Antagonistic effects of finerenone and spironolactone on the aldosterone-regulated transcriptome of human kidney cells.

Aldosterone, the main mineralocorticoid hormone in humans, plays a pivotal role in the control of water and salt reabsorption via activation of the mineralocorticoid receptor (MR). Alterations in MR signaling pathway lead to renal dysfunction, including chronic kidney disease and renal fibrosis, that can be prevented or treated with mineralocorticoid receptor antagonists (MRAs). Here, we used RNA-Sequencing to analyze effects of two MRAs, spironolactone and finerenone, on the aldosterone-induced transcriptome of a human renal cell line stably expressing the MR. Bioinformatics analysis of the data set reveals the identity of hundreds of genes induced or repressed by aldosterone. Their regulation is modulated in a time-dependent manner and, for the induced genes, depends on the aldosterone-driven direct binding of the MR onto its genomic targets that we have previously characterized. Although both MRAs block aldosterone-induced as well as aldosterone-repressed genes qualitatively similarly, finerenone has a quantitatively more efficient antagonism on some aldosterone-induced genes. Our data provide the first complete transcriptome for aldosterone on a human renal cell line and identifies pro-inflammatory markers (IL6, IL11, CCL7, and CXCL8) as aldosterone-repressed genes.

Chemoprobe-based assays of histone lysine demethylase 1A target occupation enable in vivo pharmacokinetics and pharmacodynamics studies of KDM1A inhibitors.

Screening of cellular activity for inhibitors of histone lysine modifiers is most frequently performed indirectly by analyzing changes in the total levels of histone marks targeted by lysine methylases/demethylases. However, inhibition of histone lysine modifiers often leads to local rather than total changes in histone marks. Also, because histone modifications can be modulated by more than one cellular enzyme, it is not always clear whether changes in histone marks are a direct or indirect consequence of the inhibitor treatment applied. Direct assessment of target occupation can provide a useful tool to quantify the fraction of an epigenetic modifier that is bound to a pharmacological inhibitor (target engagement). Here, we developed and used a novel chemoprobe-based immunoassay to quantify target engagement in cells. Quantification of the fraction of free KDM1A was made possible, in an immune-based assay, by coupling a biotinylated chemoprobe to a warhead capable of selectively and irreversibly binding to the free active form of KDM1A. The results obtained confirmed that this approach is able to determine the degree of target engagement in a dose-dependent manner. Furthermore, the assay can be also used on tissue extracts to analyze the in vivo pharmacokinetics and pharmacodynamics relationship of KDM1A inhibitors, as has been exemplified with ORY-1001 (iadademstat), a potent and irreversible inhibitor of KDM1A. The principle of this assay may be applied to other targets, and the KDM1A probe may be employed in chemoproteomic analyses.

Epidermal glucocorticoid and mineralocorticoid receptors act cooperatively to regulate epidermal development and counteract skin inflammation.

Endogenous and synthetic glucocorticoids (GCs) regulate epidermal development and combat skin inflammatory diseases. GC actions can be mediated through the GC receptor (GR) and/or the mineralocorticoid receptor (MR), highly homologous ligand-activated transcription factors. While the role of GR as a potent anti-inflammatory mediator is well known, that of MR is not as clear, nor is whether these receptors cooperate or antagonize each other in the epidermis. To address this, we generated mice with epidermal-specific loss of both receptors (double knockout, DKO), and analyzed the phenotypical and functional consequences relative to single KOs or controls (CO). At birth, DKO epidermis displayed a phenotype of defective differentiation and inflammation, which was more severe than in either single KO, featuring neutrophil-containing infiltrates, and gene dysregulation characteristic of human psoriatic lesions. This phenotype resolved spontaneously. However, in adulthood, single or combined loss of GC receptors increased susceptibility to inflammation and hyperproliferation triggered by phorbol ester which, different to CO, was not effectively counteracted by GC treatment. Also, DKOs were more susceptible to imiquimod-induced psoriasis than CO showing severe defective epidermal differentiation and microabcesses while single KOs showed an intermediate response. Immortalized DKO keratinocytes featured increased proliferation kinetics and reduced cell size, a unique phenotype relative to single KO cells. The lack of GR and MR in keratinocytes, individual or combined, caused constitutive increases in p38 and ERK activities, which were partially reversed upon reinsertion of receptors into DKO cells. DKO keratinocytes also displayed significant increases in AP-1 and NF-κB transcriptional activities, which were partially rescued by ERK and p38 inhibition, respectively. Reinsertion of GR and MR in DKO keratinocytes resulted in physical and cooperative functional interactions that restored the transcriptional response to GCs. In conclusion, our data have revealed that epidermal GR and MR act cooperatively to regulate epidermal development and counteract skin inflammation.

