Nuclear Retinoic Acid Receptors Research Articles

Identification of the Natural Steroid Sapogenin Diosgenin as a Direct Dual-Specific RORα/γ Inverse Agonist.

The steroid sapogenin diosgenin is a well-known natural product with a plethora of described pharmacological activities including the amelioration of T helper 17 (Th17)-driven pathologies. However, the exact underlying mode of action of diosgenin leading to a dampened Th17 response is still largely unknown and specific molecular targets have yet to be identified. Here, we show that diosgenin acts as a direct ligand and inverse agonist of the nuclear receptor retinoic acid receptor (RAR)-related orphan receptor (ROR)α and RORγ, which are key transcription factors involved in Th17 cell differentiation and metabolism. IC50 values determined by luciferase reporter gene assays, employing constructs for either RORγ-Gal4 fusion proteins or full length receptors, were in the low micromolar range at around 2 µM. To highlight the functional consequences of this RORα/γ inverse agonism, we determined gene expression levels of important ROR target genes, i.e., IL-17A and glucose-6-phosphatase, in relevant cellular in vitro models of Jurkat T and HepG2 cells, respectively, by RT-qPCR (reverse transcription quantitative PCR). Thereby, it was shown that diosgenin leads to a dose-dependent decrease in target gene expressions consistent with its potent cellular ROR inverse agonistic activity. Additionally, in silico dockings of diosgenin to the ROR ligand-binding domain were performed to determine the underlying binding mode. Taken together, our results establish diosgenin as a novel, direct and dual-selective RORα/γ inverse agonist. This finding establishes a direct molecular target for diosgenin for the first time, which can further explain reported amendments in Th17-driven diseases by this compound.

Resistance of breast cancer cells to all-trans retinoic acid is associated with a decrease in the basal level of nuclear receptor <i>RARα</i> expression and induction of cytochrome <i>CYP26A1</i> and <i>CYP26B1</i> expression

Introduction. Retinoic acid (RA) is a key regulator of cell differentiation and a critical player in such systemic processes in the body as embryonic development, immune system cell maturation and functioning, tissue remodeling and several others. This compound displays an antitumor activity due to its ability to stimulate differentiation, induce apoptosis and inhibit proliferation of malignant cells. The rapid acquisition of resistance to RA and its analogues by solid tumor cells is one of the main problems limiting the widespread use of retinoids in the therapy of malignant neoplasms. The mechanisms of RA-resistance are still poorly understood.The study objective – assessment of the relationship between the basal expression level of the nuclear RARα receptor and the RA-induced expression of the cytochromes CYP26A1and CYP26B1 with the resistance of breast cancer cells to the action of all-trans-retinoic acid.Materials and methods. Cell lines were cultured, the sensitivity of breast cancer cells to the action of fully trans-retinoic acid, RNA isolation, reverse transcription reaction and real-time polymerase chain reaction were analyzed).Results. In present study, using an experimental model represented by 9 breast cancer cell lines with different level of sensitivity to RA, we showed that the expression of the RA nuclear receptor RARα, as well as the level of mRNA induction of CYP26A1 and CYP26B1 cytochromes in response to RA treatment correlate with RA-sensitivity.Conclusion. Thus, a decrease of RARα expression as well as the reduced ability to catabolize RA are factors associated with RA-resistance of breast cancer cells.

