Dominant-negative Retinoic Acid Receptor Research Articles

Inverse agonists of retinoic acid receptor/retinoid X receptor signaling as lineage-specific antitumor agents against human adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a lethal malignancy of exocrine glands, characterized by the coexistence within tumor tissues of 2 distinct populations of cancer cells, phenotypically similar to the myoepithelial and ductal lineages of normal salivary epithelia. The developmental relationship linking these 2 cell types, and their differential vulnerability to antitumor treatments, remains unknown. Using single-cell RNA sequencing, we identified cell-surface markers (CD49f, KIT) that enabled the differential purification of myoepithelial-like (CD49fhigh/KITneg) and ductal-like (CD49flow/KIT+) cells from patient-derived xenografts (PDXs) of human ACCs. Using prospective xenotransplantation experiments, we compared the tumor-initiating capacity of the 2 cell types and tested whether one could differentiate into the other. Finally, we searched for signaling pathways with differential activation between the 2 cell types and tested their role as lineage-specific therapeutic targets. Myoepithelial-like cells displayed higher tumorigenicity than ductal-like cells and acted as their progenitors. Myoepithelial-like and ductal-like cells displayed differential expression of genes encoding for suppressors and activators of retinoic acid signaling, respectively. Agonists of retinoic acid receptor (RAR) or retinoid X receptor (RXR) signaling (all-trans retinoic acid, bexarotene) promoted myoepithelial-to-ductal differentiation, whereas suppression of RAR/RXR signaling with a dominant-negative RAR construct abrogated it. Inverse agonists of RAR/RXR signaling (BMS493, AGN193109) displayed selective toxicity against ductal-like cells and in vivo antitumor activity against PDX models of human ACC. In human ACCs, myoepithelial-like cells act as progenitors of ductal-like cells, and myoepithelial-to-ductal differentiation is promoted by RAR/RXR signaling. Suppression of RAR/RXR signaling is lethal to ductal-like cells and represents a new therapeutic approach against human ACCs.

Retinoid Signaling in Intestinal Epithelial Cells Is Essential for Early Survival From Gastrointestinal Infection.

Vitamin A deficiency (A–) increases morbidity and mortality to gastrointestinal (GI) infection. Blocking retinoid signaling (dominant negative retinoic acid receptor, dnRAR) in intestinal epithelial cells (IEC, IECdnRAR) had no effect on vitamin A absorption, the expression of tight junction proteins or the integrity of the barrier. Immune cells in the gut were present in normal frequencies in the IECdnRAR mice, with the exception of the T cell receptor (TCR)αβ+/CD8αα cells, which were significantly lower than in wildtype littermates. Challenging the IECdnRAR mice with dextran sodium sulfate to induce colitis or Citrobacter rodentium infection resulted in similar disease to wildtype littermates. Feeding mice vitamin A deficient diets reduced vitamin A status and the A– IECdnRAR mice developed more severe colitis and C. rodentium infection. In particular, retinoid signaling in the IEC was crucial for the A– host to survive early infection following C. rodentium. Treating A– mice with retinoic acid (RA) beginning on the day of infection protects most mice from early lethality. However, RA treatment of the A– IECdnRAR mice was ineffective for preventing lethality following C. rodentium infection. Retionid signaling in IEC is critical, especially when there are reduced levels of dietary vitamin A. IEC are direct targets of vitamin A for mounting early defense against infection.

Retinoic acid signalling in fibro/adipogenic progenitors robustly enhances muscle regeneration.

