X-associated Protein Research Articles

Establishment and validation of exhausted CD8+ T cell feature as a prognostic model of HCC.

The exhausted CD8+T (Tex) cells are a unique cell population of activated T cells that emerges in response to persistent viral infection or tumor antigens. Tex cells showed the characteristics of aging cells, including weakened self-renewal ability, effector function inhibition, sustained high expression of inhibitory receptors including PD-1, TIGIT, TIM-3, and LAG-3, and always accompanied by metabolic and epigenetic reprogramming. Tex cells are getting more and more attention in researching immune-related diseases and tumor immunotherapy. However, studies on Tex-related models for tumor prognosis are still lacking. We hope to establish a risk model based on Tex-related genes for HCC prognosis. Tex-related GEO datasets from different pathologic factors (chronic HBV, chronic HCV, and telomere shortening) were analyzed respectively to acquire differentially expressed genes (DEGs) by the 'limma' package of R. Genes with at least one intersection were incorporated into Tex-related gene set. GO, KEGG, and GSEA enrichment analyses were produced. Hub genes and the PPI network were established and visualized by the STRING website and Cytoscape software. Transcription factors and targeting small molecules were predicted by the TRUST and CLUE websites. The Tex-related HCC prognostic model was built by Cox regression and verified based on different datasets. Tumor immune dysfunction and exclusion (TIDE) and SubMap algorithms tested immunotherapy sensitivity. Finally, qRT-PCR and Flow Cytometry was used to confirm the bioinformatic results. Hub genes such as AKT1, CDC6, TNF and their upstream transcription factor ILF3, Regulatory factor X-associated protein, STAT3, JUN, and RELA/NFKB1 were identified as potential motivators for Tex. Tex-related genes SLC16A11, CACYBP, HSF2, and ATG10 built the HCC prognostic model and helped with Immunotherapy sensitivity prediction. Our study demonstrated that Tex-related genes might provide accurate prediction for HCC patients in clinical decision-making, prognostic assessment, and immunotherapy. In addition, targeting the hub genes or transcription factors may help to reverse T cell function and enhance the effect of tumor immunotherapy.

A caspase-2-RFXANK interaction and its implication for MHC class II expression

Despite recent achievements implicating caspase-2 in tumor suppression, the enzyme stands out from the apoptotic caspase family as a factor whose function requires further clarification. To specify enzyme characteristics through the definition of interacting proteins in apoptotic or non-apoptotic settings, a yeast 2-hybrid (Y2H) screen was performed using the full-length protein as bait. The current report describes the analysis of a captured prey and putative novel caspase-2 interacting factor, the regulatory factor X-associated ankyrin-containing protein (RFXANK), previously associated with CIITA, the transactivator regulating cell-type specificity and inducibility of MHC class II gene expression. The interaction between caspase-2 and RFXANK was verified by co-immunoprecipitations using both exogenous and endogenous proteins, where the latter approach suggested that binding of the components occurs in the cytoplasm. Cellular co-localization was confirmed by transfection of fluorescently conjugated proteins. Enhanced caspase-2 processing in RFXANK-overexpressing HEK293T cells treated with chemotherapeutic agents further supported Y2H data. Yet, no distinct differences with respect to MHC class II expression were observed in plasma membranes of antigen-presenting cells derived from wild type and caspase-2−/− mice. In contrast, increased levels of the total MHC class II protein was evident in protein lysates from caspase-2 RNAi-silenced leukemia cell lines and B-cells isolated from gene-targeted mice. Together, these data identify a novel caspase-2-interacting factor, RFXANK, and indicate a potential non-apoptotic role for the enzyme in the control of MHC class II gene regulation.

Paeoniflorin induces G2/M cell cycle arrest and caspase-dependent apoptosis through the upregulation of Bcl-2 X-associated protein and downregulation of B-cell lymphoma 2 in human osteosarcoma cells.

