRT.

The use of topical and oral adenosine derivatives in HIV prevention that need to be maintained in tissues and cells at effective levels to prevent transmission prompted us to ask whether estradiol could influence the regulation of catabolic nucleotidase enzymes in epithelial cells and fibroblasts from the upper and lower female reproductive tract (FRT) as these might affect cellular TFV-DP levels. Epithelial cells and fibroblasts were isolated from endometrium (EM), endocervix (CX) and ectocervix (ECX) tissues from hysterectomy patients, grown to confluence and treated with or without estradiol prior to RNA isolation. The expression of nucleotidase (NT) genes was measurable by RT-PCR in epithelial cells and fibroblasts from all FRT tissues. To determine if sex hormones have the potential to regulate NT, we evaluated NT gene expression and NT biological activity in FRT cells following hormone treatment. Estradiol increased expression of Cytosolic 5'-nucleotidase after 2 or 4 h in endometrial epithelial cells but not epithelial cells or fibroblasts from other sites. In studies using a modified 5'-Nucleotidase biological assay for nucleotidases, estradiol increased NT activity in epithelial cells and fibroblasts from the EM, CX and ECX at 24 and 48 h. In related studies, HUVEC primary cells and a HUVEC cell line were unresponsive to estradiol in terms of nucleotidase expression or biological activity. Our findings of an increase in nucleotidase expression and biological activity induced by estradiol do not directly assess changes in microbicide metabolism. However, they do suggest that when estradiol levels are elevated during the menstrual cycle, FRT epithelial cells and fibroblasts from the EM, CX and ECX have the potential to influence microbicide levels that could enhance protection of HIV-target cells (CD4+T cells, macrophages and dendritic cells) throughout the FRT.

Tenofovir (TFV) has been widely used for pre-exposure prophylaxis of HIV-1 infection with mixed results. While the use of TFV in uninfected individuals for prevention of HIV-1 acquisition is actively being investigated, the possible consequences of TFV exposure for the HIV-target cells and the mucosal microenvironment are unknown. In the current study, we evaluated the effects of TFV treatment on blood-derived CD4+ T cells, monocyte-derived macrophages and dendritic cells (DC). Purified HIV-target cells were treated with different concentrations of TFV (0.001-1.0 mg/ml) for 2 to 24hr. RNA was isolated and RT-PCR was performed to compare the levels of mRNA expression of nucleotidases and pro-inflammatory cytokine genes (MIP3α, IL-8 and TNFα) in the presence or absence of TFV. We found that TFV increases 5’-ecto-nucleotidase (NT5E) and inhibits mitochondrial nucleotidase (NT5M) gene expression and increases 5’ nucleotidase activity in macrophages. We also observed that TFV stimulates the expression and secretion of IL-8 by macrophages, DC, and activated CD4+ T cells and increases the expression and secretion of MIP3α by macrophages. In contrast, TFV had no effect on TNFα secretion from macrophages, DC and CD4+ T cells. Our results demonstrate that TFV alters innate immune responses in HIV-target cells with potential implications for increased inflammation at mucosal surfaces. As new preventive trials are designed, these findings should provide a foundation for understanding the effects of TFV on HIV-target cells in microbicide trials.

CitationFahey JV, Bodwell JE, Hickey DK, Ghosh M, Muia MN, Wira CR. New approaches to making the microenvironment of the female reproductive tract hostile to HIV. Am J Reprod Immunol 2011; 65: 334–343AbstractThe studies presented in this review explore three distinct areas with potential for inhibiting HIV infection in women. Based on emerging information from the physiology, endocrinology and immunology of the female reproductive tract (FRT), we propose unique ‘works in progress’ for protecting women from HIV. Various aspects of FRT immunity are suppressed by estradiol during the menstrual cycle, making women more susceptible to HIV infection. By engineering commensal Lactobacillus to secrete the anti‐HIV molecule Elafin as estradiol levels increase, women could be protected from HIV infection. Selective estrogen response modifiers enhance barrier integrity and enhance secretion of protective anti‐HIV molecules. Finally, understanding the interactions and regulation of FRT endogenous antimicrobials, proteases, antiproteases, etc., all of which are under hormonal control, will open new avenues to therapeutic manipulation of the FRT mucosal microenvironment. By seeking new alternatives to preventing HIV infection in women, we may finally disrupt the HIV pandemic.

