p53 directly represses human LINE1 transposons.

p53 is a potent tumor suppressor and commonly mutated in human cancers. Recently, we demonstrated that p53 genes act to restrict retrotransposons in germline tissues of flies and fish but whether this activity is conserved in somatic human cells is not known. Here we show that p53 constitutively restrains human LINE1s by cooperatively engaging sites in the 5'UTR and stimulating local deposition of repressive histone marks at these transposons. Consistent with this, the elimination of p53 or the removal of corresponding binding sites in LINE1s, prompted these retroelements to become hyperactive. Concurrently, p53 loss instigated chromosomal rearrangements linked to LINE sequences and also provoked inflammatory programs that were dependent on reverse transcriptase produced from LINE1s. Taken together, our observations establish that p53 continuously operates at the LINE1 promoter to restrict autonomous copies of these mobile elements in human cells. Our results further suggest that constitutive restriction of these retroelements may help to explain tumor suppression encoded by p53, since erupting LINE1s produced acute oncogenic threats when p53 was absent.

Anti-Inflammatory Chromatinscape Suggests Alternative Mechanisms of Glucocorticoid Receptor Action.

Despite the widespread use of glucocorticoids (GCs), their anti-inflammatory effects are not understood mechanistically. Numerous investigations have examined the effects of glucocorticoid receptor (GR) activation prior to inflammatory challenges. However, clinical situations are emulated by a GC intervention initiated in the midst of rampant inflammatory responses. To characterize the effects of a late GC treatment, we profiled macrophage transcriptional and chromatinscapes with Dexamethasone (Dex) treatment before or after stimulation by lipopolysaccharide (LPS). The late activation of GR had a similar gene-expression profile as from GR pre-activation, while ameliorating the disruption of metabolic genes. Chromatin occupancy of GR was not predictive of Dex-regulated gene expression, contradicting the "trans-repression by tethering" model. Rather, GR activation resulted in genome-wide blockade of NF-κB interaction with chromatin and directly induced inhibitors of NF-κB and AP-1. Our investigation using GC treatments with clinically relevant timing highlights mechanisms underlying GR actions for modulating the "inflamed epigenome."

Glucocorticoid Receptor:MegaTrans Switching Mediates the Repression of an ERα-Regulated Transcriptional Program.

The molecular mechanisms underlying the opposing functions of glucocorticoid receptors (GRs) and estrogen receptor α (ERα) in breast cancer development remain poorly understood. Here we report that, in breast cancer cells, liganded GR represses a large ERα-activated transcriptional program by binding, in trans, to ERα-occupied enhancers. This abolishes effective activation of these enhancers and their cognate target genes, and it leads to the inhibition of ERα-dependent binding of components of the MegaTrans complex. Consistent with the effects of SUMOylation on other classes of nuclear receptors, dexamethasone (Dex)-induced trans-repression of the estrogen E2 program appears to depend on GR SUMOylation, which leads to stable trans-recruitment of the GR-N-CoR/SMRT-HDAC3 corepressor complex on these enhancers. Together, these results uncover a mechanism by which competitive recruitment of DNA-binding nuclear receptors/transcription factors in trans to hot spot enhancers serves as an effective biological strategy for trans-repression, with clear implications for breast cancer and other diseases.

Synthesis and Anti-Cancer Activity of the Novel Selective Glucocorticoid Receptor Agonists of the Phenylethanolamine Series.

Glucocorticoids (GCs) are widely used for treating hematological malignancies despite their multiple adverse effects. The biological response to GCs relies on glucocorticoid receptor (GR) transrepression (TR) that mediates the anticancer effects and transactivation (TA) associated with the side effects. Selective GR agonists (SEGRAs) preferentially activating GR TR could offer greater benefits in cancer treatment. One of the well-characterized SEGRAs, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride (CpdA), exhibited anticancer activity; however, its translational potential is limited due to chemical instability. To overcome this limitation, we obtained CpdA derivatives, CpdA-01-CpdA-08, employing two synthetic strategies and studied their anti-tumor activity: 4-(1-hydroxy-2-(piperidin-1-yl)ethyl)phenol or CpdA-03 demonstrated superior GR affinity and stability compared to CpdA. In lymphoma Granta and leukemia CEM cell lines, CpdA-03 ligand exhibited typical SEGRA properties, inducing GR TR without triggering GR TA. CpdA-03 effects on cell viability, growth, and apoptosis were similar to the reference GR ligand, dexamethasone (Dex), and the source compound CpdA. In vivo testing of CpdA-03 activity against lymphoma on the transplantable P388 murine lymphoma model showed that CpdA-03 reduced tumor volume threefold, outperforming Dex and CpdA. In conclusion, in this work, we introduce a novel SEGRA CpdA-03 as a promising agent for lymphoma treatment with fewer side effects.

