In vitro and in vivo analysis of aryl hydrocarbon receptor regulation by the hepatitis B virus X-associated protein 2
Open Access
- Author:
- Hollingshead, Brett David
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 08, 2006
- Committee Members:
- Gary H Perdew, Committee Chair/Co-Chair
Jeffrey Maurice Peters, Committee Member
Ross Cameron Hardison, Committee Member
Ming Tien, Committee Member
Scott Trent Feldman, Committee Member - Keywords:
- aryl hydrocarbon receptor
XAP2
HSP90
gene regulation
chaperone proteins
AIP - Abstract:
- The aryl hydrocarbon receptor (AHR) is a soluble ligand-activated transcription factor that regulates the expression of a battery of metabolically important genes. Sustained activation of the AHR resulting from exposure to the high affinity and metabolically resistant environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a myriad of deleterious responses in humans and rodents. The AHR is also physiologically involved in liver development and immune system homeostasis. Therefore, obtaining a full mechanistic appreciation of AHR properties is imperative to understand how the AHR exerts its physiologic properties. In its inactive form the AHR resides in the cytoplasm as a multi-protein complex consisting of one AHR molecule and a dimer of the 90 kDa heat shock protein (HSP90). Additionally, the co-chaperone proteins p23 and the hepatitis B virus X-associated protein 2 (XAP2) can be found in the unliganded receptor complex. The research presented in this thesis investigates the regulation of AHR activity by XAP2 in cell culture as well as in transgenic mouse models. Through studies in cell culture utilizing a point mutant form of the AHR that is unable to functionally interact with XAP2 it is demonstrated that XAP2 acts as a repressor of AHR transcriptional activity. The absence of endogenous XAP2 binding to the mutant receptor results in higher transcriptional activity than the non-mutant AHR. Therefore, it is concluded that XAP2 modulates AHR activity, but its presence in the mature unliganded AHR complex is not essential for the formation of a transcriptionally competent receptor. XAP2 protein levels are low in liver compared to many other tissues. This led to the hypothesis that XAP2 levels in the liver are not sufficient to maximally regulate AHR transcriptional activity. Two lines of transgenic mice were generated that exhibited high, hepatocyte-specific expression of XAP2. Gene expression experiments were performed in mice using a broad dose range of the AHR agonist beta- naphthoflavone. Surprisingly, little difference in gene expression was observed between wild type and transgenic mouse lines. It was determined that although the liver expression of XAP2 was much higher in transgenic as compared to wild type mice, no significant stoichiometric increase in XAP2 binding within the unliganded AHR complex occurred. Consequently, it is concluded that the seemingly “low” levels of expressed XAP2 in liver are sufficient to maximally associate with AHR-HSP90 complexes and regulate AHR transcriptional activation. Finally, preliminary experiments demonstrate that the carcinogenic diol epoxide forms of select polycyclic aromatic hydrocarbons are capable of interacting with the AHR. They may utilize the AHR binding pocket to gain access to the nuclear DNA by way of receptor nucleocytoplasmic shuttling, whereby they can form potentially cancer-inducing DNA adducts. These introductory experiments set a foundation for future analysis of the AHR’s contribution to DNA damage resulting from exposure to polycyclic aromatic hydrocarbons.