Retinoic Acid Receptor Activation Research Articles

Retinoic acid receptor activation reprograms senescence response and enhances anti-tumor activity of natural killer cells

Cellular senescence can exert dual effects in tumors, either suppressing or promoting tumor progression. The senescence-associated secretory phenotype (SASP), released by senescent cells, plays a crucial role in this dichotomy. Consequently, the clinical challenge lies in developing therapies that safely enhance senescence in cancer, favoring tumor-suppressive SASP factors over tumor-promoting ones. Here, we identify the retinoic-acid-receptor (RAR) agonist adapalene as an effective pro-senescence compound in prostate cancer (PCa). Reactivation of RARs triggers a robust senescence response and a tumor-suppressive SASP. In preclinical mouse models of PCa, the combination of adapalene and docetaxel promotes a tumor-suppressive SASP that enhances natural killer (NK) cell-mediated tumor clearance more effectively than either agent alone. This approach increases the efficacy of the allogenic infusion of human NK cells in mice injected with human PCa cells, suggesting an alternative therapeutic strategy to stimulate the anti-tumor immune response in "immunologically cold" tumors.

Heparin-binding epidermal growth factor and fibroblast growth factor 2 rescue Müller glia-derived progenitor cell formation in microglia- and macrophage-ablated chick retinas.

Recent studies have demonstrated the impact of pro-inflammatory signaling and reactive microglia/macrophages on the formation of Müller glial-derived progenitor cells (MGPCs) in the retina. In chick retina, ablation of microglia/macrophages prevents the formation of MGPCs. Analyses of single-cell RNA-sequencing chick retinal libraries revealed that quiescent and activated microglia/macrophages have a significant impact upon the transcriptomic profile of Müller glia (MG). In damaged monocyte-depleted retinas, MG fail to upregulate genes related to different cell signaling pathways, including those related to Wnt, heparin-binding epidermal growth factor (HBEGF), fibroblast growth factor (FGF) and retinoic acid receptors. Inhibition of GSK3β, to simulate Wnt signaling, failed to rescue the deficit in MGPC formation, whereas application of HBEGF or FGF2 completely rescued the formation of MGPCs in monocyte-depleted retinas. Inhibition of Smad3 or activation of retinoic acid receptors partially rescued the formation of MGPCs in monocyte-depleted retinas. We conclude that signals produced by reactive microglia/macrophages in damaged retinas stimulate MG to upregulate cell signaling through HBEGF, FGF and retinoic acid, and downregulate signaling through TGFβ/Smad3 to promote the reprogramming of MG into proliferating MGPCs.

The Endogenous Retinoic Acid Receptor Pathway Is Exploited by Mycobacterium tuberculosis during Infection, Both InVitro and InVivo.

Retinoic acid (RA) is a fundamental vitamin A metabolite involved in regulating immune responses through the nuclear RA receptor (RAR) and retinoid X receptor. While performing experiments using THP-1 cells as a model for Mycobacterium tuberculosis infection, we observed that serum-supplemented cultures displayed high levels of baseline RAR activation in the presence of live, but not heat-killed, bacteria, suggesting that M. tuberculosis robustly induces the endogenous RAR pathway. Using invitro and invivo models, we have further explored the role of endogenous RAR activity in M. tuberculosis infection through pharmacological inhibition of RARs. We found that M. tuberculosis induces classical RA response element genes such as CD38 and DHRS3 in both THP-1 cells and human primary CD14+ monocytes via a RAR-dependent pathway. M. tuberculosis-stimulated RAR activation was observed with conditioned media and required nonproteinaceous factor(s) present in FBS. Importantly, RAR blockade by (4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthalen-2-yl]ethenyl]benzoic acid), a specific pan-RAR inverse agonist, in a low-dose murine model of tuberculosis significantly reduced SIGLEC-F+CD64+CD11c+high alveolar macrophages in the lungs, which correlated with 2× reduction in tissue mycobacterial burden. These results suggest that the endogenous RAR activation axis contributes to M. tuberculosis infection both invitro and invivo and reveal an opportunity for further investigation of new antituberculosis therapies.

