Activity Of Nuclear Hormone Receptors Research Articles

Vitamin A and vitamin D induced nuclear hormone receptor activation and its impact on B cell differentiation and immunoglobulin production

Vitamin A and vitamin D metabolites are ligands to nuclear receptors – namely RAR, RXR and VDR. The activation of these receptors in human B cells impacts B cell maturation and function. In this review, we discuss how 9-cis retinoic acid (9cRA) and 1,25-dihydroxyvitamin D3 (calcitriol) individually or in conjunction, signal through their nuclear receptors and thereby impact B cell differentiation, immunoglobulin class switching to IgA at the expense of IgE, and also B cell migration and homing. Impact of the vitamin metabolites individually on B cell survival factors are well elucidated, be it the regulation of BAFF and APRIL, the induction of TGF-β or suppression of NF-κB. Very little is known about the impact of 9cRA and calcitriol together on B cells. Recently our group revealed that 9cRA and calcitriol together in the context of the B cell differentiation, induces naïve B cell differentiation into IgA+ plasmablasts, the functional and underlying molecular regulations however require further investigation. In conclusion, the conjunctional impact of these nuclear receptor ligands on B cell functionality is important to better understand B cell dependent clinical outcomes in allergy and autoimmunity. Within this review, we hypothesize that a balance between both vitamins is of utmost importance to provide a robust humoral immune response and a better treatment of disorders characterised by dysregulated immune responses such as IgE-dependent allergy or autoimmunity such as lupus erythematosus.

Effects of cortisol, epinephrine, and bisphenol contaminants on the transcriptional landscape of marine mammal blubber.

Top ocean predators such as marine mammals are threatened by intensifying anthropogenic activity, and understanding the combined effects of multiple stressors on their physiology is critical for conservation efforts. We investigated potential interactions between stress hormones and bisphenol contaminants in a model marine mammal, the northern elephant seal (NES). We exposed precision-cut adipose tissue slices (PCATS) from blubber of weaned NES pups to cortisol (CORT), epinephrine (EPI), bisphenol A (BPA), bisphenol S (BPS), or their combinations (CORT-EPI, BPA-EPI, and BPS-EPI) ex vivo and identified hundreds of genes that were differentially regulated in response to these treatments. CORT altered expression of genes associated with lipolysis and adipogenesis, whereas EPI and CORT-EPI-regulated genes were associated with responses to hormones, lipid and protein turnover, immune function, and transcriptional and epigenetic regulation of gene expression, suggesting that EPI has wide-ranging and prolonged impacts on the transcriptional landscape and function of blubber. Bisphenol treatments alone had a weak impact on gene expression compared with stress hormones. However, the combination of EPI with bisphenols altered expression of genes associated with inflammation, cell stress, DNA damage, regulation of nuclear hormone receptor activity, cell cycle, mitochondrial function, primary ciliogenesis, and lipid metabolism in blubber. Our results suggest that CORT, EPI, bisphenols, and their combinations impact cellular, immune, and metabolic homeostasis in marine mammal blubber, which may affect the ability of marine mammals to sustain prolonged fasting during reproduction and migration, renew tissues, and mount appropriate responses to immune challenges and additional stressors.

Preserving and enhancing mitochondrial function after stroke to protect and repair the neurovascular unit: novel opportunities for nanoparticle-based drug delivery.

The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca2+) uptake supports NVU function by buffering Ca2+ and stimulating energy production. However, excessive mitochondrial Ca2+ uptake causes toxic mitochondrial Ca2+ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca2+ uptake and efflux in the brain are mediated by the mitochondrial Ca2+ uniporter complex (MCUcx) and sodium/Ca2+/lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCUcx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca2+ overloading. These findings suggest that combining MCUcx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCUcx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCUcx, or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.