The transcriptional activity of progestins used in contraception and menopausal hormone therapy via progesterone receptor A is dependent on the density of the receptor.

Studies directly comparing the efficacies and potencies of multiple progestins used in contraception and menopausal hormone therapy (MHT) in parallel via human progesterone receptor isoform A (PR-A) in the same model system are limited, and how these parameters are influenced by the density of PR-A are unclear. This is surprising as it is known that the expression levels of PR-A vary in different tissues and diseases. We thus determined for the first time the relative efficacies and potencies for transactivation of the natural PR ligand, progesterone (P4), the PR-specific agonist promegestone (R5020), and selected progestins from all four generations in parallel via different densities of PR-A overexpressed in the MDA-MB-231 breast cancer cell line. Comparative dose-response analysis showed that P4, R5020, the 1st generation progestins medroxyprogesterone acetate and norethisterone, 2nd generation progestin levonorgestrel, 3rd generation progestin gestodene, as well as 4th generation progestins nesterone, nomegestrol acetate and drospirenone display differential agonist efficacies and potencies via PR-A. Moreover, we showed that the agonist efficacies and potencies of the progestins via PR-A were modulated in a density- and progestin-specific manner. Our finding that the potencies of the progestins via PR-A, at all densities, do not exceed reported progestin serum concentrations in women, suggest that these progestins are likely to elicit similar effects in vivo. We are the first to report that P4 and the selected progestins display similar agonist activity for transrepression via PR-A, and that the density of PR-A enhances the transrepression activity of some, but not all progestogens. Collectively, our findings provide proof of concept that the effects of the selected progestins via PR-A is progestin-specific and dependent on the density of the receptor, suggesting differential progestin responses in women using these progestins in contraception and MHT.

Discovery of a novel nonsteroidal selective glucocorticoid receptor modulator by virtual screening and bioassays.

Synthetic glucocorticoids (GCs) have been widely used in the treatment of a broad range of inflammatory diseases, but their clinic use is limited by undesired side effects such as metabolic disorders, osteoporosis, skin and muscle atrophies, mood disorders and hypothalamic-pituitary-adrenal (HPA) axis suppression. Selective glucocorticoid receptor modulators (SGRMs) are expected to have promising anti-inflammatory efficacy but with fewer side effects caused by GCs. Here, we reported HT-15, a prospective SGRM discovered by structure-based virtual screening (VS) and bioassays. HT-15 can selectively act on the NF-κB/AP1-mediated transrepression function of glucocorticoid receptor (GR) and repress the expression of pro-inflammation cytokines (i.e., IL-1ß, IL-6, COX-2, and CCL-2) as effectively as dexamethasone (Dex). Compared with Dex, HT-15 shows less transactivation potency that is associated with the main adverse effects of synthetic GCs, and no cross activities with other nuclear receptors. Furthermore, HT-15 exhibits very weak inhibition on the ratio of OPG/RANKL. Therefore, it may reduce the side effects induced by normal GCs. The bioactive compound HT-15 can serve as a starting point for the development of novel therapeutics for high dose or long-term anti-inflammatory treatment.

Regulation of Cyclooxygenase-2 Expression in Human T Cells by Glucocorticoid Receptor-Mediated Transrepression of Nuclear Factor of Activated T Cells.