Osteoblastic RAR Inhibition Causes VAD-Like Craniofacial Skeletal Deformity

Retinoid signaling disorders cause craniofacial deformity, among which infants with maternal vitamin A deficiency (VAD) exhibited malformation of the eye, nose, palate, and parietal and jaw bone. Previous research uncovered the pathogenesis of eye defect and cleft palate of VAD in mice, but the studies on craniofacial skeletal deformity met obstacles, and the cell/lineage and underlying mechanism remain unclear. The retinoic acid receptor (RAR) is the key transcription factor in retinoid signaling, but individual knockout cannot simulate pathway inhibition. Here, we conditionally expressed dominant-negative RARα mutation (dnRARα) in osteoblasts to specifically inhibit the transcription activity of RAR in mice, which mimics the craniofacial deformities caused by VAD in clinical cases: hypomineralization of cranial bones, mandibular deformity, and clavicular hypoplasia. Furthermore, we performed 3-dimensional reconstruction based on micro–computed tomography and confirmed the abnormalities in the shape, size, and ossification of craniofacial bones due to osteoblastic RAR inhibition. Histological analysis indicated that inhibition of RAR in osteoblasts impaired both bone formation and bone resorption, which was confirmed by transcriptome sequencing of the calvaria. Furthermore, mechanism investigation showed that inhibition of RAR in osteoblasts directly decreased osteoblast differentiation in a cell-autonomous manner by impairing osteogenic gene transcription and also inhibited osteoclast differentiation via osteoblast–osteoclast crosstalk by impairing Rankl transcription. In summary, osteoblastic RAR activity is critical to craniofacial skeletal development, and its dysfunction leads to skeletal deformities mimicking VAD craniofacial defects, providing a new insight for VAD pathogenesis.

Retinoic acid receptor regulation of decision-making for cell differentiation.

All-trans retinoic acid (ATRA) activation of retinoic acid receptors (RARs) is crucial to an organism's proper development as established by findings for mouse foetuses from dams fed a vitamin A-deficient diet. ATRA influences decision-making by embryonic stem (ES) cells for differentiation including lineage fate. From studies of knockout mice, RARα and RARγ regulate haematopoiesis whereby active RARα modulates the frequency of decision-making for myeloid differentiation, but is not essential for myelopoiesis, and active RARγ supports stem cell self-renewal and maintenance. From studies of zebrafish embryo development, active RARγ plays a negative role in stem cell decision-making for differentiation whereby, in the absence of exogenous ATRA, selective agonism of RARγ disrupted stem cell decision-making for differentiation patterning for development. From transactivation studies, 0.24nM ATRA transactivated RARγ and 19.3nM (80-fold more) was needed to transactivate RARα. Therefore, the dose of ATRA that cells are exposed to in vivo, from gradients created by cells that synthesize and metabolize, is important to RARγ versus RARα and RARγ activation and balancing of the involvements in modulating stem cell maintenance versus decision-making for differentiation. RARγ activation favours stemness whereas concomitant or temporal activation of RARγ and RARα favours differentiation. Crosstalk with signalling events that are provoked by membrane receptors is also important.

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.

An Isochroman Analog of CD3254 and Allyl-, Isochroman-Analogs of NEt-TMN Prove to Be More Potent Retinoid-X-Receptor (RXR) Selective Agonists Than Bexarotene.

Bexarotene is an FDA-approved drug for the treatment of cutaneous T-cell lymphoma (CTCL); however, its use provokes or disrupts other retinoid-X-receptor (RXR)-dependent nuclear receptor pathways and thereby incites side effects including hypothyroidism and raised triglycerides. Two novel bexarotene analogs, as well as three unique CD3254 analogs and thirteen novel NEt-TMN analogs, were synthesized and characterized for their ability to induce RXR agonism in comparison to bexarotene (1). Several analogs in all three groups possessed an isochroman ring substitution for the bexarotene aliphatic group. Analogs were modeled for RXR binding affinity, and EC50 as well as IC50 values were established for all analogs in a KMT2A-MLLT3 leukemia cell line. All analogs were assessed for liver-X-receptor (LXR) activity in an LXRE system to gauge the potential for the compounds to provoke raised triglycerides by increasing LXR activity, as well as to drive LXRE-mediated transcription of brain ApoE expression as a marker for potential therapeutic use in neurodegenerative disorders. Preliminary results suggest these compounds display a broad spectrum of off-target activities. However, many of the novel compounds were observed to be more potent than 1. While some RXR agonists cross-signal the retinoic acid receptor (RAR), many of the rexinoids in this work displayed reduced RAR activity. The isochroman group did not appear to substantially reduce RXR activity on its own. The results of this study reveal that modifying potent, selective rexinoids like bexarotene, CD3254, and NEt-TMN can provide rexinoids with increased RXR selectivity, decreased potential for cross-signaling, and improved anti-proliferative characteristics in leukemia models compared to 1.

