Regulation of the Intestinal Extra-Adrenal Steroidogenic Pathway Component LRH-1 by Glucocorticoids in Ulcerative Colitis.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) and can be treated with glucocorticoids (GC), although some patients are unresponsive to this therapy. The transcription factor LRH-1/NR5A2 is critical to intestinal cortisol production (intestinal steroidogenesis), being reduced in UC patients. However, the relationship between LRH-1 expression and distribution with altered corticosteroid responses is unknown. To address this, we categorized UC patients by their steroid response. Here, we found that steroid-dependent and refractory patients presented reduced glucocorticoid receptor (GR)-mediated intestinal steroidogenesis compared to healthy individuals and responder patients, possibly related to increased colonic mucosa GR isoform beta (GRß) content and cytoplasmic LRH-1 levels in epithelial and lamina propria cells. Interestingly, an intestinal epithelium-specific GR-induced knockout (GRiKO) dextran sodium sulfate (DSS)-colitis mice model presented decreased epithelial LRH-1 expression, whilst it increased in the lamina propria compared to DSS-treated control mice. Mechanistically, GR directly induced NR5A2 gene expression in CCD841CoN cells and human colonic organoids. Furthermore, GR bound to two glucocorticoid-response elements within the NR5A2 promoter in dexamethasone-stimulated CCD841CoN cells. We conclude that GR contributes to intestinal steroidogenesis by inducing LRH-1 in epithelial cells, suggesting LRH-1 as a potential marker for glucocorticoid-impaired response in UC. However, further studies with a larger patient cohort will be necessary to confirm role of LRH-1 as a therapeutic biomarker.

Chronic restraint stress produces sex-specific behavioral and molecular outcomes in the dorsal and ventral rat hippocampus.

Stress-related disorders display differences at multiple levels according to sex. While most studies have been conducted in male rodents, less is known about comparable outcomes in females. In this study, we found that the chronic restraint stress model (2.5 h/day for 14 days) triggers different somatic responses in male and female adult rats. Chronic restraint produced a loss in sucrose preference and novel location preference in male rats. However, chronic restraint failed to produce loss of sucrose preference in females, while it improved spatial performance. We then characterized the molecular responses associated with these behaviors in the hippocampus, comparing the dorsal and ventral poles. Notably, sex- and hippocampal pole-specific transcriptional signatures were observed, along with a significant concordance between the female ventral and male dorsal profiles. Functional enrichment analysis revealed both shared and specific terms associated with each pole and sex. By looking into signaling pathways that were associated with these terms, we found an ample array of sex differences in the dorsal and, to a lesser extent, in the ventral hippocampus. These differences were mainly present in synaptic TrkB signaling, Akt pathway, and glutamatergic receptors. Unexpectedly, the effects of stress on these pathways were rather minimal and mostly dissociated from the sex-specific behavioral outcomes. Our study suggests that female rats are resilient and males susceptible to the restraint stress exposure in the sucrose preference and object location tests, while the activity of canonical signaling pathways is primarily determined by sex rather than stress in the dorsal and ventral hippocampus.

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.

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.

Inhibition of miR-378a-3p by Inflammation Enhances IL-33 Levels: A Novel Mechanism of Alarmin Modulation in Ulcerative Colitis.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by mucosa damage associated with an uncontrolled inflammatory response. This immunological impairment leads to altered inflammatory mediators such as IL-33, which is shown to increase in the mucosa of active UC (aUC) patients. MicroRNAs present a distorted feature in inflamed colonic mucosa and are potential IL-33 regulating candidates in UC. Therefore, we studied the microRNA and mRNA profiles in inflamed colonic samples of UC patients, evaluating the effect of a microRNA (selected by in silico analysis and its expression in UC patients), on IL-33 under inflammatory conditions. We found that inflamed mucosa (n = 8) showed increased expression of 40 microRNAs and 2,120 mRNAs, while 49 microRNAs and 1,734 mRNAs were decreased, as determined by microarrays. In particular, IL-33 mRNA showed a 3.8-fold increase and eight members of a microRNA family (miR-378), which targets IL-33 mRNA in the 3'UTR, were decreased (-3.9 to -3.0 times). We selected three members of the miR-378 family (miR-378a-3p, miR-422a, and miR-378c) according to background information and interaction energy analysis, for further correlation analyses with IL-33 expression through qPCR and ELISA, respectively. We determined that aUC (n = 24) showed high IL-33 levels, and decreased expression of miR-378a-3p and miR-422a compared to inactive UC (n = 10) and controls (n = 6). Moreover, both microRNAs were inversely correlated with IL-33 expression, while miR-378c does not show a significant difference. To evaluate the effect of TNFα on the studied microRNAs, aUC patients with anti-TNF therapy were compared to aUC receiving other treatments. The levels of miR-378a-3p and miR-378c were higher in aUC patients with anti-TNF. Based on these findings, we selected miR-378a-3p to exploring the molecular mechanism involved by in vitro assays, showing that over-expression of miR-378a-3p decreased the levels of an IL-33 target sequence ß-gal-reporter gene in HEK293 cells. Stable miR-378a-3p over-expression/inhibition inversely modulated IL-33 content and altered viability of HT-29 cells. Additionally, in an inflammatory context, TNFα decreased miR-378a-3p levels in HT-29 cells enhancing IL-33 expression. Together, our results propose a regulatory mechanism of IL-33 expression exerted by miR-378a-3p in an inflammatory environment, contributing to the understanding of UC pathogenesis.

