Molecular characterization and functional analysis of the ecdysone receptor isoform (EcR) from the oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

20-Hydroxyecdysone (20E) plays a vital role in a series of biological processes, via the nuclear receptors, EcR/USP by activating the ecdysone regulatory cascade. To clarify the role of EcR during the development of Grapholita molesta, the complementary DNA of ecdysone receptor isoform B1 (GmEcR-B1) was obtained from the transcriptome of G. molesta and verified by PCR. Alignment analysis revealed that the deduced protein sequence of GmEcR-B1 was highly homologous to EcR proteins identified in other lepidopteran species, especially the EcR-B1 isoform in Spodoptera litura. Quantitative real-time PCR showed that GmEcRs was expressed at all test developmental stages, and the expression level of GmEcRs was relatively higher during the period of the 3rd day of fifth instar larvae to 2nd of pupa than those in other stages. Moreover, the messenger RNA of GmEcRs was much more strongly expressed in the Malpighian tubule and epidermis than those in other tissues, which suggests that this gene may function in a tissue-specific manner during larval development. Silencing of GmEcRs could significantly downregulate the transcriptional level of ecdysone-inducible genes and result in increased mortality during metamorphosis and prolonged prepupal duration. Taken together, the present results indicate that GmEcRs may directly or indirectly affect the development of G. molesta.

A GREB1-steroid receptor feedforward mechanism governs differential GREB1 action in endometrial function and endometriosis.

Cellular responses to the steroid hormones, estrogen (E2), and progesterone (P4) are governed by their cognate receptor's transcriptional output. However, the feed-forward mechanisms that shape cell-type-specific transcriptional fulcrums for steroid receptors are unidentified. Herein, we found that a common feed-forward mechanism between GREB1 and steroid receptors regulates the differential effect of GREB1 on steroid hormones in a physiological or pathological context. In physiological (receptive) endometrium, GREB1 controls P4-responses in uterine stroma, affecting endometrial receptivity and decidualization, while not affecting E2-mediated epithelial proliferation. Of mechanism, progesterone-induced GREB1 physically interacts with the progesterone receptor, acting as a cofactor in a positive feedback mechanism to regulate P4-responsive genes. Conversely, in endometrial pathology (endometriosis), E2-induced GREB1 modulates E2-dependent gene expression to promote the growth of endometriotic lesions in mice. This differential action of GREB1 exerted by a common feed-forward mechanism with steroid receptors advances our understanding of mechanisms that underlie cell- and tissue-specific steroid hormone actions.

Inter-organ steroid hormone signaling promotes myoblast fusion via direct transcriptional regulation of a single key effector gene.

Steroid hormones regulate tissue development and physiology by modulating the transcription of a broad spectrum of genes. In insects, the principal steroid hormones, ecdysteroids, trigger the expression of thousands of genes through a cascade of transcription factors (TFs) to coordinate developmental transitions such as larval molting and metamorphosis. However, whether ecdysteroid signaling can bypass transcriptional hierarchies to exert its function in individual developmental processes is unclear. Here, we report that a single non-TF effector gene mediates the transcriptional output of ecdysteroid signaling in Drosophila myoblast fusion, a critical step in muscle development and differentiation. Specifically, we show that the 20-hydroxyecdysone (commonly referred to as "ecdysone") secreted from an extraembryonic tissue, amnioserosa, acts on embryonic muscle cells to directly activate the expression of antisocial (ants), which encodes an essential scaffold protein enriched at the fusogenic synapse. Not only is ants transcription directly regulated by the heterodimeric ecdysone receptor complex composed of ecdysone receptor (EcR) and ultraspiracle (USP) via ecdysone-response elements but also more strikingly, expression of ants alone is sufficient to rescue the myoblast fusion defect in ecdysone signaling-deficient mutants. We further show that EcR/USP and a muscle-specific TF Twist synergistically activate ants expression in vitro and in vivo. Taken together, our study provides the first example of a steroid hormone directly activating the expression of a single key non-TF effector gene to regulate a developmental process via inter-organ signaling and provides a new paradigm for understanding steroid hormone signaling in other developmental and physiological processes.

Fecundity is optimized by levels of nutrient signal-dependent expression of Dve and EcR in Drosophila male accessory gland.