Cyclooxygenase (COX) is the key enzyme in prostanoid synthesis from arachidonic acid (AA). Two isoforms, named COX-1 and COX-2, are expressed in mammalian tissues. The expression of COX-2 isoform is induced by several stimuli including cytokines and mitogens, and this induction is inhibited by glucocorticoids (GCs). We have previously shown that the transcriptional induction of COX-2 occurs early after T cell receptor (TCR) triggering, suggesting functional implications of this enzyme in T cell activation. Here, we show that dexamethasone (Dex) inhibits nuclear factor of activated T cells (NFAT)-mediated COX-2 transcriptional induction upon T cell activation. This effect is dependent on the presence of the GC receptor (GR), but independent of a functional DNA binding domain, as the activation-deficient GRLS7 mutant was as effective as the wild-type GR in the repression of NFAT-dependent transcription. Dex treatment did not disturb NFAT dephosphorylation, but interfered with activation mediated by the N-terminal transactivation domain (TAD) of NFAT, thus pointing to a negative cross-talk between GR and NFAT at the nuclear level. These results unveil the ability of GCs to interfere with NFAT activation and the induction of pro-inflammatory genes such as COX-2, and explain some of their immunomodulatory properties in activated human T cells.

Transposons, p53 and Genome Security.

p53, the most commonly mutated tumor suppressor, is a transcription factor known to regulate proliferation, senescence, and apoptosis. Compelling studies have found that p53 may prevent oncogenesis through effectors that are unrelated to these canonical processes and recent findings have uncovered ancient roles for p53 in the containment of mobile elements. Together, these developments raise the possibility that some p53-driven cancers could result from unrestrained transposons. Here, we explore evidence linking conserved features of p53 biology to the control of transposons. We also show how p53-deficient cells can be exploited to probe the behavior of transposons and illustrate how unrestrained transposons incited by p53 loss might contribute to human malignancies.

Structural insights into glucocorticoid receptor function.

The glucocorticoid receptor (GR) is a steroid hormone-activated transcription factor that binds to various glucocorticoid response elements to up- or down- regulate the transcription of thousands of genes involved in metabolism, development, stress and inflammatory responses. GR consists of two domains enabling interaction with glucocorticoids, DNA response elements and coregulators, as well as a large intrinsically disordered region that mediates condensate formation. A growing body of structural studies during the past decade have shed new light on GR interactions, providing a new understanding of the mechanisms driving context-specific GR activity. Here, we summarize the established and emerging mechanisms of action of GR, primarily from a structural perspective. This minireview also discusses how the current state of knowledge of GR function may guide future glucocorticoid design with an improved therapeutic index for different inflammatory disorders.

Nuclear receptor HNF4A transrepresses CLOCK:BMAL1 and modulates tissue-specific circadian networks.

Either expression level or transcriptional activity of various nuclear receptors (NRs) have been demonstrated to be under circadian control. With a few exceptions, little is known about the roles of NRs as direct regulators of the circadian circuitry. Here we show that the nuclear receptor HNF4A strongly transrepresses the transcriptional activity of the CLOCK:BMAL1 heterodimer. We define a central role for HNF4A in maintaining cell-autonomous circadian oscillations in a tissue-specific manner in liver and colon cells. Not only transcript level but also genome-wide chromosome binding of HNF4A is rhythmically regulated in the mouse liver. ChIP-seq analyses revealed cooccupancy of HNF4A and CLOCK:BMAL1 at a wide array of metabolic genes involved in lipid, glucose, and amino acid homeostasis. Taken together, we establish that HNF4A defines a feedback loop in tissue-specific mammalian oscillators and demonstrate its recruitment in the circadian regulation of metabolic pathways.

Natural and synthetic compounds as dissociated agonists of glucocorticoid receptor.

Glucocorticoid receptor (GR) belongs to the superfamily of steroid hormone receptors. The dissociated or selective GR modulators (SEGRMs), preferring the transrepression rather than the transactivation, might exhibit anti-inflammatory activities with fewer side effects. This work presents a review of the molecular mechanism of GR involved in regulation of inflammation. As complementary or alternative therapeutic agents, the botanical compounds have been extensively used in the treatment of various diseases. Hence, this work reviews the botanical compounds as well as the synthetic compounds currently known to be potential SEGRMs. High-throughput virtual screening of SEGRMs from natural products has also been summarized.

Discovery of the potent non-steroidal glucocorticoid receptor modulator BAY 1003803 as clinical candidate.

