Retinoic Acid Receptor Signaling Research Articles

Inhibition of retinoic acid signaling in proximal tubular epithelial cells protects against acute kidney injury.

Retinoic acid receptor (RAR) signaling is essential for mammalian kidney development but, in the adult kidney, is restricted to occasional collecting duct epithelial cells. We now show that there is widespread reactivation of RAR signaling in proximal tubular epithelial cells (PTECs) in human sepsis-associated acute kidney injury (AKI) and in mouse models of AKI. Genetic inhibition of RAR signaling in PTECs protected against experimental AKI but was unexpectedly associated with increased expression of the PTEC injury marker Kim1. However, the protective effects of inhibiting PTEC RAR signaling were associated with increased Kim1-dependent apoptotic cell clearance, or efferocytosis, and this was associated with dedifferentiation, proliferation, and metabolic reprogramming of PTECs. These data demonstrate the functional role that reactivation of RAR signaling plays in regulating PTEC differentiation and function in human and experimental AKI.

P16-08: Development of the NPC1_RAR_GR assay to identify putative endocrine disrupting chemicals (EDCs) disturbing glucocorticoid receptor (GR) or retinoic acid receptor (RAR) signaling in vitro

P16-08: Development of the NPC1_RAR_GR assay to identify putative endocrine disrupting chemicals (EDCs) disturbing glucocorticoid receptor (GR) or retinoic acid receptor (RAR) signaling in vitro

MTHFD1 is critical for the negative regulation of retinoic acid receptor signalling in anencephaly.

Neural tube defects are the most severe congenital malformations that result from failure of neural tube closure during early embryonic development, and the underlying molecular mechanisms remain elusive. Retinoic acid, an active derivative of vitamin A, is critical for neural system development, and retinoic acid receptor (RAR) signalling malfunctions have been observed in human neural tube defects. However, retinoic acid-retinoic acid receptor signalling regulation and mechanisms in neural tube defects are not fully understood. The mRNA expression of RARs and retinoid X receptors in the different human neural tube defect phenotypes, including 11 pairs of anencephaly foetuses, 10 pairs of hydrocephalus foetuses and nine pairs of encephalocele foetuses, was investigated by NanoString nCounter technology. Immunoprecipitation-mass spectrometry was performed to screen the potential interacting targets of retinoic acid receptor γ. The interactions between proteins were confirmed by co-immunoprecipitation and immunofluorescence laser confocal microscopy. Luciferase and chromatin immunoprecipitation with quantitative real-time polymerase chain reaction assays were used to clarify the underlying mechanism. Moreover, a neural tube defect animal model, constructed using excess retinoic acid, was used for further analysis with established molecular biology technologies. We report that level of retinoic acid receptor γ (RARγ) mRNA was significantly upregulated in the brain tissues of human foetuses with anencephaly. To further understand the actions of retinoic acid receptor γ in neural tube defects, methylenetetrahydrofolate dehydrogenase 1 was identified as a specific retinoic acid receptor γ target from IP-MS screening. Additionally, methylenetetrahydrofolate dehydrogenase 1 negatively regulated retinoic acid receptor γ transcription factor activity. Furthermore, low expression of methylenetetrahydrofolate dehydrogenase 1 and activation of retinoic acid receptor signalling were further determined in human anencephaly and a retinoic acid-induced neural tube defect mouse model. This study reveals that methylenetetrahydrofolate dehydrogenase 1, the rate-determining enzyme in the one-carbon cycle, might be a specific regulator of retinoic acid receptors; these findings provide new insights into the functional linkage between nuclear folate metabolism and retinoic acid receptor signalling in neural tube defect pathology.

Single-cell analyses reveal distinct expression patterns and roles of long non-coding RNAs during hESC differentiation into pancreatic progenitors

