Retinoic Acid Signaling Pathway Research Articles

MiR-1 as a Key Epigenetic Regulator in Early Differentiation of Cardiac Sinoatrial Region.

A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA sequence itself. In particular, miR-1 is considered the first essential microRNA in cardiac development. In this study, miR-1 potential role in early cardiac chamber differentiation was analyzed through specific signaling pathways. For this, we performed in chick embryos functional experiments by means of miR-1 microinjections into the posterior cardiac precursors-of both primitive endocardial tubes-committed to sinoatrial region fates. Subsequently, embryos were subjected to whole mount in situ hybridization, immunohistochemistry and RT-qPCR analysis. As a relevant novelty, our results revealed that miR-1 increased Amhc1, Tbx5 and Gata4, while this microRNA diminished Mef2c and Cripto expressions during early differentiation of the cardiac sinoatrial region. Furthermore, we observed in this developmental context that miR-1 upregulated CrabpII and Rarß and downregulated CrabpI, which are three crucial factors in the retinoic acid signaling pathway. Interestingly, we also noticed that miR-1 directly interacted with Hdac4 and Calm1/Calmodulin, as well as with Erk2/Mapk1, which are three key factors actively involved in Mef2c regulation. Our study shows, for the first time, a key role of miR-1 as an epigenetic regulator in the early differentiation of the cardiac sinoatrial region through orchestrating opposite actions between retinoic acid and Mef2c, fundamental to properly assign cardiac cells to their respective heart chambers. A better understanding of those molecular mechanisms modulated by miR-1 will definitely help in fields applied to therapy and cardiac regeneration and repair.

MiR-1 modulates HDAC4, calmodulin and MAPK1/Erk2 during early atrial differentiation

Abstract Funding Acknowledgements None. Introduction A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA sequence itself. In particular, miR-1 is considered the first essential microRNA in cardiac development. Purpose The aim of this work is to analyze the miR-1 modulation role in cardiac chamber differentiation through specific signaling pathways. Methods By means of microinjections in both primitive endocardial tubes of chick embryo culture, gain- and loss-of-function experiments were performed with premiR-1 and anti-miR-1, respectively. Subsequently, embryos were subjected to whole mount in situ hybridization with Tbx5, Gata4 and AMHC1 probes, as well as immunohistochemistry with Mef2c, HDAC4, Calmodulin/Calm1 and MAPK1/Erk2 antibodies. Also, we carried out RT-qPCR analysis of control and experimental embryos, including CRABPI, CRABPII and retinoic acid receptors (RAR and RXR). Results Our results reveal that miR-1 increases specific atrial gene expression such as Tbx5, Gata4 and AMHC1, while this microRNA diminishes Mef2c expression. Furthermore, we observed that miR-1 upregulates CRABPII and RARß, and downregulates CRABPI, which are three crucial factors in retinoic acid signaling pathway. Interestingly, we also observed that miR-1 actively interacts with HDAC4, Calmodulin and Erk2/MAPK1, key factors involved in Mef2c regulation. Conclusion All these data suggest that miR-1 functions as an epigenetic factor modulating complementary actions performed by retinoic acid and Mef2c, which are required to properly assign cells as sinoatrial precursors.

Association of the retinol to all-trans retinoic acid pathway with autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex group of neurodevelopmental disorders. Research has highlighted a close association between the retinoic acid (RA) signaling pathway and ASD. This study investigates alterations in the vitamin A (VA, retinol) to RA metabolic pathway in children with ASD and speculates on the underlying reasons for these changes. We propose a subtype characterized by downregulated RA signaling in ASD, laying the groundwork for precise diagnosis and treatment research. We included 489 children with ASD and 280 typically developing (TD) children. Those with ASD underwent evaluations of core symptoms and neuro-developmental levels, which were conducted by professional developmental behavior physicians using assessment scales. Serum VA and all-trans RA (atRA) levels were determined by high-performance liquid chromatography and ultra-high-performance liquid chromatography-tandem mass spectrometry. The expression levels and concentrations of enzyme molecules such as retinol dehydrogenase 10 were assessed using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Children with ASD exhibited reduced serum atRA, accompanied by a downregulation of atRA synthesis enzymes. The reduction in serum atRA levels was linked not only to VA levels but also to the aberrant expression of metabolic enzymes responsible for atRA. Furthermore, the serum atRA levels in children with ASD were more strongly correlated with core symptoms and neurodevelopmental levels than VA levels. Children with ASD exhibited a dual regulation of reduced serum atRA levels, influenced by both VA levels and abnormal expression of atRA metabolic enzymes.

