Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Bingfang Yan

Abstract

The pregnane X receptor is a nuclear receptor, functioning as a ligand activated transcriptional factor. PXR is recognized as a major regulator of xenobiotic metabolism. Several important Phase I and Phase II enzymes, and drug transporters are regulated by PXR. PXR is also a master regulator of CYP3A4, which is one of the most important enzymes in drug metabolism. Thus, knowledge of regulation of PXR expression is crucial to understand alterations in drug metabolism. Many liver diseases such as steatosis and inflammatory liver diseases alter the expression of drug metabolizing enzymes and transporters. CYP3A4 is down-regulated in these diseases. Endoplasmic reticulum (ER) stress is recognized as one important cause of the diseases and it also accompanies with other diseases such as viral infections. ER stress is also interconnected with inflammation. CYP3A4 and some other P450 enzymes are also down-regulated in inflammation. Since PXR is the master regulator of CYP3A4, this dissertation investigated the effect of ER stress on the PXR expression and its consequence on PXR-mediated CYP3A4 induction. Thapsigargin and brefeldin A were used to induce ER stress in cell cultures. ER stress down-regulated the expression of PXR in primary hepatocytes and HepG2 cells by repressing transcription. A promoter study revealed that the HNF4α protein level decreased in ER stress whereas C/EBPβ LIP increased. HNF4α and C/EBPβ were bound to the promoter of PXR suggested that both HNF4α and C/EBPβ involved in down-regulation of PXR by ER stress. PXR-mediated CYP3A4 induction significantly decreased in ER stress. ER stress also induced the expression of IL-6 in primary hepatocytes. This finding established an interconnection between ER stress and inflammation. Interestingly, IL-6 also repressed PXR expression at the same regulatory sequences as thapsigargin. Furthermore, it has been shown elsewhere that some other nuclear receptors are regulated by microRNAs (miRNAs). miRNAs regulate gene expression at the post-transcriptional level, adding complexity to the gene regulation. This dissertation identified a miRNA that regulated the expression of PXR. miR-30c-1 down-regulated PXR. miR-30c-1 interacted at the 3’-UTR of PXR and decreased the PXR mRNA level. The potential binding site of miR-30c-1 on the 3’-UTR of PXR was located.

In summary, ER stress, which is the cause and consequence of several diseases, down-regulated the expression of PXR. As a result, CYP3A4 induction by PXR was reduced. Since CYP3A4 and some others drug metabolizing enzymes regulated by PXR are crucial in drug metabolism, ER stress may alter metabolism of drugs, leading to increasing drug toxicity or decreasing drug efficacy. miR-30c-1 regulated the expression of PXR, adding complexity to the regulation on PXR. Although the regulation on the expression of miR-30c-1 is not known, miR-30c-1 might be related to diseases or biological conditions that could eventually affect PXR expression.

Share

COinS