Nongenomic Signaling Research Articles

30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Nongenomic effects via the mineralocorticoid receptor

The mineralocorticoid receptor (MR) belongs to the steroid hormone receptor family and classically functions as a ligand-dependent transcription factor. It is involved in water-electrolyte homeostasis and blood pressure regulation but independent from these effects also furthers inflammation, fibrosis, hypertrophy and remodeling in cardiovascular tissues. Next to genomic effects, aldosterone elicits very rapid actions within minutes that do not require transcription or translation and that occur not only in classical MR epithelial target organs like kidney and colon but also in nonepithelial tissues like heart, vasculature and adipose tissue. Most of these effects can be mediated by classical MR and its crosstalk with different signaling cascades. Near the plasma membrane, the MR seems to be associated with caveolin and striatin as well as with receptor tyrosine kinases like EGFR, PDGFR and IGF1R and G protein-coupled receptors like AT1 and GPER1, which then mediate nongenomic aldosterone effects. GPER1 has also been named a putative novel MR. There is a close interaction and functional synergism between the genomic and the nongenomic signaling so that nongenomic signaling can lead to long-term effects and support genomic actions. Therefore, understanding nongenomic aldosterone/MR effects is of potential relevance for modulating genomic aldosterone effects and may provide additional targets for intervention.

Targeting to the non-genomic activity of retinoic acid receptor-gamma by acacetin in hepatocellular carcinoma

We recently demonstrated that retinoic acid receptor-γ (RARγ) is overexpressed and acts as a tumor promoter in hepatocellular carcinoma (HCC). The oncogenic activity of RARγ is mainly attributed to its physiological interaction with p85α regulatory subunit of PI3K leading to constitutive activation of AKT. Here we report RARγ as a negative regulator of p53 signaling and thus extend the oncogenic potential of RARγ to a new role in controlling the balance between AKT and p53. A natural flavonoid acacetin is then identified to be capable of modulating RARγ-dependent AKT-p53 network. It specifically binds to RARγ and inhibits all-trans retinoic acid (atRA) stimulation of RARγ transactivation. However, the anticancer action of acacetin is independent on its modulation of RARγ-driven transcriptional activity. Acacetin induces cancer cell apoptosis through antagonizing the non-genomic effect of RARγ on AKT and p53. When bound to RARγ, acacetin prevents RARγ from its activation of AKT followed by recovery of the normal p53 signaling. Given the implication of AKT-p53 dysregulation in most HCC, targeting the non-genomic signaling of RARγ that switches AKT-p53 from a pro-survival to a pro-apoptotic program in cancer cells should be a promising strategy for developing novel anti-HCC drugs.

Genomic and Non-genomic Aldosterone Signaling

Genomic and Non-genomic Aldosterone Signaling

Mammary epithelial cell phenotype disruption in vitro and in vivo through ERalpha36 overexpression.

Estrogen receptor alpha 36 (ERα36) is a variant of the canonical estrogen receptor alpha (ERα66), widely expressed in hormone sensitive cancer cells and whose high expression level correlates with a poor survival prognosis for breast cancer patients. While ERα36 activity have been related to breast cancer progression or acquired resistance to treatment, expression level and location of ERα36 are poorly documented in the normal mammary gland. Therefore, we explored the consequences of a ERα36 overexpression in vitro in MCF-10A normal mammary epithelial cells and in vivo in a unique model of MMTV-ERα36 transgenic mouse strain wherein ERα36 mRNA was specifically expressed in the mammary gland. By a combination of bioinformatics and computational analyses of microarray data, we identified hierarchical gene networks, downstream of ERα36 and modulated by the JAK2/STAT3 signaling pathway. Concomitantly, ERα36 overexpression lowered proliferation rate but enhanced migration potential and resistance to staurosporin-induced apoptosis of the MCF-10A cell line. In vivo, ERα36 expression led to duct epithelium thinning and disruption in adult but not in prepubescent mouse mammary gland. These phenotypes correlated with a loss of E-cadherin expression. Here, we show that an enhanced expression of ERα36 is sufficient, by itself, to disrupt normal breast epithelial phenotype in vivo and in vitro through a dominant-positive effect on nongenomic estrogen signaling pathways. These results also suggest that, in the presence of adult endogenous steroid levels, ERα36 overexpression in vivo contributes to alter mammary gland architecture which may support pre-neoplastic lesion and augment breast cancer risk.

