Choosing the right partner in hormone-dependent gene regulation: Glucocorticoid and progesterone receptors crosstalk in breast cancer cells.

Steroid hormone receptors (SHRs) belong to a large family of ligand-activated nuclear receptors that share certain characteristics and possess others that make them unique. It was thought for many years that the specificity of hormone response lay in the ligand. Although this may be true for pure agonists, the natural ligands as progesterone, corticosterone and cortisol present a broader effect by simultaneous activation of several SHRs. Moreover, SHRs share structural and functional characteristics that range from similarities between ligand-binding pockets to recognition of specific DNA sequences. These properties are clearly evident in progesterone (PR) and glucocorticoid receptors (GR); however, the biological responses triggered by each receptor in the presence of its ligand are different, and in some cases, even opposite. Thus, what confers the specificity of response to a given receptor is a long-standing topic of discussion that has not yet been unveiled. The levels of expression of each receptor, the differential interaction with coregulators, the chromatin accessibility as well as the DNA sequence of the target regions in the genome, are reliable sources of variability in hormone action that could explain the results obtained so far. Yet, to add further complexity to this scenario, it has been described that receptors can form heterocomplexes which can either compromise or potentiate the respective hormone-activated pathways with its possible impact on the pathological condition. In the present review, we summarized the state of the art of the functional cross-talk between PR and GR in breast cancer cells and we also discussed new paradigms of specificity in hormone action.

SOX2 Modulates the Nuclear Organization and Transcriptional Activity of the Glucocorticoid Receptor.

Steroid receptors (SRs) are ligand-dependent transcription factors (TFs) relevant to key cellular processes in both physiology and pathology, including some types of cancer. SOX2 is a master TF of pluripotency and self-renewal of embryonic stem cells, and its dysregulation is also associated with various types of human cancers. A potential crosstalk between these TFs could be relevant in malignant cells yet, to the best of our knowledge, no formal study has been performed thus far. Here we show, by quantitative live-cell imaging microscopy, that ectopic expression of SOX2 disrupts the formation of hormone-dependent intranuclear condensates of many steroid receptors (SRs), including those formed by the glucocorticoid receptor (GR). SOX2 also reduces GR's binding to specific DNA targets and modulates its transcriptional activity. SOX2-driven effects on GR condensates do not require the intrinsically disordered N-terminal domain of the receptor and, surprisingly, neither relies on GR/SOX2 interactions. SOX2 also alters the intranuclear dynamics and compartmentalization of the SR coactivator NCoA-2 and impairs GR/NCoA-2 interactions. These results suggest an indirect mechanism underlying SOX2-driven effects on SRs involving this coactivator. Together, these results highlight that the transcriptional program elicited by GR relies on its nuclear organization and is intimately linked to the distribution of other GR partners, such as the NCoA-2 coactivator. Abnormal expression of SOX2, commonly observed in many tumors, may alter the biological action of GR and, probably, other SRs as well. Understanding this crosstalk may help to improve steroid hormone-based therapies in cancers with elevated SOX2 expression.

Insights into estrogen receptor alpha modulation by cholestenoic acids.

Oxysterols are a family of over 25 cholesterol metabolites naturally produced by enzymatic or radical oxidation. They are involved in many physiological and pathological pathways. Although their activity has been mainly attributed to the modulation of the Liver X Receptors (LXR), it is currently accepted that oxysterols are quite promiscuous compounds, acting at several targets at the same time. The promiscuity of the oxysterols with the Estrogen Receptor α (ERα) is crucial in several pathologies such as ER+ breast cancer, inflammation and atherosclerosis. Regarding this matter, we have previously reported the synthesis, LXR activity and binding mode of a family of cholestenoic acid analogs with a modified side chain. Here we report the transcriptional activity on the ERα triggered by these compounds and details on the molecular determinants involved in their activities in order to establish structure-activity relationships to shed light over the molecular basis of the promiscuity of these compounds on ER/LXR responses. Our results show that 3ß-hydroxy-5-cholestenoic acid can interact with the ERα receptor in a way similar to 26-hydroxycholesterol and is an agonist of the receptor. Using molecular dynamics simulations, we were able to predict the ERα activity of a set of cholestenoic acid analogs with changes in the flexibility and/or steric requirements of the side chain, some of which exhibited selective activation of ERα or LXR.

