Novel Vitamin D3 Hydroxymetabolites Require Involvement of the Vitamin D Receptor or Retinoic Acid-Related Orphan Receptors for Their Antifibrogenic Activities in Human Fibroblasts.

We investigated multiple signaling pathways activated by CYP11A1-derived vitamin D3 hydroxymetabolites in human skin fibroblasts by assessing the actions of these molecules on their cognate receptors and by investigating the role of CYP27B1 in their biological activities. The actions of 20(OH)D3, 20,23(OH)2D3, 1,20(OH)2D3 and 1,20,23(OH)3D3 were compared to those of classical 1,25(OH)2D3. This was undertaken using wild type (WT) fibroblasts, as well as cells with VDR, RORs, or CYP27B1 genes knocked down with siRNA. Vitamin D3 hydroxymetabolites had an inhibitory effect on the proliferation of WT cells, but this effect was abrogated in cells with silenced VDR or RORs. The collagen expression by WT cells was reduced upon secosteroid treatment. This effect was reversed in cells where VDR or RORs were knocked down where the inhibition of collagen production and the expression of anti-fibrotic genes in response to the hydroxymetabolites was abrogated, along with ablation of their anti-inflammatory action. The knockdown of CYP27B1 did not change the effect of either 20(OH)D3 or 20,23(OH)2D3, indicating that their actions are independent of 1α-hydroxylation. In conclusion, the expression of the VDR and/or RORα/γ receptors in fibroblasts is necessary for the inhibition of both the proliferation and fibrogenic activity of hydroxymetabolites of vitamin D3, while CYP27B1 is not required.

GLIS3 expression in the thyroid gland in relation to TSH signaling and regulation of gene expression.

Loss of GLI-Similar 3 (GLIS3) function in mice and humans causes congenital hypothyroidism (CH). In this study, we demonstrate that GLIS3 protein is first detectable at E15.5 of murine thyroid development, a time at which GLIS3 target genes, such as Slc5a5 (Nis), become expressed. This, together with observations showing that ubiquitous Glis3KO mice do not display major changes in prenatal thyroid gland morphology, indicated that CH in Glis3KO mice is due to dyshormonogenesis rather than thyroid dysgenesis. Analysis of GLIS3 in postnatal thyroid suggested a link between GLIS3 protein expression and blood TSH levels. This was supported by data showing that treatment with TSH, cAMP, or adenylyl cyclase activators or expression of constitutively active PKA enhanced GLIS3 protein stability and transcriptional activity, indicating that GLIS3 activity is regulated at least in part by TSH/TSHR-mediated activation of PKA. The TSH-dependent increase in GLIS3 transcriptional activity would be critical for the induction of GLIS3 target gene expression, including several thyroid hormone (TH) biosynthetic genes, in thyroid follicular cells of mice fed a low iodine diet (LID) when blood TSH levels are highly elevated. Like TH biosynthetic genes, the expression of cell cycle genes is suppressed in ubiquitous Glis3KO mice fed a LID; however, in thyroid-specific Glis3 knockout mice, the expression of cell cycle genes was not repressed, in contrast to TH biosynthetic genes. This indicated that the inhibition of cell cycle genes in ubiquitous Glis3KO mice is dependent on changes in gene expression in GLIS3 target tissues other than the thyroid.

Circadian regulation of Ca V 1.2 expression by RORα in the mouse heart.

