Evidence of selective activation of aryl hydrocarbon receptor nongenomic calcium signaling by pyrene

Abstract

In its classical genomic mode of action, the aryl hydrocarbon receptor (AhR) acts as a ligand activated transcription factor regulating expression of target genes such as CYP1A1 and CYP1B1. Some ligands may also trigger more rapid nongenomic responses through AhR, including calcium signaling (Ca 2+). In the present study we observed that pyrene induced a relatively rapid increase in intracellular Ca 2+-concentrations ([Ca 2+ ] i) in human microvascular endothelial cells (HMEC-1) and human embryonic kidney cells (HEK293) that was attenuated by AhR-inhibitor treatment and/or transient AhR knockdown by RNAi. In silico molecular docking based on homology models, suggested that pyrene is not able to bind to the human AhR in the agonist conformation. Instead, pyrene docked in in the antagonist conformation of the the AhR PAS-B binding pocket, although the interaction differed from antagonists such as GNF-351 and CH223191. Accordingly, pyrene did not induce CYP1A1 or CYP1B1, but suppressed CYP1-expression by benzo[a]pyrene (B[a]P) in HMEC-1 cells, confirming that pyrene act as an antagonist of AhR-induced gene expression. Use of pharmacological inhibitors and Ca 2+-free medium indicated that the pyrene-induced AhR nongenomic [Ca 2+ ] i increase was initiated by Ca 2+-release from intracellular stores followed by a later phase of extracellular Ca 2+influx, consistent with store operated calcium entry (SOCE). These effects was accompanied by an AhR-dependent reduction in ordered membrane lipid domains, as determined by di-4-ANEPPDHQ staining. Addition of cholesterol inhibited both the pyrene-induced [Ca 2+ ] i-increase and alterations in membrane lipid order. In conclusion, we propose that pyrene binds to AhR, act as an antagonist of the canonical genomic AhR/Arnt/CYP1-pathway, reduces ordered membrane lipid domains, and activates AhR nongenomic Ca 2+-signaling from intracellular stores.

Potential role of polycyclic aromatic hydrocarbons in air pollution-induced non-malignant respiratory diseases

“…Other studies suggest that AhR-activation in immune and non-immune cells in general may contribute to development and progression of COPD as well as asthma [30]. Differences in the ligands with regard to metabolism and induced toxicity, binding to AhR and subsequent activation of classic or non-classic genomicor non-genomic pathways, illustrate that the role of AhR in respiratory diseases is multifaceted [103,105].…”

“…However, the relationship between ligand binding, AhR-activation, and downstream signaling appears to be more complex than previously considered. Recent data suggest that pyrene may bind to AhR with comparable affinity as B[a]P without inducing CYP1-expression, but activating AhRdependent Ca 2+ -signaling instead [105]. Conceivably, binding of different AhR-ligands may lead to differences in the receptor conformation resulting in different activation of downstream signaling.…”

Potential role of polycyclic aromatic hydrocarbons as mediators of cardiovascular effects from combustion particles

“…Especially pyrene appeared to be a potent inducer of Ca 2+ -signaling, triggering approximately 2-fold higher [Ca 2+ ] i -responses compared to B[ a ]P or chrysene [112]. Surprisingly, pyrene-induced [Ca 2+ ] i seemed to depend on a selective activation of non-genomic AhR-signaling, occurring in absence of effects on CYP1A1 expression [119]. This differs distinctly from B[ a ]P-induced Ca 2+ -signaling through β2ADR [112], and suggest that PAHs may affect [Ca 2+ ] i -regulation in endothelial cell through different mechanisms.…”

“…This differs distinctly from B[ a ]P-induced Ca 2+ -signaling through β2ADR [112], and suggest that PAHs may affect [Ca 2+ ] i -regulation in endothelial cell through different mechanisms. Notably, studies on effect of lipophilic organic chemicals extracted from DEP, suggest that their induction of [Ca 2+ ] i could involve both AhR- and β2ADR-dependent responses [119, 195].…”

“…Transporters, channels and receptors cluster in membrane micro domains [113], and their activity may also be affected by their presence inside or outside such ordered domains [114, 115]. Several xenobiotics including DEP-extracts and PAHs have been found to affect membrane microstructure, thus possibly affecting [Ca 2+ ] i or other signaling mechanisms by altering the membrane physiology [116–119].…”