We report on the discovery of the new clinical candidate BAY 1003803 as glucocorticoid receptor agonist for the topical treatment of psoriasis or severe atopic dermatitis. In the course of optimizing the amino alcohol series as a highly potent new non-steroidal lead structure, considerations were made as to how physicochemical properties and safety concerns relate to structural motifs. BAY 1003803 demonstrates strong anti-inflammatory activity in vitro paired with a pharmacokinetic profile suitable for topical application.

New insights into the novel anti-inflammatory mode of action of glucocorticoids.

Inflammation is a physiological intrinsic host response to injury meant for removal of noxious stimuli and maintenance of homeostasis. It is a defensive body mechanism that involves immune cells, blood vessels and molecular mediators of inflammation. Glucocorticoids (GCs) are steroidal hormones responsible for regulation of homeostatic and metabolic functions of body. Synthetic GCs are the most useful anti-inflammatory drugs used for the treatment of chronic inflammatory diseases such as asthma, chronic obstructive pulmonary disease (COPD), allergies, multiple sclerosis, tendinitis, lupus, atopic dermatitis, ulcerative colitis, rheumatoid arthritis and osteoarthritis whereas, the long term use of GCs are associated with many side effects. The anti-inflammatory and immunosuppressive (desired) effects of GCs are usually mediated by transrepression mechanism whereas; the metabolic and toxic (undesired) effects are usually manifested by transactivation mechanism. Though GCs are most potent anti-inflammatory and immunosuppressive drugs, the common problem associated with their use is GC resistance. Several research studies are rising to comprehend these mechanisms, which would be helpful in improving the GC resistance in asthma and COPD patients. This review aims to focus on identification of new drug targets in inflammation which will be helpful in the resolution of inflammation. The ample understanding of GC mechanisms of action helps in the development of novel anti-inflammatory drugs for the treatment of inflammatory and autoimmune disease with reduced side effects and minimal toxicity.

Glucocorticoid receptor inhibits Müller glial galectin-1 expression via DUSP1-dependent and -independent deactivation of AP-1 signalling.

Galectin-1/LGALS1 is a hypoxia-induced angiogenic factor associated with diabetic retinopathy (DR). Recently, we elucidated a hypoxia-independent pathway to produce galectin-1 in Müller glial cells stimulated by interleukin (IL)-1ß. Here we revealed glucocorticoid receptor (GR)-mediated inhibitory mechanisms for Müller glial galectin-1/LGALS1 expression. Activator protein (AP)-1 site in the LGALS1 enhancer region, to which activating transcription factor2, c-Fos and c-Jun bind, was shown to be essential for IL-1ß-induced galectin-1/LGALS1 expression in Müller cells. Ligand (dexamethasone or triamcinolone acetonide)-activated GR induced dual specificity phosphatase (DUSP)1 expression via the glucocorticoid response element and attenuated IL-1ß-induced galectin-1/LGALS1 expression by reducing phosphorylation of these AP-1 subunits following AKT and extracellular signal-regulated kinase (ERK)1/2 deactivation. Moreover, activated GR also caused DUSP1-independent down-regulation of IL-1ß-induced LGALS1 expression via its binding to AP-1. Administration of glucocorticoids to mice attenuated diabetes-induced retinal galectin-1/Lgals1 expression together with AKT/AP-1 and ERK/AP-1 pathways. Supporting these in vitro and in vivo findings, immunofluorescence analyses showed co-localization of galectin-1 with GR and phosphorylated AP-1 in DUSP1-positive glial cells in fibrovascular tissues from patients with DR. Our present data demonstrated the inhibitory effects of glucocorticoids on glial galectin-1 expression via DUSP1-dependent and -independent deactivation of AP-1 signalling (transactivation and transrepression), highlighting therapeutic implications for DR.

Evidence that peroxisome proliferator-activated receptor γ suppresses squamous carcinogenesis through anti-inflammatory signaling and regulation of the immune response.