Abstract LB518: Osteochondromas respond and its development is inhibited by retinoic acid nuclear receptor gamma agonist treatment

Abstract Osteochondromas (OC) are cartilage-capped tumors that arise near growing physis and are considered the most common benign bone tumor in children. They can lead to skeletal deformity, pain, loss of motion and neurovascular compression. Occasionally, malignant degeneration can occur. OC is thought to arise by mysregulation of chondrogenesis or chondrocyte differentiation, leading to ectopic cartilage formation. OC can occur as a solitary or multiple OC (MO). MO can occur from a hereditary cancer syndrome in which EXT1 and EXT2 are the major causative genes. Currently, treatment is limited to surgical resection only. There are no available FDA-approved therapies for MO. Previous translational research suggested that retinoic acid nuclear receptor gamma agonist (RARγ) suppresses ectopic cartilage formation including OC in rodent models. These preclinical studies led to a clinical trial to study the efficacy and safety of Palovarotene, a RARγ agonist, for the systemic treatment of MO (NCT03442985). The clinical trial was terminated due to concerns of skeletal toxicity observed in pediatric patients. To overcome this adverse action, we should elucidate the molecular action of RARγ agonists on osteochondromas and refine the treatment regimen. The purposes of our study are to examine the mechanism in which RARγ elicits osteochondroma growth and to determine whether the responsiveness to RARγ agonists is different between OC and growth plate. Treatment of RARγ agonists on mouse chondrocytes stimulated cell death induced by hydrogen peroxide (H2O2). In addition, human OC explants treated with RARγ agonists contained more apoptotic cells than vehicle-treated OC explants. OCs were induced in AcanCreER; Ext1e2neofl/e2neofl MO mouse model via tamoxifen induction at P5. After two weeks, mice were treated with Palovarotene (1.76 mg/Kg) or corn oil, and 24hrs after treatment, forelimbs were harvested. In-situ hybridization analysis revealed that Palovarotene-treated OC had much more transcripts of Cyp26b1 (retinoid target gene) as compared with the corn oil-treated OC and that OCs had significantly higher response to Palovarotene compared to the adjacent growth plate. When Palovarotene (1.76 mg/Kg, daily) treatment continued for 2 weeks, OC development was greatly suppressed compared to vehicle control in the wrists and ribs as determined by histological staining and radiological assessments, whereas the Palovarotene-treated growth plate did not show histological abnormality. Together, these findings indicate that OC have higher sensitivity to RARγ agonist compared to the growth plate and RARγ agonists rapidly inhibits OC development without a significant negative effect on growth plate and may exert anti-tumor function on OC by inducing cell death via apoptosis. Citation Format: Sonia Garcia, Hongying Tian, John Herzenberg, Joshua Abzug, Vincent Ng, Ashley Cellini, Masahiro Iwamoto, Motomi Enomoto-Iwamoto. Osteochondromas respond and its development is inhibited by retinoic acid nuclear receptor gamma agonist treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB518.

Abstract LB518A: Osteochondromas respond and its development is inhibited by retinoic acid nuclear receptor gamma agonist treatment

