Analysis of Aryl Hydrocarbon Receptor-Mediated Signaling during Physiological Hypoxia Reveals Lack of Competition for the Aryl Hydrocarbon Nuclear Translocator Transcription Factor
- Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina
Abstract
The aryl hydrocarbon nuclear translocator (ARNT) protein functions as a transcription factor after dimerization with other basic helix-loop-helix proteins. Thus, dimerization of ARNT within one pathway may limit the availability of this protein to others. To investigate this issue, aryl hydrocarbon receptor (AHR)-mediated signaling was investigated in mouse (Hepa-1), rat (H4IIE), and human (HepG2) hepatoma cell lines undergoing physiologically induced hypoxia (<1% O2). Basal levels of ARNT protein were not affected by hypoxia in any cell line, and ARNT remained exclusively nuclear. Furthermore, quantitative Western blotting revealed that the concentration of ARNT sequestered during hypoxia represented a small fraction of the total ARNT protein pool (12 and 15% in Hepa-1 and H4 cells, respectively). When the AHR-mediated signaling pathway was activated during hypoxia by 2,3,7,8-tetrachlorodibenzo-p-dioxin, the induction of P4501A1 protein was reduced by 55% without changes in the level of mRNA in Hepa-1 cells, whereas the levels of induction of both P4501A1 protein and CYP1A1 mRNA were reduced by >80% in the H4 cell line. Importantly, gel mobility shift analysis and Western blotting showed that the same level of AHR/ARNT complexes could be detected in cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin during hypoxia and normoxia. These data suggest that the effects of hypoxia on AHR-mediated gene regulation occur distal to the formation of AHR/ARNT complexes and imply that functional interference between hypoxia and AHR-mediated signaling does not occur through competition for ARNT protein.
Footnotes
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Send reprint requests to: Dr. Richard S. Pollenz, Department of Biochemistry and Molecular Biology, MUSC, 171 Ashley Ave., Charleston, SC 29403. E-mail: pollenzr{at}musc.edu
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This work was supported in part by Grant ES08980 from the National Institute of Environmental Health Sciences. Portions of the work were presented at the Mechanisms of Toxicity Gordon Research Conference, July 1998, and the Society of Toxicology Meeting, March 1999.
- Abbreviations:
- ARNT
- aryl hydrocarbon nuclear translocator
- AHR
- aryl hydrocarbon receptor
- bHLH
- basic helix-loop-helix
- HIF-1α
- hypoxia-inducible factor-1α
- TCDD
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- PER
- periodicity
- SIM
- single minded
- PAS
- PER-ARNT-SIM
- GAR-HRP
- goat anti-rabbit horseradish peroxidase
- ECL
- enhanced chemiluminescence
- DFO
- desferrioxamine
- TTBS
- Tris-buffered saline with Tween 20
- CYP or P450
- cytochrome P-450
- PAGE
- polyacrylamide gel electrophoresis
- EMSA
- electrophoretic mobility shift assay
- XRE
- xenobiotic-responsive element
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- Received April 15, 1999.
- Accepted August 25, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
