Functional redundancy between glucocorticoid and mineralocorticoid receptors in mature corticotropin-releasing hormone neurons protects from obesity.

OBJECTIVE: Here, we aimed to investigate the role of glucocorticoid and mineralocorticoid receptors (GRs and MRs, respectively) in the regulation of energy homeostasis. METHODS: We used three mouse models with simultaneous deletion of GRs and MRs in either forebrain neurons, the paraventricular nucleus, or corticotropin-releasing hormone (CRH) neurons and compared them with wild-type controls or isolated knockout groups. In addition to body weight, food intake, energy expenditure, insulin sensitivity, fat/lean mass distribution, and plasma corticosterone levels, we also performed transcriptomic analysis of CRH neurons and assessed their response to melanocortinergic stimulation. RESULTS: Similar to global double-knockout models, deletion of GRs and MRs specifically in mature CRH neurons resulted in obesity. Importantly, the latter was accompanied by insulin resistance, but not increased plasma corticosterone levels. Transcriptomic analysis of these neurons revealed upregulation of several genes involved in postsynaptic signal transduction, including the Ptk2b gene, which encodes proline-rich tyrosine kinase 2. Knockout of both nuclear receptors leads to upregulation of Ptk2b in CRH neurons, which results in their diminished responsiveness to melanocortinergic stimulation. CONCLUSIONS: Our data demonstrate the functional redundancy of GRs and MRs in CRH neurons to maintain energy homeostasis and prevent obesity. Simultaneous targeting of both receptors might represent an unprecedented approach to counteract obesity.

Mineralocorticoid Receptor Blocker Prevents Mineralocorticoid Receptor-Mediated Inflammation by Modulating Transcriptional Activity of Mineralocorticoid Receptor-p65-Signal Transducer and Activator of Transcription 3 Complex.

BACKGROUND: Mineralocorticoid receptor (MR) induces cardiac inflammation cooperatively with nuclear factor-κB and signal transducer and activator of transcription 3 (STAT3); MR blockers exert anti-inflammatory effects. However, the underlying mechanism remains unclear. We investigated the anti-inflammatory effect of esaxerenone, a novel MR blocker, in experimental myocardial infarction (MI) and its underlying mechanisms. METHODS AND RESULTS: Male C57BL/6J mice subjected to ligation of the left anterior descending artery were randomly assigned to either the vehicle or esaxerenone group. Esaxerenone was provided with a regular chow diet. The mice were euthanized at either 4 or 15 days after MI. Cardiac function, fibrosis, and inflammation were evaluated. Esaxerenone significantly improved cardiac function and attenuated cardiac fibrosis at 15 days after MI independently of its antihypertensive effect. Inflammatory cell infiltration, inflammatory-related gene expression, and elevated serum interleukin-6 levels at 4 days after MI were significantly attenuated by esaxerenone. In vitro experiments using mouse macrophage-like cell line RAW264.7 cells demonstrated that esaxerenone- and spironolactone-attenuated lipopolysaccharide-induced interleukin-6 expression without altering the posttranslational modification and nuclear translocation of p65 and STAT3. Immunoprecipitation assays revealed that MR interacted with both p65 and STAT3 and enhanced the p65-STAT3 interaction, leading to a subsequent increase in interleukin-6 promoter activity, which was reversed by esaxerenone. CONCLUSIONS: Esaxerenone ameliorated postinfarct remodeling in experimental MI through its anti-inflammatory properties exerted by modulating the transcriptional activity of the MR-p65-STAT3 complex. These results suggest that the MR-p65-STAT3 complex can be a novel therapeutic target for treating MI.

Mineralocorticoid Receptor Signaling in Peripheral Blood Cells in Patients with Multiple Sclerosis.