Gene Expression, Morphology, and Electrophysiology During the Dynamic Development of Human Induced Pluripotent Stem Cell-Derived Atrial- and Ventricular-Like Cardiomyocytes.

Gene expression, morphology, and electrophysiological combination are essential for assessing the dynamic development of human induced pluripotent stem cell-derived atrial- and ventricular-like cardiomyocytes (iPS-AM and iPS-VM, respectively). For iPS-AM/VM differentiation, we performed the small molecule-based temporal modulation of the retinoic acid and bone morphogenetic protein signaling pathways. We investigated the gene expression and morphology using immunofluorescence, quantitative real-time polymerase chain reaction, flow cytometry, and transmission electron microscopy as well as registered electrophysiological functions using a whole-cell patch clamp on days 20, 30, and 60 post-differentiations. Pan-cardiomyocyte marker, including troponin T2 (TNNT2) and alpha-actinin-2 (ACTN2), expressions increased both in iPS-AMs and iPS-VMs. Similarly, the mRNA expression of both iPS-AM-specific markers, ie, natriuretic peptide A (NPPA), myosin light chain 7 (MYL7), and K+ channel Kir3.4 (KCNJ5), and iPS-VM-specific markers, ie, gap junction α-1 (GJA1), myosin light chain 2 (MYL2), and alpha-1-subunit of a voltage-dependent L-type calcium channel (CACNA1C), increased from 0 to 20 days, and then decreased from 30 to 60 days. Concerning morphology, cardiac troponin-T (cTnT) arrangement was progressively organized and developed from a disorderly myofibrillar distribution to an organized sarcomere pattern both in iPS-AMs and iPS-VMs. Mitochondrial numbers gradually increased and those of lipid droplets decreased during dynamic development. Regarding physiological function, the resting and action potential amplitudes remained statistically indifferent in both cell types, and the action potential duration was prolonged during the development. IPS-AMs/VMs displayed dynamic development concerning their gene expression, morphology, and electrophysiological function. The discoveries of this study could provide novel insights into heart development and encourage further research.

Retinol and retinol binding protein 4 levels and COVID-19: a Mendelian randomization study

BackgroundThe Corona Virus Disease 2019 (COVID-19) pandemic has struck globally. Whether the related proteins of retinoic acid (RA) signaling pathway are causally associated with the risk of COVID-19 remains unestablished. We conducted a two-sample Mendelian randomization (MR) study to assess the associations of retinol, retinol binding protein 4 (RBP4), retinol dehydrogenase 16 (RDH16) and cellular retinoic acid binding protein 1 (CRABP1) with COVID-19 in European population.MethodsThe outcome utilized the summary statistics of COVID-19 from the COVID-19 Host Genetics Initiative. The exposure data were obtained from public genome wide association study (GWAS) database. We extracted SNPs from exposure data and outcome data. The inverse variance weighted (IVW), MR-Egger and Wald ratio methods were employed to assess the causal relationship between exposure and outcome. Sensitivity analyses were performed to ensure the validity of the results.ResultsThe MR estimates showed that retinol was associated with lower COVID-19 susceptibility using IVW (OR: 0.69, 95% CI: 0.53–0.90, P: 0.0065), whereas the associations between retinol and COVID-19 hospitalization or severity were not significant. RBP4 was associated with lower COVID-19 susceptibility using the Wald ratio (OR: 0.83, 95% CI: 0.72–0.95, P: 0.0072). IVW analysis showed RDH16 was associated with increased COVID-19 hospitalization (OR: 1.10, 95% CI: 1.01–1.18, P: 0.0199). CRABP1 was association with lower COVID-19 susceptibility (OR: 0.95, 95% CI: 0.91–0.99, P: 0.0290) using the IVW.ConclusionsWe found evidence of possible causal association of retinol, RBP4, RDH16 and CRABP1 with the susceptibility, hospitalization and severity of COVID-19. Our study defines that retinol is significantly associated with lower COVID-19 susceptibility, which provides a reference for the prevention of COVID-19 with vitamin A supplementation.

Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.

