5 research outputs found
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
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
Gli-similar (Glis) Krüppel-like zinc finger proteins: insights into their physiological functions and critical roles in neonatal diabetes and cystic renal disease
GLI-similar (Glis) 1-3 proteins constitute a
subfamily of the Krüppel-like zinc finger transcription
factors that are closely related to the Gli family. Glis1-3
play critical roles in the regulation of a number of
physiological processes and have been implicated in
several pathologies. Mutations in GLIS2 have been
linked to nephronophthisis, an autosomal recessive
cystic kidney disease. Loss of Glis2 function leads to
renal atrophy and fibrosis that involves epithelialmesenchymal
transition (EMT) of renal tubule epithelial
cells. Mutations in human GLIS3 have been implicated
in a syndrome characterized by neonatal diabetes and
congenital hypothyroidism (NDH) and in some patients
accompanied by polycystic kidney disease, glaucoma,
and liver fibrosis. In addition, the GLIS3 gene has been
identified as a susceptibility locus for the risk of type 1
and 2 diabetes. Glis3 plays a key role in pancreatic
development, particularly in the generation of ß-cells
and in the regulation of insulin gene expression. Glis2
and Glis3 proteins have been demonstrated to localize to
the primary cilium, a signaling organelle that has been
implicated in several pathologies, including cystic renal
diseases. This association suggests that Glis2/3 are part
of primary cilium-associated signaling pathways that
control the activity of Glis proteins. Upon activation in
the primary cilium, Glis proteins may translocate to the
nucleus where they subsequently regulate gene
transcription by interacting with Glis-binding sites in the
promoter regulatory region of target genes. In this
review, we discuss the current knowledge of the Glis
signaling pathways, their physiological functions, and
their involvement in several human pathologies