Human nuclear hormone receptor activity contributes to malaria parasite liver stage development

Chemical genetic approaches have had a transformative impact on discovery of drug targets for malaria but have primarily been used for parasite targets. To identify human pathways required for intrahepatic development of parasite, we implemented multiplex cytological profiling of malaria infected hepatocytes treated with liver stage active compounds. Some compounds, including MMV1088447 and MMV1346624, exhibited profiles similar to cells treated with nuclear hormone receptor (NHR) agonist/antagonists. siRNAs targeting human NHRs, or their signaling partners identified eight genes that were critical for Plasmodium berghei infection. Knockdown of NR1D2, a host NHR, significantly impaired parasite growth by downregulation of host lipid metabolism. Importantly, treatment with MMV1088447 and MMV1346624 but not other antimalarials, phenocopied the lipid metabolism defect of NR1D2 knockdown. Our data underlines the use of high-content imaging for host-cellular pathway deconvolution, highlights host lipid metabolism as a drug-able human pathway and provides new chemical biology tools for studying host-parasite interactions.

Expression and activation of nuclear hormone receptors result in neuronal differentiation and favorable prognosis in neuroblastoma

BackgroundNeuroblastoma (NB), a childhood tumor derived from the sympathetic nervous system, presents with heterogeneous clinical behavior. While some tumors regress spontaneously without medical intervention, others are resistant to therapy, associated with an aggressive phenotype. MYCN-amplification, frequently occurring in high-risk NB, is correlated with an undifferentiated phenotype and poor prognosis. Differentiation induction has been proposed as a therapeutic approach for high-risk NB. We have previously shown that MYCN maintains an undifferentiated state via regulation of the miR-17 ~ 92 microRNA cluster, repressing the nuclear hormone receptors (NHRs) estrogen receptor alpha (ERα) and the glucocorticoid receptor (GR).MethodsCell viability was determined by WST-1. Expression of differentiation markers was analyzed by Western blot, RT-qPCR, and immunofluorescence analysis. Metabolic phenotypes were studied using Agilent Extracellular Flux Analyzer, and accumulation of lipid droplets by Nile Red staining. Expression of angiogenesis, proliferation, and neuronal differentiation markers, and tumor sections were assessed by immunohistochemistry. Gene expression from NB patient as well as adrenal gland cohorts were analyzed using GraphPad Prism software (v.8) and GSEA (v4.0.3), while pseudo-time progression on post-natal adrenal gland cells from single-nuclei transcriptome data was computed using scVelo.ResultsHere, we show that simultaneous activation of GR and ERα potentiated induction of neuronal differentiation, reduced NB cell viability in vitro, and decreased tumor burden in vivo. This was accompanied by a metabolic reprogramming manifested by changes in the glycolytic and mitochondrial functions and in lipid droplet accumulation. Activation of the retinoic acid receptor alpha (RARα) with all-trans retinoic acid (ATRA) further enhanced the differentiated phenotype as well as the metabolic switch. Single-cell nuclei transcriptome analysis of human adrenal glands indicated a sequential expression of ERα, GR, and RARα during development from progenitor to differentiated chromaffin cells. Further, in silico analysis revealed that patients with higher combined expression of GR, ERα, and RARα mRNA levels had elevated expression of neuronal differentiation markers and a favorable outcome.ConclusionTogether, our findings suggest that combination therapy involving activation of several NHRs could be a promising pharmacological approach for differentiation treatment of NB patients.

1-deoxysphingolipids bind to COUP-TF to modulate lymphatic and cardiac cell development.

Identification of physiological modulators of nuclear hormone receptor (NHR) activity is paramount for understanding the link between metabolism and transcriptional networks that orchestrate development and cellular physiology. Using libraries of metabolic enzymes alongside their substrates and products, we identify 1-deoxysphingosines as modulators of the activity of NR2F1 and 2 (COUP-TFs), which are orphan NHRs that are critical for development of the nervous system, heart, veins, and lymphatic vessels. We show that these non-canonical alanine-based sphingolipids bind to the NR2F1/2 ligand-binding domains (LBDs) and modulate their transcriptional activity in cell-based assays at physiological concentrations. Furthermore, inhibition of sphingolipid biosynthesis phenocopies NR2F1/2 deficiency in endothelium and cardiomyocytes, and increases in 1-deoxysphingosine levels activate NR2F1/2-dependent differentiation programs. Our findings suggest that 1-deoxysphingosines are physiological regulators of NR2F1/2-mediated transcription.