Abstract 5430: Discovery of CYP26A1 as a link between WNT and retinoic acid signaling pathways that modulates retinoid-induced differentiation of APC-mutant colorectal cancer cells

Abstract APC mutation is the main driver mechanism of CRC development and leads to constitutively activated WNT signaling, overpopulation of ALDH+ stem cells (SCs), and incomplete differentiation. We previously reported that retinoic acid (RA) receptors are selectively expressed in ALDH+ SCs, which provides a way to target cancer SCs with retinoids to induce differentiation. We hypothesize that a functional link exists between WNT and RA pathways, and APC mutation generates a WNT:RA imbalance that decreases retinoid-induced differentiation and increases ALDH+ SCs. Accordingly, to restore parity in WNT:RA signaling, we induced wt-APC expression in APC-mutant CRC cells and assessed the ability of all-trans-retinoic acid (ATRA) to induce differentiation. Notably, ATRA substantially increased expression of the WNT-target gene, CYP26A1, and inducing wt-APC reduced this expression by 50%. Thus, RA and WNT pathways crosstalk to modulate CYP26A1 metabolism of retinoids and retinoid agents. We found that inducing wt-APC augments ATRA-induced cell differentiation by: i) decreasing cell proliferation; ii) suppressing ALDH1A1 expression; iii) decreasing ALDH+ SCs; and iv) increasing neuroendocrine cell differentiation. NanoString profiling and bioinformatics also identified a novel CYP26A1-based network that links WNT and RA signaling. Moreover, in an effort to translate manipulation of the WNT/RA imbalance as a therapeutic approach in vitro, we treated HT29, SW480, and HCT116 CRC cells with inhibitors against CYP26A1 enzyme activity and WNT signaling. We found that CYP26A1 inhibitors sensitized CRC cells to the anti-proliferative effect of drugs that downregulate WNT signaling. Furthermore, decreased CYP26A1 expression in tumor vs normal tissue was determined to be a predictor of patient survival in patients who have wt-APC. Thus, by inducing wt-APC expression and exogenously activating RA signaling, we found a way to increase differentiation of SCs along the NEC (and other) lineage(s). Translating this strategy in vivo might lead to new, more effective treatments involving retinoids for CRC patients. Citation Format: Caroline O. Facey, Victoria O. Hunsu, Chi Zhang, Brian T. Osmond, Lynn M. Opdenaker, Bruce M. Boman. Discovery of CYP26A1 as a link between WNT and retinoic acid signaling pathways that modulates retinoid-induced differentiation of APC-mutant colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5430.

Abstract 5452: AXIN2 differentially modulates stem cell subpopulations providing a potential therapeutic opportunity for targeting colorectal cancer stem cells

Abstract Constitutively activated WNT-signaling promotes stem cell (SC) overpopulation which drives most colorectal cancer (CRC) development. It is not known, however, how WNT-signaling activity regulates different SC sub-populations. We previously reported that SC population size decreases as AXIN2 is knocked down, and increases when AXIN2 is overexpressed. Changes in cell cycle and apoptotic rate were also observed. Here, we present our recent results on changes between different SC subpopulations (ALDH+ vs. LGR5+) that occur due to AXIN2 modulation. We hypothesize that modulation of AXIN2 promotes differential expansion between different CRC SC subpopulations (ALDH+ and LGR5+). We observed that AXIN2 not only increases LGR5+ SC population size, but also expands the ALDH+ SC population to a greater extent as shown by an increased ALDH+-to-LGR5+ SCs ratio. This indicates that AXIN2, via WNT signaling, regulates SC population size, particularly ALDH+ SCs. Since the ALDH SC marker is also a key component in the retinoic acid (RA) signaling pathway, we surmised that increased AXIN2 would not only lead to elevated ALDH levels, but also upregulate the RA-signaling pathway components in CRC cells. To this end, we conducted NanoString mRNA profiling to study the effects of AXIN2 knockdown and overexpression in HT29 and SW480 CRC cells. The results showed that increased AXIN2 significantly up-regulated several key components of the RA-signaling pathway. Taken together, our results indicate that AXIN2 drives a shift in SC subpopulation sizes favoring the ALDH+ SCs. It also promotes the RA-signaling component expression - a pathway known to regulate SC differentiation. As a potential treatment strategy for CRC, therapeutically inducing high levels of AXIN2 could shift the balance between SC populations towards ALDH+ SCs, which may provide a way to target cancer SCs with retinoids to induce their differentiation. Citation Format: Chi Zhang, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. AXIN2 differentially modulates stem cell subpopulations providing a potential therapeutic opportunity for targeting colorectal cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5452.