Abstract Osteochondromas (OC) are cartilage-capped tumors that arise near growing physis and are considered the most common benign bone tumor in children. They can lead to skeletal deformity, pain, loss of motion and neurovascular compression. Occasionally, malignant degeneration can occur. OC is thought to arise by mysregulation of chondrogenesis or chondrocyte differentiation, leading to ectopic cartilage formation. OC can occur as a solitary or multiple OC (MO). MO can occur from a hereditary cancer syndrome in which EXT1 and EXT2 are the major causative genes. Currently, treatment is limited to surgical resection only. There are no available FDA-approved therapies for MO. Previous translational research suggested that retinoic acid nuclear receptor gamma agonist (RARγ) suppresses ectopic cartilage formation including OC in rodent models. These preclinical studies led to a clinical trial to study the efficacy and safety of Palovarotene, a RARγ agonist, for the systemic treatment of MO (NCT03442985). The clinical trial was terminated due to concerns of skeletal toxicity observed in pediatric patients. To overcome this adverse action, we should elucidate the molecular action of RARγ agonists on osteochondromas and refine the treatment regimen. The purposes of our study are to examine the mechanism in which RARγ elicits osteochondroma growth and to determine whether the responsiveness to RARγ agonists is different between OC and growth plate. Treatment of RARγ agonists on mouse chondrocytes stimulated cell death induced by hydrogen peroxide (H2O2). In addition, human OC explants treated with RARγ agonists contained more apoptotic cells than vehicle-treated OC explants. OCs were induced in AcanCreER; Ext1e2neofl/e2neofl MO mouse model via tamoxifen induction at P5. After two weeks, mice were treated with Palovarotene (1.76 mg/Kg) or corn oil, and 24hrs after treatment, forelimbs were harvested. In-situ hybridization analysis revealed that Palovarotene-treated OC had much more transcripts of Cyp26b1 (retinoid target gene) as compared with the corn oil-treated OC and that OCs had significantly higher response to Palovarotene compared to the adjacent growth plate. When Palovarotene (1.76 mg/Kg, daily) treatment continued for 2 weeks, OC development was greatly suppressed compared to vehicle control in the wrists and ribs as determined by histological staining and radiological assessments, whereas the Palovarotene-treated growth plate did not show histological abnormality. Together, these findings indicate that OC have higher sensitivity to RARγ agonist compared to the growth plate and RARγ agonists rapidly inhibits OC development without a significant negative effect on growth plate and may exert anti-tumor function on OC by inducing cell death via apoptosis. Citation Format: Sonia Garcia, Hongying Tian, John Herzenberg, Joshua Abzug, Vincent Ng, Ashley Cellini, Masahiro Iwamoto, Motomi Enomoto-Iwamoto. Osteochondromas respond and its development is inhibited by retinoic acid nuclear receptor gamma agonist treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB518A.

CRABPs Alter all-trans-Retinoic Acid Metabolism by CYP26A1 via Protein-Protein Interactions.

Cellular retinoic acid binding proteins (CRABP1 and CRABP2) bind all-trans-retinoic acid (atRA), the active metabolite of vitamin A, with high affinity. CRABP1 and CRABP2 have been shown to interact with the atRA-clearing cytochrome P450 enzymes CYP26B1 and CYP26C1 and with nuclear retinoic acid receptors (RARs). We hypothesized that CRABP1 and CRABP2 also alter atRA metabolism and clearance by CYP26A1, the third key atRA-metabolizing enzyme in the CYP26 family. Based on stopped-flow experiments, atRA bound CRABP1 and CRABP2 with Kd values of 4.7 nM and 7.6 nM, respectively. The unbound atRA Km values for 4-OH-atRA formation by CYP26A1 were 4.7 ± 0.8 nM with atRA, 6.8 ± 1.7 nM with holo-CRABP1 and 6.1 ± 2.7 nM with holo-CRABP2 as a substrate. In comparison, the apparent kcat value was about 30% lower (0.71 ± 0.07 min−1 for holo-CRABP1 and 0.75 ± 0.09 min−1 for holo-CRABP2) in the presence of CRABPs than with free atRA (1.07 ± 0.08 min−1). In addition, increasing concentrations in apo-CRABPs decreased the 4-OH-atRA formation rates by CYP26A1. Kinetic analyses suggest that apo-CRABP1 and apo-CRABP2 inhibit CYP26A1 (Ki = 0.39 nM and 0.53 nM, respectively) and holo-CRABPs channel atRA for metabolism by CYP26A1. These data suggest that CRABPs play a critical role in modulating atRA metabolism and cellular atRA concentrations.

1,5-Disubstituted Acylated 2-Amino-4,5-dihydroimidazoles as a New Class of Retinoic Acid Receptor-Related Orphan Receptor (ROR) Inhibitors.