Dos hermanos con leucemia linfoblástica aguda: ¿casualidad o herencia? / Two siblings with acute lymphoblastic leukaemia: chance or genetics?

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Overexpression of Glucocorticoid-induced Leucine Zipper (GILZ) increases susceptibility to Imiquimod-induced psoriasis and involves cutaneous activation of TGF-ß1.

Psoriasis vulgaris is a chronic inflammatory skin disease affecting millions of people. Its pathophysiology is complex and involves a skin compartment with epidermal and immune cells which produce cytokines, e.g. belonging to the IL-23-Th17-cell axis. Glucocorticoids (GCs) are the most common therapeutics used in cutaneous inflammatory disorders and GC-induced leucine zipper (GILZ) has emerged as a mediator of GCs due to its anti-inflammatory actions, theoretically lacking GC side-effects. We evaluated whether GILZ may provide a better therapeutic index in comparison to GCs during the onset and progression of psoriasis by generating and characterizing a mouse model with generalized overexpression of this protein (GILZ-Tg mice) and the imiquimod (IMQ) psoriasis model. Unexpectedly, in GILZ-Tg mice, the severity of IMQ-induced psoriasis-like skin lesions as well as induction of cytokines commonly up-regulated in human psoriasis (Il-17, Il-22, Il-23, Il-6, S100a8/a9, and Stat3) was significantly more pronounced relative to GILZ-Wt mice. The increased susceptibility to IMQ-induced psoriasis of GILZ-Tg mice was significantly associated with skin-specific over-activation of TGF-ß1-mediated signaling via SMAD2/3. Our findings demonstrate that GILZ may behave as pro-inflammatory protein in certain tissues and that, similar to prolonged GC therapy, GILZ as an alternative treatment for psoriasis may also have adverse effects.

Epidermal Mineralocorticoid Receptor Plays Beneficial and Adverse Effects in Skin and Mediates Glucocorticoid Responses.

Glucocorticoids (GCs) regulate skin homeostasis and combat cutaneous inflammatory diseases; however, adverse effects of chronic GC treatments limit their therapeutic use. GCs bind and activate the GC receptor and the mineralocorticoid receptor (MR), transcription factors that recognize identical hormone responsive elements. Whether epidermal MR mediates beneficial or deleterious GC effects is of great interest for improving GC-based skin therapies. MR epidermal knockout mice exhibited increased keratinocyte proliferation and differentiation and showed resistance to GC-induced epidermal thinning. However, crucially, loss of epidermal MR rendered mice more sensitive to inflammatory stimuli and skin damage. MR epidermal knockout mice showed increased susceptibility to phorbol 12-myristate 13-acetate-induced inflammation with higher cytokine induction. Likewise, cultured MR epidermal knockout keratinocytes had increased phorbol 12-myristate 13-acetate-induced NF-κB activation, highlighting an anti-inflammatory function for MR. GC-induced transcription was reduced in MR epidermal knockout keratinocytes, at least partially due to decreased recruitment of GC receptor to hormone responsive element-containing sequences. Our results support a role for epidermal MR in adult skin homeostasis and demonstrate nonredundant roles for MR and GC receptor in mediating GC actions.

Advances in the development of histone lysine demethylase inhibitors.

The covalent modification of histones is closely associated with regulation of gene transcription. Chromatin modifications have been suggested to represent an epigenetic code that is dynamically 'written' and 'erased' by specialized proteins, and 'read', or interpreted, by proteins that translate the code into gene expression changes. Initially thought to be an irreversible process, histone methylation is now known to be reversed by demethylases, FAD dependent amineoxidases and by iron(II)-alpha-ketoglutarate dependent deoxygenases of the Jumonji family. Altered histone demethylase activities have been associated with human disease, including cancer. The first wave of novel investigational drugs directed against KDM1A has recently entered the clinic, and the first specific inhibitor targeting a Jumonji KDM is advancing in preclinical regulatory studies.

Glucocorticoid receptor and Klf4 co-regulate anti-inflammatory genes in keratinocytes.

The glucocorticoid (GC) receptor (GR) and Kruppel-like factor Klf4 are transcription factors that play major roles in skin homeostasis. However, whether these transcription factors cooperate in binding genomic regulatory regions in epidermal keratinocytes was not known. Here, we show that in dexamethasone-treated keratinocytes GR and Klf4 are recruited to genomic regions containing adjacent GR and KLF binding motifs to control transcription of the anti-inflammatory genes Tsc22d3 and Zfp36. GR- and Klf4 loss of function experiments showed total GR but partial Klf4 requirement for full gene induction in response to dexamethasone. In wild type keratinocytes induced to differentiate, GR and Klf4 protein expression increased concomitant with Tsc22d3 and Zfp36 up-regulation. In contrast, GR-deficient cells failed to differentiate or fully induce Klf4, Tsc22d3 and Zfp36 correlating with increased expression of the epithelium-specific Trp63, a known transcriptional repressor of Klf4. The identified transcriptional cooperation between GR and Klf4 may determine cell-type specific regulation and have implications for developing therapies for skin diseases.

