Bioinformatics Analyses of Spatial Peripheral Circadian Clock-Mediated Gene Expression of Glucocorticoid Receptor-Related Genes.

Glucocorticoids are ubiquitous, pleotropic steroid hormones secreted from the cortices of the adrenal glands in a circadian fashion under the strong influence of the central Clock center located in the suprachiasmatic nuclei (SCN) of the hypothalamus. In previous work, we reported that the circadian transcription factor CLOCK and its heterodimer partner BMAL1 suppress the transcriptional activity of the glucocorticoid receptor (GR) by acetylating a lysine cluster located in its hinge region between the DNA- and ligand-binding domains. This regulation of GR transcriptional activity by CLOCK/BMAL1 functions as a counter-regulatory loop against the diurnally fluctuating circulating glucocorticoids. Here, we have performed further analyses of our data using bioinformatics and computational methods. Gene expression data were analyzed using unsupervised machine learning methods, such as hierarchical clustering, k-means, Naïve Bayes classification, and polynomial regression analyses. We determined expression patterns of Clock-related genes, unraveled the dynamics of spatial data, and defined the temporal function of Clock-mediated GR-regulated genes. Gene expressions manifested nonlinear dynamics, possibly because we obtained dynamic results from stationary measurements. The mechanics of the circadian rhythms are still obscure, and more studies are required to understand how such rhythms influence mammalian physiology.

A-Kinase Anchoring Protein 13 (AKAP13) Augments Progesterone Signaling in Uterine Fibroid Cells.

Context: Uterine leiomyomata (fibroids) are prevalent sex hormone‒dependent tumors with an altered response to mechanical stress. Ulipristal acetate, a selective progesterone receptor (PR) modulator, significantly reduces fibroid size in patients. However, PR signaling in fibroids and its relationship to mechanical signaling are incompletely understood. Objective: Our prior studies revealed that A-kinase anchoring protein 13 (AKAP13) was overexpressed in fibroids and contributed to altered mechanotransduction in fibroids. Because AKAP13 augmented nuclear receptor signaling in other tissues, we sought to determine whether AKAP13 might influence PR signaling in fibroids. Methods and Results: Fibroid samples from patients treated with ulipristal acetate or placebo were examined for AKAP13 expression by using immunohistochemistry. In immortalized uterine fibroid cell lines and COS-7 cells, we observed that AKAP13 increased ligand-dependent PR activation of luciferase reporters and endogenous progesterone-responsive genes for PR-B but not PR-A. Inhibition of ERK reduced activation of PR-dependent signaling by AKAP13, but inhibition of p38 MAPK had no effect. In addition, glutathione S-transferase‒binding assays revealed that AKAP13 was bound to PR-B through its carboxyl terminus. Conclusion: These data suggest an intersection of mechanical signaling and PR signaling involving AKAP13 through ERK. Further elucidation of the integration of mechanical and hormonal signaling pathways in fibroids may provide insight into fibroid development and suggest new therapeutic strategies for treatment.

Mice deficient in AKAP13 (BRX) develop compulsive-like behavior and increased body weight.

OBJECTIVE: Hormonal contributions to the sex-dependent development of both obsessive-compulsive disorder (OCD) and obesity have been described, but the underlying mechanisms are incompletely understood. A-kinase anchoring protein 13 (AKAP13) significantly augments ligand-dependent activation of estrogen receptors alpha and beta. The hypothalamus and pituitary gland are implicated in the development and exacerbation of OCD and obesity and have strong AKAP13 expression. The AKAP13 localization pattern observed in these key brain regions together with its effects on sex steroid action suggest a potential role for AKAP13 in compulsive-like behaviors. Here we tested the role of AKAP13 in compulsive-like behavior and body weight using an Akap13 haploinsufficient murine model. MATERIALS AND METHODS: Targeted deletion of the Akap13 gene generated haploinsufficient (Akap13+/-) mice in a C57BL6/J genetic background. Established behavioral assays were conducted, video recorded, and scored blindly to assess compulsive-like behavior based on genotype and gender. Tests included: marble-burying, grooming, open- field and elevated plus-maze. Brain and body weights were also obtained. Mean levels of test outcomes were compared using multi-way ANOVA to test for genotype, sex, genotype*sex, and genotype*sex*age interaction effects with Bonferroni adjustment for multiple comparisons, to further explain any significant interactions. RESULTS: The marble-burying and grooming assays revealed significant sex-dependent increases in perseverative, compulsive-like behaviors in female Akap13 haploinsufficient mice compared to female wild type (WT) mice by demonstrating increased marble-burying activity (p = .0025) and a trend towards increased grooming behavior (p = .06). Male Akap13 haploinsufficient mice exhibited no behavioral changes (p > 0.05). Elevated plus-maze and open-field test results showed no overt anxiety-like behavior in Akap13 haploinsufficient mice irrespective of sex (p > 0.05, both). No differences in brain weight were found in Akap13 haploinsufficient mice compared to WT mice (p > 0.05). However, female Akap13 haploinsufficient mice weighed more than female WT mice in the 4 to <7 months age range (p = .0051). Male Akap13 haploinsufficient mice showed no differences in weight compared to male WT mice (p = >0.05) at any age range examined. CONCLUSION: Akap13 haploinsufficiency led to sex-dependent, compulsive-like behavioral changes in a murine model. Interestingly, Akap13 haploinsufficiency also led to a sex-dependent increase in body weight. These results revealed a requirement for AKAP13 in murine behavior, particularly in female mice, and is the first report of AKAP13 involvement in murine behavior. Future studies may examine the involvement of AKAP13 in the pathophysiology of OCD in female humans and may contribute to a better understanding of the role of AKAP13 and sex hormones in the development and exacerbation of OCD.