Paeoniflorin (PF), extracted from the peony root, has been proved to possess antineoplastic activity in different cancer cell lines. However, it remains unclear whether PF has an antineoplastic effect against osteosarcoma cells. The present study investigated the effects and the specific mechanism of PF on various human osteosarcoma cell lines. Using the multiple methods to detect the activity of PF on HOS and Saos-2 human osteosarcoma cell lines, including an MTS assay, flow cytometry, transmission electron microscopy and western blotting, it was demonstrated that PF induces inhibition of proliferation, G2/M phase cell cycle arrest and apoptosis in the osteosarcoma cell lines in vitro, and activation of cleaved-caspase-3 and cleaved-poly (ADPribose) polymerase in a dose-dependent manner. Furthermore, the pro-apoptotic factors Bcl-2 X-associated protein and BH3 interacting domain death agonist were uregulated, while the anti-apoptotic factors B-cell lymphoma 2 (Bcl-2) and Bcl-2-extra large were downregulated. In conclusion, these results demonstrated that PF has a promising therapeutic potential in for osteosarcoma.

IFN-γ induces the upregulation of RFXAP via inhibition of miR-212-3p in pancreatic cancer cells: A novel mechanism for IFN-γ response.

Previous studies have demonstrated that pancreatic cancer-derived microRNA (miR)-212-3p can inhibit the expression of regulatory factor X-associated protein (RFXAP), an important transcription factor for major histocompatibility complex (MHC) class II, and thereby lead to downregulation of MHC class II in dendritic cells. It has also been established that interferon (IFN)-γ can increase the expression of MHC class II in immune cells. It was therefore hypothesized that IFN-γ can inhibit miR-212-3p expression in pancreatic cancer, leading to the upregulation of RFXAP and MHC class II expression. This may represent a novel molecular mechanism underlying the use of IFN-γ in immunotherapy. Data from the present study revealed that miR-212-3p was inhibited by IFN-γ in a dose and time-dependent manner in the pancreatic ductal adenocarcinoma cell line PANC-1. RFXAP and MHC class II expression were increased following IFN-γ stimulation. A luciferase assay was performed to validate RFXAP as a target gene of miR-212-3p. The expression levels of RFXAP and MHC class II were decreased by miR-212-3p mimics and increased by miR-212-3p inhibitors. In PANC-1 cells transfected with miR-212-3p mimics, IFN-γ stimulation could not increase the RFXAP and MHC class II. The results from the present study suggest that IFN-γ increases RFXAP and MHC class II expression by inhibiting miR-212-3p. To the best of our knowledge, this is the first report of this novel molecular mechanism underlying the effects of IFN-γ on pancreatic cancer, which may aid with the development of immunotherapies for patients with pancreatic cancer.

Novel Epigenetic Biomarkers Mediating Bisphenol A Exposure and Metabolic Phenotypes in Female Mice.

There is compelling evidence that epigenetic modifications link developmental environmental insults to adult disease susceptibility. Animal studies have associated perinatal bisphenol A (BPA) exposure to altered DNA methylation, but these studies are often limited to candidate gene and global non-loci-specific approaches. By using an epigenome-wide discovery platform, we elucidated epigenetic alterations in liver tissue from adult mice offspring (10 months) following perinatal BPA exposure at human physiologically relevant doses (50-ng, 50-μg, and 50-mg BPA/kg diet). Biological pathway analysis identified an enrichment of significant differentially methylated regions in metabolic pathways among females. Furthermore, through the use of top enriched biological pathways, 4 candidate genes were chosen to assess DNA methylation as a mediating factor linking the association of perinatal BPA exposure to metabolic phenotypes previously observed in female offspring. DNA methylation status at Janus kinase-2 (Jak-2), retinoid X receptor (Rxr), regulatory factor x-associated protein (Rfxap), and transmembrane protein 238 (Tmem238) was used within a mediational regression analysis. DNA methylation in all four of the candidate genes was identified as a mediator in the mechanistic pathway of developmental BPA exposure and female-specific energy expenditure, body weight, and body fat phenotypes. Data generated from this study are crucial for deciphering the mechanistic role of epigenetics in the pathogenesis of chronic disease and the development of epigenetic-based prevention and therapeutic strategies for complex human disease.