MicroRNA Profiling and Bioinformatics Target Analysis in Dorsal Hippocampus of Chronically Stressed Rats: Relevance to Depression Pathophysiology.

Studies conducted in rodents subjected to chronic stress and some observations in humans after psychosocial stress, have allowed to establish a link between stress and the susceptibility to many complex diseases, including mood disorders. The studies in rodents have revealed that chronic exposure to stress negatively affects synaptic plasticity by triggering changes in the production of trophic factors, subunit levels of glutamate ionotropic receptors, neuron morphology, and neurogenesis in the adult hippocampus. These modifications may account for the impairment in learning and memory processes observed in chronically stressed animals. It is plausible then, that stress modifies the interplay between signal transduction cascades and gene expression regulation in the hippocampus, therefore leading to altered neuroplasticity and functioning of neural circuits. Considering that miRNAs play an important role in post-transcriptional-regulation of gene expression and participate in several hippocampus-dependent functions; we evaluated the consequences of chronic stress on the expression of miRNAs in dorsal (anterior) portion of the hippocampus, which participates in memory formation in rodents. Here, we show that male rats exposed to daily restraint stress (2.5 h/day) during 7 and 14 days display a differential profile of miRNA levels in dorsal hippocampus and remarkably, we found that some of these miRNAs belong to the miR-379-410 cluster. We confirmed a rise in miR-92a and miR-485 levels after 14 days of stress by qPCR, an effect that was not mimicked by chronic administration of corticosterone (14 days). Our in silico study identified the top-10 biological functions influenced by miR-92a, nine of which were shared with miR-485: Nervous system development and function, Tissue development, Behavior, Embryonic development, Organ development, Organismal development, Organismal survival, Tissue morphology, and Organ morphology. Furthermore, our in silico study provided a landscape of potential miRNA-92a and miR-485 targets, along with relevant canonical pathways related to axonal guidance signaling and cAMP signaling, which may influence the functioning of several neuroplastic substrates in dorsal hippocampus. Additionally, the combined effect of miR-92a and miR-485 on transcription factors, along with histone-modifying enzymes, may have a functional relevance by producing changes in gene regulatory networks that modify the neuroplastic capacity of the adult dorsal hippocampus under stress.

LPS regulates the expression of glucocorticoid receptor α and ß isoforms and induces a selective glucocorticoid resistance in vitro.

BACKGROUND: This study was aimed to evaluate the effect of LPS in glucocorticoid receptor (GR) isoforms expression on different cell lines and PBMC from healthy donors in vitro and glucocorticoid sensitivity of PBMC in vitro. METHODS: U-2 OS cell lines expressing GR isoforms, different cell lines (CEM, RAJI, K562 and HeLa) or PBMC from healthy donors, were cultured or not with LPS. The expression of GRα and GRß was evaluated by Western blot. Glucocorticoid sensitivity was evaluated in PBMC treated with LPS, testing genes which are transactivated or transrepressed by glucocorticoid. For transactivated genes (MKP1, FKBP5) PBMC were treated with Dexamethasone 100 nM for 6 h. The mRNA expression was measured by RT-PCR. For transrepressed genes (IL-8, GM-CSF), PBMC were cultured in Dexamethasone 100 nM and LPS 10 µg/ml for 6 h and protein expression was measure by ELISA. RESULTS: GR isoforms were induced in U-2 OS cells with a greater effect on GRα expression. Both isoforms were also induced in CEM cells with a tendency to a greater effect on GRß. LPS induced only the expression of GRα in Raji and HeLa cells, and in PBMC, with no effect in K562 cells. LPS induced a loss of glucocorticoid inhibitory effect only on the secretion of GM-CSF. CONCLUSION: LPS in vitro differentially modulates the expression of GR isoforms in a cell specific manner. In PBMC from healthy donors LPS induces an approximately two times increase in the expression of GRα and a loss of the glucocorticoid inhibitory effect on the secretion of GM-CSF, without affecting other glucocorticoid responses evaluated.