Progesterone-Mediated Non-Classical Signaling.

Progesterone is essential for pregnancy maintenance and menstrual cycle regulation. Hormone action has been primarily ascribed to the well-characterized classical signaling pathway involving ligand binding, activation of nuclear progesterone receptors (PRs), and subsequent activation of genes containing progesterone response elements (PREs). Recent studies have revealed progesterone actions via non-classical signaling pathways, often mediated by non-genomic signaling. Progesterone signaling, in conjunction with growth factor signaling, impacts on the function of growth factors and regulates important physiological actions such as cell growth and remodeling, as well as apoptosis. This review focuses on non-classical progesterone signaling pathways, both including and excluding PR, and highlights how research in this area will provide a better understanding of progesterone actions and may inform novel therapeutic strategies.

Heterodimeric IL15 Treatment Enhances Tumor Infiltration, Persistence, and Effector Functions of Adoptively Transferred Tumor-specific T Cells in the Absence of Lymphodepletion.

Purpose: Adoptive cell transfer (ACT) is a promising immunotherapeutic approach for cancer. Host lymphodepletion is associated with favorable ACT therapy outcomes, but it may cause detrimental effects in humans. We tested the hypothesis that IL15 administration enhances ACT in the absence of lymphodepletion. We previously showed that bioactive IL15 in vivo comprises a stable complex of the IL15 chain with the IL15 receptor alpha chain (IL15Rα), termed heterodimeric IL15 (hetIL15).Experimental Design: We evaluated the effects of the combination regimen ACT + hetIL15 in the absence of lymphodepletion by transferring melanoma-specific Pmel-1 T cells into B16 melanoma-bearing mice.Results: hetIL15 treatment delayed tumor growth by promoting infiltration and persistence of both adoptively transferred Pmel-1 cells and endogenous CD8+ T cells into the tumor. In contrast, persistence of Pmel-1 cells was severely reduced following irradiation in comparison with mice treated with hetIL15. Importantly, we found that hetIL15 treatment led to the preferential enrichment of Pmel-1 cells in B16 tumor sites in an antigen-dependent manner. Upon hetIL15 administration, tumor-infiltrating Pmel-1 cells showed a "nonexhausted" effector phenotype, characterized by increased IFNγ secretion, proliferation, and cytotoxic potential and low level of PD-1. hetIL15 treatment also resulted in an improved ratio of Pmel-1 to Treg in the tumor.Conclusions: hetIL15 administration improves the outcome of ACT in lymphoreplete hosts, a finding with significant implications for improving cell-based cancer immunotherapy strategies. Clin Cancer Res; 23(11); 2817-30. ©2016 AACR.

Differential effects of IL-15 on the generation, maintenance and cytotoxic potential of adaptive cellular responses induced by DNA vaccination.