Integrative genetic, genomic and transcriptomic analysis of heat shock protein and nuclear hormone receptor gene associations with spontaneous preterm birth

Heat shock proteins are involved in the response to stress including activation of the immune response. Elevated circulating heat shock proteins are associated with spontaneous preterm birth (SPTB). Intracellular heat shock proteins act as multifunctional molecular chaperones that regulate activity of nuclear hormone receptors. Since SPTB has a significant genetic predisposition, our objective was to identify genetic and transcriptomic evidence of heat shock proteins and nuclear hormone receptors that may affect risk for SPTB. We investigated all 97 genes encoding members of the heat shock protein families and all 49 genes encoding nuclear hormone receptors for their potential role in SPTB susceptibility. We used multiple genetic and genomic datasets including genome-wide association studies (GWASs), whole-exome sequencing (WES), and placental transcriptomics to identify SPTB predisposing factors from the mother, infant, and placenta. There were multiple associations of heat shock protein and nuclear hormone receptor genes with SPTB. Several orthogonal datasets supported roles for SEC63, HSPA1L, SACS, RORA, and AR in susceptibility to SPTB. We propose that suppression of specific heat shock proteins promotes maintenance of pregnancy, whereas activation of specific heat shock protein mediated signaling may disturb maternal–fetal tolerance and promote labor.

Integrative Analysis of TP53INP2 in Head and Neck Squamous Cell Carcinoma.

TP53INP2 plays an important role in regulating gene transcription and starvation-induced autophagy, however, its function in head and neck squamous cell carcinoma (HNSCC) remains unclear. Therefore, we assessed the expression and prognostic value of TP53INP2. In addition, RNAseq, miRNAseq, copy number variation, and mutation profiles from The Cancer Genome Atlas (TCGA) dataset were applied to evaluate the distinctive genomic patterns related to TP53INP2 expression. We found that TP53INP2 expression was lower in HNSCC compared with normal controls. Patients with higher TP53INP2 expression had longer survival time. Knockdown of TP53INP2 promoted cell viability. Functional analysis exhibited that TP53INP2 was linked to DNA replication, DNA repair, cell cycle, and multiple metabolic pathways. Moreover, TP53INP2 might affect the expression of multiple genes via enhancing the transcriptional activity of nuclear hormone receptors. A competing endogenous RNA (ceRNA) network consisting of 33 lncRNAs, eight miRNAs, and 13 mRNAs was constructed based on the expression of TP53INP2. Taken together, our study highlights the potential value of TP53INP2 in predicting the survival of HNSCC and its important role in the genesis and development of HNSCC.

Gestational perfluoroalkyl substance exposures and early adolescent bone mineral density in the HOME Study

Background/Aim: Perfluoroalkyl substances (PFAS) may impair bone health via endocrine disruption and nuclear hormone receptor activation. Bone mineral accrual during adolescence is important for long-term bone health, but human studies of PFAS and bone mineral density are primarily in adults or use a cross-sectional study design. We estimated associations of gestational PFAS exposures with early adolescent areal bone mineral density (aBMD) in a prospective cohort study. Methods: We examined 225 pregnant mothers enrolled from 2003-2006 in Cincinnati, OH and their children in the HOME Study. We measured concentrations of perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorononaoic acid (PFNA), and perfluorohexanesulfonic acid (PFHxS) in maternal serum collected at 16 weeks gestation. When children were 12 years old, we measured lean body mass and total body, spine, total hip, femoral neck, and distal forearm (1/3 radius) aBMD with dual x-ray absorptiometry and calculated height-, age-, sex-, and race-specific aBMD z-scores. Using linear regression, we estimated covariate-adjusted associations in aBMD z-scores per doubling of gestational PFAS concentrations overall and stratified by sex. We estimated the proportion of total effects that were mediated by lean mass using structural equation modeling. Results: In adjusted models, higher PFOA concentrations were associated with lower aBMD z-scores of the forearm (β: -0.28, 95% CI: -0.49, -0.08), total hip (β: -0.28, 95% CI: -0.54, -0.02), and spine (β: -0.34, 95% CI: -0.61, -0.06) in males but not females. Lean mass mediated 29-50% of these associations. Higher PFHxS and PFNA concentrations were associated with lower aBMD z-scores of the forearm (PFHxS β: -0.11, 95% CI: -0.23, 0.00; PFNA β: -0.14, 95% CI: -0.35, 0.06), with no modification by sex or mediation by lean mass. PFOS was not associated with aBMD measures in either sex. Conclusions: Gestational PFAS exposures may reduce bone accrual in early adolescence, possibly in a sex-dependent manner.