Steroid hormones play various physiological roles including metabolism and reproduction. Steroid hormones in insects are ecdysteroids, and the major form in Drosophila melanogaster is ecdysone. In Drosophila males, the accessory gland is responsive to nutrient-dependent regulation of fertility/fecundity. The accessory gland is composed of two types of binucleated epithelial cells: a main cell and a secondary cell (SC). The transcription factors Defective proventriculus (Dve), Abdominal-B, and Ecdysone receptors (EcRs) are strongly expressed in adult SCs. We show that this EcR expression is regulated by parallel pathways of nutrient signaling and the Dve activity. Induction of Dve expression is also dependent on nutrient signaling, and it becomes nutrient signal-independent during a restricted period of development. Forced dve expression during the restricted period significantly increased the number of SCs. Here, we provide evidence that the level of nutrient signal-dependent Dve expression during the restricted period determines the number of SCs, and that ecdysone signaling is also crucial to optimize male fecundity through nutrient signal-dependent survival and maturation of SCs.

The effects of bisphenol S exposure on the growth, physiological and biochemical indices, and ecdysteroid receptor gene expression in red swamp crayfish, Procambarus clarkii.

The experiment was conducted to investigate the effects of Bisphenol S (BPS) on growth, physiological and biochemical indices, and the expression of ecdysteroid receptor (ECR) of the red swamp crayfish (Procambarus clarkii). The gene encoding ECR was isolated from red swamp crayfish by homologous cloning and rapid amplification of cDNA ends (RACE). The ECR transcripts were 1757 bp long and encoded proteins of 576 amino acids. The quantitative real-time PCR (qRT-PCR) analysis showed that the ECR gene was expressed in various tissues under normal conditions, and the highest level was observed in the ovary and the lowest level was observed in the muscle (P < 0.05). Then, the experiment was designed with four different BPS concentrations (0, 1, 10, and 100 µg/L), BPS exposure for 14 days, three parallel groups, and a total of 240 red swamp crayfish. At 100 µg/L BPS, the survival rate, weight gain rate, and relative length rate were decreased significantly (P < 0.05). Malonaldehyde (MDA) content reached the highest level at 100 µg/L BPS. When BPS concentration was higher than 10 µg/L, the activities of superoxide dismutase (SOD) and catalase (CAT) were significantly lower than those of the control group (P < 0.05). The expression levels of the ECR gene in ovary, intestinal, gill, and hepatopancreas tissues were significantly increased after BPS exposure (P < 0.05). The ECR gene expression in ovaries and Y-organs was significantly higher than other groups in 10 µg/L BPS (P < 0.05). The expressions of the tumor necrosis factor -α (TNF-α) and interleukin-6 (IL-6) genes in the hepatopancreas gradually increased, and the highest expression was observed exposed in 100 µg/L BPS (P < 0.05). This research will provide novel insights into the health risk assessment of BPS in aquatic organisms.

A phylogenetics-based nomenclature system for steroid receptors in teleost fishes.

Teleost fishes have emerged as tractable models for studying the neuroendocrine regulation of social behavior via molecular genetic techniques, such as CRISPR/Cas9 gene editing. Moreover, teleosts provide an opportunity to investigate the evolution of steroid receptors and their functions, as species within this lineage possess novel steroid receptor paralogs that resulted from a teleost-specific whole genome duplication. Although teleost fishes have grown in popularity as models for behavioral neuroendocrinology, there is not a consistent nomenclature system for steroid receptors and their genes, which may impede a clear understanding of steroid receptor paralogs and their functions. Here, we used a phylogenetic approach to assess the relatedness of protein sequences encoding steroid receptor paralogs in 18 species from 12 different orders of the Infraclass Teleostei. While most similarly named sequences grouped based on the established phylogeny of the teleost lineage, our analysis revealed several inconsistencies in the nomenclature of steroid receptor paralogs, particularly for sequences encoding estrogen receptor beta (ERß). Based on our results, we propose a nomenclature system for teleosts in which Greek symbols refer to proteins and numbers refer to genes encoding different subtypes of steroid receptors within the five major groups of this nuclear receptor subfamily. Collectively, our results bridge a critical gap by providing a cohesive naming system for steroid receptors in teleost fishes, which will serve to improve communication, promote collaboration, and enhance our understanding of the evolution and function of steroid receptors across vertebrates.

