Cardiac GR Mediates the Diurnal Rhythm in Ventricular Arrhythmia Susceptibility.

BACKGROUND: Ventricular arrhythmias (VAs) demonstrate a prominent day-night rhythm, commonly presenting in the morning. Transcriptional rhythms in cardiac ion channels accompany this phenomenon, but their role in the morning vulnerability to VAs and the underlying mechanisms are not understood. We investigated the recruitment of transcription factors that underpins transcriptional rhythms in ion channels and assessed whether this mechanism was pertinent to the heart's intrinsic diurnal susceptibility to VA. METHODS AND RESULTS: Assay for transposase-accessible chromatin with sequencing performed in mouse ventricular myocyte nuclei at the beginning of the animals' inactive (ZT0) and active (ZT12) periods revealed differentially accessible chromatin sites annotating to rhythmically transcribed ion channels and distinct transcription factor binding motifs in these regions. Notably, motif enrichment for the glucocorticoid receptor (GR; transcriptional effector of corticosteroid signaling) in open chromatin profiles at ZT12 was observed, in line with the well-recognized ZT12 peak in circulating corticosteroids. Molecular, electrophysiological, and in silico biophysically-detailed modeling approaches demonstrated GR-mediated transcriptional control of ion channels (including Scn5a underlying the cardiac Na+ current, Kcnh2 underlying the rapid delayed rectifier K+ current, and Gja1 responsible for electrical coupling) and their contribution to the day-night rhythm in the vulnerability to VA. Strikingly, both pharmacological block of GR and cardiomyocyte-specific genetic knockout of GR blunted or abolished ion channel expression rhythms and abolished the ZT12 susceptibility to pacing-induced VA in isolated hearts. CONCLUSIONS: Our study registers a day-night rhythm in chromatin accessibility that accompanies diurnal cycles in ventricular myocytes. Our approaches directly implicate the cardiac GR in the myocyte excitability rhythm and mechanistically link the ZT12 surge in glucocorticoids to intrinsic VA propensity at this time.

Imbalanced glucocorticoid and mineralocorticoid stress hormone receptor function has sex-dependent and independent regulatory effects in the mouse hippocampus.

Many stress-related neuropsychiatric disorders display pronounced sex differences in their frequency and clinical symptoms. Glucocorticoids are primary stress hormones that have been implicated in the development of these disorders but whether they contribute to the observed sex bias is poorly understood. Glucocorticoids signal through two closely related nuclear receptors, the glucocorticoid (GR) and mineralocorticoid receptor (MR). To elucidate the sex-specific and independent actions of glucocorticoids in the hippocampus, we developed knockout mice lacking hippocampal GR, MR, or both GR and MR. Mice deficient in hippocampal MR or both GR and MR showed an altered molecular phenotype of CA2 neurons and reduced anxiety-like behavior in both sexes, but altered stress adaptation behavior only in females and enhanced fear-motivated cue learning only in males. All three knockout mouse models displayed reduced sociability but only in male mice. Male and female mice deficient in both hippocampal GR and MR exhibited extensive neurodegeneration in the dentate gyrus. Global transcriptomic analysis revealed a marked expansion in the number of dysregulated genes in the hippocampus of female knockout mice compared to their male counterparts; however, the overall patterns of gene dysregulation were remarkably similar in both sexes. Within and across sex comparisons identified key GR and MR target genes and associated signaling pathways underlying the knockout phenotypes. These findings define major sex-dependent and independent effects of GR/MR imbalances on gene expression and functional profiles in the hippocampus and inform new strategies for treating men and women with stress-related neuropsychiatric disorders.

Impaired glucocorticoid receptor function attenuates herpes simplex virus 1 production during explant-induced reactivation from latency in female mice.

IMPORTANCE: A correlation exists between stress and increased episodes of human alpha-herpes virus 1 reactivation from latency. Stress increases corticosteroid levels; consequently, the glucocorticoid receptor (GR) is activated. Recent studies concluded that a GR agonist, but not an antagonist, accelerates productive infection and reactivation from latency. Furthermore, GR and certain stress-induced transcription factors cooperatively transactivate promoters that drive the expression of infected cell protein 0 (ICP0), ICP4, and VP16. This study revealed female mice expressing a GR containing a serine to alanine mutation at position 229 (GRS229A) shed significantly lower levels of infectious virus during explant-induced reactivation compared to male GRS229A or wild-type parental C57BL/6 mice. Furthermore, female GRS229A mice contained fewer VP16 + TG neurons compared to male GRS229A mice or wild-type mice during the early stages of explant-induced reactivation from latency. Collectively, these studies revealed that GR transcriptional activity has female-specific effects, whereas male mice can compensate for the loss of GR transcriptional activation.