2-D chemical structure image-based in silico model to predict agonist activity for androgen receptor

BackgroundAbnormal activation of human nuclear hormone receptors disrupts endocrine systems and thereby affects human health. There have been machine learning-based models to predict androgen receptor agonist activity. However, the models were constructed based on limited numerical features such as molecular descriptors and fingerprints.ResultIn this study, instead of the numerical features, 2-D chemical structure images of compounds were used to build an androgen receptor toxicity prediction model. The images may provide unknown features that were not represented by conventional numerical features. As a result, the new strategy resulted in a construction of highly accurate prediction model: Mathews correlation coefficient (MCC) of 0.688, positive predictive value (PPV) of 0.933, sensitivity of 0.519, specificity of 0.998, and overall accuracy of 0.981 in 10-fold cross-validation. Validation on a test dataset showed MCC of 0.370, sensitivity of 0.211, specificity of 0.991, PPV of 0.882, and overall accuracy of 0.801. Our chemical image-based prediction model outperforms conventional models based on numerical features.ConclusionOur constructed prediction model successfully classified molecular images into androgen receptor agonists or inactive compounds. The result indicates that 2-D molecular mimetic diagram would be used as another feature to construct molecular activity prediction models.

Phosphorylation of steroid receptor coactivator-3 (SRC-3) at serine 857 is regulated by the p38MAPK-MK2 axis and affects NF-\u03baB-mediated transcription

Steroid receptor coactivator-3 (SRC-3) regulates the activity of both nuclear hormone receptors and a number of key transcription factors. It is implicated in the regulation of cell proliferation, inflammation and in the progression of several common cancers including breast, colorectal and lung tumors. Phosphorylation is an important regulatory event controlling the activities of SRC-3. Serine 857 is the most studied phospho-acceptor site, and its modification has been reported to be important for SRC-3-dependent tumor progression. In this study, we show that the stress-responsive p38MAPK-MK2 signaling pathway controls the phosphorylation of SRC-3 at S857 in a wide range of human cancer cells. Activation of the p38MAPK-MK2 pathway results in the nuclear translocation of SRC-3, where it contributes to the transactivation of NF-kB and thus regulation of IL-6 transcription. The identification of the p38MAPK-MK2 signaling axis as a key regulator of SRC-3 phosphorylation and activity opens up new possibilities for the development and testing of novel therapeutic strategies to control both proliferative and metastatic tumor growth.

Consumption of “Cerrado” Cashew Pulp Affects Physiological Parameters and Gene Expression in Obese C57BL/6J High Fat-Fed Mice

Abstract Objectives The pseudofruits of A. othonianum Rizz., the “Cerrado” cashew, are a good source of vitamin C; high in phenolic compounds; contain flavonoids such as vitexin and hesperindin, anthocyanins such as cyanindin, delphinidin, pelargonidin and peonidin; and high antioxidant activity. The objective of this work was to evaluate the beneficial health effects observed with the addition of “Cerrado” cashew pulp to an obesogenic diet in male C57BL/6J mice. Methods Groups of C57BL/6J mice were provided low-fat diet (LF,10% kcal fat), high-fat diet (HF, 45% kcal fat), or HF plus “Cerrado” cashew pulp (HF + CP, 2% of total kcal) during the 10-week feeding period. Mice were housed four per cage in a room maintained at a constant temperature (22 ± 2°C) with a 12-hour light/dark cycle and given free access to food and distilled water. Body weights, food intake, and fasting glucose were determined. At the end of study mice were euthanized via cervical dislocation. Physiological parameters were measured during the study and post-mortem, serum and tissue parameters were measured, along with analysis of hepatic gene expression via Clarium gene arrays. Results At the end of week 10, body weights for LF-fed mice were lower than mice fed HF and HF + CP (P < 0.05). Body weights, food efficiencies, and serum MCP-1 concentrations were not different between the HF- and HF + CP-fed groups. At week 9, HF-fed mice had significantly higher baseline glucose concentrations vs. the LF-fed group (P < 0.05). The HF + CP-fed group had an intermediate serum glucose level that was not different than the LF-fed mice. In RNA-Sequencing analysis, 4669 genes were identified. Canonical pathways most regulated with CP intake included Complement Activation, MAPK signaling, Nuclear Hormone Receptor Activation, and PPAR Signaling. Conclusions The intake of “Cerrado” cashew together with the obesogenic diet improved baseline glucose concentrations compared to HF-fed mice. Analysis of hepatic gene expression shows a number of metabolic pathways influenced by CP intake, including cell signaling and nuclear hormone activation. Funding Sources National Council for Scientific and Technological Development – CNPq and IF Goiano, Brazil.