Multiple sclerosis (MS) is associated with alterations in neuroendocrine function, primarily the hypothalamic-pituitary-adrenal axis, including lower expression of the glucocorticoid receptor (GR) and its target genes in peripheral blood mononuclear cells (PBMC) or full blood. We previously found reduced mineralocorticoid receptor (MR) expression in MS patients' peripheral blood. MS is being treated with a widening variety of disease-modifying treatments (DMT), some of which have similar efficacy but different mechanisms of action; body-fluid biomarkers to support the choice of the optimal initial DMT and/or to indicate an unsatisfactory response before clinical activity are unavailable. Using cell culture of volunteers' PBMCs and subsequent gene expression analysis (microarray and qPCR validation), we identified the mRNA expression of OTUD1 to represent MR signaling. The MR and MR target gene expression levels were then measured in full blood samples. In 119 MS (or CIS) patients, the expression of both MR and OTUD1 was lower than in 42 controls. The expression pattern was related to treatment, with the MR expression being particularly low in patients treated with fingolimod. While MR signaling may be involved in the therapeutic effects of some disease-modifying treatments, MR and OTUD1 expression can complement the neuroendocrine assessment of MS disease course. If confirmed, such assessment may support clinical decision-making.

Novel Evolution of Mineralocorticoid Receptor in Humans Compared to Chimpanzees, Gorillas, and Orangutans.

We identified five distinct full-length human mineralocorticoid receptor (MR) genes containing either 984 amino acids (MR-984) or 988 amino acids (MR-988), which can be distinguished by the presence or absence of Lys, Cys, Ser, and Trp (KCSW) in their DNA-binding domain (DBD) and mutations at codons 180 and 241 in their amino-terminal domain (NTD). Two human MR-KCSW genes contain either (Val-180, Val-241) or (Ile-180, Val-241) in their NTD, and three human MR-984 genes contain either (Ile-180, Ala-241), (Val-180, Val-241), or (Ile-180, Val-241). Human MR-KCSW with (Ile-180, Ala-241) has not been cloned. In contrast, chimpanzees contain four MRs: two MR-988s with KCSW in their DBD, or two MR-984s without KCSW in their DBD. Chimpanzee MRs only contain (Ile180, Val-241) in their NTD. A chimpanzee MR with either (Val-180, Val-241) or (Ile-180, Ala-241) in the NTD has not been cloned. Gorillas and orangutans each contain one MR-988 with KCSW in the DBD and one MR-984 without KCSW, and these MRs only contain (Ile-180, Val-241) in their NTD. A gorilla MR or orangutan MR with either (Val-180, Val-241) or (Ile-180, Ala-241) in the NTD has not been cloned. Together, these data suggest that human MRs with (Val-180, Val-241) or (Ile-180, Ala-241) in the NTD evolved after humans and chimpanzees diverged from their common ancestor. Considering the multiple functions in human development of the MR in kidney, brain, heart, skin, and lungs, as well as MR activity in interaction with the glucocorticoid receptor, we suggest that the evolution of human MRs that are absent in chimpanzees may have been important in the evolution of humans from chimpanzees. Investigation of the physiological responses to corticosteroids mediated by the MR in humans, chimpanzees, gorillas, and orangutans may provide insights into the evolution of humans and their closest relatives.

An in Vitro triple screen model for human mineralocorticoid receptor activity.

The mineralocorticoid receptor (MR, NR3C2) mediates ion and water homeostasis in epithelial cells of the distal nephron and other tissues. Aldosterone, the prototypical mineralocorticoid, regulates electrolyte and fluid balance. Cortisol binds to MR with equal affinity to aldosterone, but many MR-expressing tissues inactivate cortisol to cortisone via 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2). Dysregulated MR activation contributes to direct cardiovascular tissue insults. Besides aldosterone and cortisol, a variety of MR agonists and/or HSD11B2 inhibitors are putative players in the pathophysiology of low-renin hypertension (LRH), and cardiovascular and metabolic pathology. We developed an in vitro human MR (hMR) model, to facilitate screening for MR agonists, antagonists, and HSD11B2 inhibitors. The CV1 monkey kidney cells were transduced with lentivirus to stably express hMR and an MR-responsive gaussia luciferase gene. Clonal populations of MR-expressing cells (CV1-MRluc) were further transduced to express HSD11B2 (CV1-MRluc-HSD11B2). CV1-MRluc and CV1-MRluc-HSD11B2 cells were treated with aldosterone, cortisol, 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18OHB), 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), progesterone, or 17-hydroxyprogesterone (17OHP). In CV1-MRLuc cells, aldosterone and DOC displayed similar potency (EC50: 0.45 nM and 0.30 nM) and maximal response (31- and 23-fold increase from baseline) on hMR; 18oxoF and 18OHB displayed lower potency (19.6 nM and 56.0 nM, respectively) but similar maximal hMR activation (25- and 27-fold increase, respectively); cortisol and corticosterone exhibited higher maximal responses (73- and 52-fold, respectively); 18OHF showed no MR activation. Progesterone and 17OHP inhibited aldosterone-mediated MR activation. In the MRluc-HSD11B2 model, the EC50 of cortisol for MR activation increased from 20 nM (CV1-MRLuc) to ∼2000 nM, while the EC50 for aldosterone remained unchanged. The addition of 18ß-glycyrrhetinic acid (18ß-GA), a HSD11B2 inhibitor, restored the potency of cortisol back to ∼70 nM in CV1-hMRLuc-HSD11B2 cells. Together, these two cell models will facilitate the discovery of novel MR-modulators, informing MR-mediated pathophysiology mechanisms and drug development efforts.