RP11-495P10.1 promotes HCC cell proliferation by regulating reprogramming of glucose metabolism and acetylation of the NR4A3 promoter via the PDK1/PDH axis.

The incidence and related death of hepatocellular carcinoma (HCC) have increased over the past decades. However, the molecular mechanisms underlying HCC pathogenesis are not fully understood. Long noncoding RNA (lncRNA) RP11-495P10.1 has been proven to be closely associated with the progression of prostate cancer, but its role and specific mechanism in HCC are still unknown. Here, we identify that RP11-495P10.1 is highly expressed in HCC tissues and cells and contributes to the proliferation of HCC cells. Moreover, this study demonstrates that RP11-495P10.1 affects the proliferation of HCC by negatively regulating the expression of nuclear receptor subfamily 4 group a member 3 (NR4A3). Glycometabolism reprogramming is one of the main characteristics of tumor cells. In this study, we discover that RP11-495P10.1 regulates glycometabolism reprogramming by changing the expression of pyruvate dehydrogenase kinase 1 (PDK1) and pyruvate dehydrogenase (PDH), thus contributing to the proliferation of HCC cells. Furthermore, knockdown of RP11-495P10.1 increases enrichment of H3K27Ac in the promoter of NR4A3 by promoting the activity of PDH and the production of acetyl-CoA, which leads to the increased transcription of NR4A3. Altogether, RP11-495P10.1 promotes HCC cell proliferation by regulating the reprogramming of glucose metabolism and acetylation of the NR4A3 promoter via the PDK1/PDH axis, which provides an lncRNA-oriented therapeutic strategy for the diagnosis and treatment of HCC.

The Drosophila ecdysone receptor promotes or suppresses proliferation according to ligand level.

The steroid hormone 20-hydroxy-ecdysone (20E) promotes proliferation in Drosophila wing precursors at low titer but triggers proliferation arrest at high doses. Remarkably, wing precursors proliferate normally in the complete absence of the 20E receptor, suggesting that low-level 20E promotes proliferation by overriding the default anti-proliferative activity of the receptor. By contrast, 20E needs its receptor to arrest proliferation. Dose-response RNA sequencing (RNA-seq) analysis of ex vivo cultured wing precursors identifies genes that are quantitatively activated by 20E across the physiological range, likely comprising positive modulators of proliferation and other genes that are only activated at high doses. We suggest that some of these "high-threshold" genes dominantly suppress the activity of the pro-proliferation genes. We then show mathematically and with synthetic reporters that combinations of basic regulatory elements can recapitulate the behavior of both types of target genes. Thus, a relatively simple genetic circuit can account for the bimodal activity of this hormone.

Knockdown of the ecdysone receptor disrupts development and causes mortality in the melon fly, Zeugodacus cucurbitae.

Cucurbits are important economic plants that are attacked by numerous pests, among which the melon fly Zeugodacus cucurbitae is extremely problematic. New sustainable pest control strategies are necessary to replace chemical insecticides that are harmful to the environment, human health and nontarget species. The RNA interference (RNAi) technology is one of the most promising tools due to high efficiency and species specificity. We developed an RNAi strategy targeting the ecdysone receptor (ECR) of Z. cucurbitae, which plays an important role in moulting and reproduction. We identified, described and isolated the ECR gene of Z. cucurbitae and measured its expression pattern across developmental stages and tissues. ZcECR knockdown via dsZcECR ingestion caused a significant larval mortality and abnormal phenotypes in pupae and adults. About 68% of larvae fed with a dsZcECR-treated diet failed to enter the pupal stage and died. In addition, ZcECR knockdown dramatically reduced pupal weight (by 3.24 mg on average) and fecundity (by about 23%). RNAi targeting the ECR gene is therefore a promising method to control Z. cucurbitae, paving the way for the development of novel sustainable and highly specific control strategies.

Long-range repression by ecdysone receptor on complex enhancers of the insulin receptor gene.