Abstract P4-15-02: Investigating the use estrogen receptor β agonists in the prevention of breast cancer using a transgenic mouse model

Abstract Breast cancer is the most common cancer among women worldwide. In the United States, 1 in 8 women will develop breast cancer during her lifetime, and in 2016 over 240,000 new cases will be diagnosed. Incidence rates differ by geographic location, with women living in Asia having the lowest rates. However, after migrating to the U.S and adopting a western diet their rates increase. This highlights the importance of lifestyle, including diet, in modulating breast cancer risk. Soy has been previously implicated as the dietary component contributing to the reduced breast cancer rates in Asian women. Compounds that can selectively activate Estrogen Receptor β have been identified in plants, including soy. Given the tumor-suppressive properties of ERβ, it may be possible to use these agonists in the chemoprevention of breast cancer. The objective of this study was to investigate the utility of using ERβ agonists in the prevention of breast cancer using a transgenic mouse model. MMTV-HER2/neu mice develop premalignant lesions at 4-5 months, and tumors starting at month 7 due to overexpression of the Her2/neu proto-oncogene. MMTV-HER2/neu mice were treated with 2 different ERβ agonists, S-equol or LY500307, for 3 months and evaluated for branching, hyperplasia and differential gene expression. When compared to controls, ERβ agonist-treated mice exhibited a significant decrease in branching and ductal hyperplasia, with no change in body weight. Differential gene expression analysis revealed 218 modified genes in response to S-equol treatment, and 258 genes modified by LY500307 treatment, with an overlap of 36 genes. Pathway analysis identified an enrichment for chemokines signaling pathways, particularly TNF, in the reversal of hyperbranching resulting from treatment with ERβ agonists. Although previous studies have demonstrated crosstalk between ER's non-genomic signaling and growth factor signal transductions pathway, this is the first study to demonstrate the impact of ERβ activation on HER2/neu mediated pre-neoplastic changes. Our study suggest that ER β agonist treatment may be a valuable therapeutic option for the chemoprevention of breast cancer in women at increased risk. Citation Format: Samayoa C, Krishnegowda NK, Vadlamudi RK, Tekmal RR. Investigating the use estrogen receptor β agonists in the prevention of breast cancer using a transgenic mouse model [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-15-02.

Abstract P3-05-02: Quantitative ERα measurements in TNBC from the I-SPY 2 TRIAL correlate with HER2-EGFR co-activation and heterodimerization

