Crosstalk interactions between transcription factors ERRα and PPARα assist PPARα-mediated gene expression.

OBJECTIVE: The peroxisome proliferator-activated receptor α (PPARα) is a transcription factor driving target genes involved in fatty acid ß-oxidation. To what extent various PPARα interacting proteins may assist its function as a transcription factor is incompletely understood. An ORFeome-wide unbiased mammalian protein-protein interaction trap (MAPPIT) using PPARα as bait revealed a PPARα-ligand-dependent interaction with the orphan nuclear receptor estrogen-related receptor α (ERRα). The goal of this study was to characterize the nature of the interaction in depth and to explore whether it was of physiological relevance. METHODS: We used orthogonal protein-protein interaction assays and pharmacological inhibitors of ERRα in various systems to confirm a functional interaction and study the impact of crosstalk mechanisms. To characterize the interaction surfaces and contact points we applied a random mutagenesis screen and structural overlays. We pinpointed the extent of reciprocal ligand effects of both nuclear receptors via coregulator peptide recruitment assays. On PPARα targets revealed from a genome-wide transcriptome analysis, we performed an ERRα chromatin immunoprecipitation analysis on both fast and fed mouse livers. RESULTS: Random mutagenesis scanning of PPARα's ligand-binding domain and coregulator profiling experiments supported the involvement of (a) bridging coregulator(s), while recapitulation of the interaction in vitro indicated the possibility of a trimeric interaction with RXRα. The PPARα·ERRα interaction depends on 3 C-terminal residues within helix 12 of ERRα and is strengthened by both PGC1α and serum deprivation. Pharmacological inhibition of ERRα decreased the interaction of ERRα to ligand-activated PPARα and revealed a transcriptome in line with enhanced mRNA expression of prototypical PPARα target genes, suggesting a role for ERRα as a transcriptional repressor. Strikingly, on other PPARα targets, including the isolated PDK4 enhancer, ERRα behaved oppositely. Chromatin immunoprecipitation analyses demonstrate a PPARα ligand-dependent ERRα recruitment onto chromatin at PPARα-binding regions, which is lost following ERRα inhibition in fed mouse livers. CONCLUSIONS: Our data support the coexistence of multiple layers of transcriptional crosstalk mechanisms between PPARα and ERRα, which may serve to finetune the activity of PPARα as a nutrient-sensing transcription factor.

Glucocorticoid receptor signaling: intricacies and therapeutic opportunities.

The glucocorticoid receptor (GR) is a major nuclear receptor (NR) drug target for the treatment of inflammatory disorders and several cancers. Despite the effectiveness of GR ligands, their systemic action triggers a plethora of side effects, limiting long-term use. Here, we discuss new concepts of and insights into GR mechanisms of action to assist in the identification of routes toward enhanced therapeutic benefits. We zoom in on the communication between different GR domains and how this is influenced by different ligands. We detail findings on the interaction between GR and chromatin, and highlight how condensate formation and coregulator confinement can perturb GR transcriptional responses. Last, we discuss the potential of novel ligands and the therapeutic exploitation of crosstalk with other NRs.

Crosstalk between glucocorticoid and mineralocorticoid receptors boosts glucocorticoid-induced killing of multiple myeloma cells.

The glucocorticoid receptor (GR) is a crucial drug target in multiple myeloma as its activation with glucocorticoids effectively triggers myeloma cell death. However, as high-dose glucocorticoids are also associated with deleterious side effects, novel approaches are urgently needed to improve GR action in myeloma. Here, we reveal a functional crosstalk between GR and the mineralocorticoid receptor (MR) that plays a role in improved myeloma cell killing. We show that the GR agonist dexamethasone (Dex) downregulates MR levels in a GR-dependent way in myeloma cells. Co-treatment of Dex with the MR antagonist spironolactone (Spi) enhances Dex-induced cell killing in primary, newly diagnosed GC-sensitive myeloma cells. In a relapsed GC-resistant setting, Spi alone induces distinct myeloma cell killing. On a mechanistic level, we find that a GR-MR crosstalk likely arises from an endogenous interaction between GR and MR in myeloma cells. Quantitative dimerization assays show that Spi reduces Dex-induced GR-MR heterodimerization and completely abolishes Dex-induced MR-MR homodimerization, while leaving GR-GR homodimerization intact. Unbiased transcriptomics analyses reveal that c-myc and many of its target genes are downregulated most by combined Dex-Spi treatment. Proteomics analyses further identify that several metabolic hallmarks are modulated most by this combination treatment. Finally, we identified a subset of Dex-Spi downregulated genes and proteins that may predict prognosis in the CoMMpass myeloma patient cohort. Our study demonstrates that GR-MR crosstalk is therapeutically relevant in myeloma as it provides novel strategies for glucocorticoid-based dose-reduction.