Pancreatic cancer-derived exosomes transfer miRNAs to dendritic cells and inhibit RFXAP expression via miR-212-3p.

It has been reported tumor-derived exosomes can transfer miRNAs to recipient cells in the tumor microenvironment, promoting tumor invasion and metastasis. The present research aimed to explore how pancreatic cancer (PC) derived exosomal miRNAs inhibited mRNA expression of dendritic cells and induced immune tolerance. Our study revealed that 9 PC-related miRNAs were increased and 208 mRNAs were inhibited in exosome-stimulated dendritic cells (exo-iDCs) compared to immature dendritic cells (iDCs). A target prediction between the 9 miRNAs and 208 mRNAs was performed by bioinformatics database analysis. From the target prediction, it was predicted and validated that regulatory factor X-associated protein (RFXAP), an important transcription factor for MHC II, was inhibited by miR-212-3p transferred from PC-secreted exosomes, resulting in decreased MHC II expression. Moreover, a clinical study showed a negative correlation between miR-212-3p and RFXAP in PC tissue. From these data, we concluded that PC-related miRNAs can be transferred to dendritic cells via exosome and inhibit target mRNA expression. More importantly, PC-derived exosomes inhibit RFXAP expression via miR-212-3p, which decrease MHC II expression and induce immune tolerance of dendritic cells. RFXAP deficiency has never been reported in solid tumors. The functions and mechanisms of RFXAP in tumors deserve future explorations.

Microarray analysis revealed that immunity-associated genes are primarily regulated by roxarsone in promoting broiler chicken (Gallus gallus domesticus) growth

Addition of roxarsone can significantly improve the growth of broiler chickens (Gallus gallus domesticus). Nevertheless, this application will lead to the contamination of the environment as well as animal products. Understanding the response of genes to roxarsone may bring about the discovery of new, safer substitutes. In this study, we monitored the expression of 8,935 genes in chicken breast muscle using microarrays. Analysis showed that 30 genes, such as the interleukin 3 regulated nuclear factor (NFIL3), the regulatory factor X-associated ankyrin-containing protein (RFXANK), the cleavage and polyadenylation-specific factor 3 (CPSF3), and the FK506 binding protein 9 (FKBP9), have consistently up or downregulated (fold change ≥1.5 or ≤0.6, P < 0.05, false discovery rate ≤0.05) throughout the medication periods. The results from microarray analysis were validated by real-time quantitative PCR. Further functional investigation showed that 13 of the identified genes are well documented, and surprisingly, 11 (85%) of these are related to immunity (5 are immunity and defense related, 4 are immunodeficiency disease related, 2 are immunosuppressive drug related), and the remaining 2 are energy metabolism related. These findings may suggest that supplement of roxarsone can improve the immunity of chickens through regulating the expression of associated genes, and as a result contribute to the growth promotion. Further research on the encoded proteins of the differentially expressed genes should provide more evidence for the potential mechanism.

Type III bare lymphocyte syndrome associated with a novel RFXAP mutation: a case report

Type III bare lymphocyte syndrome (BLS) is a severe combined immunodeficiency disease caused by the absence of MHC Class II expression associated with low expression of class I molecules. Here, we report a case with type III BLS who lacked RFXAP (Regulatory factor X-associated protein) expression as a result from a novel mutation introducing a premature stopcodon in DE-region at amino acid 73.