Retinoic acid receptor activity is required for the maintenance of type 1 innate lymphoid cells.

Group 1 innate lymphoid cells (G1-ILCs) are innate immune effectors critical for the response to intracellular pathogens and tumors. G1-ILCs comprise circulating natural killer (NK) cells and tissue-resident type 1 ILCs (ILC1s). ILC1s mainly reside in barrier tissues and provide the initial sources of interferon-γ (IFN-γ) to prime the protecting responses against infections, which are followed by the response of recruited NK cells. Despite such distribution differences, whether local environmental factors influence the behavior of NK cells and ILC1s is unclear. Here, we show that the signaling of retinoic acid (RA), active metabolites of vitamin A, is essential for the maintenance of ILC1s in the periphery. Mice expressing RARα403, a truncated form of retinoic acid receptor α (RARα) that exerts dominant negative activity, in a lymphoid cell- or G1-ILC-specific manner showed remarkable reductions of peripheral ILC1s while NK cells were unaffected. Lymphoid cell-specific inhibition of RAR activity resulted in the reduction of PD-1+ ILC progenitors (ILCPs), but not of common lymphoid progenitors (CLPs), suggesting the impaired commitment and differentiation of ILC1s. Transcriptome analysis revealed that RARα403-expressing ILC1s exhibited impaired proliferative states and declined expression of effector molecules. Thus, our findings demonstrate that cell-intrinsic RA signaling is required for the homeostasis and the functionality of ILC1s, which may present RA as critical environmental cue targeting local type 1 immunity against infection and cancer.

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.

A non-retinol retinoic acid receptor-γ (RAR-γ/NR1B3) selective agonist, tectorigenin, can effectively inhibit the ultraviolet A-induced skin damage.

Long-term ultraviolet (UV) exposure can cause inflammation, pigmentation and photoaging. All-trans retinoic acid (ATRA/tretinoin) is a commonly used retinoic acid receptor (RAR) agonist in the clinical treatment of UV-induced skin problems. However, the use of such drugs is often accompanied by systemic adverse reactions caused by nonspecific activation of RARs. Therefore, this study was designed to screen for a novel RAR-γ-selective agonist with high safety. Molecular docking, dynamic simulation and Biacore were used to screen and identify novel RAR-γ-selective agonists. RT-PCR, ELISA, western blotting, immunofluorescence staining, flow cytometry and proteomic analysis were used to detect the effects of these novel RAR-γ selective agonists on UVA-induced inflammation and photoaging cell models. UVA-induced mouse models were used to evaluate the effects of tectorigenin on skin repair, ageing and inflammation. Tectorigenin is a novel RAR-γ-selective agonist, which inhibits UV-induced oxidative damage, inflammatory factor release and matrix metalloproteinase (MMP) production. Tectorigenin can also reverse the UVA-induced loss of collagen. The results of the signalling pathway research showed that tectorigenin mainly affects the MAPK/JNK/AP-1 pathway. In animal experiments, tectorigenin showed better anti-inflammatory and anti-photoaging effects, and caused less skin irritation than ATRA. Nano-particle loaded tectorigenin significantly improved the utilization of tectorigenin. Tectorignen is a non-retinol RAR-γ-selective agonist that can inhibit UV-induced skin damage and could be developed as a safe pharmaceutical component for the prevention of photoaging and skin inflammation.

Retinoic Acid Receptor Activation Reduces Metastatic Prostate Cancer Bone Lesions by Blocking the Endothelial-to-Osteoblast Transition.