Active Surveillance of Asymptomatic, Presymptomatic, and Oligosymptomatic SARS-CoV-2-Infected Individuals in Communities Inhabiting Closed or Semi-closed Institutions.

Background: The high COVID-19 dissemination rate demands active surveillance to identify asymptomatic, presymptomatic, and oligosymptomatic (APO) SARS-CoV-2-infected individuals. This is of special importance in communities inhabiting closed or semi-closed institutions such as residential care homes, prisons, neuropsychiatric hospitals, etc., where risk people are in close contact. Thus, a pooling approach-where samples are mixed and tested as single pools-is an attractive strategy to rapidly detect APO-infected in these epidemiological scenarios. Materials and Methods: This study was done at different pandemic periods between May 28 and August 31 2020 in 153 closed or semi-closed institutions in the Province of Buenos Aires (Argentina). We setup pooling strategy in two stages: first a pool-testing followed by selective individual-testing according to pool results. Samples included in negative pools were presumed as negative, while samples from positive pools were re-tested individually for positives identification. Results: Sensitivity in 5-sample or 10-sample pools was adequate since only 2 Ct values were increased with regard to single tests on average. Concordance between 5-sample or 10-sample pools and individual-testing was 100% in the Ct ≤ 36. We tested 4,936 APO clinical samples in 822 pools, requiring 86-50% fewer tests in low-to-moderate prevalence settings compared to individual testing. Conclusions: By this strategy we detected three COVID-19 outbreaks at early stages in these institutions, helping to their containment and increasing the likelihood of saving lives in such places where risk groups are concentrated.

Phasing the intranuclear organization of steroid hormone receptors.

Steroid receptors (SRs) encompass a family of transcription factors that regulate the expression of thousands of genes upon binding to steroid hormones and include the glucocorticoid, androgen, progesterone, estrogen and mineralocorticoid receptors. SRs control key physiological and pathological processes, thus becoming relevant drug targets. As with many other nuclear proteins, hormone-activated SRs concentrate in multiple discrete foci within the cell nucleus. Even though these foci were first observed ∼25 years ago, their exact structure and function remained elusive. In the last years, new imaging methodologies and theoretical frameworks improved our understanding of the intranuclear organization. These studies led to a new paradigm stating that many membraneless nuclear compartments, including transcription-related foci, form through a liquid-liquid phase separation process. These exciting ideas impacted the SR field by raising the hypothesis of SR foci as liquid condensates involved in transcriptional regulation. In this work, we review the current knowledge about SR foci formation under the light of the condensate model, analyzing how these structures may impact SR function. These new ideas, combined with state-of-the-art techniques, may shed light on the biophysical mechanisms governing the formation of SR foci and the biological function of these structures in normal physiology and disease.

Unraveling the molecular interactions involved in phase separation of glucocorticoid receptor.

BACKGROUND: Functional compartmentalization has emerged as an important factor modulating the kinetics and specificity of biochemical reactions in the nucleus, including those involved in transcriptional regulation. The glucocorticoid receptor (GR) is a ligand-activated transcription factor that translocates to the nucleus upon hormone stimulation and distributes between the nucleoplasm and membraneless compartments named nuclear foci. While a liquid-liquid phase separation process has been recently proposed to drive the formation of many nuclear compartments, the mechanisms governing the heterogeneous organization of GR in the nucleus and the functional relevance of foci formation remain elusive. RESULTS: We dissected some of the molecular interactions involved in the formation of GR condensates and analyzed the GR structural determinants relevant to this process. We show that GR foci present properties consistent with those expected for biomolecular condensates formed by a liquid-liquid phase separation process in living human cells. Their formation requires an initial interaction of GR with certain chromatin regions at specific locations within the nucleus. Surprisingly, the intrinsically disordered region of GR is not essential for condensate formation, in contrast to many nuclear proteins that require disordered regions to phase separate, while the ligand-binding domain seems essential for that process. We finally show that GR condensates include Mediator, a protein complex involved in transcription regulation. CONCLUSIONS: We show that GR foci have properties of liquid condensates and propose that active GR molecules interact with chromatin and recruit multivalent cofactors whose interactions with additional molecules lead to the formation of a focus. The biological relevance of the interactions occurring in GR condensates supports their involvement in transcription regulation.