Both endothelial mineralocorticoid receptor expression and hyperleptinemia are required for clinical characteristics of placental ischemia in mice.

One of the initiating events in preeclampsia (PE) is placental ischemia. Rodent models of placental ischemia do not present with vascular endothelial dysfunction, a hallmark of PE. We previously demonstrated a role for leptin in endothelial dysfunction in pregnancy in the absence of placental ischemia. We hypothesized that placental ischemia requires hyperleptinemia and endothelial mineralocorticoid receptor (ECMR) expression to induce PE-associated endothelial dysfunction in pregnant mice. We induced placental ischemia via the reduced uterine perfusion pressure (RUPP) procedure in pregnant ECMR-intact (ECMR+/+) and ECMR deletion (ECMR-/-) mice at gestational day (GD) 13. ECMR+/+ RUPP pregnant mice also received concurrent leptin infusion via miniosmotic pump (0.9 mg/kg/day). RUPP increased blood pressure via radiotelemetry and decreased fetal growth in ECMR+/+ pregnant mice. Both increases in blood pressure and reduced fetal growth were abolished in RUPP ECMR-/- mice. Placental ischemia did not decrease endothelial-dependent relaxation to acetylcholine (ACh) but increased phenylephrine (Phe) contraction in mesenteric arteries of pregnant mice, which was ablated by ECMR deletion. Addition of leptin to RUPP mice significantly reduced ACh relaxation in ECMR+/+ pregnant mice, accompanied by an increase in soluble FMS-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PLGF) ratio. In conclusion, our data indicate that high leptin levels drive endothelial dysfunction in PE and that ECMR is required for clinical characteristics of hypertension and fetal growth restriction in placental ischemia PE. Collectively, we show that both ECMR and leptin play a role to mediate PE.NEW & NOTEWORTHY Leptin is a key feature of preeclampsia that initiates vascular endothelial dysfunction in preeclampsia characterized by placental ischemia. Endothelial mineralocorticoid receptor (ECMR) deletion in placental ischemia protects pregnant mice from elevations in blood pressure and fetal growth restriction in pregnancy. Increases in leptin production mediate the key pathological feature of endothelial dysfunction in preeclampsia in rodents. ECMR activation contributes to the increase in blood pressure and fetal growth restriction in preeclampsia.

Early life stress unravels epistatic genetic associations of cortisol pathway genes with depression.

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis represents one of the most consistent pathophysiological findings in depressive disorders. Cortisol signaling is affected by proteins that mediate its cellular responses or alters its availability to mineralocorticoid and glucocorticoid receptors. In our study, we evaluated candidate genes that may influence the risk for depression and suicide due to its involvement in cortisol signaling. The aim of the study was to assess whether the genotypes of these genes are associated with the risk for depression, severity of depressive symptoms, suicidal ideation, and suicide attempts. And whether there is interaction between genes and early-life stress. In this study, 100 healthy controls and 140 individuals with depression were included. The subjects were clinically assessed using the 21-item GRID-Hamilton questionnaires (GRID-HAMD-21), Beck Scale for Suicidal Ideation (BSI), and the Childhood Trauma Questionnaire (CTQ). A robust multifactorial dimensionality reduction analysis was used to characterize the interactions between the genes HSD11B1, NR3C1, NR3C2, and MDR1 and early-life stress. It was found a significant association of the heterozygous genotype of the MDR1 gene rs1128503 polymorphism with reduced risk of at least one suicide attempt (OR: 0.08, p = 0.003*) and a reduction in the number of suicide attempts (ß = -0.79, p = 0.006*). Furthermore, it was found that the MDR1 rs1228503 and NR3C2 rs2070951 genes interact with early-life stress resulting in a strong association with depression (p = 0.001). Our findings suggest that polymorphisms in the MDR1 and NR3C2 genes and their interaction with childhood trauma may be important biomarkers for depression and suicidal behaviors.

