Abstract
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to mediate toxic responses to dioxin. However, the role of the AhR in the regulation of cellular physiology has only recently been appreciated, including its ability to control cell cycle progression and apoptosis by unknown mechanisms. We hypothesized that the AhR enhances the activation of the AKT serine/threonine kinase (Akt) pathway to promote cell survival. Utilizing AhR knock-out (Ahr−/−) and wild-type (Ahr+/+) mouse lung fibroblasts (MLFs), we found that Ahr−/− MLFs have significantly higher basal Akt phosphorylation but that AhR did not affect Akt phosphorylation in MLFs exposed to growth factors or AhR ligands. Basal Akt phosphorylation was dependent on PI3K but was unaffected by changes in intracellular glutathione (GSH) or p85α. There was no significant decrease in cell viability in Ahr−/− MLFs treated with LY294002—a PI3K inhibitor—although LY294002 did attenuate MTT reduction, indicating an affect on mitochondrial function. Using a mass spectrometry (MS)-based approach, we identified several proteins that were differentially phosphorylated in the Ahr−/− MLFs compared to control cells, including proteins involved in the regulation of extracellular matrix (ECM), focal adhesion, cytoskeleton remodeling and mitochondrial function. In conclusion, Ahr ablation increased basal Akt phosphorylation in MLFs. Our results indicate that AhR may modulate the phosphorylation of a variety of novel proteins not previously identified as AhR targets, findings that help advance our understanding of the endogenous functions of AhR.
Highlights
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to mediate toxic responses to dioxin
In the canonical AhR signaling pathway, activated AhR translocates to the nucleus where it dimerizes with AhR nuclear translocator (ARNT), a class II protein of the basic helixloop-helix (bHLH)-PAS family, in order to bind to the xenobiotic responsive elements (XREs) on DNA
AKT serine/ threonine kinase (Akt) has two main activating phosphorylation sites—the threonine 308 residue (T308) which is phosphorylated by phosphoinositide-dependent kinase-1 (PDK1) and the serine 473 residue (S473) which is phosphorylated by mammalian target of rapamycin complex 210
Summary
All cells were seeded at 10,000 cells/cm[2], grown to approximately 80–90% confluence and cultured in serum-free medium for 18 h before conducting the experiments unless otherwise indicated. Smoke from one cigarette was bubbled through 15 ml of serum-free medium and the optical density (OD) of the CSE was measured using a SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, Hercules, CA). Protein quantification was performed using a Thermo Scientific Pierce Bicinchoninic Acid (BCA) protein assay kit (Thermo Fisher Scientific) according to manufacturer’s instructions and the absorbance was measured with an iMark microplate reader (Bio-Rad Laboratories, Hercules, CA). Cells were incubated in the transfection medium for one hour after which serum-containing medium was added. The final samples contained pooled protein extracts utilizing cells from two different Ahr+/+ and Ahr−/− mice. Values with P < 0.05 were considered significantly different
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