A growing body of evidence suggests a pathogenic role for pro-inflammatory T helper 17 cells (Th17) in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type I diabetes, and psoriasis—diseases for which no curative treatment is currently available. The nuclear retinoic acid receptor–related orphan receptors alpha and gamma (RORα/γ), in particular the truncated isoform RORγt that is specifically expressed in the thymus, play a critical role in the activation of a pro-inflammatory Th17 response, and RORγ inverse agonists have shown promise as negative regulators of Th17 for the treatment of autoimmune diseases. Our study underscores the screening of a large combinatorial library of 1,5-disubstituted acylated 2-amino-4,5-dihydroimidazoles using a demonstrated synthetic and screening approach and the utility of the positional scanning libraries strategy for the rapid identification of a novel class of ROR inhibitors. We identified compound 1295-273 with the highest activity against RORγ (3.3 µM IC50) in this series, and almost a two-fold selectivity towards this receptor isoform, with 5.3 and 5.8 µM IC50 against RORα and RORβ cells, respectively.

Selective Retinoic Acid Receptor γ Antagonist 7C is a Potent Enhancer of BMP-Induced Ectopic Endochondral Bone Formation.

Bone morphogenetic proteins (BMPs) have been clinically applied for induction of bone formation in musculoskeletal disorders such as critical-sized bone defects, nonunions, and spinal fusion surgeries. However, the use of supraphysiological doses of BMP caused adverse events, which were sometimes life-threatening. Therefore, safer treatment strategies for bone regeneration have been sought for decades. Systemic administration of a potent selective antagonist of retinoic acid nuclear receptor gamma (RARγ) (7C) stimulated BMP-induced ectopic bone formation. In this study, we developed 7C-loaded poly lactic nanoparticles (7C-NPs) and examined whether local application of 7C enhances BMP-induced bone regeneration. The collagen sponge discs that absorbed recombinant human (rh) BMP-2 were implanted into the dorsal fascia of young adult mice to induce ectopic bone. The combination of rhBMP-2 and 7C-NP markedly increased the total bone volume and thickness of the bone shell of the ectopic bone in a dose-dependent manner compared to those with rhBMP-2 only. 7C stimulated sulfated proteoglycan production, expression of chondrogenic marker genes, and Sox9 reporter activity in both chondrogenic cells and MSCs. The findings suggest that selective RARγ antagonist 7C or the related compounds potentiate the bone inductive ability of rhBMP-2, as well as support any future research to improve the BMP-2 based bone regeneration procedures in a safe and efficient manner.

Retinoic Acid Receptor Alpha Is Essential in Postnatal Sertoli Cells but Not in Germ Cells.

Retinoic acid signaling is indispensable for the completion of spermatogenesis. It is known that loss of retinoic acid nuclear receptor alpha (RARA) induces male sterility due to seminiferous epithelium degeneration. Initial genetic studies established that RARA acts in Sertoli cells, but a recent paper proposed that RARA is also instrumental in germ cells. In the present study, we have re-assessed the function of RARA in germ cells by genetically ablating the Rara gene in spermatogonia and their progenies using a cell-specific conditional mutagenesis approach. We show that loss of Rara in postnatal male germ cells does not alter the histology of the seminiferous epithelium. Furthermore, RARA-deficient germ cells differentiate normally and give rise to normal, living pups. This establishes that RARA plays no crucial role in germ cells. We also tested whether RARA is required in Sertoli cells during the fetal period or after birth. For this purpose, we deleted the Rara gene in Sertoli cells at postnatal day 15 (PN15), i.e., after the onset of the first spermatogenic wave. To do so, we used temporally controlled cell-specific mutagenesis. By comparing the testis phenotypes generated when Rara is lost either at PN15 or at embryonic day 13, we show that RARA exerts all of its functions in Sertoli cells not at the fetal stage but from puberty.

Towards a Better Vision of Retinoic Acid Signaling during Eye Development.