BackgroundDuring muscle regeneration, excessive formation of adipogenic and fibrogenic tissues, from their respective fibro/adipogenic progenitors (FAPs), impairs functional recovery. Intrinsic mechanisms controlling the proliferation and differentiation of FAPs remain largely unexplored.MethodsHere, we investigated the role of retinoic acid (RA) signalling in regulating FAPs and the subsequent effects on muscle restoration from a cardiotoxin-induced injury. Blockage of retinoic acid receptor (RAR) signalling was achieved through dominant negative retinoic acid receptor α (RARα403) expression specific in PDGFRα+ FAPs in vivo and by BMS493 treatment in vitro. Effects of RAR-signalling on FAP cellularity and muscle regeneration were also investigated in a high-fat diet-induced obese mice model.FindingsSupplementation of RA increased the proliferation of FAPs during the early stages of regeneration while suppressing FAP differentiation and promoting apoptosis during the remodelling stage. Loss of RAR-signalling caused ectopic adipogenic differentiation of FAPs and impaired muscle regeneration. Furthermore, obesity disrupted the cellular transition of FAPs and attenuated muscle regeneration. Supplementation of RA to obese mice not only rescued impaired muscle fibre regeneration, but also inhibited infiltration of fat and fibrotic tissues during muscle repair. These beneficial effects were abolished after blocking RAR-signalling in FAPs of obese mice.InterpretationThese data suggest that RAR-signalling in FAPs is a critical therapeutic target for suppressing differentiation of FAPs and facilitating the regeneration of muscle and other tissues.FundingThis study was supported by grants from the National Institutes of Health (R01-HD067449 and R21-AG049976) to M.D.

Divergent roles of retinoic acid signaling in T regulatory cell function

Abstract Regulatory T cells (Tregs) modulate immune responses to maintain immune homeostasis. Retinoic acid (RA) signaling promotes Treg generation and stability. Paradoxically, we observed that dominant negative RA receptor α expressed only in donor T cells (DNRARαfl/flCD4Cre) dampened inflammation and graft-versus-host disease (GVHD), while increasing Tregs. We found that diminishing RA signaling significantly enhanced their in vitro suppressive capacity. In vivo the adoptive transfer of DNRARαfl/flCD4Cre Tregs at the time of transplant was significantly (p<0.01) more effective than wild type (WT) controls in ameliorating GVHD. Mechanistically, enhanced suppression was associated with elevated expression of suppressive (CTLA-4, GITR, CD39) and fitness (CD25, pSTAT5) markers. To evaluate the intrinsic requirement of RA signaling in Tregs, we generated transgenic mice with conditional DNRARα expression in Tregs (DNRARαfl/fl x Foxp3YFPCre). Surprisingly, the extent of ablation of RA signaling resulted in divergent phenotypes. Heterozygous ablation (DNRARαfl/wt) was permissive of enhanced Treg suppression. In striking contrast and unexpectedly, homozygous ablation (DNRARαfl/fl) impaired Treg suppressive function and caused multi-organ (lung, liver) autoimmunity in >90% of females and males by 3 months of age. Poor suppression is not cell intrinsic as DNRARαfl/fl Tregs isolated from mixed bone marrow chimeras of congenic WT and DNRARαfl/fl x Foxp3YFP Cre had enhanced function. Our studies indicate that RA signaling in Tregs can be targeted to prevent autoimmunity by enhancing Treg suppression or alternatively, promote antitumor responses by reducing Treg function.

Vitamin A signaling regulates pathological epithelial cell shedding via IL-18

Abstract The intestinal epithelium is under continuous renewal via a tightly controlled process of cell proliferation and cell shedding. During infection and sepsis, the rate of cell shedding increases dramatically as a protective response. However, the mechanisms through which epithelial turnover rate is controlled in the gut remain elusive. Here we identify diet-mediated control of epithelial cell shedding as an important mechanism in defense against infectious disease. We show that abrogating vitamin A signaling by expressing a dominant negative retinoic acid receptor exclusively in intestinal epithelial cells (dnRARVillin-Cre) makes mice resistant to pathological epithelial cell shedding and cell death in an LPS model of sepsis. In a Salmonella infection model, dnRARVillin-Cremice had increased intracellular loads of the pathogen in the intestine as compared to control mice. This trend was observed even at early time points of the infection and was associated with drastically reduced epithelial cell shedding compared to dnRARfl/fl mice. Intestinal epithelium RNA-seq and protein analysis uncovered that levels of the pro-inflammatory cytokine IL-18 are severely reduced in epithelia of dnRARVillin-Cre mice. LPS and pathogen induced cell death were both rescued upon IL-18 reconstitution in dnRARVillin-Cre mice. Furthermore, even dietary vitamin A could modulate epithelial IL-18 expression. Our results thus reveal a novel mechanism whereby dietary vitamin A modulates cell shedding response in the epithelium via IL-18, affecting barrier function and infection susceptibility in the gut.