The insulin signalling pathway is evolutionarily conserved throughout metazoans, playing key roles in development, growth, and metabolism. Misregulation of this pathway is associated with a multitude of disease states including diabetes, cancer, and neurodegeneration. The human insulin receptor gene (INSR) is widely expressed throughout development and was previously described as a 'housekeeping' gene. Yet, there is abundant evidence that this gene is expressed in a cell-type specific manner, with dynamic regulation in response to environmental signals. The Drosophila insulin-like receptor gene (InR) is homologous to the human INSR gene and was previously shown to be regulated by multiple transcriptional elements located primarily within the introns of the gene. These elements were roughly defined in ~1.5 kbp segments, but we lack an understanding of the potential detailed mechanisms of their regulation. We characterized the substructure of these cis-regulatory elements in Drosophila S2 cells, focusing on regulation through the ecdysone receptor (EcR) and the dFOXO transcription factor. By identifying specific locations of activators and repressors within 300 bp subelements, we show that some previously identified enhancers consist of relatively compact clusters of activators, while others have a distributed architecture not amenable to further reduction. In addition, these assays uncovered a long-range repressive action of unliganded EcR. The complex transcriptional circuitry likely endows InR with a highly flexible and tissue-specific response to tune insulin signalling. Further studies will provide insights to demonstrate the impact of natural variation in this gene's regulation, applicable to human genetic studies.

Inhibition of ecdysone receptor (DcEcR) and ultraspiracle (DcUSP) expression in Diaphorina citri increased susceptibility to pesticides.

Diaphorina citri Kuwayama is of great concern because of its ability to transmit devastating citrus greening illness (Huanglongbing). One strategy for controlling HLB may involve limiting the spread of D. citri. Insecticides using dsRNA target genes may be a useful option to control D. citri. The ecdysone receptor (EcR) and ultraspiracle (USP) are crucial for the growth and reproduction of insects. This study identified the genes for D. citri ecdysone receptor (DcEcR) and ultraspiracle (DcUSP). According to the qPCR data, DcUSP peaked at the 5th-instar nymph stage, while DcEcR peaked at the adult stage. Females expressed DcEcR and DcUSP at much higher levels than males. RNAi was used to examine DcEcR and DcUSP function. The findings demonstrated that inhibition of DcEcR and DcUSP delayed nymph development and decreased survival and eclosion rates. dsEcR caused adults to develop deformed wings, and dsUSP caused nymphs to wither and die. Female adult ovaries developed slowly, and the females laid fewer eggs. Additionally, DcEcR and DcUSP were inhibited, increasing D. citri susceptibility to pesticides. These findings suggest that DcEcR and DcUSP are critical for D. citri development, growth, and reproduction and may serve as potential targets for D. citri management.

ncBAF enhances PXR-mediated transcriptional activation in the human and mouse liver.

Pregnane X receptor (PXR) is one of the key regulators of drug metabolism, gluconeogenesis, and lipid synthesis in the human liver. Activation of PXR by drugs such as rifampicin, simvastatin, and efavirenz causes adverse reactions such as drug-drug interaction, hyperglycemia, and dyslipidemia. The inhibition of PXR activation has merit in preventing such adverse events. Here, we demonstrated that bromodomain containing protein 9 (BRD9), a component of non-canonical brahma-related gene 1-associated factor (ncBAF), one of the chromatin remodelers, interacts with PXR. Rifampicin-mediated induction of CYP3A4 expression was attenuated by iBRD9, an inhibitor of BRD9, in human primary hepatocytes and CYP3A/PXR-humanized mice, indicating that BRD9 enhances the transcriptional activation of PXR in vitro and in vivo. Chromatin immunoprecipitation assay reveled that iBRD9 treatment resulted in attenuation of the rifampicin-mediated binding of PXR to the CYP3A4 promoter region, suggesting that ncBAF functions to facilitate the binding of PXR to its response elements. Efavirenz-induced hepatic lipid accumulation was attenuated by iBRD9 in C57BL/6J mice, suggesting that the inhibition of BRD9 would be useful to reduce the risk of efavirenz-induced hepatic steatosis. Collectively, we found that inhibitors of BRD9, a component of ncBAF that plays a role in assisting transactivation by PXR, would be useful to reduce the risk of PXR-mediated adverse reactions.

Melatonin Receptor 1B and Corticosteroid Receptor Polymorphisms in Infertile Women with Implantation Failure and Miscarriages.