Abstract Background: We have previously described that TNBC patients whose tumors have both HER2 Y1248 phosphorylation (pHER2) “high” and phospho-EGFR Y1173 (pEGFR) “high” have increased response (pCR) to neratinib in the I-SPY2 TRIAL. We hypothesize that the paradoxical finding of a response prediction signature comprised of HER2 activation in a HER2 IHC/FISH-negative population means there must be a ligand-driven biochemical event responsible for the HER2 phosphorylation because HER2 mutations were also not found to be significant. Exploratory analysis of additional cellular signaling events and protein expression levels in pre-treatment, LCM-purified tumor epithelium by reverse phase protein microarray (RPPA) included semi-quantitative measurement of total levels of estrogen receptor alpha (ERα), which has been previously shown to be able to act as a membrane non-genomic signaling molecule through direct interaction with various tyrosine kinases including EGFR and HER2. Since ERα has been previously shown to act as a ligand and co-stimulate (activate) HER2 and EGFR when present at low levels, we investigated whether or not RPPA-measured ERα levels in the TNBC cohort analyzed to date were higher in tumors with both pHER2 “high” and pEGFR “high” levels and thus provide evidence explaining how HER2-EGFR activation is occurring in TNBC. Methods: Using RPPA analysis, we measured 118 analytes in lysates of LCM tumor epithelium obtained from the pre-treatment biopsy samples of 86 TNBC (Allred=0) patients in the I-SPY2 TRIAL analyzed to date. Cutpoints for pEGFR and pHER2 were determined previously by ROC analysis for pCR correlation in the neratinib treated TNBC population, and used here to dichotomize the pHER2 and pEGFR data in the larger TNBC population. Wilcoxon Rank Sum testing was performed using the continuous variable total ERα data and compared the TNBC that were both pHER2 and pEGFR “high” (N=39) to the rest of the TNBC population (N=47). Total ERα values were then divided into “high” and “low” groups based on the TNBC population median value in order to determine frequency/percentages within each class. Our study is exploratory with no claims for generalizability of the data, and calculations are descriptive (e.g. p-values are measures of distance with no inferential content). Results: Total ERα values were obtained in 84/86 TNBC tumors analyzed. Total levels of ERα were higher (p< 0.006) in TNBC tumors with pHER2 and pEGFR “high” levels. 68% (26/38) of tumors in the pHER2 and pEGFR “high” group had ERα levels above the population median compared to 35% (16/46) in the rest of the TNBC population. Conclusion: Our exploratory analysis reveals that ERα levels are significantly higher in TNBC with pHER2 and pEGFR activation and may be behaving as a direct signaling ligand in TNBC and driving HER2-EGFR signaling. This ERα-pHER2/pEGFR association was missed by current ER and HER2 clinical laboratory testing techniques, and if validated in larger independent study sets could suggest that utilization of new protein-based techniques defining ER more quantitatively could be helpful to understand tumor biology and therapeutic response prediction, especially in the context of TNBC that are ostensibly ER negative. Citation Format: Gallagher RI, Yau C, Wolf DM, Dong T, Hirst G, Brown-Swigart L, ISPY-2 TRIAL Investigators, Buxton M, DeMichele A, van't Veer L, Yee D, Paoloni M, Esserman L, Berry D, Park J, Petricoin EF, Wulfkuhle JD. Quantitative ERα measurements in TNBC from the I-SPY 2 TRIAL correlate with HER2-EGFR co-activation and heterodimerization [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-02.

Anti-androgen 2-hydroxyflutamide modulates cadherin, catenin and androgen receptor phosphorylation in androgen-sensitive LNCaP and androgen-independent PC3 prostate cancer cell lines acting via PI3K/Akt and MAPK/ERK1/2 pathways

This study aimed to investigate rapid effect of anti-androgen 2-hydroxyflutamide (HF) on cadherin/catenin complex and androgen receptor (AR) phosphorylation in prostate cancer cell lines. In addition, a role of PI3K/Akt and MAPK/ERK1/2 pathways in mediating these effects was explored. We have demonstrated that in androgen-sensitive LNCaP cells HF induced rapid increase of E-cadherin phosphorylation at Ser 838/840 (p<0.05) in MAPK/ERK1/2-dependent manner, whereas phosphorylation of β-catenin at Tyr 654 was unchanged. Concomitantly, the reduction of the level of AR phosphorylated at Ser210/213 was found (p<0.01). In androgen-independent PC3 cells HF decreased Tyr 860 N-cadherin and Tyr 645 β-catenin phosphorylation (p<0.01), acting via both MAPK/ERK1/2 and PI3K/Akt pathways. Further, we evidenced that MAPK/ERK1/2 and PI3K/Akt pathways were differentially influenced by HF in LNCaP and PC3 cells. In LNCaP cells, both Akt (p<0.01) and ERK1/2 (p<0.001) phosphorylation were negatively regulated and this effect was mediated by Raf-1 (p<0.05). In contrast, in PC3 cells HF stimulated Akt (p<0.001) and ERK1/2 (p<0.001) activation, but had no effect on the crosstalk between PI3K/Akt and MEK/ERK1/2 pathways at the Raf-1 kinase level. Our findings expand the role of anti-androgen into non-genomic signaling, creating a link between anti-androgen action and phosphorylation of adherens junction proteins in prostate cancer cells.