Background: In addition to show autonomous beating rhythmicity, the physiological functions of the heart present daily periodic oscillations. Notably the ventricular repolarization itself varies throughout the circadian cycle which was mainly related to the periodic expression of K + channels. However, the involvement of the L-type Ca 2+ channel (Ca V 1.2 encoded by Cacna1c gene) in these circadian variations remains elusive. Methods: We used a transgenic mouse model (PCa-luc) that expresses the luciferase reporter under the control of the cardiac Cacna1c promoter and analyzed promoter activity by bioluminescent imaging, qPCR, immunoblot, Chromatin immunoprecipitation assay (ChIP) and Ca V 1.2 activity. Results: Under normal 12:12h light-dark cycle, we observed in vivo a biphasic diurnal variation of promoter activities peaking at 9 and 19.5 Zeitgeber time (ZT). This was associated with a periodicity of Cacna1c mRNA levels preceding 24-h oscillations of Ca V 1.2 protein levels in ventricle (with a 1.5 h phase shift) but not in atrial heart tissues. The periodicity of promoter activities and Ca V 1.2 proteins, which correlated with biphasic oscillations of L-type Ca 2+ current conductance, persisted in isolated ventricular cardiomyocytes from PCa-Luc mice over the course of the 24-h cycle, suggesting an endogenous cardiac circadian regulation. Comparison of 24-h temporal patterns of clock gene expressions in ventricles and atrial tissues of the same mice revealed conserved circadian oscillations of the core clock genes except for the retinoid-related orphan receptor α gene (RORα), which remained constant throughout the course of a day in atrial tissues. In vitro we found that RORα is recruited to two specific regions on the Cacna1c promoter and that incubation with specific RORα inhibitor disrupted 24-h oscillations of ventricular promoter activities and Ca V 1.2 protein levels. Similar results were observed for pore forming subunits of the K + transient outward currents, K V 4.2 and K V 4.3. Conclusions: These findings raise the possibility that the RORα-dependent rhythmic regulation of cardiac Ca V 1.2 and K V 4.2/4.3 throughout the daily cycle may play an important role in physiopathology of heart function.

Melatonin and Its Metabolites Can Serve as Agonists on the Aryl Hydrocarbon Receptor and Peroxisome Proliferator-Activated Receptor Gamma.

Melatonin is widely present in Nature. It has pleiotropic activities, in part mediated by interactions with high-affinity G-protein-coupled melatonin type 1 and 2 (MT1 and MT2) receptors or under extreme conditions, e.g., ischemia/reperfusion. In pharmacological concentrations, it is given to counteract the massive damage caused by MT1- and MT2-independent mechanisms. The aryl hydrocarbon receptor (AhR) is a perfect candidate for mediating the latter effects because melatonin has structural similarity to its natural ligands, including tryptophan metabolites and indolic compounds. Using a cell-based Human AhR Reporter Assay System, we demonstrated that melatonin and its indolic and kynuric metabolites act as agonists on the AhR with EC50's between 10-4 and 10-6 M. This was further validated via the stimulation of the transcriptional activation of the CYP1A1 promoter. Furthermore, melatonin and its metabolites stimulated AhR translocation from the cytoplasm to the nucleus in human keratinocytes, as demonstrated by ImageStream II cytometry and Western blot (WB) analyses of cytoplasmic and nuclear fractions of human keratinocytes. These functional analyses are supported by in silico analyses. We also investigated the peroxisome proliferator-activated receptor (PPAR)γ as a potential target for melatonin and metabolites bioregulation. The binding studies using a TR-TFRET kit to assay the interaction of the ligand with the ligand-binding domain (LBD) of the PPARγ showed agonistic activities of melatonin, 6-hydroxymelatonin and N-acetyl-N-formyl-5-methoxykynuramine with EC50's in the 10-4 M range showing significantly lower affinities that those of rosiglitazone, e.g., a 10-8 M range. These interactions were substantiated by stimulation of the luciferase activity of the construct containing PPARE by melatonin and its metabolites at 10-4 M. As confirmed by the functional assays, binding mode predictions using a homology model of the AhR and a crystal structure of the PPARγ suggest that melatonin and its metabolites, including 6-hydroxymelatonin, 5-methoxytryptamine and N-acetyl-N-formyl-5-methoxykynuramine, are excellent candidates to act on the AhR and PPARγ with docking scores comparable to their corresponding natural ligands. Melatonin and its metabolites were modeled into the same ligand-binding pockets (LBDs) as their natural ligands. Thus, functional assays supported by molecular modeling have shown that melatonin and its indolic and kynuric metabolites can act as agonists on the AhR and they can interact with the PPARγ at high concentrations. This provides a mechanistic explanation for previously reported cytoprotective actions of melatonin and its metabolites that require high local concentrations of the ligands to reduce cellular damage under elevated oxidative stress conditions. It also identifies these compounds as therapeutic agents to be used at pharmacological doses in the prevention or therapy of skin diseases.

International Union of Basic and Clinical Pharmacology CXIII: Nuclear Receptor Superfamily-Update 2023.