Klotho is highly expressed in the chief sites of regulated potassium secretion, and it is stimulated by potassium intake.

Klotho regulates many pathways in the aging process, but it remains unclear how it is physiologically regulated. Because Klotho is synthesized, cleaved, and released from the kidney; activates the chief urinary K+ secretion channel (ROMK) and stimulates urinary K+ secretion, we explored if Klotho protein is regulated by dietary K+ and the potassium-regulatory hormone, Aldosterone. Klotho protein along the nephron was evaluated in humans and in wild-type (WT) mice; and in mice lacking components of Aldosterone signaling, including the Aldosterone-Synthase KO (AS-KO) and the Mineralocorticoid-Receptor KO (MR-KO) mice. We found the specific cells of the distal nephron in humans and mice that are chief sites of regulated K+ secretion have the highest Klotho protein expression along the nephron. WT mice fed K+-rich diets increased Klotho expression in these cells. AS-KO mice exhibit normal Klotho under basal conditions but could not upregulate Klotho in response to high-K+ intake in the K+-secreting cells. Similarly, MR-KO mice exhibit decreased Klotho protein expression. Together, i) Klotho is highly expressed in the key sites of regulated K+ secretion in humans and mice, ii) In mice, K+-rich diets increase Klotho expression specifically in the potassium secretory cells of the distal nephron, iii) Aldosterone signaling is required for Klotho response to high K+ intake.

The multifaceted role of the mineralocorticoid receptor in cancers.

The mineralocorticoid receptor (MR/NR3C2) is a member of the family of steroid receptors (SR) which also includes the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and glucocorticoid receptor (GR). They function primarily as nuclear receptors to regulate gene expression. While the other steroid hormone receptors are known to play important roles in the pathogenesis and progression of many cancers, relatively little is understood about the role of MR in cancer biology. This review focuses on examining new insights into the potential roles and mechanisms of action of MR in cancers.

Pregestational Prediabetes Induces Maternal Hypothalamic-Pituitary-Adrenal (HPA) Axis Dysregulation and Results in Adverse Foetal Outcomes.

Maternal type 2 diabetes mellitus (T2DM) has been shown to result in foetal programming of the hypothalamic-pituitary-adrenal (HPA) axis, leading to adverse foetal outcomes. T2DM is preceded by prediabetes and shares similar pathophysiological complications. However, no studies have investigated the effects of maternal prediabetes on foetal HPA axis function and postnatal offspring development. Hence, this study investigated the effects of pregestational prediabetes on maternal HPA axis function and postnatal offspring development. Pre-diabetic (PD) and non-pre-diabetic (NPD) female Sprague Dawley rats were mated with non-prediabetic males. After gestation, male pups born from the PD and NPD groups were collected. Markers of HPA axis function, adrenocorticotropin hormone (ACTH) and corticosterone, were measured in all dams and pups. Glucose tolerance, insulin and gene expressions of mineralocorticoid (MR) and glucocorticoid (GR) receptors were further measured in all pups at birth and their developmental milestones. The results demonstrated increased basal concentrations of ACTH and corticosterone in the dams from the PD group by comparison to NPD. Furthermore, the results show an increase basal ACTH and corticosterone concentrations, disturbed MR and GR gene expression, glucose intolerance and insulin resistance assessed via the Homeostasis Model Assessment (HOMA) indices in the pups born from the PD group compared to NPD group at all developmental milestones. These observations reveal that pregestational prediabetes is associated with maternal dysregulation of the HPA axis, impacting offspring HPA axis development along with impaired glucose handling.

Lysine-Cysteine-Serine-Tryptophan inserted into the DNA-binding domain of human mineralocorticoid receptor increases transcriptional activation by aldosterone.