Retinoic acid (RA) functions as an essential signal for development of the vertebrate eye by controlling the transcriptional regulatory activity of RA receptors (RARs). During eye development, the optic vesicles and later the retina generate RA as a metabolite of vitamin A (retinol). Retinol is first converted to retinaldehyde by retinol dehydrogenase 10 (RDH10) and then to RA by all three retinaldehyde dehydrogenases (ALDH1A1, ALDH1A2, and ALDH1A3). In early mouse embryos, RA diffuses to tissues throughout the optic placode, optic vesicle, and adjacent mesenchyme to stimulate folding of the optic vesicle to form the optic cup. RA later generated by the retina is needed for further morphogenesis of the optic cup and surrounding perioptic mesenchyme; loss of RA at this stage leads to microphthalmia and cornea plus eyelid defects. RA functions by binding to nuclear RARs at RA response elements (RAREs) that either activate or repress transcription of key genes. Binding of RA to RARs regulates recruitment of transcriptional coregulators such as nuclear receptor coactivator (NCOA) or nuclear receptor corepressor (NCOR), which in turn control binding of the generic coactivator p300 or the generic corepressor PRC2. No genes have been identified as direct targets of RA signaling during eye development, so future studies need to focus on identifying such genes and their RAREs. Studies designed to learn how RA normally controls eye development in vivo will provide basic knowledge valuable for determining how developmental eye defects occur and for improving strategies to treat eye defects.

Methods for assessing the interaction of apocarotenoids with vertebrate nuclear receptors.

Apocarotenoids occur in the diets of human and other vertebrates and form from the metabolism of intact carotenoids. The metabolism of the provitamin A carotenoid, β-carotene, produces β-apo-15-carotenoic acid or retinoic acid. Retinoic acid functions as a hormone by binding to and activating nuclear retinoic acid receptors to modulate gene transcription. It is likely that other apocarotenoids may similarly modulate other ligand-activated nuclear receptors. This chapter describes in vitro biochemical and biophysical methods to characterize the direct binding of apocarotenoids to nuclear receptors and to assess their effect on binding of transcriptional coactivators to the receptor. It also provides a few widely used methods to study the consequences of nuclear receptor activation in intact cells. The effects of β-apocarotenoids on retinoid receptors provide examples.

Retinoid Agonists in the Targeting of Heterotopic Ossification.

Retinoids are metabolic derivatives of vitamin A and regulate the function of many tissues and organs both prenatally and postnatally. Active retinoids, such as all trans-retinoic acid, are produced in the cytoplasm and then interact with nuclear retinoic acid receptors (RARs) to up-regulate the transcription of target genes. The RARs can also interact with target gene response elements in the absence of retinoids and exert a transcriptional repression function. Studies from several labs, including ours, showed that chondrogenic cell differentiation and cartilage maturation require (i) the absence of retinoid signaling and (ii) the repression function by unliganded RARs. These and related insights led to the proposition that synthetic retinoid agonists could thus represent pharmacological agents to inhibit heterotopic ossification (HO), a process that recapitulates developmental skeletogenesis and involves chondrogenesis, cartilage maturation, and endochondral ossification. One form of HO is acquired and is caused by injury, and another severe and often fatal form of it is genetic and occurs in patients with fibrodysplasia ossificans progressiva (FOP). Mouse models of FOP bearing mutant ACVR1R206H, characteristic of most FOP patients, were used to test the ability of the retinoid agonists selective for RARα and RARγ against spontaneous and injury-induced HO. The RARγ agonists were found to be most effective, and one such compound, palovarotene, was selected for testing in FOP patients. The safety and effectiveness data from recent and ongoing phase II and phase III clinical trials support the notion that palovarotene may represent a disease-modifying treatment for patients with FOP. The post hoc analyses showed substantial efficacy but also revealed side effects and complications, including premature growth plate closure in some patients. Skeletally immature patients will need to be carefully weighed in any future regulatory indications of palovarotene as an important therapeutic option in FOP.