Intestinal epithelium intrinsic RARα signaling regulates gut barrier integrity via IL-18

Abstract Intestinal epithelium, overlying mucus layer and anti-microbial peptides together form a physico-chemical barrier crucial for homeostasis with the gut microbiota. Intestinal epithelial cells (IECs) are at the frontline of host-microbe interactions in the gut, yet how they modulate barrier function in response to dietary cues is poorly understood. Vitamin A and its key metabolite retinoic acid (RA), are crucial regulators of gut immune homeostasis. In this study, we employ a mouse model that expresses a dominant negative retinoic acid receptor exclusively in IECs (dnRARVillin-Cre) to establish a novel role for RA signaling in regulating the mucus barrier. The absence of RA signaling in colonic epithelium resulted in significant increase in mucus layer thickness indicating that RA regulates goblet cell function. Concomitant with this, we observed that in dnRARVillin-Cre mice there are more goblet cells and an enhanced spatial separation between host and the gut microbiota. Further, we showed that dnRARVillin-Cre mice have a diminished sensitivity to chemically induced colitis in comparison to wild-type mice. Quantitative RNA and protein analysis uncovered that gene transcription and protein activation and secretion of key pro-inflammatory cytokine IL-18 was severely impaired in epithelium of dnRARVillin-Cre mice. IL-18 has previously been shown to act as a negative regulator of goblet cell function in the intestinal epithelium. We now show that intestinal epithelium intrinsic RA signaling can modulate IL-18 levels to regulate the mucus barrier. Our results have thus disclosed a novel link between a diet derive metabolite and intestinal epithelial cell function that can be harnessed to modulate mucosal immunity.

Role of retinoic acid in the stability of the T-helper-type 1 lineage and implications for autoimmunity

BackgroundCD4 T cells with features of both T-helper-type 1 (Th1) and 17 (Th17) cells have been implicated in several autoimmune diseases suggesting that plasticity among CD4 T-cell lineages is potentially pathogenic. However, the factors that regulate T-cell lineage stability are largely unknown. Retinoic acid (RA) is synthesised at sites of inflammation. We hypothesised that retinoic acid, a profound epigenetic modifier, could regulate T-cell lineage stability. MethodsWe used a mouse model in which retinoic acid signalling is specifically ablated within the T-cell compartment through overexpression of a dominant negative retinoic acid receptor α (RARα) (dnRARα mice) to investigate its role in the regulation of Th1 lineage stability. Genome-wide ChIP-seq analysis was done to identify RARα targets. In parallel, we performed global mapping of regulatory regions, termed enhancers, to gain mechanistic insight into retinoic acid regulation of T-cell fate. The in-vivo relevance of our findings was determined in a model of oral antigen-induced intestinal inflammation. FindingsWe found that retinoic acid is crucial for maintenance of the Th1 lineage. Abrogation of retinoic acid signalling in Th1 cells resulted in loss of T-bet expression and STAT4 activity. Th1 cells from dnRARα mice showed enhanced plasticity with the emergence of hybrid Th1–Th17 and Th17 effector cells. Global analysis of RARα binding and enhancer mapping revealed that RA–RARα directly regulated enhancer activity at Th1 lineage defining genes while repressing genes that regulate Th17 cell fate. Retinoic acid inhibition of Th1 plasticity was essential for maintaining appropriate Th cell responses in vivo and preventing autoimmune intestinal inflammation. InterpretationOur study has identified RA–RARα as a key component of the regulatory network governing maintenance and plasticity of Th1 cells and defines a new pathway for the development of pathogenic Th17 cells. Retinoids might be novel therapeutic agents for Th17-associated autoimmune diseases. FundingWellcome Trust.

Retinoic acid receptor signaling is required to maintain glucose-stimulated insulin secretion and β-cell mass.