BACKGROUND: The development of assisted reproductive techniques has significantly improved fertility chances in many women, but recurrent implantation failure (RIF) and miscarriages (RM) might preclude successful pregnancy. Alterations in the intrinsic secretory patterns of melatonin and cortisol influence reproduction in humans, and imperfection of receptor - dependent signaling may additionally compromise the hormonal effects. Therefore, the present study aims to investigate the influence of certain melatonin and cortisol receptor polymorphisms in infertile women. METHODS: A total of 111 female infertile patients suffering from implantation failure and/or miscarriages were genotyped for MTNR1B rs1562444, MTNR1Brs10830962, GCCR rs41423247, and GCCR ER22/23EK variants. Additionally, 106 female volunteers were genotyped for the same polymorphisms. RESULTS: The allele and genotype distribution of the investigated polymorphisms did not differ between infertile women and the control group. Significantly more women with history of RIF have MTNR1B rs1562444 G-allele-containing genotypes in comparison to AA carriers (19.3% vs. 3.6%, p = 0.004). The minor allele of the ER22/23EK variant was more frequent in infertile patients with three or more unsuccessful implantation attempts than in other women (12.5% vs. 2.4%, p = 0.025). CONCLUSIONS: Melatonin receptor 1B polymorphisms might affect embryo implantation and early pregnancy loss, while their influence on late pregnancy complications needs further evaluation. The possible association between the cortisol receptor ER22/23EK variant and recurrent implantation failure might help to differentiate women who could benefit from corticosteroid treatment.

Coregulators Reside within Drosophila Ecdysone-Inducible Loci before and after Ecdysone Treatment.

Ecdysone signaling in Drosophila remains a popular model for investigating the mechanisms of steroid action in eukaryotes. The ecdysone receptor EcR can effectively bind ecdysone-response elements with or without the presence of a hormone. For years, EcR enhancers were thought to respond to ecdysone via recruiting coactivator complexes, which replace corepressors and stimulate transcription. However, the exact mechanism of transcription activation by ecdysone remains unclear. Here, we present experimental data on 11 various coregulators at ecdysone-responsive loci of Drosophila S2 cells. We describe the regulatory elements where coregulators reside within these loci and assess changes in their binding levels following 20-hydroxyecdysone treatment. In the current study, we detected the presence of some coregulators at the TSSs (active and inactive) and boundaries marked with CP190 rather than enhancers of the ecdysone-responsive loci where EcR binds. We observed minor changes in the coregulators' binding level. Most were present at inducible loci before and after 20-hydroxyecdysone treatment. Our findings suggest that: (1) coregulators can activate a particular TSS operating from some distal region (which could be an enhancer, boundary regulatory region, or inactive TSS); (2) coregulators are not recruited after 20-hydroxyecdysone treatment to the responsive loci; rather, their functional activity changes (shown as an increase in H3K27 acetylation marks generated by CBP/p300/Nejire acetyltransferase). Taken together, our findings imply that the 20-hydroxyecdysone signal enhances the functional activity of coregulators rather than promoting their binding to regulatory regions during the ecdysone response.

Comprehensive in vitro analysis evaluating the variable drug-drug interaction risk of rifampicin compared to rifabutin.

Compared to rifampicin (600 mg/day), standard doses of rifabutin (300 mg/day) have a lower risk of drug-drug interactions due to induction of cytochrome P450 3A4 (CYP3A4) or P-glycoprotein (Pgp/ABCB1) mediated by the pregnane X receptor (PXR). However, clinical comparisons with equal rifamycin doses or in vitro experiments respecting actual intracellular concentrations are lacking. Thus, the genuine pharmacological differences and the potential molecular mechanisms of the discordant perpetrator effects are unknown. Consequently, the cellular uptake kinetics (mass spectrometry), PXR activation (luciferase reporter gene assays), and impact on CYP3A4 and Pgp/ABCB1 expression and activity (polymerase chain reaction, enzymatic assays, flow cytometry) were evaluated in LS180 cells after treatment with different rifampicin or rifabutin concentrations for variable exposure times and eventually normalized to actual intracellular concentrations. In addition, inhibitory effects on CYP3A4 and Pgp activities were investigated. While rifampicin is poorly taken up by LS180 cells, it strongly activates PXR and leads to enhanced expression and activity of CYP3A4 and Pgp. In contrast, rifabutin is a significantly less potent and less efficient PXR activator and gene inducer, despite sixfold to eightfold higher intracellular accumulation. Finally, rifabutin is a potent inhibitor of Pgp (IC50 = 0.3 µM) compared to rifampicin (IC50 = 12.9 µM). Together, rifampicin and rifabutin significantly differ by their effects on the regulation and function of CYP3A4 and Pgp, even when controlled for intracellular concentrations. Rifabutin's concurrent Pgp inhibitory action might partly compensate the inducing effects, explaining its weaker clinical perpetrator characteristics.