IL-15 is an important cytokine for the regulation of lymphocyte homeostasis. However, the role of IL-15 in the generation, maintenance and cytotoxic potential of antigen specific T cells is not fully understood. Because the route of antigenic delivery and the vaccine modality could influence the IL-15 requirement for mounting and preserving cytotoxic T cell responses, we have investigated the immunogenicity of DNA-based vaccines in IL-15 KO mice. DNA vaccination with SIV Gag induced antigen-specific CD4(+) and CD8(+) T cells in the absence of IL-15. However, the absolute number of antigen-specific CD8(+) T cells was decreased in IL-15 KO mice compared to WT animals, suggesting that IL-15 is important for the generation of maximal number of antigen-specific CD8(+) T cells. Interestingly, antigen-specific memory CD8 cells could be efficiently boosted 8 months after the final vaccination in both WT and KO strains of mice, suggesting that the maintenance of antigen-specific long-term memory T cells induced by DNA vaccination is comparable in the absence and presence of IL-15. Importantly, boosting by DNA 8-months after vaccination revealed severely reduced granzyme B content in CD8(+) T cells of IL-15 KO mice compared to WT mice. This suggests that the cytotoxic potential of the long-term memory CD8(+) T cells is impaired. These results suggest that IL-15 is not essential for the generation and maintenance of adaptive cellular responses upon DNA vaccination, but it is critical for the preservation of maximal numbers and for the activity of cytotoxic CD8(+) T cells.

Viral infection increases glucocorticoid-induced interleukin-10 production through ERK-mediated phosphorylation of the glucocorticoid receptor in dendritic cells: potential clinical implications.

The hypothalamic-pituitary-adrenal axis plays a central role in the adaptive response to stress including infection of pathogens through glucocorticoids. Physical and/or mental stress alter susceptibility to viral infection possibly by affecting this regulatory system, thus we explored potential cellular targets and mechanisms that underlie this phenomenon in key immune components dendritic cells (DCs). Dexamethasone (DEX) treatment and subsequent Newcastle disease virus (NDV) infection most significantly and cooperatively stimulated mRNA expression of the interleukin (IL)-10 in murine bone marrow-derived DCs among 89 genes involved in the Toll-like receptor signaling pathways. NDV increased DEX-induced IL-10 mRNA and protein expression by 7- and 3-fold, respectively, which was observed from 3 hours after infection. Conventional DCs (cDCs), but not plasmacytoid DCs (pDCs) were major sources of IL-10 in bone marrow-derived DCs treated with DEX and/or infected with NDV. Murine cytomegalovirus and DEX increased serum IL-10 cooperatively in female mice. Pre-treatment of DCs with the extracellular signal-regulated kinase (ERK) inhibitor U0126 abolished cooperative induction of IL-10 by DEX and NDV. Further, ERK overexpression increased IL-10 promoter activity stimulated by wild-type human GR but not by its mutant defective in serine 203, whereas ERK knockdown abolished NDV/DEX cooperation on IL-10 mRNA and phosphorylation of the mouse GR at serine 213. NDV also increased DEX-induced mRNA expression of three known glucocorticoid-responsive genes unrelated to the Toll-like receptor signaling pathways in DCs. These results indicate that virus and glucocorticoids cooperatively increase production of anti-inflammatory cytokine IL-10 by potentiating the transcriptional activity of GR in DCs, through which virus appears to facilitate its own propagation in infected hosts. The results may further underlie in part known exacerbation of IL-10/T helper-2-related allergic disorders by stress and viral infection.

Liver x receptors regulate the transcriptional activity of the glucocorticoid receptor: implications for the carbohydrate metabolism.

GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXRα/RXRα binding to GREs. Endogenous LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly and gene-specifically attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism.

Peripheral CLOCK regulates target-tissue glucocorticoid receptor transcriptional activity in a circadian fashion in man.

CONTEXT AND OBJECTIVE: Circulating cortisol fluctuates diurnally under the control of the "master" circadian CLOCK, while the peripheral "slave" counterpart of the latter regulates the transcriptional activity of the glucocorticoid receptor (GR) at local glucocorticoid target tissues through acetylation. In this manuscript, we studied the effect of CLOCK-mediated GR acetylation on the sensitivity of peripheral tissues to glucocorticoids in humans. DESIGN AND PARTICIPANTS: We examined GR acetylation and mRNA expression of GR, CLOCK-related and glucocorticoid-responsive genes in peripheral blood mononuclear cells (PBMCs) obtained at 8 am and 8 pm from 10 healthy subjects, as well as in PBMCs obtained in the morning and cultured for 24 hours with exposure to 3-hour hydrocortisone pulses every 6 hours. We used EBV-transformed lymphocytes (EBVLs) as non-synchronized controls. RESULTS: GR acetylation was higher in the morning than in the evening in PBMCs, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized EBVLs, however, some of these genes did not show the expected diurnal mRNA fluctuations in PBMCs in vivo. Instead, their mRNA oscillated in a Clock- and a GR acetylation-dependent fashion in naturally synchronized PBMCs cultured ex vivo in the absence of the endogenous glucocorticoid, suggesting that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo. CONCLUSIONS: Peripheral CLOCK-mediated circadian acetylation of the human GR may function as a target-tissue, gene-specific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night.