Inhibition of Heme Oxygenase-1 Partially Reverses the Arsenite-Mediated Decrease of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 Catalytic Activity in Isolated Rat Hepatocytes

Heme oxygenase (HO-1), the rate-limiting enzyme in the physiological breakdown of heme, is ubiquitous, and its expression can be increased by arsenite [As(III)], and similar other stimuli that induce cellular oxidative stress. Interestingly, it has been shown that the As(III)-induced HO-1 is inversely correlated with a decrease in cytochromes P450 (P450s) activity; however, the direct role for HO-1 in the inhibition of P450 enzymes remains unknown. Our results showed that As(III) at a concentration of 5 μM decreased the constitutive and inducible expression of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 at the mRNA, protein, and catalytic activity levels. Moreover, As(III) decreased the nuclear accumulation of aryl hydrocarbon receptor (AhR) and pregnane X receptor without increasing their degradation. As(III) also increased the binding of cytosolic AhR to heat shock protein 90 and hepatitis B virus X-associated protein 2. In the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin as an inducer for CYP1A and rifampin as an inducer for CYP3A, As(III) decreased the enzymatic activity of the four P450s more than it decreased their mRNA or protein expression levels. It is noteworthy that treatment with the competitive HO-1 inhibitor, tin-mesoporphyrin, or supplementing external heme partially reversed the As(III)-mediated decrease in activities of the four P450s. In conclusion, the current study provides the first evidence that As(III) decreases CYP1A1, CYP1A2, CYP3A23, and CYP3A2 expression in freshly isolated rat primary hepatocytes. Furthermore, inhibiting the As(III)-mediated induction of HO-1 partially restores the enzymatic activity of these P450s that was initially decreased by As(III), confirming the direct role of HO-1 in the inhibition of P450s.

The Immunophilin-Like Protein XAP2 Is a Negative Regulator of Estrogen Signaling through Interaction with Estrogen Receptor α

XAP2 (also known as aryl hydrocarbon receptor interacting protein, AIP) is originally identified as a negative regulator of the hepatitis B virus X-associated protein. Recent studies have expanded the range of XAP2 client proteins to include the nuclear receptor family of transcription factors. In this study, we show that XAP2 is recruited to the promoter of ERα regulated genes like the breast cancer marker gene pS2 or GREB1 and negatively regulate the expression of these genes in MCF-7 cells. Interestingly, we show that XAP2 downregulates the E2-dependent transcriptional activation in an estrogen receptor (ER) isoform-specific manner: XAP2 inhibits ERα but not ERβ-mediated transcription. Thus, knockdown of intracellular XAP2 levels leads to increased ERα activity. XAP2 proteins, carrying mutations in their primary structures, loose the ability of interacting with ERα and can no longer regulate ER target gene transcription. Taken together, this study shows that XAP2 exerts a negative effect on ERα transcriptional activity and may thus prevent ERα-dependent events.

Differential impact of tetratricopeptide repeat proteins on the steroid hormone receptors.

BackgroundTetratricopeptide repeat (TPR) motif containing co-chaperones of the chaperone Hsp90 are considered control modules that govern activity and specificity of this central folding platform. Steroid receptors are paradigm clients of Hsp90. The influence of some TPR proteins on selected receptors has been described, but a comprehensive analysis of the effects of TPR proteins on all steroid receptors has not been accomplished yet.Methodology and Principal FindingsWe compared the influence of the TPR proteins FK506 binding proteins 51 and 52, protein phosphatase-5, C-terminus of Hsp70 interacting protein, cyclophillin 40, hepatitis-virus-B X-associated protein-2, and tetratricopeptide repeat protein-2 on all six steroid hormone receptors in a homogeneous mammalian cell system. To be able to assess each cofactor's effect on the transcriptional activity of on each steroid receptor we employed transient transfection in a reporter gene assay. In addition, we evaluated the interactions of the TPR proteins with the receptors and components of the Hsp90 chaperone heterocomplex by coimmunoprecipitation. In the functional assays, corticosteroid and progesterone receptors displayed the most sensitive and distinct reaction to the TPR proteins. Androgen receptor's activity was moderately impaired by most cofactors, whereas the Estrogen receptors' activity was impaired by most cofactors only to a minor degree. Second, interaction studies revealed that the strongly receptor-interacting co-chaperones were all among the inhibitory proteins. Intriguingly, the TPR-proteins also differentially co-precipitated the heterochaperone complex components Hsp90, Hsp70, and p23, pointing to differences in their modes of action.Conclusion and SignificanceThe results of this comprehensive study provide important insight into chaperoning of diverse client proteins via the combinatorial action of (co)-chaperones. The differential effects of the TPR proteins on steroid receptors bear on all physiological processes related to steroid hormone activity.