Discovery of N-(2-benzyl-4-oxochroman-7-yl)-2-(5-(ethylsulfonyl) pyridin-2-yl) acetamide (b12) as a potent, selective, and orally available novel retinoic acid receptor-related orphan receptor γt inverse agonist

The nuclear receptor retinoic acid receptor-related orphan receptor γ (RORγ, NR1F3, or RORc) exists in two isoforms, with one isoform (RORγ or RORc1) widely expressed in a variety of tissues, and the expression of the second isoform (RORγt or RORc2) restricted to the thymus and cells of the immune system. RORγt is a key regulator of the development and functions of T-helper 17 (Th17) cells. Clinical proof-of-concept (PoC) with small molecule inverse agonists of RORγt has been achieved with VTP-43742 (Phase II) for the treatment of psoriasis, and pre-clinical PoC for this mechanism has also been established for the treatment of autoimmune diseases. A series of aryl sulfonyl derivatives as novel RORγt inverse agonists were designed and synthesized based on VTP-43742. We conducted structural modifications that improved the activity profile. In pharmacodynamic (PD) studies, oral administration of compound b12 showed robust and dose-dependent inhibition of IL-6 and IL-17A cytokine expression. The ability of compound b12 to reduce the levels of IL-6 and IL-17A in vivo after oral dosing in mice, and a corresponding reduction in skin inflammation further supports the potential of small molecule RORγt modulation as a therapeutic target for the treatment of inflammatory diseases.

Olfactory Receptor OR7A17 Expression Correlates with All-Trans Retinoic Acid (ATRA)-Induced Suppression of Proliferation in Human Keratinocyte Cells

Olfactory receptors (ORs), which belong to the G-protein-coupled receptor family, have been widely studied as ectopically expressed receptors in various human tissues, including the skin. However, the physiological functions of only a few OR types have been elucidated in skin cells. All-trans retinoic acid (ATRA) is a well-known medication for various skin diseases. However, many studies have shown that ATRA can have adverse effects, resulting from the suppression of cell proliferation. Here, we investigated the involvement of OR7A17 in the ATRA-induced suppression of human keratinocyte (HaCaT) proliferation. We demonstrated that OR7A17 is expressed in HaCaT keratinocytes, and its expression was downregulated by ATRA. The ATRA-induced downregulation of OR7A17 was attenuated via RAR α or RAR γ antagonist treatment, indicating that the effects of ATRA on OR7A17 expression were mediated through nuclear retinoic acid receptor signaling. Moreover, we found that the overexpression of OR7A17 induced the proliferation of HaCaT cells while counteracting the antiproliferative effect of ATRA. Mechanistically, OR7A17 overexpression reversed the ATRA-induced attenuation of Ca2+ entry. Our findings indicated that ATRA suppresses cell proliferation through the downregulation of OR7A17 via RAR α- and γ-mediated retinoid signaling. Taken together, OR7A17 is a potential therapeutic target for ameliorating the anti-proliferative effects of ATRA.

Discovery of tert-amine-based RORγt agonists.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma-t (RORγt) is a transcription factor regulating Th17cell differentiation and proliferation from naive CD4+ T cells. Since Th17cells have demonstrated the antitumor efficacy by eliciting remarkable activation of CD8+ T cells, RORγt agonists could be applied as potential small molecule therapeutics for cancer immunotherapy. Based on the previously reported RORγt agonist 1 and its resolved co-crystal structure, a series of new tertiary amines were designed, synthesized and biologically evaluated, yielding optimal moieties with improved chemical properties and biological responses. The combination of these optimal moieties resulted in identification of novel RORγt agonists such as 8b with further elevated RORγt agonism responses at a target-based level as well as in cell-based assays, which provided some structural knowledge for further optimization of RORγt agonists as small molecule therapeutics for cancer immunotherapy.