A variety of evidence suggests that peroxisome proliferator-activated receptor (PPAR)γ agonists may represent a potential pharmacologic target in the prevention or treatment of skin cancer. In particular, recent reports suggest that PPARγ activation may exert at least some of its anti-neoplastic effects through the suppression of tumor promoting chronic inflammation as well as by strengthening antitumor immune responses. This activity is thought to occur through a distinct mode of ligand interaction with PPARγ that causes transrepression of transcription factors that are involved in inflammatory and immunomodulatory signaling. However, current thiazolidinedione (TZD)-type PPARγ agonists have significant safety concerns that limit their usefulness as a preventive or therapeutic option. Due to the relatively large ligand binding pocket of PPARγ, a diverse group of ligands can be seen to interact with distinct modes of binding to PPARγ, leading to the phenomenon of partial agonist activity and selective PPARγ modulators (SPPARγM). This has led to the development of ligands that are tailored to deliver desired pharmacologic activity, but lack some of the negative side effects associated with full agonists, such as the currently utilized TZD-type PPARγ agonists. In addition, there is evidence that a number of phytochemicals that are currently being touted as antineoplastic nutraceuticals also possess PPARγ activity that may partially explain their pharmacologic activity. We propose that one or more of these partial agonists, SPPARγMs, or putative phytochemical PPARγ ligands could presumably be used as a starting point to design more efficacious anti-neoplastic PPARγ ligands that lack adverse pharmacological effects.

Understanding Peroxisome Proliferator-Activated Receptors: From the Structure to the Regulatory Actions on Metabolism.

Peroxisome proliferator-activated receptors (PPARs) are multi-domains proteins, belonging to the superfamily of nuclear receptors, which mainly act as ligand-activated transcription factors. A variety of lipophilic molecules, including long-chain polyunsaturated fatty acids and eicosanoids, are capable of binding to PPAR, although the nature of the physiological ligands is still under debate. PPARs regulate the expression of a set of genes involved in glucose and lipid metabolism as well as in the control of inflammatory responses. Herein we review the main molecular and cellular events associated with the activation of PPARs and their effects on metabolism.

GR SUMOylation and formation of an SUMO-SMRT/NCoR1-HDAC3 repressing complex is mandatory for GC-induced IR nGRE-mediated transrepression.

Unique among the nuclear receptor superfamily, the glucocorticoid (GC) receptor (GR) can exert three distinct transcriptional regulatory functions on binding of a single natural (cortisol in human and corticosterone in mice) and synthetic [e.g., dexamethasone (Dex)] hormone. The molecular mechanisms underlying GC-induced positive GC response element [(+)GRE]-mediated activation of transcription are partially understood. In contrast, these mechanisms remain elusive for GC-induced evolutionary conserved inverted repeated negative GC response element (IR nGRE)-mediated direct transrepression and for tethered indirect transrepression that is mediated by DNA-bound NF-κB/activator protein 1 (AP1)/STAT3 activators and instrumental in GC-induced anti-inflammatory activity. We demonstrate here that SUMOylation of lysine K293 (mouse K310) located within an evolutionary conserved sequence in the human GR N-terminal domain allows the formation of a GR-small ubiquitin-related modifiers (SUMOs)-NCoR1/SMRT-HDAC3 repressing complex mandatory for GC-induced IR nGRE-mediated direct repression in vitro, but does not affect transactivation. Importantly, these results were validated in vivo: in K310R mutant mice and in mice ablated selectively for nuclear receptor corepressor 1 (NCoR1)/silencing mediator for retinoid or thyroid-hormone receptors (SMRT) corepressors in skin keratinocytes, Dex-induced direct repression and the formation of repressing complexes on IR nGREs were impaired, whereas transactivation was unaffected. In mice selectively ablated for histone deacetylase 3 (HDAC3) in skin keratinocytes, GC-induced direct repression, but not bindings of GR and of corepressors NCoR1/SMRT, was abolished, indicating that HDAC3 is instrumental in IR nGRE-mediated repression. Moreover, we demonstrate that the binding of HDAC3 to IR nGREs in vivo is mediated through interaction with SMRT/NCoR1. We also show that the GR ligand binding domain (LBD) is not required for SMRT-mediated repression, which can be mediated by a LBD-truncated GR, whereas it is mandatory for NCoR1-mediated repression through an interaction with K579 in the LBD.

Analysis of Rice Proteins with DLN Repressor Motif/S.