BackgroundDeep understanding the differentiation process of human embryonic stem cells (hESCs) is essential for developing cell-based therapeutic strategy. Substantial efforts have been made to investigate protein-coding genes, yet it remains lacking comprehensive characterization of long non-coding RNAs (lncRNAs) during this process.MethodshESCs were passaged every 5–6 days and had maintained stable karyotype even until the 50th generation. Pancreatic progenitor specification of in vitro differentiation from hESCs was performed and modified. The nuclei were stained with 4,6-Diamidino-2-phenylindole (DAPI). Droplet-based platform (10X Genomics) was applied to generate the single-cell RNA sequencing (scRNA-seq) data. The quality of the filtered read pairs was evaluated by using FastQC. Batch effects were removed using the size factor method. Dimension reduction and unsupervised clustering analyses were performed using Seurat R package. The Monocle 2 and MetaCell algorithms were used to order single cells on a pseudotime course and partition the scRNA-seq data into metacells, respectively. Co-expression network was constructed using WGCNA. Module- and hub-based methods were adopted to predict the functions of lncRNAs.ResultsA total of 77,382 cells during the differentiation process of hESCs toward pancreatic progenitors were sequenced. According to the single-cell map, the cells from different time points were authenticated to constitute a relatively homogeneous population, in which a total of 7382 lncRNAs could be detected. Through further analyzing the time course data, conserved and specific expression features of lncRNAs during hESC differentiation were revealed. Based upon pseudotime analysis, 52 pseudotime-associated lncRNAs that grouped into three distinct expression patterns were identified. We also implemented MetaCell algorithm and network-based methods to explore the functional mechanisms of these lncRNAs. Totally, 464 lncRNAs, including 49 pseudotime-associated lncRNAs were functionally annotated by either module-based or hub-based methods. Most importantly, we demonstrated that the lncRNA HOTAIRM1, which co-localized and co-expressed with several HOX genes, may play crucial role in the generation of pancreatic progenitors through regulation of exocytosis and retinoic acid receptor signaling pathway.ConclusionsOur single-cell analyses provide valuable data resources for biological researchers and novel insights into hESC differentiation processes, which will guide future endeavors to further elucidate the roles of lncRNAs.

Abstract P6-12-09: Retinoic acid receptor (RAR) signalling plays a role in neratinib (NER) resistance in HER2+ breast cancer (BC) cell lines

Abstract Introduction: NER is a pan-HER tyrosine kinase inhibitor (TKI) approved for the treatment of HER2+ BC in the adjuvant setting following trastuzumab and in combination with capecitabine for advanced disease. Resistance to small molecule TKIs like NER can develop in the clinic. Pre-clinical studies have highlighted that retinoic acid can inhibit BC growth and modulate HER2 signalling pathways. The RAR family of nuclear transcription factors consists of RARα, RARβ and RARγ. The synthetic retinoic acid fenretinide (FN) acts as a pan-RAR agonist, while AGN194310 (AG) acts as a pan-RAR antagonist. In order to investigate the anti-proliferative potential of co-targeting RAR and HER2 pathways in sensitive and resistant BC cell line models, we examined the effect of FN and AG in combination with NER in two HER2+, estrogen receptor-negative, trastuzumab-resistant cell lines HCC1569 and HCC1954, and their NER-resistant (NR) sub-lines HCC1569-NR and HCC1954-NR. Methods: HCC1569 and HCC1954 cell lines were cultured in RPMI/10% FCS at 370C/5% CO2. NR cell lines were generated by continuous exposure to 150nM NER for 6 months. 10 mM stock solutions of FN (H7779-Sigma), AG (SML2665-Sigma) and NER (supplied by Puma Biotechnology, Inc) were made in DMSO. Proliferation was measured as percentage growth versus DMSO control using an acid phosphatase based assay after 5 days drug exposure. The half-maximal inhibitory concentration (IC50) was calculated for each drug using CalcuSyn. The combination assays were performed using fixed ratios. The combination index (CI) values were calculated at the effective dose that inhibits 50% growth (ED50) using CalcuSyn. Values < 1 represent a synergistic effect, a value of 1 is additive and values > 1 represent an antagonistic effect. All data presented as the mean of biological triplicate experiments ± standard deviation. Results: This research found that the NR cell lines were >10-fold resistant to NER (HCC1569-NR IC50 0.44 ± 0.1 μM, HCC1954-NR IC50 0.198 ± 0.019 μM) compared to the parental HCC1569 (IC50 0.018 ± 0.015 μM) and HCC1954 (IC50 0.017 ± 0.001 μM) cell lines. Pan-RAR agonism by FN had a potent anti-proliferative effect in the HCC1569 (FN IC50 0.22 ± 0.02 μM) and the HCC1569-NR cell lines (FN IC50 0.28 ± 0.13 μM), with the HCC1954 and HCC1954-NR cell lines proving less sensitive (IC50 6.47 ± 1.3 μM and 1.9 ± 0.2 μM, respectively). When combined with NER, FN produced a strong antagonistic effect in the HCC1569 cell line (CI value: 15.63 ± 9.5) and a strong synergistic effect in the HCC1954 cell line (CI value: 0.42 ± 0.06). For the NR cell line models, the NER/FN combination proved synergistic (HCC1569-NR, CI value: 0.84 ± 0.46) or additive (HCC1954-NR, CI value: 0.97 ± 0.15). Next, we wanted to assess the impact of antagonising rather than activating RAR activity in our cell line models. All four cell lines were less sensitive to antagonist AG (IC50 >8μM for all cell lines) compared to FN. The addition of AG to NER resulted in responses diametrically opposed to the FN/NER combination. The AG/NER combination produced a strong synergistic effect in the HCC1569 cell line (CI value: 0.52 ± 0.17), an antagonistic effect in the HCC1954 cell line (CI value: 2.1 ± 0.4) and an antagonistic effect in both NR cell lines (HCC1954-NR, CI value: 2.69 ± 0.6 and HCC1569-NR, CI value: 1.58 ± 0.12). Conclusions: This pre-clinical study suggests involvement of the RAR signalling pathway in response to NER and the development of NR. Results also suggest pan-RAR agonism, rather than pan-RAR antagonism, as a potential therapeutic strategy to overcome NR. Further investigation is warranted to determine how targeting the RAR signalling pathway may assist in the treatment of HER2+ BC. Citation Format: Debbie O’Reilly, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. Retinoic acid receptor (RAR) signalling plays a role in neratinib (NER) resistance in HER2+ breast cancer (BC) cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-09.