Arsenic (As) contamination of drinking water is considered a serious worldwide environmental health threat that is associated with increased disease risks including skin, lung, bladder, and other cancers; type 2 diabetes; vascular and cardiovascular diseases; reproductive and developmental effects; and neurological and cognitive effects. Increased health risks may occur at as low as 10-50 ppb, while biological effects have been observed in experimental animal and cell culture systems at much lower levels. We previously reported that As is a potent endocrine disruptor, altering gene regulation by the closely related glucocorticoid, mineralocorticoid, progesterone, and androgen steroid receptors (SRs) at concentrations as low as 0.01 microM ( approximately 0.7 ppb). Very low doses enhanced hormone-mediated gene transcription, whereas slightly higher but still noncytotoxic doses were suppressive. We report here that As also disrupts the more distally related estrogen receptor (ER) both in vivo and in cell culture. At noncytotoxic doses (1-50 micromol/kg arsenite) As strongly suppressed ER-dependent gene transcription of the 17beta-estradiol (E2)-inducible vitellogenin II gene in chick embryo liver in vivo. In cell culture, noncytotoxic levels (0.25-3 microM, approximately 20-225 ppb) of As significantly inhibited E2-mediated gene activation of an ER-regulated reporter gene and the native ER-regulated GREB1 gene in human breast cancer MCF-7 cells. While the effects of As on ER-dependent gene regulation were generally similar to As effects on the other SRs, there were specific differences, particularly the lack of significant enhancement at the lowest doses, that may provide insights into possible mechanisms.

Chronic intake of arsenic (As) has been associated with increased risk of cancer, diabetes, developmental and reproductive problems, and cardiovascular disease. Recent studies suggest increased health risks with drinking water levels as low as 5-10 ppb. We previously reported that As disrupts glucocorticoid receptor (GR) mediated transcription in a very complex fashion. Low As levels (0.1-0.7 microM) stimulated transcription, whereas slightly higher levels (1-3 microM) were inhibitory. The DNA binding domain (DBD) was the minimal region of GR required for the response to As. Mutations in the DBD that alter the conformation of the dimerization domain (D-loop) to a DNA-bound GR conformation abolished the stimulatory effect and enhanced the inhibitory response to As. Here we report that receptors for progesterone (PR) and mineralocorticoids display a complex As response similar to that of the GR, suggesting a common mechanism for this effect. The complex response to As is not due to altered steroid or receptor levels. Moreover, a well-characterized GR dimerization mutant displayed a wild-type biphasic response to As for several divergent reporter genes, suggesting that dimerization is not critical for the response to As. Fluorescence polarization studies with purified PR and GR demonstrated that the specific PR/GR-DNA interaction is not altered in the presence of As. These results indicate that the numerous and diverse human health effects associated with As exposure may be mediated, at least in part, through its ability to simultaneously disrupt multiple hormone receptor systems.

Arsenic (As) contamination of drinking water is considered a principal environmental health threat throughout the world. Chronic intake is associated with an increased risk of cancer, diabetes, and cardiovascular disease, and recent studies suggest increased health risks at levels as low as 5-10 ppb. We report here that 0.05-1 microM (6-120 ppb) As showed stimulatory effects on glucocorticoid receptor (GR)-mediated gene activation in rat EDR3 hepatoma cells of both the endogenous tyrosine aminotransferase (TAT) gene and the reporter genes containing TAT glucocorticoid response elements. At slightly higher concentrations (1-3 microM), the effects of As became inhibitory. Thus, over this narrow concentration range, the effects of As changed from a 2- to 4-fold stimulation to a greater than 2-fold suppression in activity. Interestingly, the inhibitory effect of GR on both AP1- and NF-kappa B-mediated gene activation was not affected by As. The magnitude of GR stimulation and inhibition by As was highly dependent on the cellular level of hormone-activated GR. Mutational deletion studies indicated that the central DNA binding domain (DBD) of GR is the minimal region required for the As effect and does not require free sulfhydryls. Point mutations located within the DBD that have known structural consequences significantly altered the GR response to As. In particular, point mutations in the DBD that confer a DNA-bound GR confirmation abolished the low dose As stimulatory effect but enhanced the inhibitory response, further indicating that the DBD is important for mediating these As effects.