Cholestenoic acid analogues as inverse agonists of the liver X receptors.

Liver X Receptors (LXRs) are ligand dependent transcription factors activated by oxidized cholesterol metabolites (oxysterols) that play fundamental roles in the transcriptional control of lipid metabolism, cholesterol transport and modulation of inflammatory responses. In the last decade, LXRs have become attractive pharmacological targets for intervention in human metabolic diseases and thus, several efforts have concentrated on the development of synthetic analogues able to modulate LXR transcriptional response. In this sense, we have previously found that cholestenoic acid analogues with a modified side chain behave as LXR inverse agonists. To further investigate the structure-activity relationships and to explore how cholestenoic acid derivatives interact with the LXRs, we evaluated the LXR biological activity of new analogues containing a C24-C25 double bond. Furthermore, a microarray assay was performed to evaluate the recruitment of coregulators to recombinant LXR LBD upon ligand binding. Also, conventional and accelerated molecular dynamics simulations were applied to gain insight on the molecular determinants involved in the inverse agonism. As LXR inverse agonists emerge as very promising candidates to control LXR activity, the cholestenoic acid analogues here depicted constitute a new relevant steroidal scaffold to inhibit LXR action.

Structural Insights into the Ligand Binding Domain of the Glucocorticoid Receptor: A Molecular Dynamics Study.

The glucocorticoid receptor (GR) is a ligand-binding dependent transcription factor that ultimately regulates vital biological processes and inflammation response through specific gene expression control, thus representing a notable drug target to explore. Structurally, its ligand binding domain (LBD) harbors the region for the ligand-dependent transcriptional activation function 2 (AF-2), a majorly hydrophobic groove formed by residues from helices H3, H4, and H12, where the H12 position plays a critical role in AF-2 spatial conformation and GR function as a whole. However, the exact mechanisms underlying how regulatory ligands control the H12 structure and dynamics are yet to be elucidated. In this work, we have explored the correlation between ligand identity and GR LBD H12 behavior through different molecular dynamics (MD) simulations. After building diverse GR LBD systems in agonist and nonagonist states, we studied each system's response in the absence or the presence of an agonist ligand (dexamethasone) or an antagonist ligand (RU486) using classical MD simulations. We complemented them with steered MD for assessing the transition between those states and with the Umbrella Sampling method for free-energy evaluation. On the one hand, successfully obtaining fully folded nonagonist GR LBD states from the partially unfolded crystal GR LBD/RU486 underlines the role of the H1 in the GR LBD folding pathway. On the other hand, our results describe the H12 as a dynamic ensemble of conformations whose relative population is in the end determined by the interacting ligand: while dexamethasone privileges only a few poses (determined by a potential energy surface with a deep minimum), RU486 favors a wider H12 conformational amplitude, as indicated by a flatter potential landscape. By characterizing the H12 conformation in different conditions, we provide novel GR LBD models that represent potential targets for rational glucocorticoid drugs design, with the aim of accurately modulating GR activity.

Glucocorticoids uncover a critical role for ASH2L on BCL-X expression regulation in leukemia cells.