Segmented filamentous bacteria impede rotavirus infection via retinoic acid receptor-mediated signaling

ABSTRACT Prevention of rotavirus (RV) infection by gut-resident segmented filamentous bacteria (SFB) is an example of the influence of gut microbiota composition on enteric viral infection. Yet, the mechanism by which SFB prevents RV infection is poorly understood. A recent report that SFB colonization of germfree mice generates retinoic acid (RA) thus activating RA receptor (RAR) signaling, which protected against Citrobacter rodentium infection, prompted us to investigate whether this pathway might contribute to SFB’s protection against RV infection. Colonization of conventional mice by SFB indeed increased intestinal RA levels and direct administration of RA partially mimicked the protection against RV infection conferred by SFB. Moreover, blockade of RAR signaling eliminated SFB’s protection against RV infection. Blockade of RAR signaling did not impact RV infection in the absence of SFB, nor did it alter the protection against RV infection conferred by bacterial flagellin, which in contrast to SFB, is dependent upon IL-22 signaling. SFB/RA-mediated prevention of RV infection was associated with an RA-dependent increase in enterocyte migration, consistent with the notion that enhanced anoikis is the ultimate means by which SFB, IL-22, and RA impede RV infection.

Timeline of Developmental Defects Generated upon Genetic Inhibition of the Retinoic Acid Receptor Signaling Pathway.

It has been established for almost 30 years that the retinoic acid receptor (RAR) signalling pathway plays essential roles in the morphogenesis of a large variety of organs and systems. Here, we used a temporally controlled genetic ablation procedure to precisely determine the time windows requiring RAR functions. Our results indicate that from E8.5 to E9.5, RAR functions are critical for the axial rotation of the embryo, the appearance of the sinus venosus, the modelling of blood vessels, and the formation of forelimb buds, lung buds, dorsal pancreatic bud, lens, and otocyst. They also reveal that E9.5 to E10.5 spans a critical developmental period during which the RARs are required for trachea formation, lung branching morphogenesis, patterning of great arteries derived from aortic arches, closure of the optic fissure, and growth of inner ear structures and of facial processes. Comparing the phenotypes of mutants lacking the 3 RARs with that of mutants deprived of all-trans retinoic acid (ATRA) synthesising enzymes establishes that cardiac looping is the earliest known morphogenetic event requiring a functional ATRA-activated RAR signalling pathway.