A splicing factor phosphorylated by protein kinase A is increased in HL60 cells treated with retinoic acid

Retinoic acid (RA) induces the differentiation of human promyelocytic leukemia HL60 cells into granulocytic cells and inhibits proliferation. Certain of actions of RA are mediated by RA nuclear receptors that regulate gene expression. However, it is also known that direct protein modification by RA (retinoylation) can occur. One such retinoylated protein in HL60 cells is a regulatory subunit of protein kinase A (PKA), which is increased in the nucleus following RA treatment and which then increases phosphorylation of other nuclear proteins. However, a complete understanding of which nuclear proteins are phosphorylated is lacking. In the current study, we employed mass spectrometry to identify one of the PKA-phosphorylated proteins as a serine/arginine-rich splicing factor 1 (SF2, SRSF1). We found that RA treatment increased the level of PKA-phosphorylated SF2 but decreased the level of SF2. While SF2 regulates myelogenous cell leukemia-1 (Mcl-1, anti-apoptotic factor), RA treatment reduced the level of Mcl-1L (full-length Mcl-1 long) and increased the level of Mcl-1S (Mcl-1 short; a short splicing variant of the Mcl-1). Furthermore, treatment with a PKA inhibitor reversed these effects on Mcl-1 and inhibited RA-induced cell differentiation. In contrast, treatment with a Mcl-1L inhibitor enhanced RA-induced cell differentiation. These results indicate that RA activates PKA in the nucleus, increases phosphorylation of SF2, raises levels of Mcl-1S and lowers levels of Mcl-1L, resulting in the induction of differentiation. RA-modified PKA may play an important role in inducing cell differentiation and suppressing cell proliferation.

Retinoic acid increases phagocytosis of myelin by macrophages.

Traumatic injuries of the central nervous system (CNS) are followed by the accumulation of cellular debris including proteins and lipids from myelinated fiber tracts. Insufficient phagocytic clearance of myelin debris influences the pathological process because it induces inflammation and blocks axonal regeneration. We investigated whether ligands of nuclear receptor families retinoic acid receptors (RARs), retinoid X receptors, peroxisome proliferator‐activated receptors, lipid X receptors, and farnesoid X receptors increase myelin phagocytosis by murine bone marrow–derived macrophages and Raw264.7 cells. Using in vitro assays with 3,3′‐dioctadecyloxacarbocyanine perchlorate‐ and pHrodo‐labeled myelin we found that the transcriptional activator all‐trans retinoic acid (RA)enhanced endocytosis of myelin involving the induction of tissue transglutaminase‐2. The RAR‐dependent increase of phagocytosis was not associated with changes in gene expression of receptors FcγR1, FcγR2b, FcγR3, TREM2, DAP12, CR3, or MerTK. The combination of RA and myelin exposure significantly reduced the expression of M1 marker genes inducible nitric oxide synthase and interleukin‐1β and increased expression of transmembrane proteins CD36 and ABC‐A1, which are involved in lipid transport and metabolism. The present results suggest an additional mechanism for therapeutic applications of RA after CNS trauma. It remains to be studied whether endogenous RA‐signaling regulates phagocytosis in vivo.

Phenotypic landscape of intestinal organoid regeneration.

The development of intestinal organoids from single adult intestinal stem cells in vitro recapitulates the regenerative capacity of the intestinal epithelium1,2. Here we unravel the mechanisms that orchestrate both organoid formation and the regeneration of intestinal tissue, using an image-based screen to assay an annotated library of compounds. We generate multivariate feature profiles for hundreds of thousands of organoids to quantitatively describe their phenotypic landscape. We then use these phenotypic fingerprints to infer regulatory genetic interactions, establishing a new approach to the mapping of genetic interactions in an emergent system. This allows us to identify genes that regulate cell-fate transitions and maintain the balance between regeneration and homeostasis, unravelling previously unknown roles for several pathways, among them retinoic acid signalling. We then characterize a crucial role for retinoic acid nuclear receptors in controlling exit from the regenerative state and driving enterocyte differentiation. By combining quantitative imaging with RNA sequencing, we show the role of endogenous retinoic acid metabolism in initiating transcriptional programs that guide the cell-fate transitions of intestinal epithelium, and we identify an inhibitor of the retinoid X receptor that improves intestinal regeneration in vivo.