Transcriptional regulation includes both activation and repression of downstream genes. In plants, a well-established class of repressors are proteins with an ERF-associated amphiphilic repression/EAR domain. They contain either DLNxxP or LxLxL as the identifying hexapeptide motif. In rice (Oryza sativa), we have identified a total of 266 DLN repressor proteins, with the former motif and its modifications thereof comprising 227 transcription factors and 39 transcriptional regulators. Apart from DLNxxP motif conservation, DLNxP and DLNxxxP motifs with variable numbers/positions of proline and those without any proline conservation have been identified. Most of the DLN repressome proteins have a single DLN motif, with higher relative percentage in the C-terminal region. We have designed a simple yeast-based experiment wherein a DLN motif can successfully cause strong repression of downstream reporter genes, when fused to a transcriptional activator of rice or yeast. The DLN hexapeptide motif is essential for repression, and at least two "DLN" residues cause maximal repression. Comparatively, rice has more DLN repressor encoding genes than Arabidopsis, and DLNSPP motif from rice is 40% stronger than the known Arabidopsis SRDX motif. The study reports a straightforward assay to analyze repressor activity, along with the identification of a strong DLN repressor from rice.

Towards the emerging crosstalk: ERBB family and steroid hormones.

Growth factors acting through receptor tyrosine kinases (RTKs) of ERBB family, along with steroid hormones (SH) acting through nuclear receptors (NRs), are critical signalling mediators of cellular processes. Deregulations of ERBB and steroid hormone receptors are responsible for several diseases, including cancer, thus demonstrating the central role played by both systems. This review will summarize and shed light on an emerging crosstalk between these two important receptor families. How this mutual crosstalk is attained, such as through extensive genomic and non-genomic interactions, will be addressed. In light of recent studies, we will describe how steroid hormones are able to fine-tune ERBB feedback loops, thus impacting on cellular output and providing a new key for understanding the complexity of biological processes in physiological or pathological conditions. In our understanding, the interactions between steroid hormones and RTKs deserve further attention. A system biology approach and advanced technologies for the analysis of RTK-SH crosstalk could lead to major advancements in molecular medicine, providing the basis for new routes of pharmacological intervention in several diseases, including cancer.

α Actinin 4 (ACTN4) Regulates Glucocorticoid Receptor-mediated Transactivation and Transrepression in Podocytes.

Glucocorticoids are a general class of steroids that possess renoprotective activity in glomeruli through their interaction with the glucocorticoid receptor. However, the mechanisms by which glucocorticoids ameliorate proteinuria and glomerular disease are not well understood. In this study, we demonstrated that α actinin 4 (ACTN4), an actin-cross-linking protein known to coordinate cytoskeletal organization, interacts with the glucocorticoid receptor (GR) in the nucleus of human podocytes (HPCs), a key cell type in the glomerulus critical for kidney filtration function. The GR-ACTN4 complex enhances glucocorticoid response element (GRE)-driven reporter activity. Stable knockdown of ACTN4 by shRNA in HPCs significantly reduces dexamethasone-mediated induction of GR target genes and GRE-driven reporter activity without disrupting dexamethasone-induced nuclear translocation of GR. Synonymous mutations or protein expression losses in ACTN4 are associated with kidney diseases, including focal segmental glomerulosclerosis, characterized by proteinuria and podocyte injury. We found that focal segmental glomerulosclerosis-linked ACTN4 mutants lose their ability to bind liganded GR and support GRE-mediated transcriptional activity. Mechanistically, GR and ACTN4 interact in the nucleus of HPCs. Furthermore, disruption of the LXXLL nuclear receptor-interacting motif present in ACTN4 results in reduced GR interaction and dexamethasone-mediated transactivation of a GRE reporter while still maintaining its actin-binding activity. In contrast, an ACTN4 isoform, ACTN4 (Iso), that loses its actin-binding domain is still capable of potentiating a GRE reporter. Dexamethasone induces the recruitment of ACTN4 and GR to putative GREs in dexamethasone-transactivated promoters, SERPINE1, ANGPLT4, CCL20, and SAA1 as well as the NF-κB (p65) binding sites on GR-transrepressed promoters such as IL-1ß, IL-6, and IL-8 Taken together, our data establish ACTN4 as a transcriptional co-regulator that modulates both dexamethasone-transactivated and -transrepressed genes in podocytes.