We utilized the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) in vivo to understand how the interaction of the glucocorticoid receptor (GR) with a nucleosome-assembled promoter allows access of factors required for the transition from a repressed promoter to a derepressed, transcriptionally competent promoter.A mutation (C644G) in the ligand binding domain (LBD) of the mouse GR has provided information regarding the steps required in the derepression/activation process and in the functional significance of the two major transcriptional activation domains, AF1 and AF2.The mutant GR activates transcription from a transiently transfected promoter that has a disordered nucleosomal structure, though significantly less well than the wild-type GR.With an integrated, replicated promoter, which is assembled in an ordered nucleosomal array, the mutant GR does not activate transcription, and it fails to induce chromatin remodeling of the MMTV LTR promoter, as indicated by nuclease accessibility assays.Together, these findings support a two-step model for the transition of a nucleosome-assembled, repressed promoter to its transcriptionally active, derepressed form.In addition, we find that the C-terminal GR mutation is dominant over the transcription activation function of the N-terminal GR activation domain.These findings suggest that the primary activation function of the C-terminal activation domain is different from the function of the N-terminal activation domain and that it is required for derepression of the chromatin-repressed MMTV promoter.

The glutamate synthetase gene (GS) contains a composite glucocorticoid response element (cGRE) comprised of a GRE and an adjacent element with features of both a cAMP-response element (CRE) and a 12-O-tetradecanoylphorbol 13-acetate (TPA) response element (TRE). The CRE/TRE element of the cGRE contributed to two modes of transcriptional activation: 1) enhancement of the response to cortisol and 2) a synergistic response to cortisol and increased cAMP. COS-7 cells transfected with a cGRE-luciferase construct show minimal expression under basal conditions or forskolin treatment. After cortisol treatment, luciferase activity from the cGRE is enhanced 4- to 8-fold greater than the GRE portion of the cGRE or a GRE from the tyrosine aminotransferase gene. Treatment with both forskolin and cortisol produced a 2- to 4-fold synergistic response over cortisol alone. Synergy is also seen with 8-bromo-cAMP, is specific for the cGRE, and occurs in a number of established cell lines. Elimination of the GRE or CRE/TRE reduces the synergy by 70-100%. Altering the CRE/TRE to GRE spacing changed both enhancement and synergy. Moving the elements 3 bp closer or extending 15 bp reduced enhancement. Synergy was markedly reduced when elements were one half of a helical turn out of phase. Western blots verified that CREB (cAMP-responsive binding protein) and ATF-1 (activating transcription factor-1) binds to the cGRE sequence. A specific dominant negative inhibitor of the CREB family, A-CREB, reduced synergy by 50%. These results suggest that the GS cGRE can potentially integrate signaling from both the cAMP and glucocorticoid receptor transduction pathways and that CREB/ATF-1 may play an important role in this process.

All steroid hormone receptors are phosphorylated and undergo hormone-induced hyperphosphorylation. Most phosphorylated residues identified so far are serines in the N-terminal domain. Other residues and domains may also be phosphorylated, e.g. the estrogen receptor is phosphorylated on tyrosine in the hormone-binding domain. Many sites lie in consensus sequences for proline-directed, cell cycle-associated kinases. In some receptors hyperphosphorylation is induced by hormone antagonists as well as agonists, and leads to new phosphorylated sites. With glucocorticoid receptors, hyperphosphorylation is specific for glucocorticoid agonists, follows receptor activation and produces no new sites. Rate studies suggest that hyperphosphorylation is due to accelerated phosphorylation rather than delayed dephosphorylation. Evidence to date indicates that steroid hormone receptor phosphorylation serves not as an on–off switch but modulates function more subtly. Mutations of phosphorylated sites to alanine have been found to decrease activity by 0 to 90%, depending on mutated site, cell type, reporter gene and hormone concentration. With glucocorticoid receptors, some alanine mutants are up to 75% less active in hormone-induced transactivation of certain reporter genes. They are also inactive in hormone-induced repression of transcription of their own gene and down regulation of the receptor protein. Furthermore, they are much less sensitive to degradation. Both basal phosphorylation and hormone-dependent hyperphosphorylation of these receptors are cell cycle-dependent, basal phosphorylation being low in S phase and high in G2/M and hyperphosphorylation the reverse, suggesting a causal relation to the cell cycle-dependence of glucocorticoid activity reported with several cell lines. Hyperphosphorylation appears to be regulated by basal phosphorylation through negative charge in the N-terminal domain, which in S phase is relatively low and permits hyperphosphorylation, but in G2/M is relatively high and blocks hyperphosphorylation.