Glucocorticoids regulate AKR1D1 activity in human liver in vitro and in vivo.

Steroid 5β-reductase (AKR1D1) is highly expressed in human liver where it inactivates endogenous glucocorticoids and catalyses an important step in bile acid synthesis. Endogenous and synthetic glucocorticoids are potent regulators of metabolic phenotype and play a crucial role in hepatic glucose metabolism. However, the potential of synthetic glucocorticoids to be metabolised by AKR1D1 as well as to regulate its expression and activity has not been investigated. The impact of glucocorticoids on AKR1D1 activity was assessed in human liver HepG2 and Huh7 cells; AKR1D1 expression was assessed by qPCR and Western blotting. Genetic manipulation of AKR1D1 expression was conducted in HepG2 and Huh7 cells and metabolic assessments were made using qPCR. Urinary steroid metabolite profiling in healthy volunteers was performed pre- and post-dexamethasone treatment, using gas chromatography-mass spectrometry. AKR1D1 metabolised endogenous cortisol, but cleared prednisolone and dexamethasone less efficiently. In vitro and in vivo, dexamethasone decreased AKR1D1 expression and activity, further limiting glucocorticoid clearance and augmenting action. Dexamethasone enhanced gluconeogenic and glycogen synthesis gene expression in liver cell models and these changes were mirrored by genetic knockdown of AKR1D1 expression. The effects of AKR1D1 knockdown were mediated through multiple nuclear hormone receptors, including the glucocorticoid, pregnane X and farnesoid X receptors. Glucocorticoids down-regulate AKR1D1 expression and activity and thereby reduce glucocorticoid clearance. In addition, AKR1D1 down-regulation alters the activation of multiple nuclear hormone receptors to drive changes in gluconeogenic and glycogen synthesis gene expression profiles, which may exacerbate the adverse impact of exogenous glucocorticoids.

Phosphorylation of the histone demethylase KDM5B and regulation of the phenotype of triple negative breast cancer

Epigenetic modifications are known to play critical roles in the expression of genes related to differentiation and dedifferentiation. Histone lysine demethylase KDM5B (PLU-1) catalyzes the demethylation of histone H3 on Lys 4 (H3K4), which results in the repression of gene expression. KDM5B is involved in regulation of luminal and basal cell specific gene expression in breast cancers. However, the mechanisms by which KDM5B is regulated in breast cancer, in particular in response to post-translational signals is not well-defined. Here, we demonstrate that KDM5B is phosphorylated at Ser1456 by the cyclin-dependent kinase 1 (CDK1). Phosphorylation of KDM5B at Ser1456 attenuated the occupancy of KDM5B on the promoters of pluripotency genes. Moreover, KDM5B inhibited the expression of pluripotency genes, SOX2 and NANOG, and decreased the stem cell population in triple-negative breast cancer cell lines (TNBC). We previously reported that the tumor suppressor HEXIM1 is a mediator of KDM5B recruitment to its target genes, and HEXIM1 is required for the inhibition of nuclear hormone receptor activity by KDM5B. Similarly, HEXIM1 is required for regulation of pluripotency genes by KDM5B.