Differential and directional estrogenic signaling pathways induced by enterolignans and their precursors.

Mammalian lignans or enterolignans are metabolites of plant lignans, an important category of phytochemicals. Although they are known to be associated with estrogenic activity, cell signaling pathways leading to specific cell functions, and especially the differences among lignans, have not been explored. We examined the estrogenic activity of enterolignans and their precursor plant lignans and cell signaling pathways for some cell functions, cell cycle and chemokine secretion. We used DNA microarray-based gene expression profiling in human breast cancer MCF-7 cells to examine the similarities, as well as the differences, among enterolignans, enterolactone and enterodiol, and their precursors, matairesinol, pinoresinol and sesamin. The profiles showed moderate to high levels of correlation (R values: 0.44 to 0.81) with that of estrogen (17β-estradiol or E2). Significant correlations were observed among lignans (R values: 0.77 to 0.97), and the correlations were higher for cell functions related to enzymes, signaling, proliferation and transport. All the enterolignans/precursors examined showed activation of the Erk1/2 and PI3K/Akt pathways, indicating the involvement of rapid signaling through the non-genomic estrogen signaling pathway. However, when their effects on specific cell functions, cell cycle progression and chemokine (MCP-1) secretion were examined, positive effects were observed only for enterolactone, suggesting that signals are given in certain directions at a position closer to cell functions. We hypothesized that, while estrogen signaling is initiated by the enterolignans/precursors examined, their signals are differentially and directionally modulated later in the pathways, resulting in the differences at the cell function level.

Non-Genomic Actions of the Androgen Receptor in Prostate Cancer.

Androgen receptor (AR) is a validated drug target for prostate cancer based on its role in proliferation, survival, and metastases of prostate cancer cells. Unfortunately, despite recent improvements to androgen deprivation therapy and the advent of better antiandrogens with a superior affinity for the AR ligand-binding domain (LBD), most patients with recurrent disease will eventually develop lethal metastatic castration-resistant prostate cancer (CRPC). Expression of constitutively active AR splice variants that lack the LBD contribute toward therapeutic resistance by bypassing androgen blockade and antiandrogens. In the canonical pathway, binding of androgen to AR LBD triggers the release of AR from molecular chaperones which enable conformational changes and protein–protein interactions to facilitate its nuclear translocation where it regulates the expression of target genes. However, preceding AR function in the nucleus, initial binding of androgen to AR LBD in the cytoplasm may already initiate signal transduction pathways to modulate cellular proliferation and migration. In this article, we review the significance of signal transduction pathways activated by rapid, non-genomic signaling of the AR during the progression to metastatic CRPC and put into perspective the implications for current and novel therapies that target different domains of AR.

Protective Effect of 3, 3-Diindolylmethane on Bisphenol A Activated GPR30, RAS, PI3K/Akt Estrogenic Signaling Pathway in Female Sprague-Dawley Rats

The present study was aimed to evaluate the protective effect of 3, 3-diindolylmethane (DIM) on bisphenol A (BPA) induced estrogen signaling in the mammary glands of female Sprague-Dawley rats. Chronic administration of BPA (10 μg/kg/bw) to rats exerts rapid estrogenic action via GPR30 and activate cascade of signaling molecule which induce cell proliferation in the mammary gland. Western blot analysis of mammary tissues shows over expression of GPR30, SRC, RAS, PI3K and Akt proteins and immunohistochemical analysis reveals over expression of PCNA and no significant changes in ERs. Further, oral administration of DIM (5 mg/kg/bw), alternative days to BPA treated rats for the period of 12 weeks show significant decrease in the expression pattern of GPR30, SRC, RAS, PI3K, Akt and PCNA. The results of our study demonstrate that BPA induces rapid action via the over expression of proteins in non-genomic estrogen signaling pathway. Administration of DIM inhibits the action of BPA by modulating the expression of proteins involved in GPR30 cascade signaling pathway mediated cell proliferation in mammary glands of female rats.

Genetic Investigation of Thyroid Hormone Receptor Function in the Developing and Adult Brain.

