Abstract
Pregnane x receptor is a ligand-activated transcription factor that regulates drug-inducible expression of several key cytochrome P450 enzymes and drug transporter proteins in liver and intestine in a species-specific manner. Activation of this receptor modulates several key biochemical pathways, including gluconeogenesis, beta-oxidation of fatty acids, fatty acid uptake, cholesterol homeostasis, and lipogenesis. It is of current interest to determine whether the interaction between pregnane x receptor and these key biochemical pathways is evolutionarily conserved. We show here that activation of the cyclic AMP-dependent protein kinase signaling pathway synergizes with pregnane x receptor-mediated gene activation in mouse hepatocytes. Conversely, cyclic AMP-dependent protein kinase signaling has a repressive effect upon pregnane x receptor-mediated gene activation in rat and human hepatocytes. We show that the human pregnane x receptor protein can serve as an effective substrate for catalytically active cyclic AMP-dependent protein kinase in vitro. Metabolic labeling of the protein in vivo indicates that human pregnane x receptor exists as a phosphoprotein and that activation of cyclic AMP-dependent protein kinase signaling modulates the phosphorylation status of pregnane x receptor. Activation of cyclic AMP-dependent protein kinase signaling also modulates the interactions of pregnane x receptor with protein cofactors. Our results define the species-specific impact of cyclic AMP-dependent protein kinase signaling on pregnane x receptor and provide a molecular explanation of cyclic AMP-dependent protein kinase-mediated repression of human pregnane x receptor activity. Taken together, our results identify a novel mode of regulation of pregnane x receptor activity and highlight prominent functional differences in the process across species.
Highlights
Including certain steroids and bile acids, a plethora of naturally occurring compounds, specific antibiotics, antifungal drugs, polychlorinated binphenyls, organochloride pesticides, and phenobarbital (PB) [2]
The same laboratory reported that forskolin treatment produces increased expression of Cyp3A1 in rat hepatocytes; induction was independent of cAMP and protein kinase (PKA)
PKA Signaling has a Species-specific Effect on PXR-mediated Gene Activation in Hepatocytes—To determine if PKA signaling has a species-specific effect upon PXR-mediated gene activation, primary cultures of mouse and rat hepatocytes were isolated and treated with vehicle or 10 M pregnenolone 16␣-carbonitrile (PCN) for 24 h in the presence of increasing concentrations (0.01, 0.1, and 1.0 mM) of 8-Br-cAMP, and Northern blotting analysis was performed (Fig. 1)
Summary
Including certain steroids and bile acids, a plethora of naturally occurring compounds, specific antibiotics, antifungal drugs, polychlorinated binphenyls, organochloride pesticides, and phenobarbital (PB) [2]. Studies from our laboratory using mouse models show that the PKA signal transduction pathway synergizes with liganddependent PXR-mediated induction of Cyp3a11 gene expression [15]. These studies revealed that the PKA-mediated synergism is a PXR-dependent phenomenon in mice. During the course of these studies, we noticed significant differences in PKA-dependent alterations of drug-inducible CYP gene expression that were dependent upon the species of rodent (mouse versus rat) used to isolate primary cultures of hepatocytes. These observations led us to initiate a systematic study of the effect of PKA signaling on druginducible CYP3A gene expression in cultured hepatocytes across three different species: mice, rats, and humans
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