Due to alternative splicing in an ancestral DNA-binding domain (DBD) of the mineralocorticoid receptor (MR), humans contain two almost identical MR transcripts with either 984 amino acids (MR-984) or 988 amino acids (MR-988), in which their DBDs differ by only four amino acids, Lys,Cys,Ser,Trp (KCSW). Human MRs also contain mutations at two sites, codons 180 and 241, in the amino terminal domain (NTD). Together, there are five distinct full-length human MR genes in GenBank. Human MR-984, which was cloned in 1987, has been extensively studied. Human MR-988, cloned in 1995, contains KCSW in its DBD. Neither this human MR-988 nor the other human MR-988 genes have been studied for their response to aldosterone and other corticosteroids. Here, we report that transcriptional activation of human MR-988 by aldosterone is increased by about 50 % compared to activation of human MR-984 in HEK293 cells transfected with the TAT3 promoter, while the half-maximal response (EC50) is similar for aldosterone activation of MR-984 and MR-988. Transcriptional activation of human MR also depends on the amino acids at codons 180 and 241. Interestingly, in HEK293 cells transfected with the MMTV promoter, transcriptional activation by aldosterone of human MR-988 is similar to activation of human MR-984, indicating that the promoter has a role in the regulation of the response of human MR-988 to aldosterone. The physiological responses to aldosterone and other corticosteroids in humans with MR genes containing KCSW and with differences at codons 180 and 241 in the NTD warrant investigation.

NR3C2 microdeletions-an underrecognized cause of pseudohypoaldosteronism type 1A: a case report and literature review.

OBJECTIVES: Pseudohypoaldosteronism type 1A (PHA1A) is caused by haploinsufficiency of the mineralocorticoid receptor (MR). Heterozygous small insertions/deletions, transitions, and/or transversions within NR3C2 comprise the majority (85%-90%) of pathogenic copy number variants. Structural chromosomal abnormalities, contiguous gene deletion syndromes, and microdeletions are infrequent. We describe a neonate with PHA1A due to a novel NR3C2 microdeletion involving exons 1-2. METHODS: Literature review identified 39 individuals with PHA1A due to NR3C2 microdeletions. Transmission modality, variant description(s), testing method(s), exon(s) deleted, and affected functional domain(s) were characterized. RESULTS: In total, 40 individuals with NR3C2 microdeletions were described: 19 involved contiguous exons encoding a single MR domain; 21 involved contiguous exons encoding multiple MR domains. Transmission modality frequency was familial (65%), de novo (20%), or unknown (15%). Sequencing (Sanger or short-read next-generation) failed to detect microdeletions in 100% of tested individuals (n = 38). All were detected using deletion/duplication testing modalities. In 2 individuals, only microarray-based testing was performed; microdeletions were detected in both cases. CONCLUSION: Initial testing for PHA1A should rely on sequencing to detect the most common genetic alterations. Deletion/duplication analysis should be performed when initial testing is nondiagnostic. Most NR3C2 microdeletions are parentally transmitted, thus highlighting the importance of familial genetic testing and counseling.

Atlas of cardiac endothelial cell enhancer elements linking the mineralocorticoid receptor to pathological gene expression.

Endothelial cells play crucial roles in physiology and are increasingly recognized as therapeutic targets in cardiovascular disease. Here, we analyzed the regulatory landscape of cardiac endothelial cells by assessing chromatin accessibility, histone modifications, and 3D chromatin organization and confirmed the functional relevance of enhancer-promoter interactions by CRISPRi-mediated enhancer silencing. We used this dataset to explore mechanisms of transcriptional regulation in cardiovascular disease and compared six different experimental models of heart failure, hypertension, or diabetes. Enhancers that regulate gene expression in diseased endothelial cells were enriched with binding sites for a distinct set of transcription factors, including the mineralocorticoid receptor (MR), a known drug target in heart failure and hypertension. For proof of concept, we applied endothelial cell-specific MR deletion in mice to confirm MR-dependent gene expression and predicted direct MR target genes. Overall, we have compiled here a comprehensive atlas of cardiac endothelial cell enhancer elements that provides insight into the role of transcription factors in cardiovascular disease.

Myosin Light Chain Phosphatase Plays an Important Role in Cardiac Fibrosis in a Model of Mineralocorticoid Receptor-Associated Hypertension.