Differentiation of Pluripotent Stem Cells for Disease Modeling: Learning from Heart Development.

Heart disease is a pressing public health problem and the leading cause of death worldwide. The heart is the first organ to gain function during embryogenesis in mammals. Heart development involves cell determination, expansion, migration, and crosstalk, which are orchestrated by numerous signaling pathways, such as the Wnt, TGF-β, IGF, and Retinoic acid signaling pathways. Human-induced pluripotent stem cell-based platforms are emerging as promising approaches for modeling heart disease in vitro. Understanding the signaling pathways that are essential for cardiac development has shed light on the molecular mechanisms of congenital heart defects and postnatal heart diseases, significantly advancing stem cell-based platforms to model heart diseases. This review summarizes signaling pathways that are crucial for heart development and discusses how these findings improve the strategies for modeling human heart disease in vitro.

Abstract PR002: Proteomic profiling of endometrial carcinomas

Abstract While endometrial cancer (EC) has an overall favorable prognosis, some patients do poorly and may benefit from refinements of current classification systems. The TCGA-inspired pragmatic molecular classification tool Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) has been integrated into international guidelines for EC risk stratification and management. ProMisE stratifies ECs into four prognostic groups: POLEmut, NSMP (no specific molecular profile), MMRd (mismatch repair deficient), and p53abn, where POLEmut has the best prognosis and p53abn has the worst prognosis. Our objective was to determine if proteomic profiling could provide additional prognostic or predictive information for EC patients, across or within ProMisE molecular subtypes. Global proteome profiling of FFPE samples, that had clinicopathologic and outcome data, was performed on 184 ECs encompassing all four ProMisE subtypes, including replicate samples of the same tumor, and both biopsy and final hysterectomy specimens. To ensure representation of each subtype, we aimed for an approximately equal distribution; 40 (27 %) MMRd, 33 (22 %) POLEmut, 42 (28 %) NSMP and 33 (22 %) p53abn, rather than the population-based distributions. There was high reproducibility in the proteomic profiles of intra-tumor replicate samples, and between matched biopsy and hysterectomy tumor samples (Pearson’s correlation >0.9). Consensus clustering generated four clusters, named ‘Adhesion’, ‘Immune’, ‘Proliferation’, and ‘Metabolic’ based on proteins enriched in each cluster. The Proliferation Cluster had the worst outcomes and the highest proportion of stage III/IV, serous, and p53abn tumors than the other clusters. The Immune Cluster had the most favorable outcomes, despite having a relatively substantial proportion of stage III/IV, serous, and p53abn tumors. We correlated protein expression with common mutations, including ARID1A mutations that cause loss of ARID1A protein expression, found in up to 60% ECs. ARID1A positive tumors also express proteins in the retinoic acid signaling pathway, while ARID1A-deficient tumors express proteins indicative of neutrophil infiltration. Comparing molecular subtypes, we found p53abn ECs were enriched in proteins associated with poor outcomes in many tumor types, such as GRB7. We validated elevated GRB7 expression in p53abn tumors using immunohistochemistry and found an association with worse disease specific survival (DSS) across the whole cohort (HR=2.39). Nucleolin expression was associated with worse prognosis in the NSMP subtype (DSS HR=9.88), while BABAM1 expression was associated with better prognosis within p53abn tumors (DSS HR=2.43). Knockout of BABAM1 (part of the BRCA complex) in cell lines resulted in increased sensitivity to PARP inhibition. Proteomic analysis of EC identifies candidate prognostic markers that may further refine current molecular classification and help guide treatment decisions. New therapeutic interventions could be developed to target proteins and pathways identified by EC proteomic profiling. Citation Format: Dawn R. Cochrane, Gian Luca Negri, Jutta Huvila, Juliana Sobral de Barros, Forouh Kalantari, Nissreen Mohammad, David Farnell, Emily Thompson, Amy Lum, Sandra E. Spencer, Amy Jamieson, Samuel Leung, Derek Chiu, Martin Koebel, Stefan Kommoss, Friedrich Kommoss, Blake Gilks, Lien Hoang, David Huntsman, Gregg B. Morin, Jessica N. McAlpine. Proteomic profiling of endometrial carcinomas [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR002.