Retinoic acid signaling is required for maintaining a range of cellular processes, including cell differentiation, proliferation, and apoptosis. We investigated the actions of all-trans-retinoic acid (atRA) signaling in pancreatic β-cells of adult mice. atRA signaling was ablated in β-cells by overexpressing a dominant-negative retinoic acid receptor (RAR)-α mutant (RARdn) using an inducible Cre-Lox system under the control of the pancreas duodenal homeobox gene promoter. Our studies establish that hypomorphism for RAR in β-cells leads to an age-dependent decrease in plasma insulin in the fed state and in response to a glucose challenge. Glucose-stimulated insulin secretion was also impaired in islets isolated from mice expressing RARdn. Among genes that are atRA responsive, Glut2 and Gck mRNA levels were decreased in isolated islets from RARdn-expressing mice. Histologic analyses of RARdn-expressing pancreata revealed a decrease in β-cell mass and insulin per β-cell 1 mo after induction of the RARdn. Our results indicate that atRA signaling mediated by RARs is required in the adult pancreas for maintaining both β-cell function and mass, and provide insights into molecular mechanisms underlying these actions.

EML Erythroid and Neutrophil Differentiation Protocols

Erythroid-Myeloid-Lymphoid cells (EML) are a multipotent haematopoietic cell line of mouse bone marrow origin capable of long-term maintenance in vitro in the presence of SCF (stem cell factor) (Tsai et al., 1994). The self-renewal capacity of the EML cell line is conferred by the presence of a dominant-negative retinoic acid receptor (RAR) originally delivered by retroviral transduction (Tsai et al., 1994), which arrests cells at an early progenitor stage blocked from normal progression into myeloid differentiation. The presence of the RAR trans-gene does not interfere with erythroid differentiation, and it is possible to capture a low percentage of early erythroid, but not myeloid, committed cells in maintenance cultures (Pina et al., 2012; Ye et al., 2005).Cells can be driven into granulocytic/neutrophil differentiation through the use of high doses of retinoic acid (RA), which overcomes the differentiation block. It should be noted that these pharmacological doses of RA are not compatible with erythroid differentiation, and it is hence not viable to obtain robust erythroid and myeloid differentiation in the same assay. Indeed, colonies scored as mixed-lineage in CFC assays are a mixture of undifferentiated and erythroid cells (Tsai et al., 1994). Nevertheless, robust single-lineage erythroid and neutrophil differentiation can be obtained in liquid culture under defined cytokine conditions, as specified below., 红细胞 - 髓样淋巴细胞（EML）是能够在SCF（干细胞因子）存在下在体外长期维持的小鼠骨髓来源的多潜能造血细胞系et al。，1994）。 EML细胞系的自我更新能力通过最初通过逆转录病毒转导递送的显性 - 负性视黄酸受体（RAR）的存在（Tsai等人，1994）来赋予，其捕获细胞在阻止从正常进展成骨髓分化的早期祖细胞阶段。 RAR转基因的存在不干扰红细胞分化，并且可能在维持培养中捕获低百分比的早期红细胞而不是骨髓，定型细胞（Pina等人， 2012; Ye等人，2005）。通过使用高剂量的视黄酸（RA），细胞可以被驱动进入粒细胞/嗜中性粒细胞分化，其克服了分化阻断。应当注意，RA的这些药理学剂量与红细胞分化不相容，因此在相同测定中获得稳定的红细胞和骨髓分化是不可行的。实际上，在CFC测定中作为混合谱系评分的集落是未分化和类红细胞的混合物（Tsai et al。，1994）。然而，在如下所述的确定的细胞因子条件下的液体培养中可以获得稳定的单谱系红细胞和嗜中性粒细胞分化。

Retinoic acid-rich microenvironment provides clonal survival cues for tumor and bacteria-specific CD8+ T cells (P1376)

Abstract While studies have implicated a role of vitamin A (VA) in host resistance to infectious disease, little is known as to the role of VA and its active metabolite, retinoic acid (RA) in host defense against cancer. Herein, it is shown for the first time that local RA production within the tumor microenvironment (TME) is increased up to 5-fold compared to naïve surrounding tissue, with a commensurate increase in RA signaling to regionally infiltrating tumor-reactive T cells. Conditional disruption of RA signaling in CD8+ T cells using a dominant negative Retinoic Acid Receptor α (dnRARα) illustrated that RA signaling is required for tumor-specific CD8+ T cell expansion/accumulation and protective anti-tumor immunity. Specific deletion of RARα, RARβ and RARγ in CD8+ T cells further demonstrated that RARα is required for CD8+ T cell survival, whereas RARβ is indispensable for bacteria-specific CD8+ T cell expansion in response to bacterial infection. These studies established a novel function of RA essential for both anti-tumor and infection CD8+ T cell immunity.