BACKGROUND: Myosin phosphatase targeting subunit 2 (MYPT2) is an important subunit of cardiac MLC (myosin light chain) phosphatase, which plays a crucial role in regulating the phosphorylation of MLC to phospho-MLC (p-MLC). A recent study demonstrated mineralocorticoid receptor-related hypertension is associated with RhoA/Rho-associated kinase/MYPT1 signaling upregulation in smooth muscle cells. Our purpose is to investigate the effect of MYPT2 on cardiac function and fibrosis in mineralocorticoid receptor-related hypertension. METHODS AND RESULTS: HL-1 murine cardiomyocytes were incubated with different concentrations or durations of aldosterone. After 24-hour stimulation, aldosterone increased CTGF (connective tissue growth factor) and MYPT2 and decreased p-MLC in a dose-dependent manner. MYPT2 knockdown decreased CTGF. Cardiac-specific MYPT2-knockout (c-MYPT2-/-) mice exhibited decreased type 1 phosphatase catalytic subunit ß and increased p-MLC. A disease model of mouse was induced by subcutaneous aldosterone and 8% NaCl food for 4 weeks after uninephrectomy. Blood pressure elevation and left ventricular hypertrophy were observed in both c-MYPT2-/- and MYPT2+/+ mice, with no difference in heart weights or nuclear localization of mineralocorticoid receptor in cardiomyocytes. However, c-MYPT2-/- mice had higher ejection fraction and fractional shortening on echocardiography after aldosterone treatment. Histopathology revealed less fibrosis, reduced CTGF, and increased p-MLC in c-MYPT2-/- mice. Basal global radial strain and global longitudinal strain were higher in c-MYPT2-/- than in MYPT2+/+ mice. After aldosterone treatment, both global radial strain and global longitudinal strain remained higher in c-MYPT2-/- mice compared with MYPT2+/+ mice. CONCLUSIONS: Cardiac-specific MYPT2 knockout leads to decreased myosin light chain phosphatase and increased p-MLC. MYPT2 deletion prevented cardiac fibrosis and dysfunction in a model of mineralocorticoid receptor-associated hypertension.

Mineralocorticoid promotes intestinal inflammation through receptor dependent IL17 production in ILC3s.

Aldosterone is a key mineralocorticoid involved in regulating the concentration of blood electrolytes and physiological volume balance. Activation of mineralocorticoid receptor (MR) has been recently reported to participate in adaptive and innate immune responses under inflammation. Here, we evaluated the role of aldosterone and MR in inflammation bowel diseases (IBD). Aldosterone elevated in the colon of DSS-induced colitis mice. Aldosterone addition induced IL17 production and ROS/RNS level in group 3 innate lymphoid cells (ILC3s) and exacerbated intestinal injury. A selective mineralocorticoid receptor antagonism, eplerenone, inhibited IL17-producing ILC3s and its ROS/RNS production, protected mice from DSS-induced colitis. Mice lacking Nr3c2 (MR coding gene) in ILC3s exhibited decreased IL17 and ROS/RNS production, which alleviated colitis and colitis-associated colorectal cancer (CAC). Further experiments revealed that MR could directly bind to IL17A promoter and facilitate its transcription, which could be enhanced by aldosterone. Thus, our findings demonstrated the critical role of aldosterone-MR-IL17 signaling in ILC3s and gut homeostasis, indicating the therapeutic strategy of eplerenone in IBD clinical trial.

Mineralocorticoid receptor promotes cardiac macrophage inflammaging.