The NR superfamily comprises 48 transcription factors in humans that control a plethora of gene network programs involved in a wide range of physiologic processes. This review will summarize and discuss recent progress in NR biology and drug development derived from integrating various approaches, including biophysical techniques, structural studies, and translational investigation. We also highlight how defective NR signaling results in various diseases and disorders and how NRs can be targeted for therapeutic intervention via modulation via binding to synthetic lipophilic ligands. Furthermore, we also review recent studies that improved our understanding of NR structure and signaling. SIGNIFICANCE STATEMENT: Nuclear receptors (NRs) are ligand-regulated transcription factors that are critical regulators of myriad physiological processes. NRs serve as receptors for an array of drugs, and in this review, we provide an update on recent research into the roles of these drug targets.

Role of GLIS3 in thyroid development and in the regulation of gene expression in thyroid specific Glis3KO mice.

Loss of GLI-Similar 3 (GLIS3) function in mice and humans causes congenital hypothyroidism (CH). In this study, we demonstrate that GLIS3 protein is first detectable at E15.5 of murine thyroid development, a time when GLIS3 target genes, such as Slc5a5 (Nis), become also expressed. We further show that Glis3KO mice do not display any major changes in prenatal thyroid gland morphology indicating that CH in Glis3KO mice is due to dyshormonogenesis rather than thyroid dysgenesis. Analysis of thyroid-specific Glis3 knockout (Glis3-Pax8Cre) mice fed either a normal or low-iodine diet (ND or LID) revealed that, in contrast to ubiquitous Glis3KO mice, thyroid follicular cell proliferation and the expression of cell cycle genes were not repressed suggesting that the inhibition of thyroid follicular cell proliferation in ubiquitous Glis3KO mice is related to loss of GLIS3 function in other cell types. However, the expression of several thyroid hormone biosynthesis-, extracellular matrix (ECM)-, and inflammation-related genes was still suppressed in Glis3-Pax8Cre mice particularly under conditions of high blood levels of thyroid stimulating hormone (TSH). We further demonstrate that treatment with TSH, protein kinase A (PKA) or adenylyl cyclase activators or expression of constitutively active PKA enhances GLIS3 protein and activity, suggesting that GLIS3 transcriptional activity is regulated in part by TSH/TSHR-mediated activation of the PKA pathway. This mechanism of regulation provides an explanation for the dramatic increase in GLIS3 protein expression and the subsequent induction of GLIS3 target genes, including several thyroid hormone biosynthetic genes, in thyroid follicular cells of mice fed a LID.

Signal Response Evaluation Applied to Untargeted Mass Spectrometry Data to Improve Data Interpretability.

Feature finding is a common way to process untargeted mass spectrometry (MS) data to obtain a list of chemicals present in a sample. Most feature finding algorithms naïvely search for patterns of unique descriptors (e.g., m/z, retention time, and mobility) and provide a list of unannotated features. There is a need for solutions in processing untargeted MS data, independent of chemical or origin, to assess features based on measurement quality with the aim of improving interpretation. Here, we report the signal response evaluation as a method by which to assess the individual features observed in untargeted MS data. The basis of this method is the ubiquitous relationship between the amount and response in all MS measurements. Three different metrics with user-defined parameters can be used to assess the monotonic or linear relationship of each feature in a dilution series or multiple injection volumes. We demonstrate this approach in metabolomics data obtained from a uniform biological matrix (NIST SRM 1950) and a variable biological matrix (murine kidney tissue). The code is provided to facilitate implementation of this data processing method.

GLIS3 regulates transcription of thyroid hormone biosynthetic genes in coordination with other thyroid transcription factors.

BACKGROUND: Loss of the transcription factor GLI-Similar 3 (GLIS3) function causes congenital hypothyroidism (CH) in both humans and mice due to decreased expression of several thyroid hormone (TH) biosynthetic genes in thyroid follicular cells. Whether and to what extent, GLIS3 regulates thyroid gene transcription in coordination with other thyroid transcriptional factors (TFs), such as PAX8, NKX2.1 and FOXE1, is poorly understood. METHODS: PAX8, NKX2.1, and FOXE1 ChIP-Seq analysis with mouse thyroid glands and rat thyrocyte PCCl3 cells was performed and compared to that of GLIS3 to analyze the co-regulation of gene transcription in thyroid follicular cells by these TFs. RESULTS: Analysis of the PAX8, NKX2.1, and FOXE1 cistromes identified extensive overlaps between these TF binding loci and those of GLIS3 indicating that GLIS3 shares many of the same regulatory regions with PAX8, NKX2.1, and FOXE1, particularly in genes associated with TH biosynthesis, induced by thyroid stimulating hormone (TSH), and suppressed in Glis3KO thyroid glands, including Slc5a5 (Nis), Slc26a4, Cdh16, and Adm2. ChIP-QPCR analysis showed that loss of GLIS3 did not significantly affect PAX8 or NKX2.1 binding and did not cause major alterations in H3K4me3 and H3K27me3 epigenetic signals. CONCLUSIONS: Our study indicates that GLIS3 regulates transcription of TH biosynthetic and TSH-inducible genes in thyroid follicular cells in coordination with PAX8, NKX2.1, and FOXE1 by binding within the same regulatory hub. GLIS3 does not cause major changes in chromatin structure at these common regulatory regions. GLIS3 may induce transcriptional activation by enhancing the interaction of these regulatory regions with other enhancers and/or RNA Polymerase II (Pol II) complexes.