Glucocorticoid Receptors Drive Breast Cancer Cell Migration and Metabolic Reprogramming via PDK4.

Corticosteroids act on the glucocorticoid receptor (GR; NR3C1) to resolve inflammation and are routinely prescribed to breast cancer patients undergoing chemotherapy treatment to alleviate side effects. Triple-negative breast cancers (TNBCs) account for 15% to 20% of diagnoses and lack expression of estrogen and progesterone receptors as well as amplified HER2, but they often express high GR levels. GR is a mediator of TNBC progression to advanced metastatic disease; however, the mechanisms underpinning this transition to more aggressive behavior remain elusive. We previously showed that tissue/cellular stress (hypoxia, chemotherapies) as well as factors in the tumor microenvironment (transforming growth factor ß [TGF-ß], hepatocyte growth factor [HGF]) activate p38 mitogen-activated protein kinase (MAPK), which phosphorylates GR on Ser134. In the absence of ligand, pSer134-GR further upregulates genes important for responses to cellular stress, including key components of the p38 MAPK pathway. Herein, we show that pSer134-GR is required for TNBC metastatic colonization to the lungs of female mice. To understand the mechanisms of pSer134-GR action in the presence of GR agonists, we examined glucocorticoid-driven transcriptomes in CRISPR knock-in models of TNBC cells expressing wild-type or phospho-mutant (S134A) GR. We identified dexamethasone- and pSer134-GR-dependent regulation of specific gene sets controlling TNBC migration (NEDD9, CSF1, RUNX3) and metabolic adaptation (PDK4, PGK1, PFKFB4). TNBC cells harboring S134A-GR displayed metabolic reprogramming that was phenocopied by pyruvate dehydrogenase kinase 4 (PDK4) knockdown. PDK4 knockdown or chemical inhibition also blocked cancer cell migration. Our results reveal a convergence of GR agonists (ie, host stress) with cellular stress signaling whereby pSer134-GR critically regulates TNBC metabolism, an exploitable target for the treatment of this deadly disease.

Overcoming apoptotic resistance afforded by Bcl-2 in lymphoid tumor cells: a critical role for dexamethasone.

Bcl-2 is an anti-apoptotic protein that promotes cell survival and resistance to cell death. Predictably, Bcl-2 as well as other anti-apoptotic Bcl-2 family members have been found to be overexpressed in a variety of human cancers. Approaches to overcome apoptotic resistance afforded by Bcl-2 in cells include anti-sense oligonucleotides, drugs that inhibit Bcl-2 function, and BH3 mimics have not been universally effective; thus, the need to understand the underlying mechanism of this resistance is vital. Glucocorticoids are stress hormones that act through their cognate receptors to control the transcription of numerous target genes, and in turn regulate a diverse array of biological processes. Synthetic glucocorticoids, such as dexamethasone, are prescribed in many chemotherapy protocols for neoplasms of lymphoid origin based on their ability to inhibit lymphocyte proliferation and promote apoptosis. However, lymphoid cells expressing Bcl-2 are resistant to glucocorticoid-induced cell death. We observed both pro- and anti-apoptotic characteristics in lymphoid cells expressing Bcl-2 following glucocorticoid treatment. These cells exhibited a profound change in their intracellular ionic composition, but a limited apoptotic ion flux and the absence of cell death. Provocatively, mimicking the loss of intracellular potassium using a low dose of a microbial toxin that acts as a potassium ionophore in combination with dexamethasone overcame the resistance afforded by Bcl-2 and killed the cells. Extending our study using other potassium ionophores revealed that direct depolarization of the mitochondria membrane potential coupled with prior treatment with glucocorticoids is the key mechanism for activating the cell death program and bypassing the resistance afforded by Bcl-2 in lymphoid cells. Finally, we show that the duration of dexamethasone pre-treatment is critical for regulating distinct genes and signaling pathways that sensitize the cells to die.

Case report: Changes in the levels of stress hormones during Takotsubo syndrome.