Thyroid hormones exert a broad influence on brain development and function, which has been extensively studied over the years. Mouse genetics has brought an important contribution, allowing precise analysis of the interplay between TRα1 and TRβ1 nuclear receptors in neural cells. However, the exact contribution of each receptor, the possible intervention of nongenomic signaling, and the nature of the genetic program that is controlled by the receptors remain poorly understood.

Stress Signals, Mediated by Membranous Glucocorticoid Receptor, Activate PLC/PKC/GSK-3β/β-catenin Pathway to Inhibit Wound Closure

Glucocorticoids (GCs), key mediators of stress signals, are also potent wound healing inhibitors. To understand how stress signals inhibit wound healing, we investigated the role of membranous glucocorticoid receptor (mbGR) by using cell-impermeable BSA-conjugated dexamethasone. We found that mbGR inhibits keratinocyte migration and wound closure by activating a Wnt-like phospholipase (PLC)/ protein kinase C (PKC) signaling cascade. Rapid activation of mbGR/PLC/PKC further leads to activation of known biomarkers of nonhealing found in patients, β-catenin and c-myc. Conversely, a selective inhibitor of PKC, calphostin C, blocks mbGR/PKC pathway, and rescues GC-mediated inhibition of keratinocyte migration invitro and accelerates wound epithelialization of human wounds exvivo. This novel signaling mechanism may have a major impact on understanding how stress response via GC signaling regulates homeostasis and its role in development and treatments of skin diseases, including wound healing. To test tissue specificity of this nongenomic signaling mechanism, we tested retinal and bronchial human epithelial cells and fibroblasts. We found that mbGR/PLC/PKC signaling cascade exists in all cell types tested, suggesting a more general role. The discovery of this nongenomic signaling pathway, in which glucocorticoids activate Wnt pathway via mbGR, provides new insights into how stress-mediated signals may activate growth signals in various epithelial and mesenchymal tissues.

Extracellular matrix proteins in healthy and retained placentas, comparing hemochorial and synepitheliochorial placentas

The placenta expresses structural and biologically active proteins. Their synthesis is mainly regulated by genomic or nongenomic signals and modulated by hormones. These protein profiles are altered during different stages of pregnancy. The biological properties of extracellular matrix (ECM) proteins were defined and described in a number of tissues including placenta. These properties enable them to be the main players in the processes of attachment or invasion into the endometrium during initial placenta formation and its timely separation after delivery and detachment. In this review, we focused on the role of ECM proteins during attachment of the placenta to the uterine wall, its timely separation, and the implications of this process on retained or pathologically attached placenta.Although the amount of published information in this area is relatively scant, some of the key proteins and processes are well defined. We focused on the available data detailing the ECM protein profiles of human (histologically thin; hemochorial) and bovine (histologically thick; epitheliochorial) placentas and compared the shared and unique ECM proteins that are relevant to placental attachment and separation.

The molecular mechanisms underlying the ER\u03b1-36-mediated signaling in breast cancer

Alterations in estrogen-mediated cellular signaling have largely been implicated in the pathogenesis of breast cancer. Here, we investigated the signaling regulation of a splice variant of the estrogen receptor, namely estrogen receptor (ERα-36), associated with a poor prognosis in breast cancers. Coupling in vitro and in vivo approaches we determined the precise sequential molecular events of a new estrogen signaling network in an ERα-negative cell line and in an original patient-derived xenograft. After estrogen treatment, ERα-36 rapidly associates with Src at the level of the plasma membrane, initiating downstream cascades, including MEK1/ERK activation and paxillin phosphorylation on S126, which in turn triggers a higher expression of cyclin D1. Of note, the direct binding of ERα-36 to ERK2 prevents its dephosphorylation by MKP3 and enhances the downstream signaling. These findings improve our understanding of the regulation of non-genomic estrogen signaling and open new avenues for personalized therapeutic approaches targeting Src or MEK in ERα-36-positive patients.