Suppression mechanisms of flavonoids on aryl hydrocarbon receptor-mediated signal transduction

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates biological and toxicological effects by binding to its agonists such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Previously we demonstrated that flavonoids suppressed the TCDD-induced DNA-binding activity of the AhR in a structure-dependent manner. In this study, we investigated the mechanisms by which flavonoids suppressed the AhR-mediated signal transduction in mouse hepatoma Hepa-1c1c7 cells. Flavones and flavonols suppressed the TCDD-induced nuclear translocation of the AhR and dissociation of its partner proteins, heat shock protein 90 and X-associated protein 2, whereas flavanones and catechins did not. Flavonoids of all these four subclasses suppressed the phosphorylation of both AhR and Arnt and the formation of a heterodimer consisting of these proteins. Since certain flavonoids are known to inhibit mitogen-activated protein kinases (MAPKs), we confirmed the contribution of MAPK/ERK kinase (MEK) to the AhR-mediated signal transduction by using U0126, an inhibitor of MEK1/2. U0126 suppressed TCDD-induced phosphorylation of the AhR and Arnt followed by the DNA-binding activity of the AhR. Flavanones and catechins suppressed the TCDD-induced phosphorylation of ERK1/2. The inhibition of MEK/ERK phosphorylation is one of the mechanisms by which flavanones and catechins suppress the AhR-mediated signal transduction in Hepa-1c1c7 cells.

(−)-Epigallocatechin-3-gallate Is a Novel Hsp90 Inhibitor

(-)-Epigallocatechin-3-gallate (EGCG), a major component of green tea, protects against certain types of cancers, although the mechanism has not yet been determined. It was previously demonstrated that EGCG blocks aryl hydrocarbon receptor (AhR)-mediated transcription induced by the potent carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Unlike other AhR antagonists that directly bind to the AhR, EGCG inhibits AhR-mediated transcription by binding to hsp90. We hypothesize that EGCG exerts anti-AhR and anticancer effects by acting as an hsp90 inhibitor. Using proteolytic footprinting, immunoprecipitation, and an ATP-agarose pull-down assay, EGCG was found to directly modulate the conformation of hsp90 and bind at or near to a C-terminal ATP binding site. Hsp90 chaperone function, as assessed by its ability to mediate refolding of denatured luciferase, was inhibited by EGCG treatment. Hsp90 dimerization, which occurs at the C-terminal end, was also inhibited by EGCG treatment. Coimmunoprecipitation studies showed that EGCG stabilizes an AhR complex that includes hsp90 and XAP2 (hepatitis B virus X-associated protein 2), and decreases the association of aryl hydrocarbon nuclear translocator (Arnt) with ligand-activated AhR. Thus, EGCG, through its ability to bind to hsp90, blocks AhR response element (AhRE) recognition. These studies indicate a novel mechanism whereby EGCG inhibits ligand-induced AhRE binding and AhR-mediated transcriptional activity. In EGCG-treated human ovarian carcinoma SKOV3 cells, decreased levels of several cancer-related hsp90 client proteins, such as ErbB2, Raf-1 and phospho-AKT, were observed. EGCG also modified the association of hsp90 with several cochaperones. Overall, these data indicate that EGCG is a novel hsp90 inhibitor. Further studies are needed to determine if this has a role in the antitumor actions of EGCG.

