Signaling of the Pregnane X Receptor

University Of Rhode Island, Kingston RI

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Abstract

DESCRIPTION (provided by applicant): Yan, Bingfang ABSTRACT This is a revision application in response to the announcement NOT-OD-09-058: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications This revision application proposes a set of studies to extend the specific aims of the parent grant. The focus of the parent grant, presented as two specific aims, is to elucidate signaling events associated with the pregnane X receptor (PXR), a master regulator on the expression of many chemical elimination genes such as cytochrome P450 3A4 (CYP3A4) in humans and CYP3A23 in rats. Interestingly, the CYP3A23 but not the CYP3A4 proximal promoter responds robustly to a PXR ligand, although a functional PXR element is present in both CYP3A4 and CYP3A23 proximal promoters. Instead, the CYP3A4 proximal promoter coordinates with other upstream PXR elements and confers transactivation activity. Sequence comparison reveals that the CYP3A4 promoter, compared with CYP3A23, has the PXR element 30 bases farther from the transcription starting site. In addition, the CYP3A4 but not CYP3A23 promoter has an HNF3/CEBP site in front of the PXR element. The PXR-mediated trans- activation, on the other hand, is markedly attenuated by cytokines such as interleukin-6 (IL-6). microRNA-511 (miR-511), highly induced by IL-6, diminishes PXR transactivation. miRs are known to silence gene expression through sequence pairing with their target mRNAs. The proposed studies are designed to test the hypotheses that the PXR transcript is a sequence-specific target of miR-511, and the physical location of the PXR element and the presence of the HNF3/CEBP site contribute significantly to the inability of the CYP3A4 proximal promoter to effectively respond to PXR-transactivation. To define the potential role of the physical location or the interference of the HNF3/CEBP site, mutants will be prepared to disrupt the HNF3/CEBP site, relocate the PXR element closer to the transcription starting site, or both. These mutants will be tested for increased ability to respond to PXR transactivation. To determine the molecular action of miR-511 to decrease PXR expression, cells will be transfected with miR-511 and the levels of PXR mRNA and its translation efficiency will be monitored. In addition, the sequence supporting the action of miR-511 will be located and the role of miR-511 in the suppression of PXR by IL-6 will be established. Overall, the studies in the revision application represent significant expansion of the original aims in the parent grant. Importantly, these studies are closely integrated into the studies in the parent grant, and together, they will provide more comprehensive understanding on the expression of drug elimination genes regarding species-dependent transactivation and altered expression during inflammation. PUBLIC HEALTH RELEVANCE: The capacity of drug elimination is altered by co-administration of other drugs and disease status such as inflammation. The revision application proposes a set of studies to reveal the underlying mechanisms on how and to which extent the drug-eliminating capacity of an individual is altered by co-administered drugs or inflammatory mediators.