Retinoid Acid Specifies Neuronal Identity through Graded Expression of Ascl1

SummaryCell diversity and organization in the neural tube depend on the integration of extrinsic signals acting along orthogonal axes. These are believed to specify distinct cellular identities by triggering all-or-none changes in expression of combinations of transcription factors [1]. Under the influence of a common dorsoventral signal, sonic hedgehog, and distinct anterior-posterior (A-P) inductive signals [2, 3], two topographically related progenitor pools that share a common transcriptional code produce serotonergic and V3 neurons in the hindbrain and spinal cord, respectively [4–7]. These neurons have different physiological properties, functions, and connectivity [8, 9]. Serotonergic involvement in neuropsychiatric diseases has prompted greater characterization of their postmitotic repertoire of fate determinants, which include Gata2, Lmx1b, and Pet1 [10], whereas V3 neurons express Sim1 [4]. How distinct serotonergic and V3 neuronal identities emerge from progenitors that share a common transcriptional code is not understood. Here, we show that changes in retinoid activity in these two progenitor pools determine their fates. Retinoids, via Notch signaling, control the expression level in progenitors of the transcription factor Ascl1, which selects serotonergic and V3 neuronal identities in a dose-dependent manner. Therefore, quantitative differences in the expression of a single component of a transcriptional code can select distinct cell fates.

A Retinoic Acid—Rich Tumor Microenvironment Provides Clonal Survival Cues for Tumor-Specific CD8+ T Cells

While vitamin A has been implicated in host resistance to infectious disease, little is known about the role of vitamin A and its active metabolite, retinoic acid (RA) in host defenses against cancer. Here, we show that local RA production within the tumor microenvironment (TME) is increased up to 5-fold as compared with naïve surrounding tissue, with a commensurate increase in RA signaling to regionally infiltrating tumor-reactive T cells. Conditional disruption of RA signaling in CD8(+) T cells using a dominant negative retinoic acid receptor α (dnRARα) established that RA signaling is required for tumor-specific CD8(+) T-cell expansion/accumulation and protective antitumor immunity. In vivo analysis of antigen-specific CD8(+) T-cell responses revealed that early T-cell expansion was RA-independent; however, late T-cell expansion and clonal accumulation was suppressed strongly in the absence of RA signaling. Our findings indicate that RA function is essential for the survival of tumor-reactive CD8(+) T cells within the TME.

Retinoids ameliorate insulin resistance in a leptin-dependent manner in mice

Transgenic mice expressing dominant-negative retinoic acid receptor (RAR) α specifically in the liver exhibit steatohepatitis, which leads to the development of liver tumors. Although the cause of steatohepatitis in these mice is unknown, diminished hepatic expression of insulin-like growth factor-1 suggests that insulin resistance may be involved. In the present study, we examined the effects of retinoids on insulin resistance in mice to gain further insight into the mechanisms responsible for this condition. Dietary administration of all-trans-retinoic acid (ATRA) significantly improved insulin sensitivity in C57BL/6J mice, which served as a model for high-fat, high-fructose diet-induced nonalcoholic fatty liver disease (NAFLD). The same effect was observed in genetically insulin-resistant KK-A(y) mice, occurring in concert with activation of leptin-signaling pathway proteins, including signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2. However, such an effect was not observed in leptin-deficient ob/ob mice. ATRA treatment significantly up-regulated leptin receptor (LEPR) expression in the livers of NAFLD mice. In agreement with these observations, in vitro experiments showed that in the presence of leptin, ATRA directly induced LEPR gene expression through RARα, resulting in enhancement of STAT3 and insulin-induced insulin receptor substrate 1 phosphorylation. A selective RARα/β agonist, Am80, also enhanced hepatic LEPR expression and STAT3 phosphorylation and ameliorated insulin resistance in KK-A(y) mice. We discovered an unrecognized mechanism of retinoid action for the activation of hepatic leptin signaling, which resulted in enhanced insulin sensitivity in two mouse models of insulin resistance. Our data suggest that retinoids might have potential for treating NAFLD associated with insulin resistance.