This study provides mechanistic insights into how RAR agonists suppress prostate cancer-induced bone formation and offers a rationale for developing RAR agonists for prostate cancer bone metastasis therapy. See related commentary by Bhowmick and Bhowmick, p. 2975.

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 & 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 < 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 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 >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 >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.

Isolation of CD35+ follicular dendritic cells and its role in the differentiation from B cells to IgA+GL7+ cells

Follicular dendritic cells (FDCs) are non-hematopoietic cells that are localized in the germinal centers (GCs) of lymph nodes (LNs) and are involved in humoral immunity. FDCs are a rare population that are sensitive to mechanical and chemical stimuli, making their isolation for analysis difficult. In Peyer's Patches, which are the main IgA-inductive sites, FDCs have been reported to be activated by retinoic acid receptor (RAR) and toll-like receptor (TLR) signals to induce IgA production. However, little is known about FDCs in mesenteric LNs (MLNs), although MLNs are also an IgA-inductive site. In this study, we efficiently isolated FDCs as CD35+ cells using anti-CD35 antibodies (Abs) and magnetic bead sorting. We found that CD35+ FDCs facilitated differentiation from B220+ B cells into IgA+GL7+ GC B-like cells but not IgA+CD138+ plasma cells. Furthermore, using CD35+ FDCs from LPS-resistant C3H/HeJ mice, the generation of IgA+GL7+ GC B-like cells was not altered significantly between wild-type and LPS-resistant mice. Moreover, the addition of RAR antagonists and agonists revealed that differentiation into IgA+GL7+ GC B-like cells required the activation of RAR, especially RAR-β, in FDCs. The differentiation of IgA+GL7+ cells was promoted by FDCs in peripheral LNs as well as MLNs in our in vitro assay. Taken together, these results indicate that magnetic bead sorting with anti-CD35 Abs enable the efficient isolation of FDCs. Our data suggested that CD35+ FDCs can support differentiation of B cells into IgA+GL7+ GC B-like cells in environments that are not limited to MLNs, which can be stimulated by retinoic acid.

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

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

Compartment-Specific Role of Retinoic Acid Receptor Activation in AKI

Compartment-Specific Role of Retinoic Acid Receptor Activation in AKI

The Interactions of Insulin and Vitamin A Signaling Systems for the Regulation of Hepatic Glucose and Lipid Metabolism.

The pandemics of obesity and type 2 diabetes have become a concern of public health. Nutrition plays a key role in these concerns. Insulin as an anabolic hormonal was discovered exactly 100 years ago due to its activity in controlling blood glucose level. Vitamin A (VA), a lipophilic micronutrient, has been shown to regulate glucose and fat metabolism. VA’s physiological roles are mainly mediated by its metabolite, retinoic acid (RA), which activates retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which are two transcription factors. The VA status and activations of RARs and RXRs by RA and synthetic agonists have shown to affect the glucose and lipid metabolism in animal models. Both insulin and RA signaling systems regulate the expression levels of genes involved in the regulation of hepatic glucose and lipid metabolism. Interactions of insulin and RA signaling systems have been observed. This review is aimed at summarizing the history of diabetes, insulin and VA signaling systems; the effects of VA status and activation of RARs and RXRs on metabolism and RAR and RXR phosphorylation; and possible interactions of insulin and RA in the regulation of hepatic genes for glucose and lipid metabolism. In addition, some future research perspectives for understanding of nutrient and hormone interactions are provided.

Adipocyte‐specific Loss of Retinoic Acid Receptor Alpha (Rarα) Exacerbates Diet‐induced Obesity and Steatohepatitis in Mice

