Distinct Roles for Aryl Hydrocarbon Receptor Nuclear Translocator and Ah Receptor in Estrogen-Mediated Signaling in Human Cancer Cell Lines

The activated AHR/ARNT complex (AHRC) regulates the expression of target genes upon exposure to environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Importantly, evidence has shown that TCDD represses estrogen receptor (ER) target gene activation through the AHRC. Our data indicates that AHR and ARNT act independently from each other at non-dioxin response element sites. Therefore, we sought to determine the specific functions of AHR and ARNT in estrogen-dependent signaling in human MCF7 breast cancer and human ECC-1 endometrial carcinoma cells. Knockdown of AHR with siRNA abrogates dioxin-inducible repression of estrogen-dependent gene transcription. Intriguingly, knockdown of ARNT does not effect TCDD-mediated repression of estrogen-regulated transcription, suggesting that AHR represses ER function independently of ARNT. This theory is supported by the ability of the selective AHR modulator 3′,4′-dimethoxy-α-naphthoflavone (DiMNF) to repress estrogen-inducible transcription. Furthermore, basal and estrogen-activated transcription of the genes encoding cathepsin-D and pS2 are down-regulated in MCF7 cells but up-regulated in ECC-1 cells in response to loss of ARNT. These responses are mirrored at the protein level with cathepsin-D. Furthermore, knock-down of ARNT led to opposite but corresponding changes in estrogen-stimulated proliferation in both MCF7 and ECC-1 cells. We have obtained experimental evidence demonstrating a dioxin-dependent repressor function for AHR and a dioxin-independent co-activator/co-repressor function for ARNT in estrogen signalling. These results provide us with further insight into the mechanisms of transcription factor crosstalk and putative therapeutic targets in estrogen-positive cancers

A novel AhR ligand, 2AI, protects the retina from environmental stress.

Various retinal degenerative diseases including dry and neovascular age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy are associated with the degeneration of the retinal pigmented epithelial (RPE) layer of the retina. This consequently results in the death of rod and cone photoreceptors that they support, structurally and functionally leading to legal or complete blindness. Therefore, developing therapeutic strategies to preserve cellular homeostasis in the RPE would be a favorable asset in the clinic. The aryl hydrocarbon receptor (AhR) is a conserved, environmental ligand-dependent, per ARNT-sim (PAS) domain containing bHLH transcription factor that mediates adaptive response to stress via its downstream transcriptional targets. Using in silico, in vitro and in vivo assays, we identified 2,2'-aminophenyl indole (2AI) as a potent synthetic ligand of AhR that protects RPE cells in vitro from lipid peroxidation cytotoxicity mediated by 4-hydroxynonenal (4HNE) as well as the retina in vivo from light-damage. Additionally, metabolic characterization of this molecule by LC-MS suggests that 2AI alters the lipid metabolism of RPE cells, enhancing the intracellular levels of palmitoleic acid. Finally, we show that, as a downstream effector of 2AI-mediated AhR activation, palmitoleic acid protects RPE cells from 4HNE-mediated stress, and light mediated retinal degeneration in mice

Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles

Ligand activation of the aryl hydrocarbon (AHR) has profound effects upon the immunological status of the gastrointestinal tract, establishing and maintaining signaling networks, which facilitate host-microbe homeostasis at the mucosal interface. However, the identity of the ligand(s) responsible for such AHR-mediated activation within the gut remains to be firmly established. Here, we combine in vitro ligand binding, quantitative gene expression, protein-DNA interaction and ligand structure activity analyses together with in silico modeling of the AHR ligand binding domain to identify indole, a microbial tryptophan metabolite, as a human-AHR selective agonist. Human AHR, acting as a host indole receptor may exhibit a unique bimolecular (2:1) binding stoichiometry not observed with typical AHR ligands. Such bimolecular indole-mediated activation of the human AHR within the gastrointestinal tract may provide a foundation for inter-kingdom signaling between the enteric microflora and the immune system to promote commensalism within the gut.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by Nature Publishing Group. The published article can be found at: http://www.nature.com/articles/srep1268

Alteration in levels and synthesis of proteins in trout hepatocytes due to dietary cyclopropenoid fatty acid[s]

Cyclopropenoid fatty acids (CPFA) are unique compounds that contain a highly strained and reactive cyclopropene ring structure. These compounds have been shown to cause a number of toxic effects in a variety of animals. Rainbow trout (Salmo gairdneri) have proven to be particularly sensitive to CPFA. Studies have revealed that CPFA are both carcinogenic and cocarcinogenic in rainbow trout. However, the mechanism(s) of these adverse biological effects are not understood. In the present report a series of studies were performed in order to determine the effect of CPFA on the levels and synthesis rates of trout hepatocyte proteins. In the first study, the influence of dietary CPFA on protein synthesis was measured via the use of amino acid double labeling experiments in isolated hepatocytes. Both the microsomal and cytosolic subcellular fractions were examined in these studies after separation by lithium dodecyl sulfate polyacrylamide gel electrophoresis. In the cytosolic fraction, the synthesis of proteins with apparent molecular weights in the range of 68,000 to 74,000 were significantly decreased. A marked depression in both the level and synthesis rate of microsomal proteins was observed for proteins that migrate in the 200,000 to 240,000 relative molecular mass region in polyacrylamide gels. These high molecular weight proteins do not appear to be membrane proteins and one of them has biotin associated with it. Using avidin-peroxidase staining, it was shown that the mass of this protein was reduced in CPFA-fed trout by 80%. The possible identity of these proteins is discussed. In the second study, initial attempts were made to use two dimensional gel electrophoresis to study alterations in individual liver microsomal polypeptides from trout fed CPFA. In order to effectively resolve membrane proteins in the first dimension (isoelectric focusing) changes in the standard techniques were needed. Replacement of the detergent nonidet-40 with 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate (CHAPS) in isoelectric focusing of trout liver microsomes have greatly increased resolution. These results have allowed effective resolution of complex polypeptide patterns for comparative purposes. In the third study, antibodies against β-napthoflavone-fed rainbow trout cytochrome P-450 (LM₂) were employed to localize the corresponding polypeptide(s) via protein blotting and immunochemical staining. Microsomes isolated from β-napthoflavone-fed trout contained only a single polypeptide. In contrast, control microsomes contained two distinct polypeptides differing only in their isoelectric points. Thus, an additional P-450 isozyme in rainbow trout was tentatively identified. CPFA treatment caused a preferential decrease in only one of the isozymes found in the control samples. The presence of concanavalin A binding glycopolypeptides was determined. The two P-450 isozymes localized on control microsomal gels were found to bind concanavalin A, suggesting that these isozymes are giycoproteins. Another result of CPFA treatment was a shift in a closely related group of membrane glycopolypeptides, labeled gp80, gp82, gp80₁, and gp82₁. A decrease in the mass of gp80 and gp82, and a corresponding increase in mass of gp80₁ and gp82₁ was observed