Targeting the apoptosis machinery is a promising therapeutic approach in myeloid malignancies. BCL2L1 is a well-known glucocorticoid-responsive gene and a key apoptosis regulator that, when over-expressed, can contribute to tumor development, progression and therapeutic resistance. Moreover, synthetic glucocorticoids, like dexamethasone, are frequently used in the treatment of hematopoietic diseases due to its pro-apoptotic properties. We report here that the trithorax protein ASH2L, considered one of the core subunits of H3K4-specific MLL/SET methyltransferase complexes, contributes to anti-apoptotic BCL-XL over-expression and cell survival in patient-derived myeloid leukemia cells. We find that the unliganded glucocorticoid receptor (uGR) and ASH2L interact in a common protein complex through a chromatin looping determined by uGR and ASH2L binding to BCL2L1 specific +58 HRE and promoter region, respectively. Upon addition of dexamethasone, GR and ASH2L recruitment is reduced, BCL-XL expression diminishes and apoptosis is induced consequently. Overall, our findings indicate that uGR and ASH2L may act as key regulatory players of BCL- XL upregulation in AML cells.

The glucocorticoid receptor interferes with progesterone receptor-dependent genomic regulation in breast cancer cells.

The glucocorticoid and progesterone receptors (GR and PR) are closely related members of the steroid receptor family. Despite sharing similar structural and functional characteristics; the cognate hormones display very distinct physiological responses. In mammary epithelial cells, PR activation is associated with the incidence and progression of breast cancer, whereas the GR is related to growth suppression and differentiation. Despite their pharmacological relevance, only a few studies have compared GR and PR activities in the same system. Using a PR+/GR+ breast cancer cell line, here we report that either glucocorticoid-free or dexamethasone (DEX)-activated GR inhibits progestin-dependent gene expression associated to epithelial-mesenchymal-transition and cell proliferation. When both receptors are activated with their cognate hormones, PR and GR can form part of the same complex according to co-immunoprecipitation, quantitative microscopy and sequential ChIP experiments. Moreover, genome-wide studies in cells treated with either DEX or R5020, revealed the presence of several regions co-bound by both receptors. Surprisingly, GR also binds novel genomic sites in cells treated with R5020 alone. This progestin-induced GR binding was enriched in REL DNA motifs and located close to genes coding for chromatin remodelers. Understanding GR behavior in the context of progestin-dependent breast cancer could provide new targets for tumor therapy.

Mapping the neurosteroid binding sites on glycine receptors.

Glycine is a major inhibitory neurotransmitter in the CNS, where it modulates both sensory and motor transduction throughout its binding to glycine receptors (GlyRs), pentameric chloride channels that share structural and functional properties with type A γ-aminobutyric acid receptors (GABAAR). A large number of structurally diverse organic compounds have been identified as GlyR and GABAAR allosteric modulators, making these receptors attractive pharmacological targets. Taking into account the recent resolved crystal structures of GABAAR/neurosteroid complexes, and due to the high sequence identity between the GABAAR and GlyR transmembrane domains, in this work we applied molecular modeling methods to explore the neurosteroid binding to GlyR. Our results indicated that neurosteroid binding sites of GABAARs are also conserved in the GlyRs. Furthermore, docking and molecular dynamics simulations predicted that neurosteroids are stably recognized at these sites, providing precise information on the molecular basis of the neurosteroid binding mode to GlyR. The comparison of how allopregnanolone and pregnanolone 3-OH moieties are recognized by the GlyR binding pocket revealed significant differences that may be associated to opposite effects of these isomers on the GlyR response.

Liver X receptor-α activation enhances cholesterol secretion in lactating mammary epithelium.

Liver X receptors (LXRs) are ligand-dependent transcription factors activated by cholesterol metabolites. These receptors induce a suite of target genes required for de novo synthesis of triglycerides and cholesterol transport in many tissues. Two different isoforms, LXRα and LXRß, have been well characterized in liver, adipocytes, macrophages, and intestinal epithelium among others, but their contribution to cholesterol and fatty acid efflux in the lactating mammary epithelium is poorly understood. We hypothesize that LXR regulates lipogenesis during milk fat production in lactation. Global mRNA analysis of mouse mammary epithelial cells (MECs) revealed multiple LXR/RXR targets upregulated sharply early in lactation compared with midpregnancy. LXRα is the primary isoform, and its protein levels increase throughout lactation in MECs. The LXR agonist GW3965 markedly induced several genes involved in cholesterol transport and lipogenesis and enhanced cytoplasmic lipid droplet accumulation in the HC11 MEC cell line. Importantly, in vivo pharmacological activation of LXR increased the milk cholesterol percentage and induced sterol regulatory element-binding protein 1c (Srebp1c) and ATP-binding cassette transporter a7 (Abca7) expression in MECs. Cumulatively, our findings identify LXRα as an important regulator of cholesterol incorporation into the milk through key nodes of de novo lipogenesis, suggesting a potential therapeutic target in women with difficulty initiating lactation.

