Phenobarbital regulates nuclear expression of HNF-4alpha in mouse and rat hepatocytes independent of CAR and PXR.
Phenobarbital is a lipophilic molecule used as a sedative and antiepileptic drug that elicits a multitude of effects in the liver, including gross liver enlargement, hepatocyte hypertrophy, and induced expression of drug-metabolizing enzymes and other liver-specific genes. The constitutive androstane receptor (CAR; NR1I3) and to a lesser extent the pregnane X receptor (PXR; NR1I2) are responsible for mediating induction of many phenobarbital-responsive genes. However, CAR-mediated transcriptional control of some genes is critically dependent on hepatocyte nuclear factor 4 alpha (HNF-4alpha; NR2A1), which itself regulates multiple liver-specific genes involved in hepatic growth, metabolism, and differentiation. We studied the effects of phenobarbital on HNF-4alpha expression in hepatocytes and provide evidence that HNF-4alpha nuclear expression is regulated in response to phenobarbital. Real-time polymerase chain reaction analyses revealed that HNF-4alpha mRNA is modestly up-regulated by phenobarbital. In addition, nuclear expression of HNF-4alpha protein is significantly elevated 3 hours after the administration of phenobarbital in wild-type, CAR-/-, and CAR-/-/PXR-/- mice. In vitro analysis revealed that phenobarbital-induced HNF-4alpha expression is both time- and dose dependent. In addition, the phosphatase inhibitor okadaic acid and the Ca2+/calmodulin-dependent protein kinase II inhibitor KN62 block nuclear induction of HNF-4alpha by phenobarbital. Furthermore, HNF-4alpha nuclear expression is enhanced by inhibition of cyclic AMP-dependent protein kinase A. In conclusion, induced nuclear expression of HNF-4alpha and CAR is an integral part of the phenobarbital response, aimed at coordinated regulation of genes involved in drug metabolism and detoxification as well as maintenance of liver function.
Bell
Aaron W
AW
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Michalopoulos
George K
GK
D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D000090702:Constitutive Androstane Receptor; D015870:Gene Expression / Q000187:drug effects*; D051557:Hepatocyte Nuclear Factor 4 / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010634:Phenobarbital / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D000077297:Pregnane X Receptor; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011916:Rats, Inbred F344; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D000927:Anticonvulsants; D000090702:Constitutive Androstane Receptor; D051557:Hepatocyte Nuclear Factor 4; C496593:Hnf4a protein, mouse; C496594:Hnf4a protein, rat; C000625853:Nr1i2 protein, mouse; C000625854:Nr1i2 protein, rat; C000716328:Nr1i3 protein, mouse; C000716329:Nr1i3 protein, rat; D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D014157:Transcription Factors; D010634:Phenobarbital