Retinoids and EZH2 inhibitors cooperate to orchestrate anti-oncogenic effects on bladder cancer cells.

The highly mutated nature of bladder cancers harboring mutations in chromatin regulatory genes opposing Polycomb-mediated repression highlights the importance of targeting EZH2 in bladder cancer. Furthermore, the critical role of the retinoic acid signaling pathway in the development and homeostasis of the urothelium, and the anti-oncogenic effects of retinoids are well established. Therefore, our aim is to simultaneously target EZH2 and retinoic acid signaling in bladder cancer to potentiate the therapeutic response. Here we report that this coordinated targeting strategy stimulates an anti-oncogenic profile, as reflected by inducing a synergistic reduction in cell viability that was associated with increased apoptosis and cell cycle arrest in a cooperative and orchestrated manner. This study characterized anti-oncogenic transcriptional reprogramming centered on the transcriptional regulator CHOP by stimulating the endoplasmic reticulum stress response. We further portrayed a molecular mechanism whereby EZH2 maintains H3K27me3-mediated repression of a subset of genes involved in unfolded protein responses, reflecting the molecular mechanism underlying this co-targeting strategy. These findings highlight the importance of co-targeting the EZH2 and retinoic acid pathway in bladder cancers and encourage the design of novel treatments employing retinoids coupled with EZH2 inhibitors in bladder carcinoma.

Zebrafish cobll1a regulates lipid homeostasis via the RA signaling pathway.

The COBLL1 gene has been implicated in human central obesity, fasting insulin levels, type 2 diabetes, and blood lipid profiles. However, its molecular mechanisms remain largely unexplored. In this study, we established cobll1a mutant lines using the CRISPR/Cas9-mediated gene knockout technique. To further dissect the molecular underpinnings of cobll1a during early development, transcriptome sequencing and bioinformatics analysis was employed. Our study showed that compared to the control, cobll1a -/- zebrafish embryos exhibited impaired development of digestive organs, including the liver, intestine, and pancreas, at 4 days post-fertilization (dpf). Transcriptome sequencing and bioinformatics analysis results showed that in cobll1a knockout group, the expression level of genes in the Retinoic Acid (RA) signaling pathway was affected, and the expression level of lipid metabolism-related genes (fasn, scd, elovl2, elovl6, dgat1a, srebf1 and srebf2) were significantly changed (p < 0.01), leading to increased lipid synthesis and decreased lipid catabolism. The expression level of apolipoprotein genes (apoa1a, apoa1b, apoa2, apoa4a, apoa4b, and apoea) genes were downregulated. Our study suggest that the loss of cobll1a resulted in disrupted RA metabolism, reduced lipoprotein expression, and abnormal lipid transport, therefore contributing to lipid accumulation and deleterious effects on early liver development.

Exposure of male adult zebrafish (Danio rerio) to triphenyl phosphate (TPhP) induces eye development disorders and disrupts neurotransmitter system-mediated abnormal locomotor behavior in larval offspring

Triphenyl phosphate (TPhP) is a widely used organophosphorus flame retardant, which has become ubiquitous in the environment. However, little information is available regarding its transgenerational effects. This study aimed to investigate the developmental toxicity of TPhP on F1 larvae offspring of adult male zebrafish exposed to various concentrations of TPhP for 28 or 60 days. The findings revealed significant morphological changes, alterations in locomotor behavior, variations in neurotransmitter, histopathological changes, oxidative stress levels, and disruption of Retinoic Acid (RA) signaling in the F1 larvae. After 28 and 60 days of TPhP exposure, the F1 larvae exhibited a myopia-like phenotype with pathological alterations in the lens and retina. The genes involved in the RA signaling pathway were down-regulated following parental TPhP exposure. Swimming speed and total distance of F1 larvae were significantly reduced by TPhP exposure, and long-term exposure to environmental levels of TPhP had more pronounced effects on locomotor behavior and neurotransmitter levels. In conclusion, TPhP induced histological and morphological alterations in the eyes of F1 larvae, leading to visual dysfunction, disruption of RA signaling and neurotransmitter systems, and ultimately resulting in neurobehavioral abnormalities. These findings highlight the importance of considering the impact of TPhP on the survival and population reproduction of wild larvae.