ERK MAP Kinase Signaling Regulates RAR Signaling to Confer Retinoid Resistance on Breast Cancer Cells

Retinoic acid (RA) and its synthetic derivatives, retinoids, have been established as promising anticancer agents based on their ability to regulate cell proliferation and survival. Clinical trials, however, have revealed that cancer cells often acquire resistance to retinoid therapy. Therefore, elucidation of underlying mechanisms of retinoid resistance has been considered key to developing more effective use of retinoids in cancer treatment. In this study, we show that constitutive activation of ERK MAP kinase signaling, which is often caused by oncogenic mutations in RAS or RAF genes, suppresses RA receptor (RAR) signaling in breast cancer cells. We show that activation of the ERK pathway suppresses, whereas its inhibition promotes, RA-induced transcriptional activation of RAR and the resultant upregulation of RAR-target genes in breast cancer cells. Importantly, ERK inhibition potentiates the tumor-suppressive activity of RA in breast cancer cells. Moreover, we also reveal that suppression of RAR signaling and activation of ERK signaling are associated with poor prognoses in breast cancer patients and represent hallmarks of specific subtypes of breast cancers, such as basal-like, HER2-enriched and luminal B. These results indicate that ERK-dependent suppression of RAR activity underlies retinoid resistance and is associated with cancer subtypes and patient prognosis in breast cancers.

Spinal cord retinoic acid receptor signaling gates mechanical hypersensitivity in neuropathic pain

Central sensitization caused by spinal disinhibition is a key mechanism of mechanical allodynia in neuropathic pain. However, the molecular mechanisms underlying spinal disinhibition after nerve injury remain unclear. Here, we show in mice that spared nerve injury (SNI), which induces mechanical hypersensitivity and neuropathic pain, triggers homeostatic reduction of inhibitory outputs from dorsal horn parvalbumin-positive (PV+) interneurons onto both primary afferent terminals and excitatory interneurons. The reduction in inhibitory outputs drives hyperactivation of the spinal cord nociceptive pathway, causing mechanical hypersensitivity. We identified the retinoic acid receptor RARα, a central regulator of homeostatic plasticity, as the key molecular mediator for this synaptic disinhibition. Deletion of RARα in spinal PV+ neurons or application of an RARα antagonist in the spinal cord prevented the development of SNI-induced mechanical hypersensitivity. Our results identify RARα as a crucial molecular effector for neuropathic pain and a potential target for its treatment.

A novel RARA-SNX15 fusion in PML-RARA-positive acute promyelocytic leukemia with t(11;17;15)(q13;q21.2;q24.1).

Acute promyelocytic leukemia (APL) is characterized by a series of retinoic acid receptor (RAR) fusion genes that lead to the dysregulation of RAR signaling and onset of APL. PML-RARA is the most common fusion generated from t(15;17)(q24;q21). In addition, the reciprocal fusion RARA-PML is present in over 80% of t(15;17) APL cases. The bcr3 types of RARA-PML and RARA-PLZF in particular are reciprocal fusions that contribute to leukemogenesis. Here, we report a variant APL case with t(11;17;15)(q13;q21.2;q24.1). Massive parallel sequencing of patient RNA detected the novel fusion transcripts RARA-SNX15 and SNX15-LINC02255 along with the bcr3 type of PML-RARA. Genetic analysis revealed that RARA-SNX15L is an in-frame fusion due to intron retention caused by RNA mis-splicing. RARA-SNX15L consisted mainly of SNX15 domains, including the Phox-homology domain, which has a critical role in protein-protein interactions among sorting nexins and with other partners. Co-immunoprecipitation analysis revealed that RARA-SNX15L is directly associated with SNX15 and with itself. Further studies are needed to evaluate the biological significance of RARA-SNX15L in APL. In conclusion, this is the first report of APL with a complex chromosomal rearrangement involving SNX15.

T Cell-Intrinsic Vitamin A Metabolism and Its Signaling Are Targets for Memory T Cell-Based Cancer Immunotherapy.

Memory T cells play an essential role in infectious and tumor immunity. Vitamin A metabolites such as retinoic acid are immune modulators, but the role of vitamin A metabolism in memory T-cell differentiation is unclear. In this study, we identified retinol dehydrogenase 10 (Rdh10), which metabolizes vitamin A to retinal (RAL), as a key molecule for regulating T cell differentiation. T cell-specific Rdh10 deficiency enhanced memory T-cell formation through blocking RAL production in infection model. Epigenetic profiling revealed that retinoic acid receptor (RAR) signaling activated by vitamin A metabolites induced comprehensive epigenetic repression of memory T cell-associated genes, including TCF7, thereby promoting effector T-cell differentiation. Importantly, memory T cells generated by Rdh deficiency and blocking RAR signaling elicited potent anti-tumor responses in adoptive T-cell transfer setting. Thus, T cell differentiation is regulated by vitamin A metabolism and its signaling, which should be novel targets for memory T cell-based cancer immunotherapy.