The Zinc-Binding Group Effect: Lessons from Non-Hydroxamic Acid Vorinostat Analogs.

Histone deacetylases (HDACs) are enzymes pursued as drug targets in various cancers and several non-oncological conditions, such as inflammation and neurodegenerative disorders. In the past decade, HDAC inhibitors (HDACi) have emerged as relevant pharmaceuticals, with many efforts devoted to the development of new representatives. However, the growing safety concerns regarding the established hydroxamic acid-based HDAC inhibitors tend to drive current research more toward the design of inhibitors bearing alternative zinc-binding groups (ZBGs). This Perspective presents an overview of all non-hydroxamic acid ZBGs that have been incorporated into the clinically approved prototypical HDACi, suberoylanilide hydroxamic acid (vorinostat). This provides the unique opportunity to compare the inhibition potential and biological effects of different ZBGs in a direct way, as the compounds selected for this Perspective differ only in their ZBG. To that end, different strategies used to select a ZBG, its properties, activity, and liabilities are discussed.

Novel assays monitoring direct glucocorticoid receptor protein activity exhibit high predictive power for ligand activity on endogenous gene targets.

Exogenous glucocorticoids are widely used in the clinic for the treatment of inflammatory disorders and auto-immune diseases. Unfortunately, their use is hampered by many side effects and therapy resistance. Efforts to find more selective glucocorticoid receptor (GR) agonists and modulators (called SEGRAMs) that are able to separate anti-inflammatory effects via gene repression from metabolic effects via gene activation, have been unsuccessful so far. In this study, we characterized a set of functionally diverse GR ligands in A549 cells, first using a panel of luciferase-based reporter gene assays evaluating GR-driven gene activation and gene repression. We expanded this minimal assay set with novel luciferase-based read-outs monitoring GR protein levels, GR dimerization and GR Serine 211 (Ser211) phosphorylation status and compared their outcomes with compound effects on the mRNA levels of known GR target genes in A549 cells and primary hepatocytes. We found that luciferase reporters evaluating GR-driven gene activation and gene repression were not always reliable predictors for effects on endogenous target genes. Remarkably, our novel assay monitoring GR Ser211 phosphorylation levels proved to be the most reliable predictor for compound effects on almost all tested endogenous GR targets, both driven by gene activation and repression. The integration of this novel assay in existing screening platforms running both in academia and industry may therefore boost chances to find novel GR ligands with an actual improved therapeutic benefit.

Identification of mercaptoacetamide-based HDAC6 inhibitors via a lean inhibitor strategy: screening, synthesis, and biological evaluation.

Non-selective inhibition of different histone deacetylase enzymes by hydroxamic acid-based drugs causes severe side effects when used as a (long-term) cancer treatment. In this work, we searched for a potent zinc-binding group able to replace the contested hydroxamic acid by employing a lean inhibitor strategy. This instructed the synthesis of a set of HDAC6-selective inhibitors containing the more desirable mercaptoacetamide moiety. Biological evaluation of these new compounds showed an IC50 in the nanomolar range, dose-dependent HDAC6 inhibition in MM1.S cells and improved genotoxicity results, rendering these new inhibitors valuable hits for applications even beyond oncology.

Approaches towards tissue-selective pharmacology of the mineralocorticoid receptor.