Change of the retinoic acid pathway in hypothalamus and pituitary damage induced by combined exposure to low-level Pb~(2+) and 1-nitropyrene in mice

To observe the expression of the retinoic acid(RA) pathway in hypothalamus and pituitary damage induced by combined exposure of low-level lead and 1-nitropyrene in mice, and to explore the relationship between the changes of RA pathway and hypothalamus and pituitary damage. A total of 84 4-week-old ICR mice were randomly divided into the control group, Pb~(2+) tainted group(0.008 mg/L), 1-NP tainted group(0.1 mg/kg), low(0.008 mg/L Pb~(2+)+0.004 mg/kg 1-NP), medium(0.008 mg/L Pb~(2+)+0.02 mg/kg 1-NP), and high-dose co-toxicity group(0.008 mg/L Pb~(2+)+0.1 mg/kg 1-NP) according to body weight, with 14 mice in each group. Among them, Pb~(2+) was provided by lead acetate, added to deionized water and ingested by mice drinking freely, 1-NP was given by intraperitoneal injection, 1-NP was administered by intraperitoneal injection. Record daily water intake and food intake. After 21 consecutive days of exposure, body mass was measured, histological changes in the hypothalamus and pituitary were observed under an optical microscope, and lead content in brain tissue was measured by atomic absorption spectrometry. The real-time fluorescence quantitative PCR was used to detect the abundance of retinoic acid pathway members and c-Jun N-terminal kinases genes(Jnks), and the western blot method was used to detect expression levels of acetaldehyde dehydrogenase 2(ALDH2), cytochrome P450 family member 26A1(CYP26a1) proteins. There was no difference in the mean weekly water intake and food intake of the mice in each group. The body weight of the high-dose co-toxicity group mice((27.4±1.9)g) was lower than that of the control group((29.8±2.3)g)(P&lt;0.05). The level of serum follicle-stimulating hormone(FSH) in the middle and high dose co-toxicity groups((265.01±2.99), (260.42±3.61)pg/mL, respectively) was lower than that in the control group((279.00±1.30)pg/mL, P&lt;0.05). The content of Pb~(2+) in the brain of each group containing Pb~(2+) was higher than that of the control group. In the hypothalamic and pituitary tissues, the abundance of Adh1, Adh2, Rar and Rxr, and ALDH2 levels in the medium and high dose co-toxicity groups were higher than those in the control group(P&lt;0.05). Cyp26a1 gene abundance and protein levels were lower in the medium and high dose co-toxicity groups than in the control group(P&lt;0.05). The abundance of Jnks in the high-dose co-toxicity group was higher than that in the control group(P&lt;0.05). Continuous exposure to 0.008 mg/L Pb~(2+)+0.1 mg/kg 1-NP for 21 days can cause damage to the hypothalamus and pituitary of mice, and activate the RA signaling pathway.

OR09-04 Ovarian Stimulation Disrupts Expression Of The Retinoic Acid (RA) Signaling Pathway In The Human Endometrium

Abstract Disclosure: Q. Zhao: None. P. Timmins: None. M. Saad-Naguib: None. C. Samuels: None. R. Loia: None. T. Wu: None. A. Chemerinski: None. S.S. Morelli: None. A.V. Babwah: None. N. Douglas: None. Ovarian stimulation (OS) is employed to recruit multiple dominant follicles and mature oocytes for in vitro fertilization (IVF). OS followed by a fresh embryo transfer (ET) is associated with decreased pregnancy rates. By better understanding the processes which prepare the endometrium for implantation, we hope to improve outcomes following OS. To understand the impact of OS on the endometrium, we performed an in-depth comparative analysis of the human endometrium under natural vs OS conditions during the periovulatory (PO) and mid-secretory (MSE) phase of the menstrual cycle. Our data showed that OS, which significantly elevated serum E2 and P4 levels, was associated with dyssynchronous glandular-stromal development, increased glandular epithelial volume, and increased macrophage and B cell abundance. We then performed bulk and single-cell RNA sequencing (RNA-seq) to identify the molecular pathways and cell-type specific transcriptomes associated with these changes. We found that OS significantly changed the expression of numerous genes in the retinoic acid (RA) signaling pathway, a pathway known to be crucial for a successful pregnancy. Our data showed that OS affected the fibroblast and epithelial cell-specific expression of genes that regulate RA signaling, with increased expression of RA synthesis genes and RA target genes, suggesting that OS increases endometrial RA signaling. RA is the metabolically active form of vitamin A which regulates the transcription of over 500 genes and adversely affects pregnancy when deficient or in excess. Thus, these findings led us to hypothesize that OS disrupts normal endometrial RA signaling and thereby endometrial differentiation that is required for implantation. We investigated whether RA is required for endometrial differentiation in humans and the establishment of pregnancy in mice. Using siRNAs against the RA receptor RARA, we observed that reduced expression of RARA mRNA in human endometrial stromal cells reduced their ability to undergo decidualization, an essential requirement for successful embryo implantation. We found that administration of BMS93, a pan RAR receptor antagonist, to pregnant dams prior to implantation from E0.5 or E2.5 until E7.5 resulted in pregnancy failure. Whereas BMS493 administration to pregnant dams from E4.5 to E7.5, resulted in pregnancies with reduced decidual vascular development in the anti-mesometrial region. Together, these data suggest that RA is required for the proper preparation of the endometrium for embryo implantation and loss of RA signaling in the endometrium, not the embryo, is a cause of pregnancy failure. Importantly, OS induced disruptions of RA signaling might contribute to reduced implantation rates in fresh ET, uncovering a novel pharmacologic target for improving pregnancy outcomes in fresh ET following OS. Presentation: Friday, June 16, 2023

