Carboxyl terminus of hsc70-interacting protein (CHIP) can remodel mature aryl hydrocarbon receptor (AhR) complexes and mediate ubiquitination of both the AhR and the 90 kDa heat-shock protein (hsp90) in vitro

The regulation of the aryl hydrocarbon receptor (AhR) protein levels has been an area of keen interest, given its important role in mediating the cellular adaptation and toxic response to several environmental pollutants. The carboxyl terminus of hsc70-interacting protein (CHIP) ubiquitin ligase was previously associated with the regulation of the aryl hydrocarbon receptor, although the mechanisms were not directly demonstrated. In this study, we established that CHIP could associate with the AhR at cellular levels of these two proteins, suggesting a potential role for CHIP in the regulation of the AhR complex. The analysis of the sucrose-gradient-fractionated in vitro translated AhR complexes revealed that CHIP can mediate hsp90 ubiquitination while cooperating with unidentified factors to promote the ubiquitination of mature unliganded AhR complexes. In addition, the immunophilin-like protein XAP2 was able to partially protect the AhR from CHIP-mediated ubiquitination in vitro. This protection required the direct interaction of the XAP2 with the AhR complex. Surprisingly, CHIP silencing in Hepa-1c1c7 cells by siRNA methods did not reveal the function of CHIP in the AhR complex, because it did not affect well-characterized activities of the AhR nor affect its steady-state protein levels. However, the presence of potential compensatory mechanisms may be confounding this particular observation. Our results suggest a model where the E3 ubiquitin ligase CHIP cooperates with other ubiquitination factors to remodel native AhR-hsp90 complexes and where co-chaperones such as the XAP2 may affect the ability of CHIP to target AhR complexes for ubiquitination.     

Identification of new aryl hydrocarbon receptor (AhR) antagonists using a zebrafish model

A new series of 1,3-diketone, heterocyclic and α,β-unsaturated derivatives were synthesized and evaluated for their AhR antagonist activity using zebrafish and mammalian cells. Compounds 1b, 2c, 3b and 5b showed significant AhR antagonist activity in a transgenic zebrafish model. Among them, compound 3b, and 5b were found to have excellent AhR antagonist activity with IC₅₀ of 3.36 nM and 8.3 nM in a luciferase reporter gene assay. In stem cell proliferation assay, compound 5b elicited marked HSC expansion.     

Lack of an absolute requirement for the native aryl hydrocarbon receptor (AhR) and AhR nuclear translocator transactivation domains in protein kinase C-mediated modulation of the AhR pathway.

Protein kinase C (PKC)-mediated modulation of the aryl hydrocarbon receptor (AhR) pathway was examined in CHOK1-derived L10.I cells stably transfected with the pGUDLUC6.1 reporter; pGUDLUC6.1 is solely controlled by four dioxin-responsive enhancer elements. Co treatment of L10.I cells with 10 nM 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and 81 nM phorbol 12-myristate 13-acetate (PMA), an activator of sn-1,2-diacylglyerol binding PKCs, enhanced transactivation of the reporter construct several-fold relative to cells treated with a saturating 10 nM TCDD dose alone; this effect was dubbed the "PMA effect." A domain swapping and deletional analysis of the native AhR and AhR nuclear translocator (ARNT) protein transactivation domains (TADs) was performed to determine if these domains are absolutely required for the AhR x ARNT dimer-mediated PMA effect in the L10.I model system; controls demonstrate the suitability of the L10.I model for these analyses and that endogenous AhR and ARNT levels are extremely low in this model. Transient coexpression of the AhR and ARNT-474-FLAG, an ARNT protein lacking the native ARNT TAD, in L10.I cells reveals the native ARNT TAD is not absolutely required for the AhR x ARNT-474-FLAG dimer to mediate the PMA effect. Transient coexpression of AhRDeltaCVP, a chimeric AhR protein in which the native AhR TAD has been replaced with the VP16 (herpes simplex virus protein 16) TAD (which control experiments demonstrate is unaffected by PMA), and ARNT in L10.I cells indicates that the native AhR TAD is not absolutely required for this AhRDeltaCVP x ARNT dimer to mediate the PMA effect. These observations strongly suggest that PKC-mediated modulation of the AhR pathway is not absolutely dependent on coactivators recruited to the AhR. ARNT dimer by the native TADs of the AhR and its heterodimerization partner ARNT.     

Impact of aryl hydrocarbon receptor (AhR) knockdown on cell cycle progression in human HaCaT keratinocytes

Abstract While activation of the aryl hydrocarbon receptor (AhR) by exogenous ligands is well investigated, its physiological function is less understood. By extending research in AhR biology, evidence appeared that the receptor generally plays an important role in cell physiology. In keratinocytes, little is known about endogenous functions of the AhR. In order to expand this knowledge, we analyzed the impact of AhR knockdown on cell cycle progression in HaCaT cells and showed that proliferation of siAhR HaCaT cells was significantly decreased. In line with that result, western blot analysis revealed that protein level of the cyclin dependent kinase inhibitor p27(KIP1) was increased, whereas protein level of the cyclin dependent kinase (CDK) 2 was reduced. CDK4 and CDK6 protein levels remained unchanged, whereas protein level of the retinoblastoma protein (pRB) was reduced. By measuring ethoxyresorufin-O-deethylase (EROD) activity we showed that endogenous cytochrome P450 1 (CYP1), especially CYP1A1 is required for normal cell cycle in HaCaT cells, as well. To the best of our knowledge, we provide evidence for the first time in human skin cells, that in the absence of exogenous ligands, the AhR promotes cell cycle progression in HaCaT cells and one can speculate that this is the physiological function of this receptor in keratinocytes.     