Abstract 1519: ALDH1A1 enhances micro homology-mediated end joining by increasing POLQ expression

Abstract Aldehyde dehydrogenase (ALDH) is a superfamily of 19 known enzymes that participated in the metabolism of endogenous and exogenous aldehydes. ALDH1A1 is a major subtype contributing to high ALDH activity out of all ALDH isoforms and plays a critical role in the maintenance of cancer stem cells. Our previous studies have shown that ALDH1A1 is able to reduce the sensitivity of BRCA2-/- ovarian cancer cells to PARP inhibitors by enhancing microhomology-mediated end joining (MMEJ). MMEJ uses 5-25 base pair micro homologous sequences to align the broken strands before joining, and this repair pathway requires DNA polymerase θ (Pol θ, encoded by POLQ). Here we show that ALDH1A1 can increase the expression of Pol θ, at both mRNA and protein levels, in ovarian cancer cell lines. ALDH1A1 is reported to catalyze the production of all-trans retinoic acid (ATRA), which can bind to the retinoic acid receptor (RAR), and promote transcription of the downstream target genes that possess retinoic acid response element (RARE) in their promoter regions. Interestingly we identified several RAREs in the promoter region of the POLQ gene. Thus, we hypothesized that ALDH1A1 promotes Pol θ expression via activation of the RAR signaling pathway. We found that ATRA and RAR agonist (CH55) increased but pan RAR antagonist (AGN193109) treatment decreased the expression of POLQ, indicating that the RAR pathway is involved in the transcription of POLQ. We also demonstrated that RARα can bind to the promoter region of Pol Q, activation of RAR signaling by treatment with ATRA or CH55 increased the enrichment of K27 acetylated histone H3 in this promoter region. Moreover, we showed that mutated ALDH1A1 lacking the enzyme activity was unable to enhance POLQ transcription. These data suggest that ALDH1A1 enhances the Pol Q expression by increasing the production of ATRA which further activates the RAR signaling pathway. Citation Format: Kousalya Lavudi, Ananya Banerjee, Shurui Cai, Na Li, Xuetao Bai, Wang Qi-En. ALDH1A1 enhances micro homology-mediated end joining by increasing POLQ expression [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 1519.

Bioinformatic Prioritization and Functional Annotation of GWAS-Based Candidate Genes for Primary Open-Angle Glaucoma.

Background: Primary open-angle glaucoma (POAG) is the most prevalent glaucoma subtype, but its exact etiology is still unknown. In this study, we aimed to prioritize the most likely ‘causal’ genes and identify functional characteristics and underlying biological pathways of POAG candidate genes. Methods: We used the results of a large POAG genome-wide association analysis study from GERA and UK Biobank cohorts. First, we performed systematic gene-prioritization analyses based on: (i) nearest genes; (ii) nonsynonymous single-nucleotide polymorphisms; (iii) co-regulation analysis; (iv) transcriptome-wide association studies; and (v) epigenomic data. Next, we performed functional enrichment analyses to find overrepresented functional pathways and tissues. Results: We identified 142 prioritized genes, of which 64 were novel for POAG. BICC1, AFAP1, and ABCA1 were the most highly prioritized genes based on four or more lines of evidence. The most significant pathways were related to extracellular matrix turnover, transforming growth factor-β, blood vessel development, and retinoic acid receptor signaling. Ocular tissues such as sclera and trabecular meshwork showed enrichment in prioritized gene expression (>1.5 fold). We found pleiotropy of POAG with intraocular pressure and optic-disc parameters, as well as genetic correlation with hypertension and diabetes-related eye disease. Conclusions: Our findings contribute to a better understanding of the molecular mechanisms underlying glaucoma pathogenesis and have prioritized many novel candidate genes for functional follow-up studies.