Inflammaging, a pro-inflammatory status that characterizes aging and primarily involving macrophages, is a master driver of age-related diseases. Mineralocorticoid receptor (MR) activation in macrophages critically regulates inflammatory and fibrotic processes. However, macrophage-specific mechanisms and the role of the macrophage MR for the regulation of inflammation and fibrotic remodeling in the aging heart have not yet been elucidated. Transcriptome profiling of cardiac macrophages from male/female young (4 months-old), middle (12 months-old) and old (18 and 24 months-old) mice revealed that myeloid cell-restricted MR deficiency prevents macrophage differentiation toward a pro-inflammatory phenotype. Pathway enrichment analysis showed that several biological processes related to inflammation and cell metabolism were modulated by the MR in aged macrophages. Further, transcriptome analysis of aged cardiac fibroblasts revealed that macrophage MR deficiency reduced the activation of pathways related to inflammation and upregulation of ZBTB16, a transcription factor involved in fibrosis. Phenotypic characterization of macrophages showed a progressive replacement of the TIMD4+MHC-IIneg/low macrophage population by TIMD4+MHC-IIint/high and TIMD4-MHC-IIint/high macrophages in the aging heart. By integrating cell sorting and transwell experiments with TIMD4+/TIMD4-macrophages and fibroblasts from old MRflox/MRLysMCre hearts, we showed that the inflammatory crosstalk between TIMD4- macrophages and fibroblasts may imply the macrophage MR and the release of mitochondrial superoxide anions. Macrophage MR deficiency reduced the expansion of the TIMD4- macrophage population and the emergence of fibrotic niches in the aging heart, thereby protecting against cardiac inflammation, fibrosis, and dysfunction. This study highlights the MR as an important mediator of cardiac macrophage inflammaging and age-related fibrotic remodeling.

The impact of parental and developmental stress on DNA methylation in the avian hypothalamic-pituitary-adrenal axis.

The hypothalamic-pituitary-adrenal (HPA) axis coordinates an organism's response to environmental stress. The responsiveness and sensitivity of an offspring's stress response may be shaped not only by stressors encountered in their early post-natal environment but also by stressors in their parent's environment. Yet, few studies have considered how stressors encountered in both of these early life environments may function together to impact the developing HPA axis. Here, we manipulated stressors in the parental and post-natal environments in a population of house sparrows (Passer domesticus) to assess their impact on changes in DNA methylation (and corresponding gene expression) in a suite of genes within the HPA axis. We found that nestlings that experienced early life stress across both life-history periods had higher DNA methylation in a critical HPA axis gene, the glucocorticoid receptor (NR3C1). In addition, we found that the life-history stage when stress was encountered impacted some genes (HSD11B1, NR3C1 and NR3C2) differently. We also found evidence for the mitigation of parental stress by post-natal stress (in HSD11B1 and NR3C2). Finally, by assessing DNA methylation in both the brain and blood, we were able to evaluate cross-tissue patterns. While some differentially methylated regions were tissue-specific, we found cross-tissue changes in NR3C2 and NR3C1, suggesting that blood is a suitable tissue for assessing DNA methylation as a biomarker of early life stress. Our results provide a crucial first step in understanding the mechanisms by which early life stress in different life-history periods contributes to changes in the epigenome of the HPA axis.

mTOR Regulates Mineralocorticoid Receptor Transcriptional Activity by ULK1-Dependent and -Independent Mechanisms.

The mineralocorticoid receptor (MR) is a transcription factor for genes mediating diverse, cell-specific functions, including trophic effects as well as promoting fluid/electrolyte homeostasis. It was reported that in intercalated cells, phosphorylation of the MR at serine 843 (S843) by Unc-51-like kinase (ULK1) inhibits MR activation and that phosphorylation of ULK1 by mechanistic target of rapamycin (mTOR) inactivates ULK1, and thereby prevents MR inactivation. We extended these findings with studies in M1 mouse cortical collecting duct cells stably expressing the rat MR and a reporter gene. Pharmacological inhibition of ULK1 dose-dependently increased ligand-induced MR transactivation, while ULK1 activation had no effect. Pharmacological inhibition of mTOR and CRISPR/gRNA gene knockdown of rapamycin-sensitive adapter protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) decreased phosphorylated ULK1 and ligand-induced activation of the MR reporter gene, as well as transcription of endogenous MR-target genes. As predicted, ULK1 inhibition had no effect on aldosterone-mediated transcription in M1 cells with the mutated MR-S843A (alanine cannot be phosphorylated). In contrast, mTOR inhibition dose-dependently decreased transcription in the MR-S843A cells, though not as completely as in cells with the wild-type MR-S843. mTOR, Raptor, and Rictor coprecipitated with the MR and addition of aldosterone increased their phosphorylated, active state. These results suggest that mTOR significantly regulates MR activity in at least 2 ways: by suppressing MR inactivation by ULK1, and by a yet ill-defined mechanism that involves direct association with MR. They also provide new insights into the diverse functions of ULK1 and mTOR, 2 key enzymes that monitor the cell's energy status.