Dysfunction of the RAR/RXR signaling pathway in the forebrain impairs hippocampal memory and synaptic plasticity

BackgroundRetinoid signaling pathways mediated by retinoic acid receptor (RAR)/retinoid × receptor (RXR)-mediated transcription play critical roles in hippocampal synaptic plasticity. Furthermore, recent studies have shown that treatment with retinoic acid alleviates age-related deficits in hippocampal long-term potentiation (LTP) and memory performance and, furthermore, memory deficits in a transgenic mouse model of Alzheimer's disease. However, the roles of the RAR/RXR signaling pathway in learning and memory at the behavioral level have still not been well characterized in the adult brain. We here show essential roles for RAR/RXR in hippocampus-dependent learning and memory. In the current study, we generated transgenic mice in which the expression of dominant-negative RAR (dnRAR) could be induced in the mature brain using a tetracycline-dependent transcription factor and examined the effects of RAR/RXR loss.ResultsThe expression of dnRAR in the forebrain down-regulated the expression of RARβ, a target gene of RAR/RXR, indicating that dnRAR mice exhibit dysfunction of the RAR/RXR signaling pathway. Similar with previous findings, dnRAR mice displayed impaired LTP and AMPA-mediated synaptic transmission in the hippocampus. More importantly, these mutant mice displayed impaired hippocampus-dependent social recognition and spatial memory. However, these deficits of LTP and memory performance were rescued by stronger conditioning stimulation and spaced training, respectively. Finally, we found that pharmacological blockade of RARα in the hippocampus impairs social recognition memory.ConclusionsFrom these observations, we concluded that the RAR/RXR signaling pathway greatly contributes to learning and memory, and LTP in the hippocampus in the adult brain.

A functional role for the histone demethylase UTX in normal and malignant hematopoietic cells

Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), an H3K27Me2/3 demethylase, has been implicated in development, self-renewal, and differentiation of various organs and embryonic stem cells through chromatin modifications and transcriptional regulation of important developmentally related genes, such as Hox genes. However, the function of UTX in hematopoiesis is not well understood. To study the role of UTX in the mammalian hematopoietic system, we used lentiviral short hairpin RNA constructs to knockdown UTX in the murine hematopoietic progenitor cell line EML, in primary murine bone marrow cells and in leukemic cell lines. We report that Utx is highly expressed in the hematopoietic compartment and that it plays an important role in cell proliferation and homeostasis of hematopoietic cells in vitro. Knockdown of UTX in EML and primary murine bone marrow cells impairs their colony-forming ability. Moreover, knockdown of UTX affects expression of key genes that regulate hematopoietic differentiation such as Mll1, Runx1, and Scl in primary murine bone marrow cells. And we further demonstrate that UTX directly associates with the promoters of the Mll1, Runx1, and Scl genes and modulate their transcription by controlling H3K27me3 marks on respective promoter regions. In addition, UTX depletion severely impaired proliferation of several human leukemia cell lines. Together, these data demonstrate a functional role for UTX in normal and malignant hematopoiesis.

Retinoic acid receptor and CNGA2 channel signaling are part of a regulatory feedback loop controlling axonal convergence and survival of olfactory sensory neurons

Little is known about the identities and functions of extracellular signaling molecules that work in concert with neuronal activity to regulate refinement and maintenance of the mouse olfactory sensory map. We show that expression of a dominant negative retinoic acid receptor (RAR) in olfactory sensory neurons (OSNs) increased the number of glomeruli that incorrectly contained OSN axons expressing different odorant receptors. This phenotype became apparent postnatally, coincided with increased cell death, and was preceded by increased Neuropilin-1 and reduced Kirrel-2 expressions. Kirrel-2-mediated cell adhesion influences odorant receptor-specific axonal convergence and is regulated by odorant receptor signaling via the olfactory cyclic nucleotide-gated (CNG) ion channel. Accordingly, we found that inhibited RAR function correlated with reduced CNG channel expression. Naris occlusion experiments and analysis of CNG channel-deficient mice further indicated that RAR-regulated CNG channel levels influenced the intrinsic neuronal activity required for cell survival in the absence of odor stimulation. Finally, we showed that CNG channel activity regulated expression of the retinoic acid-degrading enzyme Cyp26B1. Combined, these results identify a novel homeostatic feedback mechanism involving retinoic acid metabolism and CNG channel activity, which influences glomerular homogeneity and maintenance of precisely connected OSNs.—Öztokatli, H., Hörnberg, M., Berghard, A., Bohm, S. Retinoic acid receptor and CNGA2 channel signaling are part of a regulatory feedback loop controlling axonal convergence and survival of olfactory sensory neurons.