Conserved function of mediator in regulating nuclear hormone receptor activation between plants and animals

ABSTRACTPerception of the plant hormone jasmonoyl-isoleucine (JA-Ile) involves the formation of a co-receptor complex between COI1, the F-box subunit of a SCF-type E3 ubiquitin ligase, and its substrates, a group of jasmonate ZIM-domain (JAZ) transcriptional repressors. In recent studies, we show that MED25, a subunit of the Arabidopsis Mediator, physically and functionally interacts with COI1 and the master transcription factor MYC2 on MYC2 target promoters. Here we provide evidence that MED25 also physically interacts with a subset of JAZ proteins. Therefore, in term of their interaction with Mediator, the JA-Ile co-receptor complex SCFCOI1-JAZs together with the master transcription factor MYC2 resembles the nuclear hormone receptor system of metazoans. In addition, we show that the plant MED25 and its animal counterpart also cooperates with similar epigenetic regulators in distinct signaling pathways. Our study reveals a scenario that plants and animals have evolved distinct, yet largely similar, mechanism for nuclear hormone receptor activation at the level of transcriptional regulation.

Dose-dependent effects on rat liver miRNAs 200a/b and 429: potential early biomarkers of liver carcinogenesis.

An increased incidence of liver tumours in the long term rodent bioassay is not an uncommon finding, invariably as a result of a non-genotoxic mode of action. Non-genotoxic liver carcinogenesis has been found to involve activation of certain nuclear hormone receptors (NHR) including the constitutive androstane receptor (CAR), peroxisome proliferator activated receptor alpha (PPARalpha) and arylhydrocarbon receptor (AHR) and more recently the induction of specific microRNAs (miRs), has also been demonstrated following CAR activation in studies up to 90 days (Koufaris et al., 2012). The stable induction of these tissue specific miRs, namely miR200a, 200b and 429, by liver non-genotoxic carcinogens may serve as early predictors (biomarkers) of heptocarcinogenic potential. To test this hypothesis we used RT-PCR to measure the levels of these miRs in the livers from Wistar rats treated with two rat hepatocarcinogenic and one non hepatocarcinogenic pyrazole carboxamide succinate dehydrogenase inhibitors, Isopyrazam, Sedaxane and Benzovindiflupyr, respectively. The miRs were quantified by RT-PCR in liver RNA samples from three 90 day repeat dose toxicity studies performed at the low, mid and high doses relative to control. In Isopyrazam treated rats a statistically significant (p < 0.01) dose-dependent increase in miR 200a, 220b and 429 in both males and females was observed, whilst for Sedaxane a significant (p < 0.05) increase in miR200b in males and females at the high dose was seen. Benzovindiflupyr treatment did not cause any dose related changes in miR 200a, 200b and 429 relative to control. Our results suggest that assessment of miR 200a/200b/429 levels has potential as a biomarker of the perturbation of pathways involved in hepatocarcinogenesis in Wistar rats. Further work is required to establish the possible relationship between miR200 cluster induction and CAR-mediated hepatocarcinogenesis in a more diverse range of compounds.

A Pyrrole-Imidazole Polyamide Is Active against Enzalutamide-Resistant Prostate Cancer

The LREX' prostate cancer model is resistant to the antiandrogen enzalutamide via activation of an alternative nuclear hormone receptor, glucocorticoid receptor (GR), which has similar DNA-binding specificity to the androgen receptor (AR). Small molecules that target DNA to interfere with protein-DNA interactions may retain activity against enzalutamide-resistant prostate cancers where ligand-binding domain antagonists are ineffective. We reported previously that a pyrrole-imidazole (Py-Im) polyamide designed to bind the consensus androgen response element half-site has antitumor activity against hormone-sensitive prostate cancer. In enzalutamide-resistant LREX' cells, Py-Im polyamide interfered with both AR- and GR-driven gene expression, whereas enzalutamide interfered with only that of AR. Genomic analyses indicated immediate interference with the AR transcriptional pathway. Long-term treatment with Py-Im polyamide demonstrated a global decrease in RNA levels consistent with inhibition of transcription. The polyamide was active against two enzalutamide-resistant xenografts with minimal toxicity. Overall, our results identify Py-Im polyamide as a promising therapeutic strategy in enzalutamide-resistant prostate cancer. Cancer Res; 77(9); 2207-12. ©2017 AACR.