Metabolic activation of tachysterol3 to biologically active hydroxyderivatives that act on VDR, AhR, LXRs, and PPARγ receptors.

CYP11A1 and CYP27A1 hydroxylate tachysterol3 , a photoproduct of previtamin D3 , producing 20S-hydroxytachysterol3 [20S(OH)T3 ] and 25(OH)T3 , respectively. Both metabolites were detected in the human epidermis and serum. Tachysterol3 was also detected in human serum at a concentration of 7.3 ± 2.5 ng/ml. 20S(OH)T3 and 25(OH)T3 inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti-oxidative genes in keratinocytes in a similar manner to 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ]. They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR coupled GFP from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay system revealed marked activation of AhR by 20S(OH)T3 , a smaller effect by 25(OH)T3 , and a minimal effect for their precursor, tachysterol3 . Tachysterol3 hydroxyderivatives showed high-affinity binding to the ligan-binding domain (LBD) of the liver X receptor (LXR) α and ß, and the peroxisome proliferator-activated receptor γ (PPARγ) in LanthaScreen TR-FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs, and PPARγ revealed high docking scores for 20S(OH)T3 and 25(OH)T3 , comparable to their natural ligands. The scores for the non-genomic-binding site of the VDR were very low indicating a lack of interaction with tachysterol3 ligands. Our identification of endogenous production of 20S(OH)T3 and 25(OH)T3 that are biologically active and interact with VDR, AhR, LXRs, and PPARγ, provides a new understanding of the biological function of tachysterol3 .

CYP11A1­derived vitamin D hydroxyderivatives as candidates for therapy of basal and squamous cell carcinomas.

Hydroxyderivatives of vitamin D3, including classical 1,25(OH)2D3 and novel CYP11A1­derived hydroxyderivatives, exert their biological activity by acting as agonists on the vitamin D receptor (VDR) and inverse agonists on retinoid­related orphan receptors (ROR)α and γ. The anticancer activities of CYP11A1­derived hydroxyderivatives were tested using cell biology, tumor biology and molecular biology methods in human A431 and SCC13 squamous (SCC)­ and murine ASZ001 basal (BCC)­cell carcinomas, in comparison with classical 1,25(OH)2D3. Vitamin D3­hydroxyderivatives with or without a C1α(OH) inhibited cell proliferation in a dose­dependent manner. While all the compounds tested had similar effects on spheroid formation by A431 and SCC13 cells, those with a C1α(OH) group were more potent in inhibiting colony and spheroid formation in the BCC line. Potent anti­tumorigenic activity against the BCC line was exerted by 1,25(OH)2D3, 1,20(OH)2D3, 1,20,23(OH)3D3, 1,20,24(OH)3D3, 1,20,25(OH)3D3 and 1,20,26(OH)3D3, with smaller effects seen for 25(OH)D3, 20(OH)D3 and 20,23(OH)2D3. 1,25(OH)2D3, 1,20(OH)2D3 and 20(OH)D3 inhibited the expression of GLI1 and ß­catenin in ASZ001 cells. In A431 cells, these compounds also decreased the expression of GLI1 and stimulated involucrin expression. VDR, RORγ, RORα and CYP27B1 were detected in A431, SCC13 and ASZ001 lines, however, with different expression patterns. Immunohistochemistry performed on human skin with SCC and BCC showed nuclear expression of all three of these receptors, as well as megalin (transmembrane receptor for vitamin D­binding protein), the level of which was dependent on the type of cancer and antigen tested in comparison with normal epidermis. Classical and CYP11A1­derived vitamin D3­derivatives exhibited anticancer­activities on skin cancer cell lines and inhibited GLI1 and ß­catenin signaling in a manner that was dependent on the position of hydroxyl groups. The observed expression of VDR, RORγ, RORα and megalin in human SCC and BCC suggested that they might provide targets for endogenously produced or exogenously applied vitamin D hydroxyderivatives and provide excellent candidates for anti­cancer therapy.

