Regulation of the Pregnane X Receptor Signaling Pathway

Liver-enriched nuclear receptors (NRs) collectively function as metabolic and toxicological `sensors' that mediate liver-specific gene-activation in mammals. NR-mediated gene-environment interaction regulates important steps in the hepatic uptake, metabolism and excretion of glucose, fatty acids, lipoproteins, cholesterol, bile acids, and xenobiotics. While it is well-recognized that ligand-binding is the primary mechanism behind activation of NRs, recent research is revealing that multiple signal transduction pathways modulate NR-function in liver. The interface between specific signal transduction pathways and NRs helps to determine their overall responsiveness to various environmental and physiological stimuli. The pregnane x receptor (PXR, NR1I2) was identified in 1998 as a member of the NR superfamily of ligand-activated transcription factors. PXR is activated by a broad range of lipophilic compounds in a species-specific manner. The primary function ascribed to PXR is the homeostatic control of steroids, bile acids, and xenobiotics. This function is mediated through PXR's ability to coordinately activate gene expression and regulate the subsequent activity of phase I and phase II metabolic enzymes, as well as several membrane transporter proteins. While PXR likely evolved primarily to protect the liver from toxic assault, its activation also represents the molecular basis for an important class of drug-drug, herb-drug, and food-drug interactions. While ligand binding is the primary mode of PXR activation, several signal transduction pathways interface with the PXR protein to determine its overall responsiveness to environmental stimuli. Multiple signaling pathways modulate the activity of PXR, likely through direct alteration of the phosphorylation status of the receptor and its protein cofactors. Therefore, specific combinations of ligand binding and cell signaling pathways affect PXR-mediated gene activation and determine the overall biological response. This dissertation contributes to the molecular understanding of the regulation of PXR by novel agonists, cAMP-dependent protein kinase (PKA) signaling, and phosphorylation. The results presented here were primarily obtained from mouse and tissue culture systems. This dissertation identifies Tian Xian, a traditional Chinese herbal anti-cancer remedy, as a novel PXR activator. This evidence suggests that Tian Xian should be used cautiously by cancer patients taking chemotherapy due to its potential to increase the metabolism of co-administered medications. In addition, data presented here show that activation of PKA signaling modulates PXR activity in a species-specific manner. It is further revealed that PXR exists as phospho-protein in vivo and that the activation of PKA signaling modulates the phospho-threonine status of PXR. Finally, the potential phosphorylation sites within the PXR protein are identified. These phosphorylation sites are characterized, using a phosphomimetic and phospho-deficient site-directed mutagenesis based approach, based on their ability to modulate PXR activity. Taken together, the work presented in this dissertation contributes to understanding the interface between ligands, signal transduction pathways and PXR activity, which is critical for the development of safe and effective therapeutic strategies

Identification of 42 genes linked to stage II colorectal cancer metastatic relapse

Colorectal cancer (CRC) is one of the leading causes of cancer mortality. Metastasis remains the primary cause of CRC death. Predicting the possibility of metastatic relapse in early-stage CRC is of paramount importance to target therapy for patients who really need it and spare those with low-potential of metastasis. Ninety-six stage II CRC cases were stratified using high-resolution array comparative genomic hybridization (aCGH) data based on a predictive survival algorithm and supervised clustering. All genes included within the resultant copy number aberrations were each interrogated independently at mRNA level using CRC expression datasets available from public repositories, which included 1820 colon cancers, and 167 normal colon tissues. Reduced mRNA expression driven by copy number losses and increased expression driven by copy number gains revealed 42 altered transcripts (29 reduced and 13 increased transcripts) associated with metastatic relapse, short disease-free or overall survival, and/or epithelial to mesenchymal transition (EMT). Resultant genes were classified based on gene ontology (GO), which identified four functional enrichment groups involved in growth regulation, genomic integrity, metabolism, and signal transduction pathways. The identified 42 genes may be useful for predicting metastatic relapse in stage II CRC. Further studies are necessary to validate these findings

LUBAC prevents lethal dermatitis by inhibiting cell death induced by TNF, TRAIL and CD95L

The linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1 and SHARPIN, is required for optimal TNF-mediated gene activation and to prevent cell death induced by TNF. Here, we demonstrate that keratinocyte-specific deletion of HOIP or HOIL-1 (E-KO) results in severe dermatitis causing postnatal lethality. We provide genetic and pharmacological evidence that the postnatal lethal dermatitis in HoipE-KO and Hoil-1E-KO mice is caused by TNFR1-induced, caspase-8-mediated apoptosis that occurs independently of the kinase activity of RIPK1. In the absence of TNFR1, however, dermatitis develops in adulthood, triggered by RIPK1-kinase-activity-dependent apoptosis and necroptosis. Strikingly, TRAIL or CD95L can redundantly induce this disease-causing cell death, as combined loss of their respective receptors is required to prevent TNFR1-independent dermatitis. These findings may have implications for the treatment of patients with mutations that perturb linear ubiquitination and potentially also for patients with inflammation-associated disorders that are refractory to inhibition of TNF alone

A Systematic Analysis of Predicted Phosphorylation Sites within the Human Pregnane X Receptor Protein

The pregnane X receptor (PXR, NR1I2) regulates the expression of genes that encode drug-metabolizing enzymes and drug transporter proteins in liver and intestine. Understanding the molecular mechanisms that modulate PXR activity is therefore critical for the development of effective therapeutic strategies. Several recent studies have implicated the activation of kinase signaling pathways in the regulation of PXR biological activity, although direct evidence and molecular mechanisms are currently lacking. We therefore sought to characterize potential phosphorylation sites within the PXR protein by use of a rational, comprehensive, and systematic site-directed mutagenesis approach to generate phosphomimetic mutations (Ser/Thr → Asp) and phospho-deficient mutations (Ser/Thr → Ala) at 18 predicted consensus kinase recognition sequences in the human PXR protein. Here, we identify amino acid residues Ser8, Thr57, Ser208, Ser305, Ser350, and Thr408 as being critical for biological activity of the PXR protein. Mutations at positions 57 and 408 abolish ligand-inducible PXR activity. Mutations in the extreme N terminus and in the PXR ligand-binding domain at positions Ser8, Ser305, Ser350, and Thr408 decrease the ability of PXR to form heterodimers with retinoid X receptor α. Mutations at positions Ser208, Ser305, Ser350, and Thr408 alter PXR-protein cofactor interactions. Finally, the subcellular localization of the PXR protein is profoundly affected by mutations at position Thr408. These data suggest that PXR activity can potentially be regulated by phosphorylation at specific amino acid residues within several predicted consensus kinase recognition sequences to differentially affect PXR biological activity