Adipocytes are the major sites of lipid storage and important for regulating lipid metabolism. Adipocytes also serve as the adipokine-releasing cells that mediates lipid and glucose homeostasis in the body. Under diet-induced conditions impaired lipid metabolism in adipocytes can lead to ectopic lipid accumulation and insulin resistance. Previous studies show that activation of retinoic acid receptors via retinoic acid signaling can inhibit adipogenesis and promote adipose tissue browning. We hypothesized that adipocyte-specific retinoic acid receptor alpha (Rarα) might play a role in protecting against diet-induced obesity. To test this hypothesis, we conducted in-vivo studies using adipocyte-specific Rarα knockout mice. Rarα floxed mice were crossed with adiponectin-Cre transgenic mice to generate adipocyte-specific knockout mice (adipo-Rarα-/-). Rarα fl/fl (control) and adipo-Rarα-/- mice were given a high fat diet for 20 weeks. During the study, the body weight of adipo-Rarα-/- significantly increased compared to control. The body composition analysis showed that loss of Rarα in adipocytes increased body fat content. Indirect calorimetry studies showed that reduced energy expenditure in adipo-Rarα-/- mice. Histological analysis showed that brown adipose tissue had increased lipid deposition. Adipo-Rarα-/- mice also had impaired glucose tolerance. In the liver, adipo-Rarα-/- mice had increased triglyceride and non-esterified free fatty acid accumulation, and fibrosis. In the plasma, adipo-Rarα-/- mice and increased free fatty acid, AST and ALT levels. Thus, our study indicates that adipocyte RARα is protective against obesity and steatohepatitis.

A transgenic reporter mouse model for in vivo assessment of retinoic acid receptor transcriptional activation.

Background: Vitamin A is essential for a wide range of life processes throughout embryogenesis to adult life. With the aim of developing an in vivo model to monitor retinoic acid receptor (RAR) transactivation real-time in intact animals, we generated transgenic mice carrying a luciferase (luc) reporter gene under the control of retinoic acid response elements (RAREs) consisting of three copies of a direct repeat with five spacing nucleotides (DR5). Methods: Transgenic mice carrying a RARE dependent luciferase reporter flanked with insulator sequence were generated by pronuclear injection. RARE dependent luciferase activity was detected by in vivo imaging or in tissue extracts following manipulations with RAR/retinoid X receptor (RXR) agonists, RAR antagonists or in vitamin A deficient mice. Results: We found a strong induction of luciferase activity in a time and dose dependent manner by retinoic acid as well as RAR agonists, but not by the RXR agonist (using n=4-6 per group; 94 mice). In addition, luciferase activity was strongly reduced in vitamin A-deficient mice (n=6-9; 30 mice). These observations confirm that luciferase activity was controlled by RAR activation in the RARE-luc mouse. Luciferase activity was detectable in various organs, with high activity especially in brain and testis, indicating strong retinoid signalling in these tissues. Conclusion: The RARE-luc transgenic mice, which enabled real-time in vivo assessment of RAR activation, will be useful in understanding the normal physiology of vitamin A, the role of retinoid signalling in pathologies as well as to evaluate pharmacological ligands for RARs.

Retinoic acid can improve autophagy through depression of the PI3K-Akt-mTOR signaling pathway via RARα to restore spermatogenesis in cryptorchid infertile rats

Cryptorchidism-caused adult infertility is a common component of idiopathic reasons for male infertility. Retinoic acid (RA) has a vital effect on the spermatogenesis process. Here, we found that the expression of c-Kit, Stra8, and Sycp3 could be up-regulated via the activation of retinoic acid receptor α (RARα) after RA supplementation in neonatal cryptorchid infertile rats. We also demonstrated that the protein expression of PI3K, p-Akt/pan-Akt, and p-mTOR/mTOR was higher in cryptorchid than in normal testes, and could be suppressed with RA in vivo. After RA treatment in infertile cryptorchid testis in vivo, the levels of the autophagy proteins LC3 and Beclin1 increased and those of P62 decreased. Biotin tracer indicated that the permeability of blood-testis barrier (BTB) in cryptorchid rats decreased after RA administration. Additionally, after blocking the RARα with AR7 (an RARα antagonist) in testicle culture in vitro, we observed that compared with normal testes, the PI3K-Akt-mTOR signaling pathway and the autophagy pathway was increased and decreased, respectively, which were coincident with cryptorchisd testes in vivo. Additionally, the appropriate concentrations of RA treatment could depress the PI3K-Akt-mTOR signaling pathway and improve the autophagy pathway. The results confirmed that RA can rehabilitate BTB function and drive key protein levels in spermatogonial differentiation through depressing the PI3K-Akt-mTOR signaling pathway via RARα.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314