Mineralocorticoid receptor antagonists (MRAs) are highly effective therapies for cardiovascular and renal disease. However, the widespread clinical use of currently available MRAs in cardiorenal medicine is hampered by an increased risk of hyperkalaemia. The mineralocorticoid receptor (MR) is a nuclear receptor responsible for fluid and electrolyte homeostasis in epithelial tissues, whereas pathophysiological MR activation in nonepithelial tissues leads to undesirable pro-inflammatory and profibrotic effects. Therefore, new strategies that selectively target the deleterious effects of the MR but spare its physiological function are needed. In this review, we discuss recent pharmacological developments starting from novel non-steroidal MRAs, such as finerenone or esaxerenone, that are now entering clinical use, to concepts arising from the current knowledge of the MR signalling pathway, aiming at receptor-coregulator interaction, epigenetics or downstream effectors of the MR. LINKED ARTICLES: This article is part of a themed issue on Emerging Fields for Therapeutic Targeting of the Aldosterone-Mineralocorticoid Receptor Signaling Pathway. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.13/issuetoc.

Latest perspectives on glucocorticoid-induced apoptosis and resistance in lymphoid malignancies.

Glucocorticoids are essential drugs in the treatment protocols of lymphoid malignancies. These steroidal hormones trigger apoptosis of the malignant cells by binding to the glucocorticoid receptor (GR), which is a member of the nuclear receptor superfamily. Long term glucocorticoid treatment is limited by two major problems: the development of glucocorticoid-related side effects, which hampers patient quality of life, and the emergence of glucocorticoid resistance, which is a gradual process that is inevitable in many patients. This emphasizes the need to reevaluate and optimize the widespread use of glucocorticoids in lymphoid malignancies. To achieve this goal, a deep understanding of the mechanisms governing glucocorticoid responsiveness is required, yet, a recent comprehensive overview is currently lacking. In this review, we examine how glucocorticoids mediate apoptosis by detailing GR's genomic and non-genomic action mechanisms in lymphoid malignancies. We continue with a discussion of the glucocorticoid-related problems and how these are intertwined with one another. We further zoom in on glucocorticoid resistance by critically analyzing the plethora of proposed mechanisms and highlighting therapeutic opportunities that emerge from these studies. In conclusion, early detection of glucocorticoid resistance in patients remains an important challenge as this would result in a timelier treatment reorientation and reduced glucocorticoid-instigated side effects.

Nuclear receptor crosstalk - defining the mechanisms for therapeutic innovation.

Nuclear receptor crosstalk can be defined as the interplay between different nuclear receptors or between their overlapping signalling pathways. A subset of nuclear receptors (such as PPARs and RARs) engage in the formation of well-characterized 'typical' heterodimers with RXR. 'Atypical' heterodimers (such as GR with PPARs, or PPAR with ERR) might form a novel class of physical complexes that might be more transient in nature. These heterodimers might harbour strong transcriptional flexibility, with no strict need for DNA binding of both partners. Direct crosstalk could stem from a pairwise physical association between atypical nuclear receptor heterodimers, either via pre-existing interaction pairs or via interactions that are newly induced with small molecules; such crosstalk might constitute an uncharted space to target nuclear receptor physiological and/or pathophysiological actions. In this Review, we discuss the emerging aspects of crosstalk in the nuclear receptor field and present various mechanistic crosstalk modes with examples that support applicability of the atypical heterodimer concept. Stabilization or disruption, in a context-dependent or cell type-dependent manner, of these more transient heterodimers is expected to fuel unprecedented translational approaches to yield novel therapeutic agents to treat major human diseases with higher precision.

Effect of combining glucocorticoids with Compound A on glucocorticoid receptor responsiveness in lymphoid malignancies.