Background: Takotsubo syndrome is an acute cardiac condition usually involving abnormal regional left ventricular wall motion and impaired left ventricular contractility. It is due mainly to hyper-stimulation of the sympathetic nerve system, inducing an excess of catecholamines, usually triggered by intense psychological or physiological stress. The relationship between Takotsubo syndrome and the circulating stress hormones cortisol and copeptin (a surrogate marker of arginine vasopressin) has not been well documented. Case summary: Here, we describe the dynamic changes in circulating cortisol and copeptin during an entire episode of Takotsubo syndrome in a post-partum woman after spontaneous vaginal delivery. The patient was diagnosed with inverted Takotsubo syndrome accompanied by HELLP syndrome. We found qualitative and quantitative changes in cortisol: a loss of circadian rhythm and a three-fold elevation in the plasma concentration of the hormone with a peak appearing several hours before circulating cardiac biomarkers began to rise. By contrast, levels of copeptin remained normal during the entire episode. Discussion: Our findings indicate that the levels of cortisol change during Takotsubo syndrome whereas those of copeptin do not. This association between elevated cortisol and Takotsubo syndrome suggests that aberrant levels of this stress hormone may contribute to the observed cardiac pathology. We conclude that biochemical assays of circulating cortisol and cardiac biomarkers may be a useful complement to the diagnosis of Takotsubo syndrome by non-invasive cardiac imaging.

Intestinal epithelial glucocorticoid receptor promotes chronic inflammation-associated colorectal cancer.

Synthetic immunosuppressive glucocorticoids (GCs) are widely used to control inflammatory bowel disease (IBD). However, the impact of GC signaling on intestinal tumorigenesis remains controversial. Here, we report that intestinal epithelial GC receptor (GR), but not whole intestinal tissue GR, promoted chronic intestinal inflammation-associated colorectal cancer in both humans and mice. In patients with colorectal cancer, GR was enriched in intestinal epithelial cells and high epithelial cell GR levels were associated with poor prognosis. Consistently, intestinal epithelium-specific deletion of GR (GR iKO) in mice increased macrophage infiltration, improved tissue recovery, and enhanced antitumor response in a chronic inflammation-associated colorectal cancer model. Consequently, GR iKO mice developed fewer and less advanced tumors than control mice. Furthermore, oral GC administration in the early phase of tissue injury delayed recovery and accelerated the formation of aggressive colorectal cancers. Our study reveals that intestinal epithelial GR signaling repressed acute colitis but promoted chronic inflammation-associated colorectal cancer. Our study suggests that colorectal epithelial GR could serve as a predictive marker for colorectal cancer risk and prognosis. Our findings further suggest that, although synthetic GC treatment for IBD should be used with caution, there is a therapeutic window for GC therapy during colorectal cancer development in immunocompetent patients.

Glucocorticoid Inhibition of Estrogen Regulation of the Serotonin Receptor 2B in Cardiomyocytes Exacerbates Cell Death in Hypoxia/Reoxygenation Injury.

Background Stress has emerged as an important risk factor for heart disease in women. Stress levels have been shown to correlate with delayed recovery and increased mortality after a myocardial infarction. Therefore, we sought to investigate if the observed sex-specific effects of stress in myocardial infarction may be partly attributed to genomic interactions between the female sex hormones, estrogen (E2), and the primary stress hormones glucocorticoids. Methods and Results Genomewide studies show that glucocorticoids inhibit estrogen-mediated regulation of genes with established roles in cardiomyocyte homeostasis. These include 5-HT2BR (cardiac serotonin receptor 2B), the expression of which is critical to prevent cardiomyocyte death in the adult heart. Using siRNA, gene expression, and chromatin immunoprecipitation assays, we found that 5-HT2BR is a primary target of the glucocorticoid receptor and the estrogen receptor α at the level of transcription. The glucocorticoid receptor blocks the recruitment of estrogen receptor α to the promoter of the 5-HT2BR gene, which may contribute to the adverse effects of stress in the heart of premenopausal women. Using immunoblotting, TUNEL (terminal deoxynucleotidal transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling), and flow cytometry, we demonstrate that estrogen decreases cardiomyocyte death by a mechanism relying on 5-HT2BR expression. In vitro and in vivo experiments show that glucocorticoids inhibit estrogen cardioprotection in response to hypoxia/reoxygenation injury and exacerbate the size of the infarct areas in myocardial infarction. Conclusions These results established a novel mechanism underlying the deleterious effects of stress on female cardiac health in the setting of ischemia/reperfusion.