The aryl hydrocarbon receptor (AhR) tyrosine 9, a residue that is essential for AhR DNA binding activity, is not a phosphoresidue but augments AhR phosphorylation

We delineate a mechanism by which dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD)-mediated formation of the aryl hydrocarbon receptor (AhR) DNA binding complex is disrupted by a single mutation at the conserved AhR tyrosine 9. Replacement of tyrosine 9 with the structurally conservative phenylalanine (AhRY9F) abolished binding to dioxin response element (DRE) D, E, and A and abrogated DRE-driven gene induction mediated by the AhR with no effect on TCDD binding, TCDD-induced nuclear localization, or ARNT heterodimerization. The speculated role for phosphorylation at tyrosine 9 was also examined. Anti-phosphotyrosine immunoblotting could not detect a major difference between the AhRY9F mutant and wild-type AhR, but a basic isoelectric point shift was detected by two-dimensional gel electrophoresis of AhRY9F. However, an antibody raised to recognize only phosphorylated tyrosine 9 (anti-AhRpY9) confirmed that AhR tyrosine 9 is not a phosphorylated residue required for DRE binding. Kinase assays using synthetic peptides corresponding to the wild-type and mutant AhR residues 1-23 demonstrated that a tyrosine at position 9 is important for substrate recognition at serine(s)/threonine(s) within this sequence by purified protein kinase C (PKC). Also, compared with AhRY9F, immunopurified full-length wild-type receptor was more rapidly phosphorylated by PKC. Furthermore, co-treatment of AhR-deficient cells that expressed AhRY9F and a DRE-driven luciferase construct with phorbol 12-myristate 13-acetate and TCDD resulted in a 30% increase in luciferase activity compared with AhRY9F treated with TCDD alone. Overall, AhR tyrosine 9, which is not a phosphorylated residue itself but is required for DNA binding, appears to play a crucial role in AhR activity by permitting proper phosphorylation of the AhR.     

Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon receptors (AHRs)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, environmental contaminants that pose a potential risk to fish populations. Both field and laboratory studies suggest that exposure of the early life stages of fish to PAH can mimic the embryotoxic effects of the planar halogenated hydrocarbons (PHHs), the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin. PHH toxicity is mediated by the aryl hydrocarbon receptor (AHR) and PHH potency is predicted by its AHR-binding affinity and CYP1A induction potency. However, the role of the AHR, if any, in mediating the developmental effects of PAH to fish remains unknown. In this study we looked at the AHR binding affinity of a test set of PAH that had been previously ranked for their potency for inducing teleost CYP1A. PAH that induced CYP1A inhibited [3H]TCDD binding to in vitro-expressed AHRs from rainbow trout and the AHR expressed in PLHC-1 fish hepatoma cells. Generally, the relative rank order for AHR binding affinity predicted the rank order of these same PAH for inducing CYP1A reported in other studies. There was a strong, positive relationship between binding to the PLHC-1 AHR (stimulus) and the EC50s for CYP1A induction (response) in whole juvenile trout and in RTL-W1 cells, but EC50s were much higher than expected for a 1:1 stimulus/response relationship. These data show that the ability of PAH to bind to teleost AHR predicts PAH potency for CYP1A induction. If PAH toxicity is receptor-mediated and predicted by induction potencies, we will have a powerful mechanistic-based tool for rapidly assessing the risk of toxicity to fish of PAH from any source.     

The aryl hydrocarbon receptor, more than a xenobiotic-interacting protein

The aryl hydrocarbon (dioxin) receptor (AhR) has been studied for several decades largely because of its critical role in xenobiotic-induced toxicity and carcinogenesis. Albeit this is a major issue in basic and clinical research, an increasing number of investigators are turning their efforts to try to understand the physiology of the AhR under normal cellular conditions. This is an exciting area that covers cell proliferation and differentiation, endogenous mechanisms of activation, gene regulation, tumor development and cell motility and migration, among others. In this review, we will attempt to summarize the studies supporting the implication of the AhR in those endogenous cellular processes.     

芳香烃受体在非酒精性脂肪性肝脏疾病中的作用

非酒精性脂肪性肝脏疾病(nonalcoholic fatty liver diseases,NAFLD)是目前备受关注的一种肝脏疾病。肥胖、2型糖尿病、高脂血症等是NAFLD的重要危险因素,但其发病机理仍不十分清楚。芳香烃受体(aryl hydrocarbon receptor,AHR)是由配体激活的转录因子,其在多种重要疾病活动中发挥了重要作用。近年来多项研究表明AHR激活促进了NAFLD的发病进展,对AHR参与NAFLD发病机制的探讨将有利于进一步阐明NAFLD的发病机理,为NAFLD的防治提出新的思路。本文就AHR与NAFLD关系的研究进展做一综述,以期为该领域的研究提供新的方向。     

The steroid-binding properties of recombinant glucocorticoid receptor: a putative role for heat shock protein hsp90

The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter. The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide approximately 70 nM). Glycerol gradient analysis of the E. coli lystate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins. However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lystate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2nM). Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor. Radiosequence analysis of the recombinant steroid-binding domain expressed in E. coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly. However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.