Aryl hydrocarbon receptor is regulated via multiple mechanisms in human keratinocytes

by Selleckchem | Sep 30 2023

Aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor activated by polycyclic aromatic hydrocarbons of synthetic and natural origin. While a number of novel AhR ligands have been recently identified, 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 N-TERT (N-TERT1) immortalized human keratinocytes, 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 the skin, it was found primarily in the cytoplasm, but not in the nucleus, suggesting its inactivity. At the same time, treatment of N-TERT cells with proteasomal inhibitor MG132 and eventual inhibition of AhR degradation resulted in nuclear AhR accumulation. Treatment of keratinocytes with AhR ligands such as TCDD, FICZ, caused near-complete disappearance of AhR, and treatment with I3C resulted in substantially diminished level of AhR possibly due to ligand-induced AhR degradation. The AhR decay was blocked by proteasome inhibition, indicating degradation-based mechanism of regulation. Additionally, AhR decay was blocked by ligand-selective AhR antagonist CH223191, implying substrate-induced mechanism of degradation. Furthermore, degradation of AhR was blocked in N-TERT cells with knockdown of AhR dimerization partner ARNT (HIF1β), suggesting that ARNT is required for AhR proteolysis. However, addition of hypoxia mimetics (HIF1 pathway activators) CoCl2 and DMOG had only minor effects on degradation of AhR. Additionally, inhibition of HDACs with Trichostatin A resulted in enhanced expression of AhR in both untreated and ligand-treated cells. These results demonstrate that in immortalized epidermal keratinocytes AhR is primarily regulated post-translationally via proteasome-mediated degradation, and suggest potential means to manipulate AhR levels and signaling in the skin. Overall, the AhR is regulated via multiple mechanisms, including proteasomal ligand- and ARNT-dependent degradation, and transcriptional regulation by HDACs, implying complex system of balancing its expression and protein stability.

Comments:

The passage you provided describes a study conducted on the Aryl hydrocarbon receptor (AhR), a transcription factor that is activated by polycyclic aromatic hydrocarbons (PAHs) of both natural and synthetic origin. The study aimed to investigate the effects of AhR ligands on the expression and stability of AhR in human keratinocytes and assess the patterns of AhR expression in human and mouse skin and its appendages.

The researchers used various techniques such as western blot, qRT-PCR, immunocytochemistry, and immunohistochemistry to analyze AhR expression and localization. Initially, they found that AhR was highly expressed in cultured keratinocytes and in the skin, but it was primarily located in the cytoplasm rather than the nucleus, indicating its inactivity.

To explore the factors influencing AhR levels and stability, the researchers treated the N-TERT cells (immortalized human keratinocytes) with different AhR ligands such as TCDD, FICZ, and I3C. They observed that treatment with these ligands resulted in the decrease or disappearance of AhR expression, possibly due to ligand-induced degradation of AhR.

The researchers then investigated the mechanism of AhR degradation and found that it was mediated by the proteasome, as treatment with a proteasomal inhibitor (MG132) prevented AhR decay and led to its accumulation in the nucleus. They also discovered that the ligand-selective AhR antagonist CH223191 blocked the degradation of AhR, suggesting a substrate-induced mechanism of degradation.

Furthermore, the study revealed that the dimerization partner of AhR, called ARNT (HIF1β), was required for the proteolysis of AhR. Knockdown of ARNT in N-TERT cells prevented the degradation of AhR. However, the addition of hypoxia mimetics (HIF1 pathway activators) had only minor effects on AhR degradation, indicating that the degradation of AhR was not strongly influenced by hypoxia.

In addition to proteasomal degradation, the researchers found that the expression of AhR could be regulated by histone deacetylases (HDACs). Inhibition of HDACs with Trichostatin A resulted in enhanced AhR expression in both untreated and ligand-treated cells, suggesting a role for transcriptional regulation in controlling AhR levels.

Overall, the study demonstrated that AhR is primarily regulated post-translationally through proteasome-mediated degradation in immortalized epidermal keratinocytes. It also suggested that the degradation of AhR is influenced by its ligands, ARNT, and HDACs, indicating a complex system for balancing the expression and stability of AhR. These findings provide insights into potential strategies to manipulate AhR levels and signaling in the skin.