A functional IL1RL1 variant regulates corticosteroid-induced sST2 expression in ulcerative colitis.

The ST2/IL33 signalling pathway has been associated with ulcerative colitis (UC). ST2, encoded by the IL1RL1 gene, is expressed as both a membrane-anchored receptor (ST2L) activated by IL33 and as a soluble receptor (sST2) with anti-inflammatory properties. In UC patients, sST2 is further increased by corticosteroid treatment; however, the glucocorticoid-mediated molecular regulation remains unknown. We therefore tested whether genetic variants in the IL1RL1 distal promoter are involved in UC and affect glucocorticoid-mediated ST2 expression. Serum ST2 levels and genetic variants in the IL1RL1 distal promoter were examined by ELISA and PCR sequencing in UC patients receiving corticosteroids. Glucocorticoid-mediated ST2 production was evaluated in intestinal mucosa cultures. Molecular regulation of glucocorticoid-mediated ST2 was assessed by RT-qPCR, ChIP assay and luciferase reporter assay. Dexamethasone effect on ST2 transcript expression was analyzed in leukocytes and related to IL1RL1 variants. Sequencing of a distal IL1RL1 promoter region demonstrated that SNPs rs6543115(C) and rs6543116(A) are associated with increased sST2 in UC patients on corticosteroids. Dexamethasone up-regulated sST2 transcription through interaction with the glucocorticoid-response element (GRE) carrying rs6543115(C) variant. Our data indicate that IL1RL1 SNPs rs6543115(C) confer susceptibility to UC and is contained in the GRE, which may modulate glucocorticoid-induced sST2 expression.

Phosphatidylinositol 3-kinase interacts with the glucocorticoid receptor upon TLR2 activation.

Airway inflammation is a common condition where glucocorticoids (GC) are a well-established therapy. It has been demonstrated that GC stimulate components of innate immunity. Specifically, GC up-regulate TLR2 expression and activation upon inflammatory stimuli; however, little is known about the signalling involved in this process. To determine the mechanism by which dexamethasone modulates TLR2-induced cytokine production this signalling pathway was monitored in a lung epithelial cell line exposed to the TLR2 synthetic agonist, Pam(3) -Cys-Ser-Lys(4) . These experiments demonstrate that phosphatidylinositol 3-kinase (PI3K) is critical for the TLR2 downstream effects of GC. Cells expressing a PI3K mutant (p85-dominant negative, DN; p85 Δ478-511) and exposed to Pam(3) -Cys-Ser-Lys(4) in the presence or absence of dexamethasone, showed enhanced tumour necrosis factor (TNF)α expression while AP-1 and NF-κB transcriptional activity were repressed. We provide experimental evidence that PI3K physically interacts with the glucocorticoid receptor (GR) through two putative PI3K recruitment consensus YxxM binding motifs in the GR, suggesting that some functions regulated by this receptor might occur through kinase interaction. Mutations of two tyrosine residues in the GR, 598 and 663, to phenylalanine significantly reduced interaction with PI3K and the GC effects on TLR2-induced TNF-α expression. However, these mutations did not alter GR transcriptional activity nor affect cellular localization of the expressed mutant GR in COS-1 cells. Therefore, the PI3K-GR interaction may contribute to the effects of GC on the TLR2 pro-inflammatory signalling cascade, thus defining a novel signalling mechanism with a profound impact on innate immune responses.

Molecular interplay between ion channels and the regulation of apoptosis.

Apoptosis is the programmed and deliberate destruction of specific cells. This process occurs during normal development and maintains cellular homeostasis. Disruption or malfunction of apoptosis is implicated in diseases like cancer, AIDS as well as neurodegenerative disorders. The movement of monovalent ions appears to set the stage for the induction of the self-destruction machinery by creating an intracellular environment that favors activation and coordinated execution of the apoptotic program. Understanding the components and steps involved in this intricate process can further our insight to diseases and reveal new approaches for therapeutic treatment.

Molecular interplay between ion channels and the regulation of apoptosis

Apoptosis is the programmed and deliberate destruction of specific cells. This process occurs during normal development and maintains cellular homeostasis. Disruption or malfunction of apoptosis is implicated in diseases like cancer, AIDS as well as neurodegenerative disorders. The movement of monovalent ions appears to set the stage for the induction of the self-destruction machinery by creating an intracellular environment that favors activation and coordinated execution of the apoptotic program. Understanding the components and steps involved in this intricate process can further our insight to diseases and reveal new approaches for therapeutic treatment