Membrane Androgen Receptor(S) and their Role in Prostate Cancer:A Review

Androgen, a steroid hormone, typically mediates its action by binding to the cytosolic Androgen Receptor (AR), via the classical or genomic pathway. Androgens can also act through a non-classical or non-genomic pathway interacting with receptors present on the plasma membrane of cells. Although the identity of the nuclear AR is well established, the identity of the membrane AR is still not clear. Through independent studies, three proteins have been identified that are present on plasma membranes of prostate cells and can mediate androgen signalling, viz, GPRC6A, AR8 and ZIP9. Although these proteins can mediate androgen signalling, the membrane receptor which is used most frequently and specifically for mediating androgen action in prostate cells is not confirmed. Recent research has shown that the non-genomic androgen signalling plays a key role in progression of prostate cancer (PCa). In this review, the potential of these three proteins for their ability to act as the membrane AR has been analysed. The use of membrane AR as a novel target for the treatment of PCa has also been discussed.

Epigenetic regulation of transcription factor promoter regions by low-dose genistein through mitogen-activated protein kinase and mitogen-and-stress activated kinase 1 nongenomic signaling

BackgroundThe phytoestrogen, genistein at low doses nongenomically activates mitogen-activated protein kinase p44/42 (MAPKp44/42) via estrogen receptor alpha (ERα) leading to proliferation of human uterine leiomyoma cells. In this study, we evaluated if MAPKp44/42 could activate downstream effectors such as mitogen- and stress-activated protein kinase 1 (MSK1), which could then epigenetically modify histone H3 by phosphorylation following a low dose (1 μg/ml) of genistein.ResultsUsing hormone-responsive immortalized human uterine leiomyoma (ht-UtLM) cells, we found that genistein activated MAPKp44/42 and MSK1, and also increased phosphorylation of histone H3 at serine10 (H3S10ph) in ht-UtLM cells. Colocalization of phosphorylated MSK1 and H3S10ph was evident by confocal microscopy in ht-UtLM cells (r = 0.8533). Phosphorylation of both MSK1and H3S10ph was abrogated by PD98059 (PD), a MEK1 kinase inhibitor, thereby supporting genistein’s activation of MSK1 and Histone H3 was downstream of MAPKp44/42. In proliferative (estrogenic) phase human uterine fibroid tissues, phosphorylated MSK1 and H3S10ph showed increased immunoexpression compared to normal myometrial tissues, similar to results observed in in vitro studies following low-dose genistein administration. Real-time RT-PCR arrays showed induction of growth-related transcription factor genes, EGR1, Elk1, ID1, and MYB (cMyb) with confirmation by western blot, downstream of MAPK in response to low-dose genistein in ht-UtLM cells. Additionally, genistein induced associations of promoter regions of the above transcription factors with H3S10ph as evidenced by Chromatin Immunoprecipitation (ChIP) assays, which were inhibited by PD. Therefore, genistein epigenetically modified histone H3 by phosphorylation of serine 10, which was regulated by MSK1 and MAPK activation.ConclusionHistone H3 phosphorylation possibly represents a mechanism whereby increased transcriptional activation occurs following low-dose genistein exposure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0141-2) contains supplementary material, which is available to authorized users.

Importance of Estrogenic Signaling and Its Mediated Receptors in Prostate Cancer.

Prostate cancer (PCa) treatment was first established by Huggins and Hodges in 1941, primarily described as androgen deprivation via interference of testicular androgen production. The disease remains incurable with relapse of hormone-refractory cancer after treatments. Epidemiological and clinical studies disclosed the importance of estrogens in PCa. Discovery of estrogen receptor ERβ prompted direct estrogenic actions, in conjunction with ERα, on PCa cells. Mechanistically, ERs upon ligand binding transactivate target genes at consensus genomic sites via interactions with various transcriptional co-regulators to mold estrogenic signaling. With animal models, Noble revealed estrogen dependencies of PCa, providing insight into potential uses of antiestrogens in the treatment. Subsequently, various clinical trials were conducted and molecular and functional consequences of antiestrogen treatment in PCa were delineated. Besides, estrogens can also trigger rapid non-genomic signaling responses initiated at the plasma membrane, at least partially via an orphan G-protein-coupled receptor GPR30. Activation of GPR30 significantly inhibited in vitro and in vivo PCa cell growth and the underlying mechanism was elucidated. Currently, molecular networks of estrogenic and antiestrogenic signaling via ERα, ERβ and GPR30 in PCa have not been fully deciphered. This crucial information could be beneficial to further developments of effective estrogen- and antiestrogen-based therapy for PCa patients.