Endogenous Hepatic Expression of the Hepatitis B Virus X-Associated Protein 2 Is Adequate for Maximal Association with Aryl Hydrocarbon Receptor-90-kDa Heat Shock Protein Complexes

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that acts as an environmental sensor by binding to a variety of xenobiotics. AHR activation serves to combat xenotoxic stress by inducing metabolic enzyme expression in the liver. The hepatitis B virus X-associated protein (XAP2) is a component of the cytosolic AHR complex and modulates AHR transcriptional properties in vitro and in cell culture and yeast systems. Expression of XAP2 is low in liver compared with other nonhepatic tissues and the AHR exhibits high ligand-induced transcriptional activity. Because XAP2 has been demonstrated to repress AHR activity, we hypothesized that XAP2 may be limiting in liver and that increasing XAP2 levels would attenuate AHR transcriptional activity. To this end, transgenic mice were generated that exhibit hepatocyte-specific elevation in XAP2 expression. Transgenic XAP2 expression was restricted to liver, and its ability to complex with the AHR was verified. Gene expression experiments were performed by inducing AHR transcriptional activity with beta-naphthoflavone via intraperitoneal injection, and mRNA quantification was done by real-time polymerase chain reaction. Wild-type and transgenic animals showed little difference in constitutive or ligand-induced CYP1A1; CYP1A2; UDP glucuronosyltransferase 1A2; NAD(P)H dehydrogenase, quinone 1; constitutive androstane receptor; or nuclear factor erythroid 2-related factor 2 mRNA expression. Sucrose density fractionation and AHR immunoprecipitation experiments found little or no stoichiometric increase in bound XAP2 to the AHR between genotypes. Gene array studies were performed to identify novel XAP2-regulated targets. Taken together, this work shows that despite the relatively low level of XAP2 in liver, it is not a limiting component in AHR regulation.

The co‐chaperone XAP2 is required for activation of hypothalamic thyrotropin‐releasing hormone transcription in vivo

Transcriptional control of hypothalamic thyrotropin-releasing hormone (TRH) integrates central regulation of the hypothalamo-hypophyseal-thyroid axis and hence thyroid hormone (triiodothyronine (T(3))) homeostasis. The two beta thyroid hormone receptors, TRbeta1 and TRbeta2, contribute to T(3) feedback on TRH, with TRbeta1 having a more important role in the activation of TRH transcription. How TRbeta1 fulfils its role in activating TRH gene transcription is unknown. By using a yeast two-hybrid screening of a mouse hypothalamic complementary DNA library, we identified a novel partner for TRbeta1, hepatitis virus B X-associated protein 2 (XAP2), a protein first identified as a co-chaperone protein. TR-XAP2 interactions were TR isoform specific, being observed only with TRbeta1, and were enhanced by T(3) both in yeast and mammalian cells. Furthermore, small inhibitory RNA-mediated knockdown of XAP2 in vitro affected the stability of TRbeta1. In vivo, siXAP2 abrogated specifically TRbeta1-mediated (but not TRbeta2) activation of hypothalamic TRH transcription. This study provides the first in vivo demonstration of a regulatory, physiological role for XAP2.

Role of Endogenous XAP2 Protein on the Localization and Nucleocytoplasmic Shuttling of the Endogenous Mouse Ahb-1 Receptor in the Presence and Absence of Ligand

Studies using transient expression systems have implicated the hepatitis B virus X-associated protein (XAP2) in the control of aryl hydrocarbon receptor (AHR) stability and subcellular location. Studies were performed in Hepa-1 cells to evaluate these functions of XAP2 on the mouse Ahb-1 receptor under endogenous stoichiometry. The Ahb-1 receptor is cytoplasmic, and it becomes predominantly nuclear after 30 to 60 min of ligand exposure with minimal degradation. During this time, XAP2 coprecipitates with the AHR, suggesting that it does not affect the nuclear localization of the liganded receptor. Overexpression of XAP2 in Hepa-1 cells does not result in increased association with the endogenous Ahb-1 complex or influence the receptors cytoplasmic localization. Knockdown of endogenous XAP2 by small interfering RNA results in >or=90% reduction in the amount of XAP2 associated with the endogenous Ahb-1 receptor complex. Despite the reduction in XAP2, the unliganded Ahb-1 receptor complex remains cytoplasmic, although inhibition of nuclear export results in accumulation of the receptor in the nucleus. Truncation of the C-terminal 305 amino acids of the Ahb-1 receptor (AHR500) results in proteins that exhibit a predominantly nuclear localization and remain associated with the same level of endogenous XAP2 as full-length AHRs. Together, these results support a model in which the majority of the unliganded Ahb-1 receptor complexes are associated with XAP2, and the association prevents dynamic nucleocytoplasmic shuttling in the unliganded state. After ligand binding, XAP2 remains associated with the Ahb-1 receptor complex, and it does not impair nuclear translocation but may function to limit receptor "transformation".