Tristetraprolin Prevents Gastric Metaplasia in Mice by Suppressing Pathogenic Inflammation.

BACKGROUND & AIMS: Aberrant immune activation is associated with numerous inflammatory and autoimmune diseases and contributes to cancer development and progression. Within the stomach, inflammation drives a well-established sequence from gastritis to metaplasia, eventually resulting in adenocarcinoma. Unfortunately, the processes that regulate gastric inflammation and prevent carcinogenesis remain unknown. Tristetraprolin (TTP) is an RNA-binding protein that promotes the turnover of numerous proinflammatory and oncogenic messenger RNAs. Here, we assess the role of TTP in regulating gastric inflammation and spasmolytic polypeptide-expressing metaplasia (SPEM) development. METHODS: We used a TTP-overexpressing model, the TTPΔadenylate-uridylate rich element mouse, to examine whether TTP can protect the stomach from adrenalectomy (ADX)-induced gastric inflammation and SPEM. RESULTS: We found that TTPΔadenylate-uridylate rich element mice were completely protected from ADX-induced gastric inflammation and SPEM. RNA sequencing 5 days after ADX showed that TTP overexpression suppressed the expression of genes associated with the innate immune response. Importantly, TTP overexpression did not protect from high-dose-tamoxifen-induced SPEM development, suggesting that protection in the ADX model is achieved primarily by suppressing inflammation. Finally, we show that protection from gastric inflammation was only partially due to the suppression of Tnf, a well-known TTP target. CONCLUSIONS: Our results show that TTP exerts broad anti-inflammatory effects in the stomach and suggest that therapies that increase TTP expression may be effective treatments of proneoplastic gastric inflammation. Transcript profiling: GSE164349.

Sex-Dependent Changes of miRNA Levels in the Hippocampus of Adrenalectomized Rats Following Acute Corticosterone Administration.

We explored sex-biased effects of the primary stress glucocorticoid hormone corticosterone on the miRNA expression profile in the rat hippocampus. Adult adrenalectomized (ADX) female and male rats received a single corticosterone (10 mg/kg) or vehicle injection, and after 6 h, hippocampi were collected for miRNA, mRNA, and Western blot analyses. miRNA profiling microarrays showed a basal sex-biased miRNA profile in ADX rat hippocampi. Additionally, acute corticosterone administration triggered a sex-biased differential expression of miRNAs derived from genes located in several chromosomes and clusters on the X and 6 chromosomes. Putative promoter analysis unveiled that most corticosterone-responsive miRNA genes contained motifs for either direct or indirect glucocorticoid actions in both sexes. The evaluation of transcription factors indicated that almost 50% of miRNA genes sensitive to corticosterone in both sexes was under glucocorticoid receptor regulation. Transcription factor-miRNA regulatory network analyses identified several transcription factors that regulate, activate, or repress miRNA expression. Validated target mRNA analysis of corticosterone-responsive miRNAs showed a more complex miRNA-mRNA interaction network in males compared to females. Enrichment analysis revealed that several hippocampal-relevant pathways were affected in both sexes, such as neurogenesis and neurotrophin signaling. The evaluation of selected miRNA targets from these pathways displayed a strong sex difference in the hippocampus of ADX-vehicle rats. Corticosterone treatment did not change the levels of the miRNA targets and their corresponding tested proteins. Our data indicate that corticosterone exerts a sex-biased effect on hippocampal miRNA expression, which may engage in sculpting the basal sex differences observed at higher levels of hippocampal functioning.

Combinatorial actions of glucocorticoid and mineralocorticoid stress hormone receptors are required for preventing neurodegeneration of the mouse hippocampus.

Chronic stress contributes to numerous human pathologies including cognition impairments and psychiatric disorders. Glucocorticoids are primary stress hormones that activate two closely related nuclear receptors, the glucocorticoid (GR) and mineralocorticoid receptor (MR), that are both highly expressed in the hippocampus. To investigate potential combinatorial actions of hippocampal GR and MR, we developed mice with conditional knockout of both GR and MR in the hippocampus and compared them to their single knockout counterparts. Mice lacking MR alone or both GR and MR in the hippocampus exhibited altered expression of multiple CA2-specific neuronal markers and enhanced cue-dependent learning in a conditioned fear test. Provocatively, in contrast to the single knockouts, mice depleted of both GR and MR showed profound neurodegeneration of the hippocampus. Neuronal death was increased and neurogenesis was reduced in the dentate gyrus of the double knockout mice. Global gene expression assays of the knockout mice revealed a synergistic increase in the number of dysregulated genes in the hippocampus lacking both GR and MR. This large cohort of genes reliant on both GR and MR for expression was strongly associated with cell death and cell proliferation pathways. GR/MR complexes were detected in CA1 and dentate gyrus neurons suggesting receptor heterodimers contribute to the joint actions of GR and MR. These findings reveal an obligate role for MR signaling in regulating the molecular phenotype of CA2 neurons and demonstrate that combinatorial actions of GR and MR are essential for preserving dentate gyrus neurons and maintaining hippocampal health.