Depletion of LONP2 unmasks differential requirements for peroxisomal function between cell types and in cholesterol metabolism

Peroxisomes play a central role in tuning metabolic and signaling programs in a tissue- and cell-type-specific manner. However, the mechanisms by which the status of peroxisomes is communicated and integrated into cellular signaling pathways are not yet understood. Herein, we report the cellular responses to peroxisomal proteotoxic stress upon silencing the peroxisomal protease/chaperone LONP2. Depletion of LONP2 triggered the accumulation of its substrate TYSND1 protease, while the overall expression of peroxisomal proteins, as well as TYSND1-dependent ACOX1 processing appeared normal, reflecting early stages of peroxisomal proteotoxic stress. Consequently, the alteration of peroxisome size and numbers, and luminal protein import failure was coupled with induction of cell-specific cellular stress responses. Specific to COS-7 cells was a strong activation of the integrated stress response (ISR) and upregulation of ribosomal biogenesis gene expression levels. Common changes between COS-7 and U2OS cell lines included repression of the retinoic acid signaling pathway and upregulation of sphingolipids. Cholesterol accumulated in the endomembrane compartments in both cell lines, consistent with evidence that peroxisomes are required for cholesterol flux out of late endosomes. These unexpected consequences of peroxisomal stress provide an important insight into our understanding of the tissue-specific responses seen in peroxisomal disorders.

Interference with the retinoic acid signalling pathway inhibits the initiation of teeth and caudal primary scales in the small-spotted catshark Scyliorhinus canicula.

The retinoic acid (RA) pathway was shown to be important for tooth development in mammals, and suspected to play a key role in tooth evolution in teleosts. The general modalities of development of tooth and "tooth-like" structures (collectively named odontodes) seem to be conserved among all jawed vertebrates, both with regard to histogenesis and genetic regulation. We investigated the putative function of RA signalling in tooth and scale initiation in a cartilaginous fish, the small-spotted catshark Scyliorhinus canicula. To address this issue, we identified the expression pattern of genes from the RA pathway during both tooth and scale development and performed functional experiments by exposing small-spotted catshark embryos to exogenous RA or an inhibitor of RA synthesis. Our results showed that inhibiting RA synthesis affects tooth but not caudal primary scale development while exposure to exogenous RA inhibited both. We also showed that the reduced number of teeth observed with RA exposure is probably due to a specific inhibition of tooth bud initiation while the observed effects of the RA synthesis inhibitor is related to a general delay in embryonic development that interacts with tooth development. This study provides data complementary to previous studies of bony vertebrates and support an involvement of the RA signalling pathway toolkit in odontode initiation in all jawed vertebrates. However, the modalities of RA signalling may vary depending on the target location along the body, and depending on the species lineage.

Targeting the retinoic acid signaling pathway as a modern precision therapy against cancers.

