Stability of the aryl hydrocarbon receptor and its regulated genes in the low activity variant of Hepa-1 cell line

Andria Humphrey-Johnson,Rawia Abukalam,Sakina E Eltom

doi:10.1016/j.toxlet.2015.01.016

Abstract

We examined the expression kinetics of some of the aryl hydrocarbon receptor (AhR)-regulated genes in LA1 variant cells compared to wild type (WT) Hepa-1 mouse hepatoma cell lines, and we investigated the stability of AhR protein as a key step in the function of this receptor. Treatment of both cell types with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in increased CYP1A1 and CYP1B1 mRNA with a subsequent down regulation of AhR. We show here that co-treatment with transcription inhibitor actinomycin D (ActD) has reversed the TCDD-induced depletion of AhR protein in WT. However, the proteolytic degradation of AhR in absence of TCDD was significantly higher in LA1 cells than in WT, and ActD treatment reduced this loss. Induction of CYP1A1 and CYP1B1 mRNA by TCDD in WT cells each exhibited bursts of activity in the initial hour which were about 3-fold greater than in LAI cells. The induced mRNA levels in LA1 exhibited a slow and sustained increase approximating the WT levels by 20h. The induction of two other AhR-regulated genes also showed comparable turnover differences between the two types of cell. Thus, altered regulation of the AhR responsive genes in LA1 may result from a difference in AhR stability.

Highlights

The aryl hydrocarbon receptor (AhR) which is a ligand-activated basic helix-loop-helix transcriptional factor (Burbach, Poland et al 1992), binds poly aromatic hydrocarbons (PAHs), including 2,3,7,8 tetrachloro-dibenzo-p-dioxin (TCDD), and mediates their toxic responses (Poland and Knutson 1982)
The AhR is transformed into a form that readily translocates to the nucleus where it forms a heterodimer with the related basic helix-loop-helix (bHLH), Ah receptor nuclear translocator (ARNT) protein (Hoffman, Reyes et al 1991)
We find this elevated CYP1A1 mRNA in LA1 disproportionate to their AhR levels (Fig. 1-A), to be associated with early passage of cells in culture, whereas culture of later passaged-LA1 cells show substantially less inducibility of CYP1A1 with TCDD treatment (Fig. 1-B)

Summary

Introduction

The aryl hydrocarbon receptor (AhR) which is a ligand-activated basic helix-loop-helix (bHLH) transcriptional factor (Burbach, Poland et al 1992), binds poly aromatic hydrocarbons (PAHs), including 2,3,7,8 tetrachloro-dibenzo-p-dioxin (TCDD), and mediates their toxic responses (Poland and Knutson 1982). The AhR is transformed into a form that readily translocates to the nucleus where it forms a heterodimer with the related bHLH, Ah receptor nuclear translocator (ARNT) protein (Hoffman, Reyes et al 1991) Binding of this heterodimer to DNA recognition motifs designated as xenobiotic-responsive elements (XREs), results in enhanced transcription of multiple genes (Jones, Galeazzi et al 1985; Denison, Fisher et al 1989). These genes known as the Ah-responsive genes include CYP1A1, CYP1A2 (Gonzalez, Mackenzie et al 1984) and CYP1B1 (Savas, Bhattacharyya et al 1994; Bhattacharyya, Brake et al 1995). The protein products of these CYPs are catalytically active in metabolizing many endogenous compounds, such as β-estradiol, and many drugs, dietary components, mutagens, carcinogens and environmental pollutants (Conney 1982)

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