XPteg ( Xenopus proximal tubules-expressed gene) is essential for pronephric mesoderm specification and tubulogenesis

Retinoic acid (RA) signaling is important for the early steps of nephrogenic cell fate specification. Here, we report a novel target gene of RA signaling named XPteg ( Xenopus proximal tubules-expressed gene) which is critical for pronephric development. XPteg starts to be expressed at the earliest stage of embryonic kidney specification and was restricted to the pronephric proximal tubules during kidney development. Anti-sense morpholino (MO)-mediated knockdown of XPteg perturbed formation of pronephros as demonstrated by reduced expression of pronephric tubule markers. Conversely, overexpression of XPteg promoted endogenous and ectopic expression of those markers and expanded pronephric tubules. Treatment of retinoic acid induced the expression of XPteg in the pronephric field without protein synthesis. Furthermore, we found that the pronephric defects caused by a dominant negative RA receptor could be rescued by coexpression of XPteg. Taken together, these results suggest that XPteg functions as a direct transcriptional target of RA signaling to regulate pronephric tubulogenesis in Xenopus early development.

Comprehensive Genomic Screens Reveal Multiple Modes of Action of the PLZF-RAR-α Oncoprotein

The t(11;17)(q23;q21) translocation is associated with a retinoic acid-insensitive form of acute promyelocytic leukemia (APL) involving the production of reciprocal fusion proteins PLZF-RARα and RARα-PLZF. These proteins mediate malignant transformation by binding to and dysregulating RARα/RXR and PLZF target genes, respectively. In order to investigate the molecular basis of PLZF-RARα induced leukemia, we performed a genome wide screen for PLZF-RARα direct target genes using a gain of function model in which PLZF-RARα was expressed in human U937 leukemia cells. Chromatin from U937/PLZF-RARα cells was immunoprecipitated using PLZF antibodies, amplified by ligation-mediated PCR and biological triplicates were hybridized to NimbleGen 2.7kB promoter arrays, which represent 24,659 human promoters. We identified 4916 genes directly bound by PLZF-RARα (2/3 biological replicates, FDR <0.2). These genes were highly enriched for ontological categories including immunity and defense (p<10-6), apoptosis (p<2×10-5), cell cycle (p<10-3) and oncogenesis (p<10-2). Gene expression profiling of U937/PLZF-RARα cells revealed that 34% of direct targets were also transcriptionally regulated in response to PLZF-RARα induction. Despite the established role of PLZF-RARα as a transcriptional repressor, 56% of genes bound by PLZF-RARα were upregulated and 44% repressed. Bioinformatic analysis of PLZF-RARα bound sequences using the MATRIXReduce algorithm identified the '-AGGTCA-' core sequence as the highest ranked position specific affinity matrix (PSAM). Comparison of this matrix with known transcription factor binding sites from the JASPAR core database revealed high similarity to the recognition sequence for the RAR-related orphan receptor A1 (RORA1) (E value: 5.2×10-3), RORA2 (3.5×10-2) and RXRA-VDR (4.4×10-2). This suggests that the natural binding site of PLZF-RARα is similar to that of other nuclear receptors. The 'GTCA' core sequence is frequently observed in canonical retinoic acid receptor response elements and this motif was only associated with genes repressed with binding by PLZF-RARα. Together these results are consistent with the idea that PLZF-RARα acts in large part as a dominant negative retinoic acid receptor. A comparison of genes bound directly by PLZF-RARα with gene expression profiles from 22 APL (4 PLZF-RARα, 18 PML-RARα) and 99 acute myeloid leukemias (AML) selected at random from the Erasmus University dataset, using gene set enrichment analysis, revealed that direct targets of PLZF-RARα were differentially repressed in APL when compared to other forms of AML. Overexpression of PLZF-RARα in murine hematopoietic progenitors and human CD34+ cord blood, blocked myeloid differentiation, an effect associated with the repression of C/EBP genes (α, β and ε), which were identified as direct targets of PLZF-RARα by ChIP-chip. Treatment of primary CD34+ cells with ATRA led to an increase in CEBPα and β, but repression of CEBPε was not relieved. Overexpression of PLZF-RARα in primary murine bone marrow led to an increase in the more primitive Sca1+ population, coincident with increased serial replating ability. Overexpression of PLZF-RARα in mouse and human progenitors led to increased proliferation with more cells in the S and G2/M phases of cell cycle. Correlating with this effect, genes with defined roles in hematopoietic stem cell self-renewal including HOXA9 and MPL were bound and activated by the induction of PLZF-RARα in U937 cells. Increased proliferation was also coincident with repressed expression of Cdkn2d (p19) a cyclin dependent kinase inhibitor, also directly bound by PLZF-RARα. PLZF-RARα appears to transform cells through three interlinked modes of action, inhibition of differentiation by direct repression of key myeloid transcription factors, stimulation of proliferation by repression of a cyclin dependent kinase inhibitor and activation of genes critical for self renewal.