Biomarkers of Tretinoin Precursors and Tretinoin Efficacy in Patients With Moderate to Severe Facial Photodamage

Topical formulations of tretinoin precursors (retinol and its ester derivatives) are widely available over the counter and may offer similar clinical benefits to those of tretinoin for treatment of photoaging. However, which of the many purported molecular effects of retinoids most strongly drives clinical improvements in tretinoin-treated skin remains unclear. To evaluate the clinical efficacy of topical tretinoin precursors (TTP) vs tretinoin (RA) in treating moderate to severe facial photodamage and to identify potential biomarkers that correlate with clinical efficacy. This randomized, double-blind, single-center, parallel-arm study of 24 patients with moderate to severe facial photodamage was conducted at an academic referral center from November 2010 to December 2011, with data analysis performed from January 2012 to December 2021. Daily topical application of 0.02% RA or 1.1% TTP formulation containing retinol, retinyl acetate, and retinyl palmitate for 24 weeks. Photoaging and tolerability were assessed by dermatologist evaluations and patient-reported outcomes. Target gene expression was assessed by real-time quantitative polymerase chain reaction of biopsied tissue from treated areas. A total of 20 White women were ultimately analyzed (9 randomized to TTP, 11 randomized to RA). At week 24, there was no significant difference in Griffiths photoaging scores among patients receiving TTP vs RA (median, 4 vs 5) (TTP - RA difference: -1; 95% CI, -2 to 1; P = .27). Treatment with TTP was associated with erythema 6 times less frequently than RA (11% vs 64%) (TTP - RA difference: -0.53; 95% CI, -0.88 to -0.17; P = .01). Target gene analysis showed significant CRABP2 messenger RNA (mRNA) induction (confirming retinoic acid receptor signaling) but no significant changes in procollagen I or MMP1/3/9 mRNA in TTP-treated samples. Instead, MMP2 mRNA, which encodes a type IV collagenase, was significantly reduced in TTP-treated samples (week 24 - baseline mRNA difference: -5; 96% CI, -33 to 1.6; P = .02), and changes in MMP2 were strongly correlated with changes in fine wrinkles (r = 0.54; 95% CI, 0.12 to 0.80; P = .01). Interestingly, patients with severe baseline wrinkles exhibited greater improvements (r = -0.74; 95% CI, -0.89 to -0.43; P < .001). This trend was mirrored in MMP2 mRNA, with initial expression strongly predicting subsequent changes (r = -0.78; 95% CI, -0.89 to -0.43; P < .001). In this randomized clinical trial, there was no significant difference in efficacy between this particular formulation of TTP and tretinoin 0.02%. However, the results of these mechanistic studies highlight MMP2 as a possible mediator of retinoid efficacy in photoaging. ClinicalTrials.gov Identifier: NCT01283464.

Retinoic acid signaling acts as a rheostat to balance Treg function.

Regulatory T cells (Tregs) promote immune homeostasis by maintaining self-tolerance and regulating inflammatory responses. Under certain inflammatory conditions, Tregs can lose their lineage stability and function. Previous studies have reported that ex vivo exposure to retinoic acid (RA) enhances Treg function and stability. However, it is unknown how RA receptor signaling in Tregs influences these processes in vivo. Herein, we employed mouse models in which RA signaling is silenced by the expression of the dominant negative receptor (DN) RARα in all T cells. Despite the fact that DNRARα conventional T cells are hypofunctional, Tregs had increased CD25 expression, STAT5 pathway activation, mTORC1 signaling and supersuppressor function. Furthermore, DNRARα Tregs had increased inhibitory molecule expression, amino acid transporter expression, and metabolic fitness and decreased antiapoptotic proteins. Supersuppressor function was observed when wild-type mice were treated with a pharmacologic pan-RAR antagonist. Unexpectedly, Treg-specific expression of DNRARα resulted in distinct phenotypes, such that a single allele of DNRARα in Tregs heightened their suppressive function, and biallelic expression led to loss of suppression and autoimmunity. The loss of Treg function was not cell intrinsic, as Tregs that developed in a noninflammatory milieu in chimeric mice reconstituted with DNRARα and wild-type bone marrow maintained the enhanced suppressive capacity. Fate mapping suggested that maintaining Treg stability in an inflammatory milieu requires RA signaling. Our findings indicate that RA signaling acts as a rheostat to balance Treg function in inflammatory and noninflammatory conditions in a dose-dependent manner.