Heme Oxygenase 1 Impairs Glucocorticoid Receptor Activity in Prostate Cancer.

Glucocorticoids are used during prostate cancer (PCa) treatment. However, they may also have the potential to drive castration resistant prostate cancer (CRPC) growth via the glucocorticoid receptor (GR). Given the association between inflammation and PCa, and the anti-inflammatory role of heme oxygenase 1 (HO-1), we aimed at identifying the molecular processes governed by the interaction between HO-1 and GR. PCa-derived cell lines were treated with Hemin, Dexamethasone (Dex), or both. We studied GR gene expression by RTqPCR, protein expression by Western Blot, transcriptional activity using reporter assays, and nuclear translocation by confocal microscopy. We also evaluated the expression of HO-1, FKBP51, and FKBP52 by Western Blot. Hemin pre-treatment reduced Dex-induced GR activity in PC3 cells. Protein levels of FKBP51, a cytoplasmic GR-binding immunophilin, were significantly increased in Hemin+Dex treated cells, possibly accounting for lower GR activity. We also evaluated these treatments in vivo using PC3 tumors growing as xenografts. We found non-significant differences in tumor growth among treatments. Immunohistochemistry analyses revealed strong nuclear GR staining in almost all groups. We did not observe HO-1 staining in tumor cells, but high HO-1 reactivity was detected in tumor infiltrating macrophages. Our results suggest an association and crossed modulation between HO-1 and GR pathways.

In Search of GABAA Receptor's Neurosteroid Binding Sites.

Neurosteroids (NS) are the main modulators of γ-aminobutyric acid type A receptors (GABAARs), which are the ligand-gated channels target of the major inhibitory neurotransmitter in vertebrates. As a consequence of their ability to modify inhibitory functions in the brain, NS have high physiological and clinical relevance. Accumulated evidence has strongly suggested that NS binding sites were located in the GABAAR transmembrane domain; however the specific localization of these sites has remained an enigma for decades. Fortunately, recent resolution of GABAARs crystal structures, together with computational strategies applied to investigate the NS binding, has paved the way to rationalizing the molecular basis of NS modulation. This work reviews from a historical perspective the road followed for establishing the GABAAR/NS binding mode, from their initial molecular modeling to the latest findings. Furthermore, a comparative analysis describing the NS binding is provided, plus a preliminary analysis of putative NS sites in other assemblies.

Structure and dynamics of neurosteroid binding to the α1ß2γ2 GABAA receptor.

Neurosteroids are the principal endogenous modulators of the γ-Aminobutyric acid receptors (GABAARs), pentameric membrane-bound proteins that can be assembled from at least 19 subunits. In the most abundant GABAAR arrangement (α1ß2γ2), neurosteroids can potentiate the GABA action as well as produce a direct activation of the channel. The recent crystal structures of neurosteroids bound to α homopentameric GABAAR reveal binding to five equivalent sites. However, these results have been obtained using receptors that are not physiologically relevant, suggesting a need to investigate neurosteroid binding to heteropentameric receptors that exist in the central nervous system. In a previous work, we predicted the neurosteroid binding site by applying molecular modeling methods on the ß3 homopentamer. Here we construct a homology model of the transmembrane domain of the heteropentameric α1ß2γ2 receptor and then, by combining docking and molecular dynamics simulations, we analyzed neurosteroid binding. Results show that the five neurosteroid cavities are conserved in the α1ß2γ2 receptor and all of them are able to bind neurosteroids. Two different binding modes were detected depending on the identity of the residue at position 241 in the transmembrane helix 1. These theoretical findings provide microscopic insights into neurosteroid binding at the heteropentameric GABAAR. The existence of two classes of sites may be associated with how neurosteroids modulate GABAAR. Our finding would represent the essential first step to reach a comprehensive understanding of how these endogenous molecules regulate the central nervous system.