Identification of ecdysone receptor target genes in the worker honey bee brains during foraging behavior.

Ecdysone signaling plays central roles in morphogenesis and female ovarian development in holometabolous insects. In the European honey bee (Apis mellifera L.), however, ecdysone receptor (EcR) is expressed in the brains of adult workers, which have already undergone metamorphosis and are sterile with shrunken ovaries, during foraging behavior. Aiming at unveiling the significance of EcR signaling in the worker brain, we performed chromatin-immunoprecipitation sequencing of EcR to search for its target genes using the brains of nurse bees and foragers. The majority of the EcR targets were common between the nurse bee and forager brains and some of them were known ecdysone signaling-related genes. RNA-sequencing analysis revealed that some EcR target genes were upregulated in forager brains during foraging behavior and some were implicated in the repression of metabolic processes. Single-cell RNA-sequencing analysis revealed that EcR and its target genes were expressed mostly in neurons and partly in glial cells in the optic lobes of the forager brain. These findings suggest that in addition to its role during development, EcR transcriptionally represses metabolic processes during foraging behavior in the adult worker honey bee brain.

Pregnane X receptor activation alleviates renal fibrosis in mice via interacting with p53 and inhibiting the Wnt7a/ß-catenin signaling.

Renal fibrosis is a common pathological feature of chronic kidney disease (CKD) with various etiologies, which seriously affects the structure and function of the kidney. Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily and plays a critical role in regulating the genes related to xenobiotic and endobiotic metabolism in mammals. Previous studies show that PXR is expressed in the kidney and has protective effect against acute kidney injury (AKI). In this study, we investigated the role of PXR in CKD. Adenine diet-induced CKD (AD) model was established in wild-type and PXR humanized (hPXR) mice, respectively, which were treated with pregnenolone-16α-carbonitrile (PCN, 50 mg/kg, twice a week for 4 weeks) or rifampicin (RIF, 10 mg·kg-1·d-1, for 4 weeks). We showed that both PCN and RIF, which activated mouse and human PXR, respectively, improved renal function and attenuated renal fibrosis in the two types of AD mice. In addition, PCN treatment also alleviated renal fibrosis in unilateral ureter obstruction (UUO) mice. On the contrary, PXR gene deficiency exacerbated renal dysfunction and fibrosis in both adenine- and UUO-induced CKD mice. We found that PCN treatment suppressed the expression of the profibrotic Wnt7a and ß-catenin in AD mice and in cultured mouse renal tubular epithelial cells treated with TGFß1 in vitro. We demonstrated that PXR was colocalized and interacted with p53 in the nuclei of tubular epithelial cells. Overexpression of p53 increased the expression of Wnt7a, ß-catenin and its downstream gene fibronectin. We further revealed that p53 bound to the promoter of Wnt7a gene to increase its transcription and ß-catenin activation, leading to increased expression of the downstream profibrotic genes, which was inhibited by PXR. Taken together, PXR activation alleviates renal fibrosis in mice via interacting with p53 and inhibiting the Wnt7a/ß-catenin signaling pathway.

NEAT1_2 and DAZAP1, Paraspeckle Components, Interact with PXR to Negatively Regulate CYP3A4 Induction.

Human pregnane X receptor (PXR) is a major nuclear receptor that upregulates the expression of drug-metabolizing enzymes such as CYP3A4. In our recent study, it was revealed that PXR interacts with DAZ-associated protein 1 (DAZAP1), which is an essential component of the paraspeckle, a membraneless nuclear body, and the interaction was disassociated by rifampicin, a ligand of PXR. The purpose of this study was to clarify the roles of paraspeckles in PXR-mediated transcriptional regulation. Immunoprecipitation assays using PXR-overexpressing HepG2 (ShP51) cells revealed that PXR interacts with not only DAZAP1 but also NEAT1_2, a long noncoding RNA included in the paraspeckle, and that the interaction between PXR and NEAT1_2 was disassociated by rifampicin. These results suggest that PXR is trapped in paraspeckles and that the activation of PXR by its ligands facilitates its disassociation from paraspeckles. Induction of CYP3A4 by rifampicin was significantly enhanced by the knockdown of NEAT1_2 or DAZAP1 in ShP51 cells and their parental HepG2 cells. A luciferase assay using a plasmid containing the PXR response elements of CYP3A4 revealed that the increased CYP3A4 induction by siNEAT1_2 or siDAZAP1 was due to the increased transactivation by PXR. These results suggest that paraspeckles play a role in trapping nuclear PXR in the absence of the ligand to negatively regulate transactivation of its downstream gene. Collectively, this is the first study to demonstrate that the paraspeckle components NEAT1_2 and DAZAP1 negatively regulate CYP3A4 induction by PXR. SIGNIFICANCE STATEMENT: This study revealed that PXR interacts with paraspeckle components NEAT1_2 and DAZAP1 to suppress CYP3A4 induction by PXR, and the interaction is dissociated by PXR ligands. This finding provides a novel concept that paraspeckles formed by liquid-liquid phase separation potentially affect drug metabolism via negative regulation of PXR function.

