2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon that is considered to be one of the most toxic synthetic compounds known to man. Exposure to TCDD is felt to be responsible for a cascade of human health problems, ranging from birth defects to cancer. The biological responses to TCDD are mediated through the actions of a regulatory gene product in the cytosol of the cell called the dioxin- or Ah receptor (AhR). TCDD binds to the AhR initiating a series of biological events that leads to displacement of the heat shock protein hsp90, followed by translocation of the receptor to the nucleus. The DNA binding or activated form of the AhR is a phosphoprotein complex composed of a ligand binding subunit and the AhR nuclear translocator (ARNT) protein. In studies with the CYP1A1 gene, a cognate DNA binding sequence has been elucidated that serves as a specific cis acting element to facilitate transcriptional activation. Therefore, the rudimentary process of ligand binding to the AhR in cytosol facilitates a cascade of events that results in the process of transcriptional activation. While this process resembles that of the classical steroid-like hormone receptors, little is actually known regarding the biological events that participate in activation of the AhR. This application will be dedicated to understanding the cellular events involved in the regulation of the mouse AhR in vivo. We have recently demonstrated that by treating mice with tumor promoters such as phorbol esters (TPA), it is possible to limit the nuclear accumulation of the AhR, and inhibit the TCDD directed transcription of the Cyp 1 genes. (Okino, S.T. et al., J. Biol. Chem. 267, 6991,1992). We believe that the actions of TPA in vivo inhibit the functional properties of the cytosolic form of the AhR. Preliminary experiments have demonstrated that ligand binding is not affected, but TPA blocks the ability of the activated form of the AhR to bind to the Cyp1a-1 enhancer sequences. Experiments outlined in this application are designed to characterize the mechanism of AhR inhibition in vivo by TPA. We will pursue a course of investigation to examine the possibility that TPA may interfere with the unmasking of DNA binding sites by hsp90, or interrupt the functional properties of the ARNT protein, which is critical for nuclear translocation and DNA binding. In addition, we will also examine the possibility that TPA downregulates specific protein kinase C (PKC) isozymes in a fashion concordant with the inhibition of AhR function. However, an argument is also presented indicating that TPA may not mimic stimulatory actions of diacylglycerol on PKC, but may function indirectly in vivo by activating humoral factors that would then lead to a depression in AhR function. While significant information is available regarding the physical composition of the AhR, the above experiments should add to our understanding of the events that control the expression of this important protein.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA037139-11
Application #
2089238
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1984-04-01
Project End
1996-06-30
Budget Start
1995-07-01
Budget End
1996-06-30
Support Year
11
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Lamb, J G; Straub, P; Tukey, R H (1994) Cloning and characterization of cDNAs encoding mouse Ugt1.6 and rabbit UGT1.6: differential induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochemistry 33:10513-20
Okino, S T; Pendurthi, U R; Tukey, R H (1993) 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces the nuclear translocation of two XRE binding proteins in mice. Pharmacogenetics 3:101-9
Tukey, R H; Pendurthi, U R; Nguyen, N T et al. (1993) Cloning and characterization of rabbit liver UDP-glucuronosyltransferase cDNAs. Developmental and inducible expression of 4-hydroxybiphenyl UGT2B13. J Biol Chem 268:15260-6
Pendurthi, U R; Okino, S T; Tukey, R H (1993) Accumulation of the nuclear dioxin (Ah) receptor and transcriptional activation of the mouse Cyp1a-1 and Cyp1a-2 genes. Arch Biochem Biophys 306:65-9
Okino, S T; Pendurthi, U R; Tukey, R H (1992) Phorbol esters inhibit the dioxin receptor-mediated transcriptional activation of the mouse Cyp1a-1 and Cyp1a-2 genes by 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Biol Chem 267:6991-8
Strom, D K; Postlind, H; Tukey, R H (1992) Characterization of the rabbit CYP1A1 and CYP1A2 genes: developmental and dioxin-inducible expression of rabbit liver P4501A1 and P4501A2. Arch Biochem Biophys 294:707-16
Tukey, R H; Okino, S T (1991) Quantitation of related gene products by nuclear run-on and northern blot analysis. Methods Enzymol 206:284-90
Hapgood, J; Cuthill, S; Soderkvist, P et al. (1991) Liver cells contain constitutive DNase I-hypersensitive sites at the xenobiotic response elements 1 and 2 (XRE1 and -2) of the rat cytochrome P-450IA1 gene and a constitutive, nuclear XRE-binding factor that is distinct from the dioxin receptor. Mol Cell Biol 11:4314-23
Pendurthi, U R; Lamb, J G; Nguyen, N et al. (1990) Characterization of the CYP2C5 gene in 21L III/J rabbits. Allelic variation affects the expression of P450IIC5. J Biol Chem 265:14662-8
Potenza, C L; Pendurthi, U R; Strom, D K et al. (1989) Regulation of the rabbit cytochrome P-450 3c gene. Age-dependent expression and transcriptional activation by rifampicin. J Biol Chem 264:16222-8

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