Synthesis and activity evaluation of a series of cholanamides as modulators of the liver X receptors.

The Liver X receptors (LXRs) are members of the nuclear receptor family, that play fundamental roles in cholesterol transport, lipid metabolism and modulation of inflammatory responses. In recent years, the synthetic steroid N,N-dimethyl-3ß-hydroxycholenamide (DMHCA) arised as a promising LXR ligand. This compound was able to dissociate certain beneficial LXRs effects from those undesirable ones involved in triglyceride metabolism. Here, we synthetized a series of DMHCA analogues with different modifications in the steroidal nucleus involving the A/B ring fusion, that generate changes in the overall conformation of the steroid. The LXRα and LXRß activity of these analogues was evaluated by using a luciferase reporter assay in BHK21 cells. Compounds were tested in both the agonist and antagonist modes. Results indicated that the agonist/antagonist profile is dependent on the steroid configuration at the A/B ring junction. Notably, in contrast to DMHCA, the amide derived from lithocholic acid (2) with an A/B cis configuration and its 6,19-epoxy analogue 4 behaved as LXRα selective agonists, while the 2,19-epoxy analogues with an A/B trans configuration were antagonists of both isoforms. The binding mode of the analogues to both LXR isoforms was assessed by using 50 ns molecular dynamics (MD) simulations. Results revealed conformational differences between LXRα- and LXRß-ligand complexes, mainly in the hydrogen bonding network that involves the C-3 hydroxyl. Overall, these results indicate that the synthetized DMHCA analogues could be interesting candidates for a therapeutic modulation of the LXRs.

Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy.

The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasm overcrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets. These properties determine that many nuclear functions such as transcription, replication, splicing or DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advanced fluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) provide complementary and exquisite information on the dynamics of fluorescent labeled molecules moving through the nuclear space and are helping us to comprehend the complexity of the nuclear structure. Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleus in live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescent tagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. In addition, we describe general rules to set the parameters for one and two-color experiments and the required controls for these experiments. Finally, we review the statistical analysis of the FCS data and summarize the use of numerical simulations as a complementary approach that helps us to understand the complex matrix of molecular interactions network within the nucleus.

21-Hydroxy-6,19-epoxyprogesterone: A Promising Therapeutic Agent and a Molecular Tool for Deciphering Glucocorticoid Action.

Glucocorticoids are steroid hormones that exert most of their effects through their binding to the glucocorticoid receptor (GR), a ligand regulated transcription factor. Although glucocorticoids are widely used in the clinic, their usage in chronic therapies provokes severe adverse reactions. In the quest for safer glucocorticoids a dissociated model was established that proposes a disconnection between GR activated pathways responsible of desired pharmacological effects and pathways involved in adverse GR reactions. Under this model, a myriad of steroidal and non-steroidal compounds has been characterized, with most of them still producing side effects. X-ray crystallographic studies followed by molecular dynamics analysis led research to insights on the receptor Ligand Binding Domain (LBD), which undergoes specific ligand dependent conformational changes that influence receptor activities. In this sense, the flexibility of the ligand structure would contribute to the final GR outcome. Here, we review different data of 21-hydroxy-6,19-epoxyprogesterone (21OH-6,19OP), a rigid steroid with potential pharmaceutical interest due to its anti-inflammatory and immunosuppressive activities, lacking several GR adverse reactions. The rigid structure endows this compound with an enhanced selectivity towards GR. Molecular characterization of the GR/21OH-6,19OP complex revealed specific intermediate conformations adopted by the receptor that would explain the influence on GR dimerization and the recruitment of a specific set of GR transcription modulators. We summarize recent data that will contribute to understand the complexity of glucocorticoid response.