Glucocorticoids and Androgens Protect From Gastric Metaplasia by Suppressing Group 2 Innate Lymphoid Cell Activation.

BACKGROUND & AIMS: The immune compartment is critical for maintaining tissue homeostasis. A weak immune response increases susceptibility to infection, but immune hyperactivation causes tissue damage, and chronic inflammation may lead to cancer development. In the stomach, inflammation damages the gastric glands and drives the development of potentially preneoplastic metaplasia. Glucocorticoids are potent anti-inflammatory steroid hormones that are required to suppress gastric inflammation and metaplasia. However, these hormones function differently in males and females. Here, we investigate the impact of sex on the regulation of gastric inflammation. METHODS: Endogenous glucocorticoids and male sex hormones were removed from mice using adrenalectomy and castration, respectively. Mice were treated with 5α-dihydrotestosterone (DHT) to test the effects of androgens on regulating gastric inflammation. Single-cell RNA sequencing of gastric leukocytes was used to identify the leukocyte populations that were the direct targets of androgen signaling. Type 2 innate lymphoid cells (ILC2s) were depleted by treatment with CD90.2 antibodies. RESULTS: We show that adrenalectomized female mice develop spontaneous gastric inflammation and spasmolytic polypeptide-expressing metaplasia (SPEM) but that the stomachs of adrenalectomized male mice remain quantitatively normal. Simultaneous depletion of glucocorticoids and sex hormones abolished the male-protective effects and triggered spontaneous pathogenic gastric inflammation and SPEM. Treatment of female mice with DHT prevented gastric inflammation and SPEM development when administered concurrent with adrenalectomy and also reversed the pathology when administered after disease onset. Single-cell RNAseq of gastric leukocytes revealed that ILC2s expressed abundant levels of both the glucocorticoid receptor (Gr) and androgen receptor (Ar). We demonstrated that DHT treatment potently suppressed the expression of the proinflammatory cytokines Il13 and Csf2 by ILC2s. Moreover, ILC2 depletion protected the stomach from SPEM development. CONCLUSIONS: Here, we report a novel mechanism by which glucocorticoids and androgens exert overlapping effects to regulate gastric inflammation. Androgen signaling within ILC2s prevents their pathogenic activation by suppressing the transcription of proinflammatory cytokines. This work revealed a critical role for sex hormones in regulating gastric inflammation and metaplasia.

Deletion of hippocampal Glucocorticoid receptors unveils sex-biased microRNA expression and neuronal morphology alterations in mice.

Sex differences in the brain have prompted many researchers to investigate the underlying molecular actors, such as the glucocorticoid receptor (GR). This nuclear receptor controls gene expression, including microRNAs (miRNAs), in non-neuronal cells. Here, we investigated sex-biased effects of GR on hippocampal miRNA expression and neuronal morphology by generating a neuron-specific GR knockout mouse (Emx1-Nr3c1 -/-). The levels of 578 mature miRNAs were assessed using NanoString technology and, in contrast to males, female Emx1-Nr3c1 -/- mice showed a substantially higher number of differentially expressed miRNAs, confirming a sex-biased effect of GR ablation. Based on bioinformatic analyses we identified several transcription factors potentially involved in miRNA regulation. Functional enrichment analyses of the miRNA-mRNA interactions revealed pathways related to neuronal arborization and both spine morphology and density in both sexes. Two recognized regulators of dendritic morphology, CAMKII-α and GSK-3ß, increased their protein levels by GR ablation in female mice hippocampus, without changes in males. Additionally, sex-specific effects of GR deletion were observed on CA1 neuronal arborization and dendritic spine features. For instance, a reduced density of mushroom spines in apical dendrites was evidenced only in females, while a decreased length in basal dendrites was noted only in males. However, length and arborization of apical dendrites were reduced by GR ablation irrespective of the sex. Overall, our study provides new insights into the sex-biased GR actions, especially in terms of miRNAs expression and neuronal morphology in the hippocampus.