Redefining the Role of the Endogenous XAP2 and C-terminal hsp70-interacting Protein on the Endogenous Ah Receptors Expressed in Mouse and Rat Cell Lines

Studies using transient expression systems have implicated the XAP2 protein in the control of aryl hydrocarbon receptor (AHR) stability and subcellular location. Thus, studies were performed in cell lines that expressed endogenous rat or mouse Ah(b-1) (C57BL/6) or Ah(b-2) (C3H) AHRs with similar levels of endogenous XAP2. Unliganded rat and mouse Ah(b-2) receptor complexes associated with reduced levels of XAP2 and exhibited dynamic nucleocytoplasmic shuttling in comparison with Ah(b-1) receptors. Rat and mouse Ah(b-2) receptors also exhibited a greater magnitude of ligand-induced degradation than Ah(b-1) receptors. Small interfering RNA reduction of endogenous XAP2 by >80% had minimal impact on the level of Ah(b-2) receptors but resulted in a 25-30% reduction of Ah(b-1) receptors. XAP2 reduction resulted in increased susceptibility of the Ah(b-1) receptor to ligand-induced degradation yet produced higher levels of endogenous CYP1A1 induction. Stable expression of the Ah(b-2) receptor in the C57BL/6 background resulted in a protein with reduced association with XAP2, dynamic nucleocytoplasmic shuttling, and increased levels of ligand-induced degradation. Small interfering RNA reduction of endogenous XAP2 in a C-terminal hsp70-interacting protein knockout mouse cell line, exhibited a 25-30% reduction in the level of endogenous Ah(b-1) AHR and showed high levels of ligand-induced degradation. Thus, endogenous XAP2 exerts a negative function on a small fraction of the endogenous Ah(b-1) receptor complex but appears to have a minimal impact on endogenous rat or Ah(b-2) receptors. This implies that the analysis of the AHR-mediated signaling via rat and mouse Ah(b-2) receptors may better represent the physiology of this signal transduction pathway.

The Transactivation Domain of the Ah Receptor Is a Key Determinant of Cellular Localization and Ligand-Independent Nucleocytoplasmic Shuttling Properties

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates transcription of a number of target genes upon binding ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Large intra- and interspecies variations exist with respect to sensitivity to TCDD, and this could, at least in part, be due to a considerable variation in the AhR amino acid sequence between species. The N-terminal half of the AhR is well-conserved across species, whereas the C-terminal half exhibits a considerable degree of degeneracy. It has previously been shown that there are differences between the mouse (mAhR) and human AhR (hAhR) in terms of cellular localization, nucleocytoplasmic shuttling, the effect of chaperone proteins on these properties, and differences in relative ligand affinity. In this study, two chimeras were generated such that each had the N-terminal half of one receptor and the C-terminal half of the other receptor. The C-terminal half of the receptor, containing the transactivation domain, determines the cellular localization of the transiently transfected receptor and regulates the ability of hepatitis B virus X-associated protein 2 (XAP2) to inhibit ligand-independent nuclear import of AhR. In addition, the transactivation domain (TAD) appears to determine the presence of XAP2 in the nuclear ligand-bound AhR/hsp90 complex prior to association with the AhR nuclear translocator protein (ARNT). However, the transactivation domain does not appear to play a role in determining relative ligand affinity of the receptor, and mAhR and hAhR have similar overall transactivation potential in a cell-based reporter system at a saturating dose of ligand. This study demonstrates for the first time that the transactivation domain of the AhR influences important biochemical properties of the N-terminal half of the AhR, and the degeneracy in the transactivation domain between the mAhR and the hAhR results in species-specific differences in receptor properties.