Vitamin A regulates phagocytosis by resident macrophages of the small intestine

Abstract Intestinal Tim4+ CD4+ macrophages are a distinctive macrophage subset that express Tim4, a receptor for phosphatidylserine on dying apoptotic cells, Unlike other macrophage subsets, they do not depend on blood monocytes for their turnover, instead self-maintained in the small intestine. The signal(s) responsible for the self-maintenance and function of Tim4+ CD4+ macrophages is not known. We have discovered that maintenance of the gut resident Tim4+ CD4+ macrophage population depends on dietary vitamin A and its derivative retinoic acid (RA). Retinoic acid receptors, which direct RA-dependent transcription, were required for maintenance of Tim4+ CD4+ macrophages. Chemical blockade of retinoic acid receptor (RAR) signaling and macrophage-specific genetic inactivation of RARs in mice further revealed that macrophage-intrinsic RARα signaling was required for Timd4 expression and maintenance of Tim4+ CD4+ macrophages. Macrophage RARα signaling was furthermore essential for phagocytosis by Tim4+ CD4+ macrophages. Ongoing studies are examining the role of Tim4+ CD4+ macrophages and vitamin A in the clearance of apoptotic intestinal epithelial cells. Our findings reveal that vitamin A provides an essential dietary signal for the maintenance and function of a gut resident macrophage subset. Supported by Welch foundation grant I-1874

Palovarotene Can Attenuate Heterotopic Ossification Induced by Tendon Stem Cells by Downregulating the Synergistic Effects of Smad and NF-κB Signaling Pathway following Stimulation of the Inflammatory Microenvironment.

Heterotopic ossification (HO) is defined as the formation of bone tissues outside the bones, such as in the muscles. Currently, the mechanism of HO is still unclear. Tendon stem cells (TSCs) play important roles in the occurrence and development of HO. The inflammatory microenvironment dominated by macrophages also plays an important role in the course of HO. The commonly used clinical treatment methods, such as nonsteroidal anti-inflammatory drugs and radiotherapy, have relatively large side effects, and more efficient treatment methods are needed in clinical practice. Under physiological conditions, retinoic acid receptor (RAR) signal transduction pathway inhibits osteogenic progenitor cell aggregation and chondrocyte differentiation. We focus on palovarotene, a retinoic acid γ-receptor activator, showing an inhibitory effect on HO mice, but the specific mechanism is still unclear. This study was aimed at exploring the specific molecular mechanism of palovarotene by blocking osteogenic differentiation and HO formation of TSCs in vitro and in vivo in an inflammatory microenvironment. We constructed a coculture model of TCSs and polarized macrophages, as well as overexpression and knockdown models of the Smad signaling pathway of TCSs. In addition, a rat model of HO, which was constructed by Achilles tendon resection, was also established. These models explored the role of inflammatory microenvironment and Smad signaling pathways in the osteogenic differentiation of TSCs which lead to HO, as well as the reversal role played by palovarotene in this process. Our results suggest that, under the stimulation of inflammatory microenvironment and trauma, the injured site was in an inflammatory state, and macrophages were highly concentrated in the injured site. The expression of osteogenic and inflammation-related proteins, as well as Smad proteins, was upregulated. Osteogenic differentiation was performed in TCSs. We also found that TCSs activated Smad and NF-κB signaling pathways, which initiated the formation of HO. Palovarotene inhibited the aggregation of osteogenic progenitor cells and macrophages and attenuated HO by blocking Smad and NF-κB signaling pathways. Therefore, palovarotene may be a novel HO inhibitor, while other drugs or antibodies targeting Smad and NF-κB signaling pathways may also prevent or treat HO. The expressions of Smad5, Id1, P65, and other proteins may predict HO formation.

Abstract P2-13-28: Inhibiting retinoic acid receptor signalling enhances the effect of neratinib in HER2 positive breast cancer cell lines

