Brian J. McMillan

The Aryl Hydrocarbon Receptor sans Xenobiotics: Endogenous Function in Genetic Model Systems

For more than 30 years, the aryl hydrocarbon receptor [Ah receptor (AHR)] has been extensively scrutinized as the cellular receptor for numerous environmental contaminants, including polychlorinated dioxins, dibenzofurans, and biphenyls. Recent evidence argues that this description is incomplete and perhaps myopic. Ah receptor orthologs have been demonstrated to mediate diverse endogenous functions in our close vertebrate relatives as well as our distant invertebrate ancestors. Moreover, these endogenous functions suggest that xenobiotic toxicity may be best understood in the context of intrinsic AHR physiology. In this literature review, we survey the emerging picture of endogenous AHR biology from work in the vertebrate and invertebrate model systems Mus musculus, Caenorhabditis elegans, and Drosophila melanogaster.

The aryl hydrocarbon receptor is activated by modified low-density lipoprotein.

Endogenous activation of the aryl hydrocarbon receptor (AHR) is required for normal vascular development. This biology led us to investigate the interplay between the AHR and vascular physiology by using an in vitro model of fluid shear stress. Using this system, we show that fluid flow induces a robust AHR-mediated increase in CYP1 expression. Furthermore, we demonstrate that incubation with sheared bovine or human sera is sufficient for AHR activation, indicating that direct cellular exposure to shear stress is not required for this response. Fractionation of sera by size and density revealed the AHR-activating factor to be low-density lipoprotein (LDL). Purified LDL (0.1 mg/ml) from sheared sera induces a 6-fold increase in AHR-mediated signaling as compared with LDL purified from static sera. Similar results were obtained by exposing a purified fraction of LDL to fluid flow, suggesting that shear stress is capable of directly modifying LDL structure and/or function. In addition, we show that LDL can be converted to an AHR-activating species by conventional methods of lipoprotein modification, such as NaOCl oxidation. Finally, we demonstrate that an increased level of AHR-activating LDL is present in the sera of AHR null mice as compared with heterozygous littermates, suggesting a role for the Ahr locus in the physiological response to modified LDL in vivo. Overall, these data demonstrate a previously undescribed relationship between LDL modification and AHR biology and provide a potential explanation for the vascular abnormalities observed in AHR null mice.

Genetic analysis in UK Biobank links insulin resistance and transendothelial migration pathways to coronary artery disease

UK Biobank is among the worlds largest repositories for phenotypic and genotypic information in individuals of European ancestry. We performed a genome-wide association study in UK Biobank testing ∼9 million DNA sequence variants for association with coronary artery disease (4,831 cases and 115,455 controls) and carried out meta-analysis with previously published results. We identified 15 new loci, bringing the total number of loci associated with coronary artery disease to 95 at the time of analysis. Phenome-wide association scanning showed that CCDC92 likely affects coronary artery disease through insulin resistance pathways, whereas experimental analysis suggests that ARHGEF26 influences the transendothelial migration of leukocytes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces premature activation of the KLF2 regulon during thymocyte development.

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) causes numerous and diverse toxic events via activation of the aryl hydrocarbon receptor, including atrophy of the thymus. Exposure to TCDD induces acute thymocyte cell loss, which occurs concomitantly with proliferation arrest and premature emigration of triple negative (TN; CD4-, CD8-, CD3-) T cell progenitors. In this report, we demonstrate that TCDD exposure results in dysregulation of KLF2 (Kruppel-like factor 2) expression in developing thymocytes. The Klf2 gene encodes an Sp1-like zinc finger transcription factor that functions as a central regulator of T lymphocyte proliferation and trafficking. During normal thymocyte development, KLF2 is expressed exclusively in CD4 and CD8 single positive T cells and promotes a nonproliferative, promigratory phenotype. In mice exposed to TCDD, however, the Klf2 gene is prematurely expressed in TN thymocytes. Administration of a 100 μg/kg dose of TCDD results in a ∼15-fold induction of KLF2 as early as the TN2 (CD44+, CD25+) stage of development and immediately precedes acute cell loss in the TN3, TN4, and double positive (CD4+, CD8+) cell stages. Induction of KLF2 occurs within 12 h of TCDD exposure and is fully dependent on expression of the aryl hydrocarbon receptor. In addition, TCDD exposure alters the expression of several factors comprising the KLF2 regulon, including Edg1/S1P1, β7 integrin, CD52, Cdkn2d (cyclin-dependent kinase inhibitor 2D), s100a4, and IL10Rα. These findings indicate that the pollutant TCDD interferes with early thymopoeisis via ectopic expression of the KLF2 regulon.

A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.

Electrostatic Interactions between Elongated Monomers Drive Filamentation of Drosophila Shrub, a Metazoan ESCRT-III Protein.

SUMMARY The endosomal sorting complex required for transport (ESCRT) is a conserved protein complex that facilitates budding and fission of membranes. It executes a key step in many cellular events, including cytokinesis and multi-vesicular body formation. The ESCRT-III protein Shrub in flies, or its homologs in yeast (Snf7) or humans (CHMP4B), is a critical polymerizing component of ESCRT-III needed to effect membrane fission. We report the structural basis for polymerization of Shrub and define a minimal region required for filament formation. The X-ray structure of the Shrub core shows that individual monomers in the lattice interact in a staggered arrangement using complementary electrostatic surfaces. Mutations that disrupt interface salt bridges interfere with Shrub polymerization and function. Despite substantial sequence divergence and differences in packing interactions, the arrangement of Shrub subunits in the polymer resembles that of Snf7 and other family homologs, suggesting that this intermolecular packing mechanism is shared among ESCRT-III proteins.