Membrane-bound glucocorticoid receptors on distinct nociceptive neurons as potential targets for pain control through rapid non-genomic effects

Glucocorticoids were long believed to primarily function through cytosolic glucocorticoid receptor (GR) activation and subsequent classical genomic pathways. Recently, however, evidence has emerged that suggests the presence of rapid non-genomic GR-dependent signaling pathways within the brain, though their existence in spinal and peripheral nociceptive neurons remains elusive. In this paper, we aim to systemically identify GR within the spinal cord and periphery, to verify their putative membrane location and to characterize possible G protein coupling and pain modulating properties. Double immunofluorescence confocal microscopy revealed that GR predominantly localized in peripheral peptidergic and non-peptidergic nociceptive C- and Aδ-neurons and existed only marginally in myelinated mechanoreceptive and proprioreceptive neurons. Within the spinal cord, GR predominantly localized in incoming presynaptic nociceptive neurons, in pre- and postsynaptic structures of the dorsal horn, as well as in microglia. GR saturation binding revealed that these receptors are linked to the cell membrane of sensory neurons and, upon activation, they trigger membrane targeted [35S]GTPγS binding, indicating G protein coupling to a putative receptor. Importantly, subcutaneous dexamethasone immediately and dose-dependently attenuated acute nociceptive behavior elicited in an animal model of formalin-induced pain hypersensitivity compared to naive rats. Overall, this study provides firm evidence for a novel neuronal mechanism of GR agonists that is rapid, non-genomic, dependent on membrane binding and G protein coupling, and acutely modulates nociceptive behavior, thus unraveling a yet unconsidered mechanism of pain relief.

Acute Stress Suppresses Synaptic Inhibition and Increases Anxiety via Endocannabinoid Release in the Basolateral Amygdala

Stress and glucocorticoids stimulate the rapid mobilization of endocannabinoids in the basolateral amygdala (BLA). Cannabinoid receptors in the BLA contribute to anxiogenesis and fear-memory formation. We tested for rapid glucocorticoid-induced endocannabinoid regulation of synaptic inhibition in the rat BLA. Glucocorticoid application to amygdala slices elicited a rapid, nonreversible suppression of spontaneous, but not evoked, GABAergic synaptic currents in BLA principal neurons; the effect was also seen with a membrane-impermeant glucocorticoid, but not with intracellular glucocorticoid application, implicating a membrane-associated glucocorticoid receptor. The glucocorticoid suppression of GABA currents was not blocked by antagonists of nuclear corticosteroid receptors, or by inhibitors of gene transcription or protein synthesis, but was blocked by inhibiting postsynaptic G-protein activity, suggesting a postsynaptic nongenomic steroid signaling mechanism that stimulates the release of a retrograde messenger. The rapid glucocorticoid-induced suppression of inhibition was prevented by blocking CB1 receptors and 2-arachidonoylglycerol (2-AG) synthesis, and it was mimicked and occluded by CB1 receptor agonists, indicating it was mediated by the retrograde release of the endocannabinoid 2-AG. The rapid glucocorticoid effect in BLA neurons in vitro was occluded by prior in vivo acute stress-induced, or prior in vitro glucocorticoid-induced, release of endocannabinoid. Acute stress also caused an increase in anxiety-like behavior that was attenuated by blocking CB1 receptor activation and inhibiting 2-AG synthesis in the BLA. Together, these findings suggest that acute stress causes a long-lasting suppression of synaptic inhibition in BLA neurons via a membrane glucocorticoid receptor-induced release of 2-AG at GABA synapses, which contributes to stress-induced anxiogenesis. We provide a cellular mechanism in the basolateral amygdala (BLA) for the rapid stress regulation of anxiogenesis in rats. We demonstrate a nongenomic glucocorticoid induction of long-lasting suppression of synaptic inhibition that is mediated by retrograde endocannabinoid release at GABA synapses. The rapid glucocorticoid-induced endocannabinoid suppression of synaptic inhibition is initiated by a membrane-associated glucocorticoid receptor in BLA principal neurons. We show that acute stress increases anxiety-like behavior via an endocannabinoid-dependent mechanism centered in the BLA. The stress-induced endocannabinoid modulation of synaptic transmission in the BLA contributes, therefore, to the stress regulation of anxiety, and may play a role in anxiety disorders of the amygdala.