The iodothyronine deiodinases are a family of oxidoreductases that catalyze the removal of iodide from thyroid hormones. Each of the three isoforms contain selenocysteine at its active site and several cysteine residues that may be important for catalytic activity. Of particular interest in the type I deiodinase (D1) is Cys(124) which is vicinal to the selenocysteine at position 126, and Cys(194), which has been conserved in all deiodinases identified to date. In the present studies, we have characterized the functional properties of C124A, C194A, and C124A/C194A. D1 mutants, which were prepared by site-directed mutagenesis and expressed in COS-7 cells. In broken cell preparations, the sensitivity of the mutants to the selective D1 inhibitors propylthiouracil and aurothioglucose were unaltered. Mutagenesis at the Cys(124) position was associated with a 7-11-fold increase in the K-m of dithiothreitol, whereas V-max values remained largely unchanged. However, both mutations resulted in marked decreases in V-max values when glutathione or a reconstituted thioredoxin cofactor system were used in the assay. In contrast to the results of these in vitro studies, no impairment in deiodinating capability was noted in intact cells expressing equivalent levels of the mutant constructs, These studies demonstrate that Cys(124) and Cys(194) influence the reactivity of the D1 with thiol cofactors in in vitro assay systems but are not determinants of the sensitivity of the enzyme to propylthiouracil and aurothioglucose. Furthermore, the observation that the cysteine mutants are fully active in intact cells demonstrates that the results of commonly used broken cell assays do not accurately predict the activity of the D1 in intact cells and suggests that glutathione and thioredoxin are not the major thiols utilized in vivo to support D1 activity.

Mouse glucocorticoid receptors (GRs) are phosphorylated in the N-terminal domain at serine/threonine residues, most lying in consensus sequences for cell cycle-associated kinases. Glucocorticoid agonists, but not antagonists, induce hyperphosphorylation. Phosphorylation of GRs overexpressed in Chinese hamster ovary (CHO) cells is cell cycle-dependent: basal phosphorylation in S phase is one third that in G2/M; glucocorticoids induce hyperphosphorylation in S but not G2/M, paralleling the reported sensitivity in S and resistance in G2/M of proliferating cells to transcriptional activation by glucocorticoids. This parallel led us to investigate what controls hyperphosphorylation. We tested three hypotheses: hyperphosphorylation is controlled by 1) negative charge due to basal GR phosphorylation, being permitted in S by low charge and blocked in G2/M by high charge; 2) presence in S and absence in G2/M of required kinases; 3) availability in S and lack in G2/M of unoccupied phosphorylatable sites. Our results are inconsistent with 2) and 3), but strongly support 1). GR mutants with alanines (A7GR) or glutamates (E7GR) replacing all but one phosphorylated site were overexpressed in CHO cells. Serine 122 remained intact to report GR phosphorylation. Consistent with hypothesis 1, with A7GRs hormone-induced hyperphosphorylation occurred in both S and G2/M (thus revealing kinase activity for hyperphosphorylation of at least serine 122 in both phases), whereas with E7GRs it occurred in neither phase. We conclude that basal GR phosphorylation controls hormone-induced GR hyperphosphorylation by modulating negative charge in the N-terminal domain and could potentially control other cell cycle-dependent GR properties.