Glucocorticoid Signaling and the Aging Heart.

A decline in normal physiological functions characterizes the aging process. While some of these changes are benign, the decrease in the function of the cardiovascular system that occurs during aging leads to the activation of pathological processes associated with an increased risk for heart disease and its complications. Imbalances in endocrine function are also common occurrences during the aging process. Glucocorticoids are primary stress hormones and are critical regulators of energy metabolism, inflammation, and cardiac function. Glucocorticoids exert their actions by binding the glucocorticoid receptor (GR) and, in some instances, to the mineralocorticoid receptor (MR). GR and MR are members of the nuclear receptor family of ligand-activated transcription factors. There is strong evidence that imbalances in GR and MR signaling in the heart have a causal role in cardiac disease. The extent to which glucocorticoids play a role in the aging heart, however, remains unclear. This review will summarize the positive and negative direct and indirect effects of glucocorticoids on the heart and the latest molecular and physiological evidence on how alterations in glucocorticoid signaling lead to changes in cardiac structure and function. We also briefly discuss the effects of other hormones systems such as estrogens and GH/IGF-1 on different cardiovascular cells during aging. We will also review the link between imbalances in glucocorticoid levels and the molecular processes responsible for promoting cardiomyocyte dysfunction in aging. Finally, we will discuss the potential for selectively manipulating glucocorticoid signaling in cardiomyocytes, which may represent an improved therapeutic approach for preventing and treating age-related heart disease.

Glucocorticoid receptors are required effectors of TGFß1-induced p38 MAPK signaling to advanced cancer phenotypes in triple-negative breast cancer.

BACKGROUND: Altered signaling pathways typify breast cancer and serve as direct inputs to steroid hormone receptor sensors. We previously reported that phospho-Ser134-GR (pS134-GR) species are elevated in triple-negative breast cancer (TNBC) and cooperate with hypoxia-inducible factors, providing a novel avenue for activation of GR in response to local or cellular stress. METHODS: We probed GR regulation by factors (cytokines, growth factors) that are rich within the tumor microenvironment (TME). TNBC cells harboring endogenous wild-type (wt) or S134A-GR species were created by CRISPR/Cas knock-in and subjected to transwell migration, invasion, soft-agar colony formation, and tumorsphere assays. RNA-seq was employed to identify pS134-GR target genes that are regulated both basally (intrinsic) or by TGFß1 in the absence of exogenously added GR ligands. Regulation of selected basal and TGFß1-induced pS134-GR target genes was validated by qRT-PCR and chromatin immunoprecipitation assays. Bioinformatics tools were used to probe public data sets for expression of pS134-GR 24-gene signatures. RESULTS: In the absence of GR ligands, GR is transcriptionally activated via p38-dependent phosphorylation of Ser134 as a mechanism of homeostatic stress-sensing and regulated upon exposure of TNBC cells to TME-derived agents. The ligand-independent pS134-GR transcriptome encompasses TGFß1 and MAPK signaling gene sets associated with TNBC cell survival and migration/invasion. Accordingly, pS134-GR was essential for TNBC cell anchorage-independent growth in soft-agar, migration, invasion, and tumorsphere formation, an in vitro readout of cancer stemness properties. Both pS134-GR and expression of the MAPK-scaffolding molecule 14-3-3ζ were essential for a functionally intact p38 MAPK signaling pathway downstream of MAP3K5/ASK1, indicative of a feedforward signaling loop wherein self-perpetuated GR phosphorylation enables cancer cell autonomy. A 24-gene pS134-GR-dependent signature induced by TGFß1 predicts shortened overall survival in breast cancer patients. CONCLUSIONS: Phospho-S134-GR is a critical downstream effector of p38 MAPK signaling and TNBC migration/invasion, survival, and stemness properties. Our studies define a ligand-independent role for GR as a homeostatic "sensor" of intrinsic stimuli as well as extrinsic factors rich within the TME (TGFß1) that enable potent activation of the p38 MAPK stress-sensing pathway and nominate pS134-GR as a therapeutic target in aggressive TNBC.

Glucocorticoids preserve the t-tubular system in ventricular cardiomyocytes by upregulation of autophagic flux.