Abstract Introduction: Treatment strategies for HER2+ breast cancer (BC) involves first line treatment with the monoclonal antibody trastuzumab, however resistance to treatment can occur. The pan-HER tyrosine kinase inhibitor neratinib is an approved adjuvant treatment for early stage HER2+ BC after one year of treatment with trastuzumab. Recent studies have highlighted the potential benefits of using retinoic acid as an adjuvant treatment for BC. The retinoic acid receptor (RAR) family of nuclear transcription factors consists of RARα, RARβ and RARγ. Interestingly ~30% of HER2+ BC have a co-amplification of the RARA gene, which codes for RARα. We examined the effect of RARα agonists (Fenretinide &amp; AM580) and antagonist (AGN194310) in combination with neratinib in two HER2+ cell lines: SKBR3, has co-amplification of ERBB2 and RARA, and HCC1569 has amplified ERBB2 but are not RARA-amplified. Methods: SKBR3 and HCC1569 cell lines were cultured in RPMI/10% FCS at 370C/5% CO2. 10mM stock concentrations of fenretinide (H7779-Sigma), AM580 (A8843-Sigma), AGN194310 (SML2665-Sigma) and neratinib (Puma Biotechnology, Inc) were prepared in DMSO. For proliferation assays, cells were seeded in 96 well plates at a density of 3 X 104 cells per well for 24h. Cells were treated with a drug alone (2X concentration) or combination of drugs (4X concentration) in 100μL of medium. Proliferation was measured using an acid phosphatase-based assay after 5 days as percentage growth versus DMSO control. The half maximal inhibitory concentration (IC50) was calculated for each drug, using CalcuSyn. The combination assays were performed using fixed ratios. The combination index (CI) values were calculated at the effective dose that inhibits 50% growth (ED50), using CalcuSyn. Values &amp;lt; 1 represent a synergistic effect, a value of 1 is additive and values &amp;gt; 1 represent an antagonistic effect. All data presented as the mean of biological triplicate experiments ± standard deviation. Results: This research has found that the synthetic retinoic acid fenretinide, a pan-RAR (α, β and γ) activator, was more potent in the HCC1569 cells compared to the SKBR3 cell line with an IC50 of 0.21 ± 0.03μM compared to 4.55 ± 0.87 μM, respectively. However, when combined with neratinib there was a strong antagonistic effect observed in the HCC1569 cell line (CI value: 15.63 ± 9.5). Conversely, fenretinide enhanced the effect of neratinib in the SKBR3 cell line (CI value: 0.83 ± 0.4). The RARα-specific activator AM580 had an IC50 &amp;gt;10μM for both cell lines. When combined with neratinib there was an additive effect observed in the RARα negative HCC1569 cells (CI value: 0.97 ± 0.4), and a synergistic effect observed in the RARα upregulated SKBR3 cell line (CI value: 0.78 ± 0.08). Next we wanted to determine if inhibiting RAR activity would have any effect on the proliferation of these cell lines. The IC50 for the pan-RAR (α, β and γ) antagonist AGN194310 was &amp;gt;10μM for each cell line. However, when combined with neratinib treatment there was a strong synergistic effect observed in the HCC1569 cell line (CI value: 0.52 ± 0.17) and the SKBR3 cell line (CI value: 0.66 ± 0.19). Conclusions: This study suggests a differential effect of RAR agonists when combined with neratinib in RARα amplified versus non-amplified HER2+ BC. Targeting RAR signalling, particularly with a RAR antagonist, in combination with the pan-HER inhibitor warrants further investigation in HER2+ BC. Citation Format: Debbie O'Reilly, Nicola Gaynor, Neil Conlon, Irmina Diala, Lisa D Eli, John Crown, Denis M Collins. Inhibiting retinoic acid receptor signalling enhances the effect of neratinib in HER2 positive breast cancer cell lines [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-28.

Dysregulated retinoic acid signaling in airway smooth muscle cells in asthma.

Vitamin A deficiency has been shown to exacerbate allergic asthma. Previous studies have postulated that retinoic acid (RA), an active metabolite of vitamin A and high-affinity ligand for RA receptor (RAR), is reduced in airway inflammatory condition and contributes to multiple features of asthma including airway hyperresponsiveness and excessive accumulation of airway smooth muscle (ASM) cells. In this study, we directly quantified RA and examined the molecular basis for reduced RA levels and RA-mediated signaling in lungs and ASM cells obtained from asthmatic donors and in lungs from allergen-challenged mice. Levels of RA and retinol were significantly lower in lung tissues from asthmatic donors and house dust mite (HDM)-challenged mice compared to non-asthmatic human lungs and PBS-challenged mice, respectively. Quantification of mRNA and protein expression revealed dysregulation in the first step of RA biosynthesis consistent with reduced RA including decreased protein expression of retinol dehydrogenase (RDH)-10 and increased protein expression of RDH11 and dehydrogenase/reductase (DHRS)-4 in asthmatic lung. Proteomic profiling of non-asthmatic and asthmatic lungs also showed significant changes in the protein expression of AP-1 targets consistent with increased AP-1 activity. Further, basal RA levels and RA biosynthetic capabilities were decreased in asthmatic human ASM cells. Treatment of human ASM cells with all-trans RA (ATRA) or the RARγ-specific agonist (CD1530) resulted in the inhibition of mitogen-induced cell proliferation and AP-1-dependent transcription. These data suggest that RA metabolism is decreased in asthmatic lung and that enhancing RAR signaling using ATRA or RARγ agonists may mitigate airway remodeling associated with asthma.

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.