GLIS1-3: Links to Primary Cilium, Reprogramming, Stem Cell Renewal, and Disease.

The GLI-Similar 1-3 (GLIS1-3) genes, in addition to encoding GLIS1-3 Krüppel-like zinc finger transcription factors, also generate circular GLIS (circGLIS) RNAs. GLIS1-3 regulate gene transcription by binding to GLIS binding sites in target genes, whereas circGLIS RNAs largely act as miRNA sponges. GLIS1-3 play a critical role in the regulation of many biological processes and have been implicated in various pathologies. GLIS protein activities appear to be regulated by primary cilium-dependent and -independent signaling pathways that via post-translational modifications may cause changes in the subcellular localization, proteolytic processing, and protein interactions. These modifications can affect the transcriptional activity of GLIS proteins and, consequently, the biological functions they regulate as well as their roles in disease. Recent studies have implicated GLIS1-3 proteins and circGLIS RNAs in the regulation of stemness, self-renewal, epithelial-mesenchymal transition (EMT), cell reprogramming, lineage determination, and differentiation. These biological processes are interconnected and play a critical role in embryonic development, tissue homeostasis, and cell plasticity. Dysregulation of these processes are part of many pathologies. This review provides an update on our current knowledge of the roles GLIS proteins and circGLIS RNAs in the control of these biological processes in relation to their regulation of normal physiological functions and disease.

Activation of retinoic acid-related orphan receptor γ(t) by parabens and benzophenone UV-filters.

Retinoic acid-related orphan receptor γt (RORγt) regulates immune responses and its impaired function contributes to inflammatory and autoimmune diseases and may promote skin cancer. Synthetic inverse RORγt agonists block the production of Th17-associated cytokines including interleukin (IL)-17A and IL-22 and are under investigation for treatment of such pathologies. Unintentional RORγt activation in skin, following exposure to environmental chemicals, may promote inflammatory skin disease. Parabens and UV-filters, frequently used as additives in cosmetics and body care products, are intensively inspected for endocrine disrupting properties. This study assessed whether such compounds can interfere with RORγ activity using a previously established tetracycline-inducible reporter gene assay in CHO cells. These transactivation experiments revealed hexylparaben, benzylparaben and benzophenone-10 as RORγ agonists (EC50 values: 144 ± 97 nM, 3.39 ± 1.74 µM and 1.67 ± 1.04 µM, respectively), and they could restore RORγ activity after suppression by an inverse agonist. Furthermore, they enhanced RORγt-dependent transcription of the pro-inflammatory IL-17A and/or IL-22 genes in the murine T-cell model EL4. Virtual screening of a cosmetics database for structurally similar chemicals and in vitro testing of the most promising hits revealed benzylbenzoate, benzylsalicylate and 4-methylphenylbenzoate as RORγ agonists (low micromolar EC50 values). Moreover, an analysis of mixtures of the newly identified RORγ agonists suggested additive effects. This study presents novel RORγ(t) agonistic structural scaffolds. By activating RORγ(t) the identified parabens and UV-filters may potentially aggravate pathophysiological conditions, especially skin diseases where highest exposure of such chemicals can be expected. Follow-up studies should assess whether such compounds, either alone or as mixtures, can reach relevant concentrations in tissues and target cells to activate RORγ(t) in vivo.

Chemical synthesis, biological activities and action on nuclear receptors of 20S(OH)D3, 20S,25(OH)2D3, 20S,23S(OH)2D3 and 20S,23R(OH)2D3.