A major contributor to contractile dysfunction in heart failure is remodelling and loss of the cardiomyocyte transverse tubular system (t-system), but underlying mechanisms and signalling pathways remain elusive. It has been shown that dexamethasone promotes t-tubule development in stem cell-derived cardiomyocytes and that cardiomyocyte-specific glucocorticoid receptor (GR) knockout (GRKO) leads to heart failure. Here, we studied if the t-system is altered in GRKO hearts and if GR signalling is required for t-system preservation in adult cardiomyocytes. Confocal and 3D STED microscopy of myocardium from cardiomyocyte-specific GRKO mice revealed decreased t-system density and increased distances between ryanodine receptors (RyR) and L-type Ca2+ channels (LTCC). Because t-system remodelling and heart failure are intertwined, we investigated the underlying mechanisms in vitro. Ventricular cardiomyocytes from failing human and healthy adult rat hearts cultured in the absence of glucocorticoids (CTRL) showed distinctively lower t-system density than cells treated with dexamethasone (EC50 1.1 nM) or corticosterone. The GR antagonist mifepristone abrogated the effect of dexamethasone. Dexamethasone improved RyR-LTCC coupling and synchrony of intracellular Ca2+ release, but did not alter expression levels of t-system-associated proteins junctophilin-2 (JPH2), bridging integrator-1 (BIN1) or caveolin-3 (CAV3). Rather, dexamethasone upregulated LC3B and increased autophagic flux. The broad-spectrum protein kinase inhibitor staurosporine prevented dexamethasone-induced upregulation of autophagy and t-system preservation, and autophagy inhibitors bafilomycin A and chloroquine accelerated t-system loss. Conversely, induction of autophagy by rapamycin or amino acid starvation preserved the t-system. These findings suggest that GR signalling and autophagy are critically involved in t-system preservation and remodelling in the heart.

Deletion of the Cardiomyocyte Glucocorticoid Receptor Leads to Sexually Dimorphic Changes in Cardiac Gene Expression and Progression to Heart Failure.

Background The contribution of glucocorticoids to sexual dimorphism in the heart is essentially unknown. Therefore, we sought to determine the sexually dimorphic actions of glucocorticoid signaling in cardiac function and gene expression. To accomplish this goal, we conducted studies on mice lacking glucocorticoid receptors (GR) in cardiomyocytes (cardioGRKO mouse model). Methods and Results Deletion of cardiomyocyte GR leads to an increase in mortality because of the development of spontaneous cardiac pathology in both male and female mice; however, females are more resistant to GR signaling inactivation in the heart. Male cardioGRKO mice had a median survival age of 6 months. In contrast, females had a median survival age of 10 months. Transthoracic echocardiography data showed phenotypic differences between male and female cardioGRKO hearts. By 3 months of age, male cardioGRKO mice exhibited left ventricular systolic dysfunction. Conversely, no significant functional deficits were observed in female cardioGRKO mice at the same time point. Functional sensitivity of male hearts to the loss of cardiomyocyte GR was reversed following gonadectomy. RNA-Seq analysis showed that deleting GR in the male hearts leads to a more profound dysregulation in the expression of genes implicated in heart rate regulation (calcium handling). In agreement with these gene expression data, cardiomyocytes isolated from male cardioGRKO hearts displayed altered intracellular calcium responses. In contrast, female GR-deficient cardiomyocytes presented a response comparable with controls. Conclusions These data suggest that GR regulates calcium responses in a sex-biased manner, leading to sexually distinct responses to stress in male and female mice hearts, which may contribute to sex differences in heart disease, including the development of ventricular arrhythmias that contribute to heart failure and sudden death.

ß-Arrestin-1 inhibits glucocorticoid receptor turnover and alters glucocorticoid signaling.