The Calreticulin control of human stress erythropoiesis is impaired by JAK2V617F in polycythemia vera.

Calreticulin (CALR) is a Ca2+-binding protein that shuttles among cellular compartments with proteins bound to its N/P domains. The knowledge that activation of the human erythropoietin receptor induces Ca2+ fluxes prompted us to investigate the role of CALR in human erythropoiesis. As shown by Western blot analysis, erythroblasts generated invitro from normal sources and JAK2V617F polycythemia vera (PV) patients expressed robust levels of CALR. However, Ca2+ regulated CALR conformation only in normal cells. Normal erythroblasts expressed mostly the N-terminal domain of CALR (N-CALR) on their cell surface (as shown by flow cytometry) and C-terminal domain (C-CALR) in their cytoplasm (as shown by confocal microscopy) and expression of both epitopes decreased with maturation. In the proerythroblast (proEry) cytoplasm, C-CALR was associated with the glucocorticoid receptor (GR), which initiated the stress response. In these cells, Ca2+ deprivation and inhibition of nuclear export increased GR nuclear localization while decreasing cytoplasmic detection of C-CALR and C-CALR/GR association and proliferation in response to the GR agonist dexamethasone (Dex). C-CALR/GR association and Dex responsiveness were instead increased by Ca2+ and erythropoietin. In contrast, JAK2V617F proErys expressed normal cell-surface levels of N-CALR but barely detectable cytoplasmic levels of C-CALR. These cells contained GR mainly in the nucleus and were Dex unresponsive. Ruxolitinib rescued cytoplasmic detection of C-CALR, C-CALR/GR association, and Dex responsiveness in JAK2V617F proErys and its effects were antagonized by nuclear export and Ca2+ flux inhibitors. These results indicates that Ca2+-induced conformational changes of CALR regulate nuclear export of GR in normal erythroblasts and that JAK2V617F deregulates this function in PV.

Synthesis of dihydroimidazole tethered imidazolinethiones and their activity as novel antagonists of the nuclear retinoic acid receptor-related orphan receptors (RORs)

Targeting the transcriptional activity of nuclear hormone receptors has proven an effective strategy to treat certain human diseases, and they have become a major focus point to develop novel therapies for the treatment of cancer, inflammation, autoimmune diseases, metabolic disorders, and others. One family of nuclear receptors that has attracted most interest in recent years is the retinoic acid receptor-related orphan receptors (RORs), in particular RORγ. RORγ is a critical regulator of the immune system and RORγ antagonists have shown activity in animal models of inflammatory autoimmune diseases. Here we present the synthesis and biological evaluation of dihydroimidazole tethered imidazolinethiones. We have identified several dual RORγ/α and pan-ROR antagonists with significant activity in cellular assays that could serve as starting points for future optimization efforts to generate potent and selective RORγ modulators.

Inhibition of Adhesion Molecule Gene Expression and Cell Adhesion by the Metabolic Regulator PGC-1α.

Cell adhesion plays an important role in determining cell shape and function in a variety of physiological and pathophysiological conditions. While links between metabolism and cell adhesion were previously suggested, the exact context and molecular details of such a cross-talk remain incompletely understood. Here we show that PGC-1α, a pivotal transcriptional co-activator of metabolic gene expression, acts to inhibit expression of cell adhesion genes. Using cell lines, primary cells and mice, we show that both endogenous and exogenous PGC-1α down-regulate expression of a variety of cell adhesion molecules. Furthermore, results obtained using mRNA stability measurements as well as intronic RNA expression are consistent with a transcriptional effect of PGC-1α on cell adhesion gene expression. Interestingly, the L2/L3 motifs of PGC-1α, necessary for nuclear hormone receptor activation, are only partly required for inhibition of several cell adhesion genes by PGC-1α. Finally, PGC-1α is able to modulate adhesion of primary fibroblasts and hepatic stellate cells to extracellular matrix proteins. Our results delineate a cross talk between a central pathway controlling metabolic regulation and cell adhesion, and identify PGC-1α as a molecular link between these two major cellular networks.