Differences in the signaling pathways of α(1A)- and α(1B)-adrenoceptors are related to different endosomal targeting.

AIMS: To compare the constitutive and agonist-dependent endosomal trafficking of α(1A)- and α(1B)-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype. METHODS: Using CypHer5 technology and VSV-G epitope tagged α(1A)- and α(1B)-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot). RESULTS AND CONCLUSIONS: Constitutive as well as agonist-induced trafficking of α(1A) and α(1B) ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α(1A)-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to ß-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α(1B)-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-ß-cyclodextrin.

Genetic predisposition to early recurrence in clinically localized prostate cancer.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Currently available nomograms to predict preoperative risk of early biochemical recurrence (EBCR) after radical prostatectomy are solely based on classic clinicopathological variables. Despite providing useful predictions, these models are not perfect. Indeed, most researchers agree that nomograms can be improved by incorporating novel biomarkers. In the last few years, several single nucleotide polymorphisms (SNPs) have been associated with prostate cancer, but little is known about their impact on disease recurrence. We have identified four SNPs associated with EBCR. The addition of SNPs to classic nomograms resulted in a significant improvement in terms of discrimination and calibration. The new nomogram, which combines clinicopathological and genetic variables, will help to improve prediction of prostate cancer recurrence. OBJECTIVES: To evaluate genetic susceptibility to early biochemical recurrence (EBCR) after radical prostatectomy (RP), as a prognostic factor for early systemic dissemination. To build a preoperative nomogram to predict EBCR combining genetic and clinicopathological factors. PATIENTS AND METHODS: We evaluated 670 patients from six University Hospitals who underwent RP for clinically localized prostate cancer (PCa), and were followed-up for at least 5 years or until biochemical recurrence. EBCR was defined as a level prostate-specific antigen >0.4 ng/mL within 1 year of RP; preoperative variables studied were: age, prostate-specific antigen, clinical stage, biopsy Gleason score, and the genotype of 83 PCa-related single nucleotide polymorphisms (SNPs). Univariate allele association tests and multivariate logistic regression were used to generate predictive models for EBCR, with clinicopathological factors and adding SNPs. We internally validated the models by bootstrapping and compared their accuracy using the area under the curve (AUC), net reclassification improvement, integrated discrimination improvement, calibration plots and Vickers' decision curves. RESULTS: Four common SNPs at KLK3, KLK2, SULT1A1 and BGLAP genes were independently associated with EBCR. A significant increase in AUC was observed when SNPs were added to the model: AUC (95% confidence interval) 0.728 (0.674-0.784) vs 0.763 (0.708-0.817). Net reclassification improvement showed a significant increase in probability for events of 60.7% and a decrease for non-events of 63.5%. Integrated discrimination improvement and decision curves confirmed the superiority of the new model. CONCLUSIONS: Four SNPs associated with EBCR significantly improved the accuracy of clinicopathological factors. We present a nomogram for preoperative prediction of EBCR after RP.

New water-soluble sulfonylphosphoramidic acid derivatives of the COX-2 selective inhibitor cimicoxib. A novel approach to sulfonamide prodrugs.

The synthesis and pharmacological evaluation of new water-soluble phosphoramidate derivatives of the COX-2 selective inhibitor cimicoxib (4) are described. The sulfonylphosphoramidic acid derivative 10 was converted to 4 in human plasma and showed excellent in vivo activity in the rat carrageenan-edema test. Pharmacokinetic evaluation in dogs indicated that 10 behaved as a prodrug, immediately converting to 4 and giving an identical profile to that of the parent compound. These results represent the first description of phosphoramidic acids as prodrugs for the sulfonamido group. Compound 10 also exhibited an important and sustained analgesic effect in the hyperalgesia test in rats and a high aqueous solubility at pH higher than 7. This profile led to the selection of 10 (UR-14048) for further development in the parenteral treatment of acute pain.

Synthesis and structure-activity relationship of a new series of COX-2 selective inhibitors: 1,5-diarylimidazoles.

The synthesis and the pharmacological activity of a series of 1,5-diarylimidazoles developed as potent and selective cyclooxygenase-2 (COX-2) inhibitors are described. The new compounds were evaluated both in vitro (COX-1 and COX-2 inhibition in human whole blood) and in vivo (carrageenan-induced paw edema, air-pouch, and hyperalgesia tests). Modification of all the positions of two regioisomeric imidazole cores led to the identification of 4-[4-chloro-5-(3-fluoro-4-methoxyphenyl)imidazol-1-yl]benzenesulfonamide (UR-8880, 51f) as the best candidate, which is now undergoing Phase I clinical trials.