20-hydroxyecdysone Upregulates Ecdysone Receptor (ECR) Gene to Promote Pupation in the Honeybee, Apis mellifera Ligustica.

A heterodimeric complex of two nuclear receptors, the ecdysone receptor (ECR) and ultraspiracle (USP), transduces 20-hydroxyecdysone (20E) signaling to modulate insect growth and development. Here, we aimed to determine the relationship between ECR and 20E during larval metamorphosis and also the specific roles of ECR during larval-adult transition in Apis mellifera. We found that ECR gene expression peaked in the 7-day-old larvae, then decreased gradually from the pupae stage. 20E slowly reduced food consumption and then induced starvation, resulting in small-sized adults. In addition, 20E induced ECR expression to regulate larval development time. Double-stranded RNAs (dsRNAs) were prepared using common dsECR as templates. After dsECR injection, larval transition to the pupal stage was delayed, and 80% of the larvae showed prolonged pupation beyond 18 h. Moreover, the mRNA levels of shd, sro, nvd, and spo, and ecdysteroid titers were significantly decreased in ECR RNAi larvae compared with those in GFP RNAi control larvae. ECR RNAi disrupted 20E signaling during larval metamorphosis. We performed rescuing experiments by injecting 20E in ECR RNAi larvae and found that the mRNA levels of ECR, USP, E75, E93, and Br-c were not restored. 20E induced apoptosis in the fat body during larval pupation, while RNAi knockdown of ECR genes reduced apoptosis. We concluded that 20E induced ECR to modulate 20E signaling to promote honeybee pupation. These results assist our understanding of the complicated molecular mechanisms of insect metamorphosis.

Cytoplasmic Hsp70s promote EcR transport into the nucleus by responding to various stimuli.

Metamorphosis is one of the most important physiological processes in insects, which is coordinated by juvenile hormone (JH) and 20-hydroxyecdysone (20E). Ecdysone receptor (EcR) is a steroid receptor (SR), which usually presents in cytoplasm and transfers into nucleus after binding to 20E. Heat shock proteins (Hsps) are suggested to be important members of the SR complex. However, their role in nucleocytoplasmic shuttle of the EcR remains unclear. In the present study, we found that apoptozole (Hsp70 inhibitor) suppressed the larval molting by decreasing the expression of ecdysone signaling genes. Two cytoplasmic (Cy) Hsp70s (Hsp72 and Hsp73) interacted with both EcR and ultraspiracle (USP, the heterodimer partner of EcR). By immunohistochemistry experiments, we revealed that CyHsp70 co-localized with EcR in the cytoplasm, and that both apoptozole and interfering of CyHsp70 significantly inhibited the process of EcR entering the nucleus under 20E induction, while reducing the expression of ecdysone signaling genes. Interestingly, the nuclear localization of EcR was also promoted by two other stimuli, including JH and heat stress, and this promotion was inhibited by apoptozole. This implies that various stimuli can induce EcR entry into the nucleus, and that this process is mediated by CyHsp70. Curiously, neither JH nor heat stress activated the ecdysone signaling genes; instead, they have a significant inhibitory effect on them. Taken together, it seems that Cytoplasmic Hsp70s promote EcR transport into the nucleus by responding to various stimuli, and that the biological effects of various stimuli passing through the EcR are different. Thus, our data provide a new viewpoint to understand the mechanism of nucleocytoplasmic shuttle of EcR.