New and more efficient routes of chemical synthesis of vitamin D3 (D3) hydroxy (OH) metabolites, including 20S(OH)D3, 20S,23S(OH)2D3 and 20S,25(OH)2D3, that are endogenously produced in the human body by CYP11A1, and of 20S,23R(OH)2D3 were established. The biological evaluation showed that these compounds exhibited similar properties to each other regarding inhibition of cell proliferation and induction of cell differentiation but with subtle and quantitative differences. They showed both overlapping and differential effects on T-cell immune activity. They also showed similar interactions with nuclear receptors with all secosteroids activating vitamin D, liver X, retinoic acid orphan and aryl hydrocarbon receptors in functional assays and also as indicated by molecular modeling. They functioned as substrates for CYP27B1 with enzymatic activity being the highest towards 20S,25(OH)2D3 and the lowest towards 20S(OH)D3. In conclusion, defining new routes for large scale synthesis of endogenously produced D3-hydroxy derivatives by pathways initiated by CYP11A1 opens an exciting era to analyze their common and differential activities in vivo, particularly on the immune system and inflammatory diseases.

GLIS3: A Critical Transcription Factor in Islet ß-Cell Generation.

Understanding of pancreatic islet biology has greatly increased over the past few decades based in part on an increased understanding of the transcription factors that guide this process. One such transcription factor that has been increasingly tied to both ß-cell development and the development of diabetes in humans is GLIS3. Genetic deletion of GLIS3 in mice and humans induces neonatal diabetes, while single nucleotide polymorphisms (SNPs) in GLIS3 have been associated with both Type 1 and Type 2 diabetes. As a significant progress has been made in understanding some of GLIS3's roles in pancreas development and diabetes, we sought to compare current knowledge on GLIS3 within the pancreas to that of other islet enriched transcription factors. While GLIS3 appears to regulate similar genes and pathways to other transcription factors, its unique roles in ß-cell development and maturation make it a key target for future studies and therapy.

An EMT-primary cilium-GLIS2 signaling axis regulates mammogenesis and claudin-low breast tumorigenesis.

The epithelial-mesenchymal transition (EMT) and primary ciliogenesis induce stem cell properties in basal mammary stem cells (MaSCs) to promote mammogenesis, but the underlying mechanisms remain incompletely understood. Here, we show that EMT transcription factors promote ciliogenesis upon entry into intermediate EMT states by activating ciliogenesis inducers, including FGFR1. The resulting primary cilia promote ubiquitination and inactivation of a transcriptional repressor, GLIS2, which localizes to the ciliary base. We show that GLIS2 inactivation promotes MaSC stemness, and GLIS2 is required for normal mammary gland development. Moreover, GLIS2 inactivation is required to induce the proliferative and tumorigenic capacities of the mammary tumor­initiating cells (MaTICs) of claudin-low breast cancers. Claudin-low breast tumors can be segregated from other breast tumor subtypes based on a GLIS2-dependent gene expression signature. Collectively, our findings establish molecular mechanisms by which EMT programs induce ciliogenesis to control MaSC and MaTIC stemness, mammary gland development, and claudin-low breast cancer formation.

GLIS1 regulates trabecular meshwork function and intraocular pressure and is associated with glaucoma in humans.

Chronically elevated intraocular pressure (IOP) is the major risk factor of primary open-angle glaucoma, a leading cause of blindness. Dysfunction of the trabecular meshwork (TM), which controls the outflow of aqueous humor (AqH) from the anterior chamber, is the major cause of elevated IOP. Here, we demonstrate that mice deficient in the Krüppel-like zinc finger transcriptional factor GLI-similar-1 (GLIS1) develop chronically elevated IOP. Magnetic resonance imaging and histopathological analysis reveal that deficiency in GLIS1 expression induces progressive degeneration of the TM, leading to inefficient AqH drainage from the anterior chamber and elevated IOP. Transcriptome and cistrome analyses identified several glaucoma- and extracellular matrix-associated genes as direct transcriptional targets of GLIS1. We also identified a significant association between GLIS1 variant rs941125 and glaucoma in humans (P = 4.73 × 10-6), further supporting a role for GLIS1 into glaucoma etiology. Our study identifies GLIS1 as a critical regulator of TM function and maintenance, AqH dynamics, and IOP.

Vitamin D and lumisterol derivatives can act on liver X receptors (LXRs).