Retinoic acid (RA) is a vital metabolite derived from vitamin A. RA plays a prominent role during development, which helps in embryological advancement and cellular differentiation. Mechanistically, RA binds to its definite nuclear receptors including the retinoic acid receptor and retinoid X receptor, thus triggering gene transcription and further consequences in gene regulation. This functional heterodimer activation later results in gene activation/inactivation. Several reports have been published related to the detailed embryonic and developmental role of retinoic acids and as an anti-cancer drug for specific cancers, including acute promyelocytic leukemia, breast cancer, and prostate cancer. Nonetheless, the other side of all-trans retinoic acid (ATRA) has not been explored widely yet. In this review, we focused on the role of the RA pathway and its downstream gene activation in relation to cancer progression. Furthermore, we explored the ways of targeting the retinoic acid pathway by focusing on the dual role of aldehyde dehydrogenase (ALDH) family enzymes. Combination strategies by combining RA targets with ALDH-specific targets make the tumor cells sensitive to the treatment and improve the progression-free survival of the patients. In addition to the genomic effects of ATRA, we also highlighted the role of ATRA in non-canonical mechanisms as an immune checkpoint inhibitor, thus targeting the immune oncological perspective of cancer treatments in the current era. The role of ATRA in activating independent mechanisms is also explained in this review. This review also highlights the current clinical trials of ATRA in combination with other chemotherapeutic drugs and explains the future directional insights related to ATRA usage.

Nervous system development related gene expression regulation in the zebrafish embryo after exposure to valproic acid and retinoic acid: A genome wide approach

The evaluation of chemical and pharmaceutical safety for humans is moving from animal studies to New Approach Methodologies (NAM), reducing animal use and focusing on mechanism of action, whilst enhancing human relevance. In developmental toxicology, the mechanistic approach is facilitated by the assessment of predictive biomarkers, which allow mechanistic pathways perturbation monitoring at the basis of human hazard assessment. In our search for biomarkers of maldevelopment, we focused on chemically-induced perturbation of the retinoic acid signaling pathway (RA-SP), a major pathway implicated in a plethora of developmental processes. A genome-wide expression screening was performed on zebrafish embryos treated with two teratogens, all-trans retinoic acid (ATRA) and valproic acid (VPA), and a non-teratogen reference compound, folic acid (FA). Each compound was found to have a specific mRNA expression profile with 248 genes commonly dysregulated by both teratogenic compounds but not by FA. These genes were implicated in several developmental processes (e.g., the circulatory and nervous system). Given the prominent response of neurodevelopmental gene sets, and the crucial need to better understand developmental neurotoxicity, our study then focused on nervous system development. We found 62 genes that are potential early neurodevelopmental toxicity biomarker candidates. These results advance NAM-based safety assessment evaluation by highlighting the usefulness of the RA-SP in providing early toxicity biomarker candidates.

ALDH1A1 promotes PARP inhibitor resistance by enhancing retinoic acid receptor-mediated DNA polymerase θ expression

Poly (ADP-ribose) Polymerase (PARP) inhibitors (PARPi) have been approved for both frontline and recurrent setting in ovarian cancer with homologous recombination (HR) repair deficiency. However, more than 40% of BRCA1/2-mutated ovarian cancer lack the initial response to PARPi treatment, and the majority of those that initially respond eventually develop resistance. Our previous study has demonstrated that increased expression of aldehyde dehydrogenase 1A1 (ALDH1A1) contributes to PARPi resistance in BRCA2-mutated ovarian cancer cells by enhancing microhomology-mediated end joining (MMEJ) but the mechanism remains unknown. Here, we find that ALDH1A1 enhances the expression of DNA polymerase θ (Polθ, encoded by the POLQ gene) in ovarian cancer cells. Furthermore, we demonstrate that the retinoic acid (RA) pathway is involved in the transcription activation of the POLQ gene. The RA receptor (RAR) can bind to the retinoic acid response element (RARE) located in the promoter of the POLQ gene, promoting transcription activation-related histone modification in the presence of RA. Given that ALDH1A1 catalyzes the biosynthesis of RA, we conclude that ALDH1A1 promotes POLQ expression via the activation of the RA signaling pathway. Finally, using a clinically-relevant patient-derived organoid (PDO) model, we find that ALDH1A1 inhibition by the pharmacological inhibitor NCT-505 in combination with the PARP inhibitor olaparib synergistically reduce the cell viability of PDOs carrying BRCA1/2 mutation and positive ALDH1A1 expression. In summary, our study elucidates a new mechanism contributing to PARPi resistance in HR-deficient ovarian cancer and shows the therapeutic potential of combining PARPi and ALDH1A1 inhibition in treating these patients.