Phosphorylation Analysis of 90 kDa Heat Shock Protein within the Cytosolic Arylhydrocarbon Receptor Complex

The arylhydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that regulates the transcription of genes encoding xenobiotic metabolizing enzymes and also mediates most of the toxic effects caused by dioxins and polycyclic aromatic hydrocarbons. The cytosolic AhR complex exists as a transcriptionally cryptic complex, consisting of the 90 kDa heat shock protein (HSP90) and the hepatitis B virus X-associated protein 2 (XAP2). The posttranslational modifications, especially phosphorylation, of the cytosolic AhR-HSP90-XAP2 complex are poorly understood, although the phosphorylation of a transcriptionally active heterodimer of AhR and an AhR nuclear translocator is critically involved in AhR function. To reveal the phosphorylation status involved in AhR function, we used mass spectrometry to determine the site-specific phosphorylation of the steady-state cytosolic AhR complex, prepared from Chinese hamster ovary cells stably expressing mouse AhR. We identified phosphorylations of the HSP90 subunits within the AhR complex at Ser225 and Ser254 of HSP90beta and Ser230 of HSP90alpha. By site-directed mutagenesis, these serine residues were substituted with alanine and glutamic acid to elucidate the role of the HSP90beta serine phosphorylations in the AhR function. Immunoprecipitation assays using COS7 transfectants showed that the replacement of Ser225 and Ser254 by Ala, S225/254A, increased the binding affinity for AhR, as compared with the Glu replacement. In a ligand-induced AhR transcription activity assay using Hepa1 transfectants, the S255/254A mutant exhibited more potent transcription activity than the S225/254E mutant, which had activity similar to that of wild-type HSP90beta. These results suggest that the phosphorylations in the charged linker region of the HSP90 molecule modulate the formation of the functional cytosolic AhR complex.

The Aryl Hydrocarbon (Ah) Receptor Transcriptional Regulator Hepatitis B Virus X-associated Protein 2 Antagonizes p23 Binding to Ah Receptor-Hsp90 Complexes and Is Dispensable for Receptor Function

To further understand the role that the hepatitis B virus X-associated protein 2 (XAP2) plays in regulating aryl hydrocarbon receptor (AhR) function, a point mutation was introduced at tyrosine 408 of the AhR, changing the residue to an alanine or lysine. These mutations resulted in the loss of AhR binding to endogenous XAP2 in COS-1 cells and reduced binding of exogenously expressed XAP2. Cellular localization of the mutant AhR-yellow fluorescent protein fusion proteins remained nuclear when XAP2 was co-expressed, while the non-mutant receptor was redistributed to the cytoplasm. XAP2 expression caused an overall repression of constitutive and ligand-induced AhR transcriptional activity. However, increased expression of XAP2 had no effect on the AhRY408A mutant transcriptional activity. Additionally the XAP2 binding-deficient AhR mutants showed overall higher transcriptional activity when compared with the non-mutant receptor. Interestingly reduced incorporation of the Hsp90 associated co-chaperone p23 in the unliganded AhR complex was observed with increasing XAP2 expression. The displacement of p23 from Hsp90 did not occur when increasing levels of XAP2 were introduced in COS-1 cells in the absence of the AhR; thus this displacement event occurs specifically within an AhR complex. Finally XAP2 itself was capable of existing in multimeric complexes, and these complexes did not require Hsp90 or AhR to form. However, it is not yet clear whether XAP2 can exist within the AhR complex in more than one copy.