Glucocorticoids (GCs) are a cornerstone in the treatment of lymphoid malignancies such as multiple myeloma (MM) and acute lymphoblastic leukemia (ALL). Yet, prolonged GC use is hampered by deleterious GC-related side effects and the emergence of GC resistance. To tackle and overcome these GC-related problems, the applicability of selective glucocorticoid receptor agonists and modulators was studied, in search of fewer side-effects and at least equal therapeutic efficacy as classic GCs. Compound A (CpdA) is a prototypical example of such a selective glucocorticoid receptor modulator and does not support GR-mediated transactivation. Here, we examined whether the combination of CpdA with the classic GC dexamethasone (Dex) may improve GC responsiveness of MM and ALL cell lines. We find that the combination of Dex and CpdA does not substantially enhance GC-mediated cell killing. In line, several apoptosis hallmarks, such as caspase 3/7 activity, PARP cleavage and the levels of cleaved-caspase 3 remain unchanged upon combining Dex with CpdA. Moreover, we monitor no additional inhibition of cell proliferation and the homologous downregulation of GR is not counteracted by the combination of Dex and CpdA. In addition, CpdA is unable to modulate Dex-liganded GR transactivation and transrepression, yet, Dex-mediated transrepression is also aberrant in these lymphoid cell lines. Together, transrepression-favoring compounds, alone or combined with GCs, do not seem a valid strategy in the treatment of lymphoid malignancies.

Coregulator profiling of the glucocorticoid receptor in lymphoid malignancies.

Coregulators cooperate with nuclear receptors, such as the glucocorticoid receptor (GR), to enhance or repress transcription. These regulatory proteins are implicated in cancer, yet, their role in lymphoid malignancies, including multiple myeloma (MM) and acute lymphoblastic leukemia (ALL), is largely unknown. Here, we report the use and extension of the microarray assay for real-time nuclear receptor coregulator interactions (MARCoNI) technology to detect coregulator associations with endogenous GR in cell lysates. We use MARCoNI to determine the GR coregulator profile of glucocorticoid-sensitive (MM and ALL) and glucocorticoid-resistant (ALL) cells, and identify common and unique coregulators for different cell line comparisons. Overall, we identify SRC-1/2/3, PGC-1α, RIP140 and DAX-1 as the strongest interacting coregulators of GR in MM and ALL cells and show that the interaction strength does not correlate with GR protein levels. Lastly, as a step towards patient samples, we determine the GR coregulator profile of peripheral blood mononuclear cells. We profile the interactions between GR and coregulators in MM and ALL cells and suggest to further explore the GR coregulator profile in hematological patient samples.

Selective glucocorticoid receptor-activating adjuvant therapy in cancer treatments.

Although adverse effects and glucocorticoid resistance cripple their chronic use, glucocorticoids form the mainstay therapy for acute and chronic inflammatory disorders, and play an important role in treatment protocols of both lymphoid malignancies and as adjuvant to stimulate therapy tolerability in various solid tumors. Glucocorticoid binding to their designate glucocorticoid receptor (GR), sets off a plethora of cell-specific events including therapeutically desirable effects, such as cell death, as well as undesirable effects, including chemotherapy resistance, systemic side effects and glucocorticoid resistance. In this context, selective GR agonists and modulators (SEGRAMs) with a more restricted GR activity profile have been developed, holding promise for further clinical development in anti-inflammatory and potentially in cancer therapies. Thus far, the research into the prospective benefits of selective GR modulators in cancer therapy limped behind. Our review discusses how selective GR agonists and modulators could improve the therapy regimens for lymphoid malignancies, prostate or breast cancer. We summarize our current knowledge and look forward to where the field should move to in the future. Altogether, our review clarifies novel therapeutic perspectives in cancer modulation via selective GR targeting.

Characterization of the interaction between the dopamine D4 receptor, KLHL12 and ß-arrestins.

Dopamine receptors are G protein-coupled receptors involved in regulation of cognition, learning, movement and endocrine signaling. The action of G protein-coupled receptors is highly regulated by multifunctional proteins, such as ß-arrestins which can control receptor desensitization, ubiquitination and signaling. Previously, we have reported that ß-arrestin 2 interacts with KLHL12, a BTB-Kelch protein which functions as an adaptor in a Cullin3-based E3 ligase complex and promotes ubiquitination of the dopamine D4 receptor. Here, we have investigated the molecular basis of the interaction between KLHL12 and ß-arrestins and questioned its functional relevance. Our data demonstrate that ß-arrestin 1 and ß-arrestin 2 bind constitutively to the most common dopamine D4 receptor polymorphic variants and to KLHL12 and that all three proteins can interact within a single macromolecular complex. Surprisingly, stimulation of the receptor has no influence on the association between these proteins or their cellular distribution. We found that Cullin3 also interacts with both ß-arrestins but has no influence on their ubiquitination. Knockout of one of the two ß-arrestins hampers neither interaction between the dopamine D4 receptor and KLHL12, nor ubiquitination of the receptor. Finally, our results indicate that p44/42 MAPK phosphorylation, the signaling pathway which is often regulated by ß-arrestins is not influenced by KLHL12, but seems to be exclusively mediated by Gαi protein upon dopamine D4 receptor stimulation.