Glucocorticoids are among the most widely used drugs to treat many autoimmune and inflammatory diseases. Although much research has been focused on investigating glucocorticoid activity, it remains unclear how glucocorticoids regulate distinct processes in different cells. Glucocorticoids exert their effects through the glucocorticoid receptor (GR), which, upon glucocorticoid binding, interacts with regulatory proteins, affecting its activity and function. These protein-protein interactions are necessary for the resolution of glucocorticoid-dependent physiological and pharmacological processes. In this study, we discovered a novel protein interaction between the glucocorticoid receptor and ß-arrestin-1, a scaffold protein with a well-established role in G protein-coupled receptor signaling. Using co-immunoprecipitation and in situ proximity ligation assays in A549 cells, we observed that ß-arrestin-1 and unliganded GR interact in the cytoplasm and that, following glucocorticoid binding, the protein complex is found in the nucleus. We show that siRNA-mediated ß-arrestin-1 knockdown alters GR protein turnover by up-regulating the E3 ubiquitin ligase Pellino-1, which catalyzes GR ubiquitination and thereby marks the receptor for proteasomal degradation. The enhanced GR turnover observed in ß-arrestin-1-deficient cells limits the duration of the glucocorticoid response on GR target genes. These results demonstrate that ß-arrestin-1 is a crucial player for the stability of the glucocorticoid receptor. The GR/ß-arrestin-1 interaction uncovered here may help unravel mechanisms that contribute to the cell type-specific activities of glucocorticoids.

Cardiomyocyte glucocorticoid and mineralocorticoid receptors directly and antagonistically regulate heart disease in mice.

Stress is increasingly associated with heart dysfunction and is linked to higher mortality rates in patients with cardiometabolic disease. Glucocorticoids are primary stress hormones that regulate homeostasis through two nuclear receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), both of which are present in cardiomyocytes. To examine the specific and coordinated roles that these receptors play in mediating the direct effects of stress on the heart, we generated mice with cardiomyocyte-specific deletion of GR (cardioGRKO), MR (cardioMRKO), or both GR and MR (cardioGRMRdKO). The cardioGRKO mice spontaneously developed cardiac hypertrophy and left ventricular systolic dysfunction and died prematurely from heart failure. In contrast, the cardioMRKO mice exhibited normal heart morphology and function. Despite the presence of myocardial stress, the cardioGRMRdKO mice were resistant to the cardiac remodeling, left ventricular dysfunction, and early death observed in the cardioGRKO mice. Gene expression analysis revealed the loss of gene changes associated with impaired Ca2+ handling, increased oxidative stress, and enhanced cell death and the presence of gene changes that limited the hypertrophic response and promoted cardiomyocyte survival in the double knockout hearts. Reexpression of MR in cardioGRMRdKO hearts reversed many of the cardioprotective gene changes and resulted in cardiac failure. These findings reveal a critical role for balanced cardiomyocyte GR and MR stress signaling in cardiovascular health. Therapies that shift stress signaling in the heart to favor more GR and less MR activity may provide an improved approach for treating heart disease.

Muscle-specific regulation of right ventricular transcriptional responses to chronic hypoxia-induced hypertrophy by the muscle ring finger-1 (MuRF1) ubiquitin ligase in mice.

BACKGROUND: We recently identified a role for the muscle-specific ubiquitin ligase MuRF1 in right-sided heart failure secondary to pulmonary hypertension induced by chronic hypoxia (CH). MuRF1-/- mice exposed to CH are resistant to right ventricular (RV) dysfunction whereas MuRF1 Tg + mice exhibit impaired function indicative of heart failure. The present study was undertaken to understand the underlying transcriptional alterations in the RV of MuRF1-/- and MuRF1 Tg + mice. METHODS: Microarray analysis was performed on RNA isolated from the RV of MuRF1-/-, MuRF1 Tg+, and wild-type control mice exposed to CH. RESULTS: MuRF1-/- RV differentially expressed 590 genes in response to CH. Analysis of the top 66 genes (> 2-fold or < - 2-fold) revealed significant associations with oxidoreductase, transcription regulation, and transmembrane component annotations. The significant genes had promoters enriched for HOXD12, HOXC13, and RREB-1 protein transcription factor binding sites. MuRF1 Tg + RV differentially expressed 150 genes in response to CH. Analysis of the top 45 genes (> 3-fold or < - 3-fold) revealed significant associations with oxidoreductase-metabolic, glycoprotein-transmembrane-integral proteins, and alternative splicing/splice variant annotations. The significant genes were enriched for promoters with ZIC1 protein transcription factor binding sites. CONCLUSIONS: The differentially expressed genes in MuRF1-/- and MuRF1 Tg + RV after CH have common functional annotations related to oxidoreductase (including antioxidant) and transmembrane component functions. Moreover, the functionally-enhanced MuRF1-/- hearts regulate genes related to transcription, homeobox proteins, and kinases/phosphorylation. These studies also reveal potential indirect effects of MuRF1 through regulating Rreb-1, and they reveal mechanisms by which MuRF1 may transcriptionally regulate anti-oxidant systems in the face of right heart failure.