Vitamin A regulates fibroblast growth factor 23 (FGF23).

Renal phosphate and vitamin D metabolism are regulated by proteohormone fibroblast growth factor 23 (FGF23), which is secreted by bone cells. FGF23 inhibits phosphate reabsorption and the production of calcitriol, active vitamin D (1,25(OH)2D3). FGF23 generated by other cells exerts further paracrine effects in the liver, heart, and immune system. The FGF23 plasma concentration is positively associated with the onset and progression of kidney and cardiovascular diseases, disclosing FGF23 as a potential disease biomarker. The effects of vitamin A on the expression of FGF23 are controversial. Vitamin A components, retinoids, are mainly effective through nuclear retinoic acid receptors (RAR) and exert different effects on bone. The aim of this study was to clarify whether vitamin A modulates the production of FGF23. We studied the relevance of vitamin A for FGF23 production. Fgf23 transcripts were determined by real-time quantitative polymerase chain reaction in UMR106 osteoblast-like cells and IDG-SW3 osteocytes. FGF23 protein in the cell culture supernatant was measured by enzyme-linked immunosorbent assay. All-trans-retinoic acid, retinyl acetate, RAR agonist TTNPB (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid), and 13-cis-retinoic acid downregulated the expression of the Fgf23 gene in a dose-dependent manner. This effect was significantly attenuated by RAR antagonist AGN193109 (4-[2-[5,6-Dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl]ethynyl]benzoic acid). The present study demonstrated that vitamin A is a potent suppressor of FGF23 production through RAR.

Recent Advances Regarding the Therapeutic Potential of Adapalene.

Adapalene (ADP) is a representative of the third retinoids generation and successfully used in first-line acne treatment. ADP binds to retinoic acid nuclear receptors. The comedolytic, anti-inflammatory, antiproliferative, and immunomodulatory are the known ADP effects. Its safety profile is an advantage over other retinoids. ADP recently was found to be effective in the treatment of several dermatological diseases and photoaging besides the utility in the treatment of acne vulgaris. New biological effects of adapalene with therapeutic potential are highlighted in this review paper. Thus, adapalene could be a valuable therapeutic drug into the treatment of several types of cancer. Additionally, some neurodegenerative diseases could be treated with a suitable formulation for intravenous administration. The antibacterial activity against methicillin-resistant Staphylococcus aureus of an analogue of ADP has been proven. In different therapeutic schemes, ADP is more effective in combination with other active substances. New topical combinations with adapalene include ketoconazole (antifungal), mometasone furoate (anti-inflammatory corticosteroid), nadifloxacin (fluoroquinolone), and alfa and beta hydroxy acids. Combination with oral drugs is a new trend that enhances the properties of topical formulations with adapalene. Several studies have investigated the effects of ADP in co-administration with azithromycin, doxycycline, faropenem, isotretinoin, and valganciclovir. Innovative formulations of ADP also aim to achieve a better bioavailability, increased efficacy, and reduced side effects. In this review, we have highlighted the current studies on adapalene regarding biological effects useful in various treatment types. Adapalene has not been exploited yet to its full biological potential.

Optimization and biological evaluation of thiazole-bis-amide inverse agonists of RORγt

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with small molecule therapeutics. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We recently described the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole scaffold. Herein we describe the successful optimization of this class by incorporation of an additional amide moiety at the 4-position of the thiazole core. In several optimization cycles, we have reduced human PXR activation, improved solubility, and increased potency while maintaining nuclear receptor selectivity. X-ray crystallographic analysis of compound 1g bound in the sterol binding site of the ligand binding domain of RORγt was largely consistent with an earlier structure, guiding further insight into the molecular mechanism for RORγt inhibition with this series. Compound 1g is orally bioavailable, potent in a human whole blood assay and proved to be efficacious in an ex-vivo IL-17A assay, and was selected for preclinical evaluation.