Aryl-hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor activated by polyaromatic hydrocarbon compounds of synthetic and natural origin. While a number of novel AhR ligands have been identified recently, little is known about their possible influence on AhR levels and stability. We used western blot, qRT-PCR and immunocytochemistry to determine the effects of AhR ligands on AhR expression in cultured human keratinocytes (N-TERT), and immunohistochemistry to assess patterns of AhR expression in human and mouse skin and skin appendages. While AhR was highly expressed in cultured keratinocytes and in skin in vivo, including epidermis and certain hair follicle compartments (IRS, ORS, hair bulb), it was found primarily in the cytoplasm, but not in the nucleus, suggesting lack of its activity. At the same time, treatment of N-TERT cells with proteasomal inhibitor MG132 and consequent inhibition of AhR degradation resulted in nuclear AhR accumulation. Treatment of keratinocytes with AhR ligands such as TCDD (dioxin), FICZ, and I3C (indole-3-carbinol), but not with kynurenine, caused near-complete disappearance of AhR with protein half-life of 2-4 hours for ligand-treated samples. The AhR decay was blocked by proteasome inhibition, indicating degradation-based mechanism of regulation. Furthermore, addition of hypoxia mimetics CoCl2 and DMOG decreased effects of AhR ligands on degradation of AhR, suggesting that the AhR dimerization partner ARNT (HIF1β) is required for degradation. Consistently, substrate-induced degradation of AhR was blocked in N-TERT cells with knockdown of ARNT. Additionally, inhibition of HDACs with Trichostatin S resulted in enhanced expression of AhR in both untreated and ligand-treated cells. Altogether, these results demonstrate that AhR is primarily regulated posttranslationally via proteasome-mediated degradation, and suggest novel ways to manipulate AhR levels and signaling.

Ligands of aryl-hydrocarbon receptor regulate its stability in ARNT-dependent manner