Virus-induced differential expression of nuclear receptors and coregulators in dendritic cells: implication to interferon production.

We investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in mouse bone marrow-derived dendritic cells (DCs) upon infection with Newcastle Disease virus (NDV) or murine cytomegalovirus (MCMV). These viruses regulated mRNA expression of some NRs among which NOR1 and LXRα were highly induced at mRNA and protein levels. Exogenous expression of the latter NRs repressed IRF3- or IRF7-induced transactivation of the interferon ß promoter and NDV infection further potentiated their repressive effect. The viral infection also significantly regulated mRNA expression of some coregulators, including HDAC1. Toll-like receptor ligands regulated NR and coregulator mRNA expression similar to the viruses. Thus, NRs and coregulators are integral components of DC-organizing anti-viral response wherein NOR1 and LXRα participate in regulating interferon production.

AMPK regulates metabolic actions of glucocorticoids by phosphorylating the glucocorticoid receptor through p38 MAPK.

Glucocorticoids play central roles in the regulation of energy metabolism by shifting it toward catabolism, whereas AMP-activated protein kinase (AMPK) is the master regulator of energy homeostasis, sensing energy depletion and stimulating pathways of increasing fuel uptake and saving on peripheral supplies. We showed here that AMPK regulates glucocorticoid actions on carbohydrate metabolism by targeting the glucocorticoid receptor (GR) and modifying transcription of glucocorticoid-responsive genes in a tissue- and promoter-specific fashion. Activation of AMPK in rats reversed glucocorticoid-induced hepatic steatosis and suppressed glucocorticoid-mediated stimulation of glucose metabolism. Transcriptomic analysis in the liver suggested marked overlaps between the AMPK and glucocorticoid signaling pathways directed mostly from AMPK to glucocorticoid actions. AMPK accomplishes this by phosphorylating serine 211 of the human GR indirectly through phosphorylation and consequent activation of p38 MAPK and by altering attraction of transcriptional coregulators to DNA-bound GR. In human peripheral mononuclear cells, AMPK mRNA expression positively correlated with that of glucocorticoid-responsive glucocorticoid-inducible leucine zipper protein, which correlated also positively with the body mass index of subjects. These results indicate that the AMPK-mediated energy control system modulates glucocorticoid action at target tissues. Because increased action of glucocorticoids is associated with the development of metabolic disorders, activation of AMPK could be a promising target for developing pharmacological interventions to these pathologies.

Signalling mechanisms regulating the activation of human eosinophils by mast-cell-derived chymase: implications for mast cell-eosinophil interaction in allergic inflammation.

Allergic diseases such as asthma and allergic dermatitis are associated with the degranulation of mast cells. Chymase, a mast-cell-specific protease, is the major component in mast cell granules that can induce eosinophil infiltration into inflammatory sites. We examined the immunopathological mechanisms for the activation of eosinophils by chymase in allergic inflammation. Cytokines were measured by cytometric bead array Flex Sets multiplex assay using flow cytometry and enzyme-linked immunosorbent assay. Adhesion molecules, migration and intracellular signalling pathways were assessed by flow cytometry, Boyden chamber assay and Western blot, respectively. Chymase suppressed the apoptosis of eosinophils and induce the release of the cytokine interleukin-6 (IL-6) and chemokines CXCL8, CCL2 and CXCL1 by eosinophils dose-dependently. It also up-regulated the surface expression of adhesion molecule CD18 and stimulated the chemokinetic migration of eosinophils. The expressions of adhesion molecules, cytokines and chemokines, and chemokinetic migration were differentially regulated by the activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, Akt, Janus-activated kinase and nuclear factor-kappaB pathways. Chymase therefore plays a pivotal immunological role in the interaction between mast cells and eosinophils in allergic diseases such as allergic dermatitis by inducing adhesion molecule-mediated chemokinetic migration and inflammatory cytokines and chemokines of eosinophils, through multiple intracellular signalling molecules and transcription factor. Our results therefore provide a further biochemical basis for the pathogenesis of allergic inflammation consequent on the interaction between mast cells and eosinophils, and give insight for the development of new therapies.