Developmental exposure to endocrine disrupting chemicals alters the epigenome: Identification of reprogrammed targets.

Endocrine disruptions induced by environmental toxicants have placed an immense burden on society to properly diagnose, treat and attempt to alleviate symptoms and disease. Environmental exposures during critical periods of development can permanently reprogram normal physiological responses, thereby increasing susceptibility to disease later in life - a process known as developmental reprogramming. During development, organogenesis and tissue differentiation occur through a continuous series of tightly-regulated and precisely-timed molecular, biochemical and cellular events. Humans may encounter endocrine disrupting chemicals (EDCs) daily and during all stages of life, from conception and fetal development through adulthood and senescence. Though puberty and perimenopausal periods may be affected by endocrine disruption due to hormonal effects, prenatal and early postnatal windows are most critical for proper development due to rapid changes in system growth. Developmental reprogramming is shown to be caused by alterations in the epigenome. Development is the time when epigenetic programs are 'installed' on the genome by 'writers', such as histone methyltransferases (HMTs) and DNA methyltransferases (DNMTs), which add methyl groups to lysine and arginine residues on histone tails and to CpG sites in DNA, respectively. A number of environmental compounds, referred to as estrogenic endocrine disruptors (EEDs), are able to bind to estrogen receptors (ERs) and interfere with the normal cellular development in target tissues including the prostate and uterus. These EEDs, including diethylstilbestrol (DES), bisphenol A (BPA), and genistein (a phytoestrogen derived from soybeans), have been implicated in the malformation of reproductive organs and later development of disease. Due to the lack of fully understanding the underlying mechanisms of how environmental toxicants and their level of exposure affect the human genome, it can be challenging to create clear clinical guidance to address the potential health effects of lower-level exposures commonly experienced within the general population. In addition, human studies concerning environmental exposures are limited in feasibility by ethical concerns for human safety. Therefore, studies in animal models provide great opportunities to reveal links between early-life exposure to EDCs and related diseases. It has been shown that developmental exposure to EDCs, such as diethylstilbestrol (DES) and genistein, during reproductive tract development increases the incidence, multiplicity and overall size of uterine fibroids in the Eker rat model, concomitantly reprogramming estrogen-responsive gene expression. Importantly, EDC exposure represses enhancer of zeste 2 (EZH2) and reduces levels of the histone 3 lysine 27 trimethylation (H3K27me3) repressive mark through Estrogen receptor / Phosphatidylinositide 3-kinases / Protein kinase B non-genomic signaling in the developing uterus. More recent research identified a developmental reprogramming target, Scbg2a1 gene, whose epigenetic status can be altered by early exposure to BPA in the rat prostate. Molecular analyses revealed markedly increased expression (greater than 100 fold) of Scgb2a1, a secretaglobin gene in response to developmental exposure to BPA. This increase in Scgb2a1 expression is concomitantly associated with increased enrichment of acetylated H3K9 (H3K9Ac representing active chromatin status) and hypomethylation of DNA for a CpG island upstream of the transcription start site of Scgb2a1. These data suggest that expression of Scgb2a1 in the adult prostate could be epigenetically reprogrammed by BPA exposure during prostate development. Further studies are needed to create more targeted preventative interventions as well as specific, effective therapeutics to decrease the incidence of diseases.