Cofactor profiling of the glucocorticoid receptor from a cellular environment.

The Microarray Assay for Realtime Coregulator-Nuclear receptor Interaction (MARCoNI) technology allows the identification of nuclear receptor-coregulator interactions via flow-through microarrays. As such, differences in the coregulator profile between distinct nuclear receptors or of a single receptor in agonist or antagonist mode can be investigated, even in a single run. In this chapter, the method how to perform these peptide microarrays with cell lysates containing the overexpressed glucocorticoid receptor is described, as well as the influence of assay parameters, variations to the protocol, and data analysis.

Compound A, a selective glucocorticoid receptor modulator, enhances heat shock protein Hsp70 gene promoter activation.

Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-κB-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1), upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated IκBα degradation and NF-κB p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA's anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells.

Mitogen- and stress-activated protein kinase 1 MSK1 regulates glucocorticoid response element promoter activity in a glucocorticoid concentration-dependent manner.

The glucocorticoid receptor is a nuclear receptor, and can be activated by glucocorticoid ligands. Mitogen- and stress-activated protein kinase (MSK1), when activated by p38 and ERK mitogen-activated protein kinases (MAPKs), plays a major role in chromatin relaxation via phosphorylation of histone H3 S10. The glucocorticoid receptor can target MSK1 as part of its anti-inflammatory mechanism. Here, we studied the converse mechanism, i.e. the impact of MSK1 on glucocorticoid receptor-mediated transactivation. Upstream MSK1-activating kinases concentration-dependently enhanced glucocorticoid response element (GRE)-regulated promoter activity. Correspondingly, MSK1 inhibition, via H89, or combined p38 and ERK MAPK inhibition, via SB203580 and U0126, diminished maximally stimulated GRE-regulated promoter activity using high concentrations of glucocorticoids. Concomitantly, the combination of these agents does not seem to alter site-specific phosphorylations of murine glucocorticoid receptor S212 or S220. Paradoxically, we reveal that a sub-maximally activated GRE-mediated promoter activity, by using lower concentrations of glucocorticoids, is consistently enhanced by H89 or a combination of SB203580 and U0126, irrespective of the GRE promoter context. Furthermore, we show that the glucocorticoid-induced nucleocytoplasmic translocation of MSK1 occurs in a glucocorticoid concentration-dependent manner. The observed glucocorticoid concentration-dependent effect of MSK1 or MAPK inhibition on glucocorticoid receptor transactivation warrants further research into the applicability of combined glucocorticoid and kinase inhibitor strategies for anti-inflammatory purposes.

The Fukui matrix: a simple approach to the analysis of the Fukui function and its positive character.

The Fukui matrix is introduced as the derivative of the one-electron reduced density matrix with respect to a change in the number of electrons under constant external potential. The Fukui matrix extends the Fukui function concept: the diagonal of the Fukui matrix is the Fukui function. Diagonalizing the Fukui matrix gives a set of eigenvectors, the Fukui orbitals, and accompanying eigenvalues. At the level of theory used, there is always one dominant eigenvector, with an eigenvalue equal to 1. The remaining eigenvalues are either zero or come in pairs with eigenvalues of the same magnitude but opposite sign. Analysis of the frontier molecular orbital coefficient in the eigenvector with eigenvalue 1 gives information on the quality of the frontier molecular orbital picture. The occurrence of negative Fukui functions can be easily interpreted in terms of the nodal character of the dominant eigenvector versus the characteristics of the remaining eigenvectors and eigenvalues.