The interactions of derivatives of lumisterol (L3) and vitamin D3 (D3) with liver X receptors (LXRs) were investigated. Molecular docking using crystal structures of the ligand binding domains (LBDs) of LXRα and ß revealed high docking scores for L3 and D3 hydroxymetabolites, similar to those of the natural ligands, predicting good binding to the receptor. RNA sequencing of murine dermal fibroblasts stimulated with D3-hydroxyderivatives revealed LXR as the second nuclear receptor pathway for several D3-hydroxyderivatives, including 1,25(OH)2D3. This was validated by their induction of genes downstream of LXR. L3 and D3-derivatives activated an LXR-response element (LXRE)-driven reporter in CHO cells and human keratinocytes, and by enhanced expression of LXR target genes. L3 and D3 derivatives showed high affinity binding to the LBD of the LXRα and ß in LanthaScreen TR-FRET LXRα and ß coactivator assays. The majority of metabolites functioned as LXRα/ß agonists; however, 1,20,25(OH)3D3, 1,25(OH)2D3, 1,20(OH)2D3 and 25(OH)D3 acted as inverse agonists of LXRα, but as agonists of LXRß. Molecular dynamics simulations for the selected compounds, including 1,25(OH)2D3, 1,20(OH)2D3, 25(OH)D3, 20(OH)D3, 20(OH)L3 and 20,22(OH)2L3, showed different but overlapping interactions with LXRs. Identification of D3 and L3 derivatives as ligands for LXRs suggests a new mechanism of action for these compounds.

Antifibrogenic Activities of CYP11A1-derived Vitamin D3-hydroxyderivatives Are Dependent on RORγ.

Previous studies showed that noncalcemic 20(OH)D3, a product of CYP11A1 action on vitamin D3, has antifibrotic activity in human dermal fibroblasts and in a bleomycin mouse model of scleroderma. In this study, we tested the role of retinoic acid-related orphan receptor γ (RORγ), which is expressed in skin, in the action of CYP11A1-derived secosteroids using murine fibroblasts isolated from the skin of wild-type (RORγ +/+), knockout (RORγ -/-), and heterozygote (RORγ +/-) mice. CYP11A1-derived 20(OH)D3, 20,23(OH)2D3, 1,20(OH)2D3, and 1,20,23(OH)3D3 inhibited proliferation of RORγ +/+ fibroblasts in a dose-dependent manner with a similar potency to 1,25(OH)2D3. Surprisingly, this effect was reversed in RORγ +/- and RORγ -/- fibroblasts, with the most pronounced stimulatory effect seen in RORγ -/- fibroblasts. All analogs tested inhibited TGF-ß1-induced collagen synthesis in RORγ +/+ fibroblasts and the expression of other fibrosis-related genes. This effect was curtailed or reversed in RORγ -/- fibroblasts. These results show that the antiproliferative and antifibrotic activities of the vitamin D hydroxy derivatives are dependent on a functional RORγ. The dramatic changes in the transcriptomes of fibroblasts of RORγ -/- versus wild-type mice following treatment with 20(OH)D3 or 1,20(OH)2D3 provide a molecular basis to explain, at least in part, the observed phenotypic differences.

Association among Vitamin D, Retinoic Acid-Related Orphan Receptors, and Vitamin D Hydroxyderivatives in Ovarian Cancer.

Vitamin D and its derivatives, acting via the vitamin D receptor (VDR) and retinoic acid-related orphan receptors γ and α (RORγ and RORα), show anticancer properties. Since pathological conditions are characterized by disturbances in the expression of these receptors, in this study, we investigated their expression in ovarian cancers (OCs), as well as explored the phenotypic effects of vitamin D hydroxyderivatives and RORγ/α agonists on OC cells. The VDR and RORγ showed both a nuclear and a cytoplasmic location, and their expression levels were found to be reduced in the primary and metastatic OCs in comparison to normal ovarian epithelium, as well as correlated to the tumor grade. This reduction in VDR and RORγ expression correlated with a shorter overall disease-free survival. VDR, RORγ, and RORα were also detected in SKOV-3 and OVCAR-3 cell lines with increased expression in the latter line. 20-Hydroxy-lumisterol3 (20(OH)L3) and synthetic RORα/RORγ agonist SR1078 inhibited proliferation only in the OVCAR-3 line, while 20-hydroxyvitamin-D3 (20(OH)D3) only inhibited SKOV-3 cell proliferation. 1,25(OH)2D3, 20(OH)L3, and SR1078, but not 20(OH)D3, inhibited spheroid formation in SKOV-3 cells. In summary, decreases in VDR, RORγ, and RORα expression correlated with an unfavorable outcome for OC, and compounds targeting these receptors had a context-dependent anti-tumor activity in vitro. We conclude that VDR and RORγ expression can be used in the diagnosis and prognosis of OC and suggest their ligands as potential candidates for OC therapy.