Retinoic Acid Receptor-Dependent Survival of Olfactory Sensory Neurons in Postnatal and Adult Mice

To address the hypothesis that retinoids produced by synthesizing enzymes present in the primary olfactory system influence the mouse olfactory sensory map, we expressed a dominant-negative retinoic acid receptor selectively in olfactory sensory neurons. We show that neurons deficient in nuclear retinoid signaling are responsive to odors and form correct odorant receptor-specific axonal projections to target neurons in the olfactory bulb of the brain. Subsequent to the formation of the map, the neurons die prematurely by retrograde-driven caspase-3 activation, which resembles the previously described mechanism of neural death after olfactory bulb ablation. This neurodegenerative event is initiated the second postnatal week and occurs in the adult animal without a compensatory increase of progenitor cell proliferation. In addition, we find that nuclear retinoid signaling is required for the expression of a retinoic acid-degrading enzyme, Cyp26B1, in a small fraction of mature neurons. Collectively, the results provide evidence for a role of locally regulated retinoid metabolism in neuroprotection and in determining population size of neurons at a late stage of neural circuit formation.

Retinoid‐mediated stimulation of steroid sulfatase activity in myeloid leukemic cell lines requires RARα and RXR and involves the phosphoinositide 3‐kinase and ERK‐MAP kinase pathways

All-trans retinoic acid and 9-cis-retinoic acid stimulate the activity of steroid sulfatase in HL60 acute myeloid leukemia cells in a concentration- and time-dependent manner. Neither of these 'natural retinoids' augmented steroid sulfatase activity in a HL60 sub-line that expresses a dominant-negative retinoic acid receptor alpha (RARalpha). Experiments with synthetic RAR and RXR agonists and antagonists suggest that RARalpha/RXR heterodimers play a role in the retinoid-stimulated increase in steroid sulfatase activity. The retinoid-driven increase in steroid sulfatase activity was attenuated by inhibition of phospholipase D (PLD), but not by inhibitors of phospholipase C. Experiments with inhibitors of protein kinase C (PKC) show that PKCalpha and PKCdelta play an important role in modulating the retinoid-stimulation of steroid sulfatase activity in HL60 cells. Furthermore, we show that pharmacological inhibition of the RAF-1 and ERK MAP kinases blocked the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells and, by contrast, inhibition of the p38-MAP kinase or JNK-MAP kinase had no effect. Pharmacological inhibitors of the phosphatidylinositol 3-kinase, Akt, and PDK-1 also abrogated the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells. These results show that crosstalk between the retinoid-stimulated genomic and non-genomic pathways is necessary to increase steroid sulfatase activity in HL60 cells.