Phenobarbital regulates nuclear expression of HNF-4α in mouse and rat hepatocytes independent of CAR and PXR

Abstract

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-4␣; NR2A1), which itself regulates multiple liver-specific genes involved in hepatic growth, metabolism, and differentiation. We studied the effects of phenobarbital on HNF-4␣ expression in hepatocytes and provide evidence that HNF-4␣ nuclear expression is regulated in response to phenobarbital. Realtime polymerase chain reaction analyses revealed that HNF-4␣ mRNA is modestly upregulated by phenobarbital. In addition, nuclear expression of HNF-4␣ 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-4␣ expression is both time-and dose dependent. In addition, the phosphatase inhibitor okadaic acid and the Ca 2؉ /calmodulin-dependent protein kinase II inhibitor KN62 block nuclear induction of HNF-4␣ by phenobarbital. Furthermore, HNF-4␣ nuclear expression is enhanced by inhibition of cyclic AMP-dependent protein kinase A. In conclusion, induced nuclear expression of HNF-4␣ 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. (HEPATOLOGY 2006;44: 186-194.) P henobarbital (PB) is an antiepileptic drug and is the prototype of a large family of lipophilic PB-like compounds that have profound effects in the liver. Its effects on liver physiology are typified by hepatic hypertrophy, hyperproliferation of the smooth endoplasmic reticulum, and induction or repression of numerous genes, especially the genes of cytochrome P450 enzymes. 1-3 Hepatic hypertrophy induced by PB is mediated by a moderate increase in hepatocyte DNA synthesis and dramatic enlargement of individual hepatocytes. 4,5 Hepatic enlargement subsides after PB withdrawal and this decrease in liver size is mediated by hepatocyte apoptosis. 6 Liver regeneration in response to partial hepatectomy is markedly diminished in PB-treated rat and mouse livers. 7,8 PB is also one of the most effective liver tumor promoters in both rats and mice. The mechanism of liver tumor promotion by PB is not understood; however, there is evidence that PB affects EGF signaling. 9 . Furthermore, TGF␤1 receptors I, II, and III are specifically down-regulated in early neoplastic lesions following treatment with PB. 10,11 It has been hypothesized that the inhibition of DNA synthesis in normal hepatocytes gives a proliferative advant...

Comparison of Immortalized Fa2N-4 Cells and Human Hepatocytes as in Vitro Models for Cytochrome P450 Induction

“…2 and 3). Among the compounds tested, rifampicin, bosentan, moricizine, and ritonavir are reported as selective PXR-activators (LeCluyse, 2001;Luo et al, 2002;van Giersbergen et al, 2002), phenobarbital, phenytoin, and efavirenz are PXR/CAR dual activators (Hariparsad et al, 2004;Wang et al, 2004;Trubetskoy et al, 2005;Bell and Michalopoulos, 2006;Faucette et al, 2007), and CITCO and artemisinin are selective CAR activators (Maglich et al, 2003;Simonsson et al, 2006). Rifampicin-mediated CYP3A4 induction was conducted in Fa2N-4 cells and four batches of human hepatocytes (DMQ, 527, 455, and LHO).…”

“…Faucette et al (2006) observed that PXR activators induced both CYP3A4 and CYP2B6, whereas CAR activators induced CYP2B6 but not CYP3A4. Thus, the expression of PXR in Fa2N-4 cells and human hepatocytes could cause the induction of CYP2B6 mRNA in Fa2N-4 cells treated with phenobarbital as this compound is also a PXR activator (Bell and Michalopoulos, 2006). This may also explain why rifampicin, which is a selective PXR activator, induced CYP2B6 (EC 50 ϭ 8.0 M and E max ϭ 4.1-fold) in Fa2N-4 cells (LeCluyse, 2001).…”

“…b Gene induction occurs via cross-talk between PXR and CAR (Wang et al, 2004;Trubetskoy et al, 2005;Bell and Michalopoulos, 2006). c Dose-response curve did not reach saturation for CYP3A4 induction within concentrations tested.…”

Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes

“…cAMP and PGC1A have been shown to regulate gene expression patterns in the liver in vivo. For example, under conditions of fasting, cAMP levels are upregulated, resulting in the rapid induction of PGC1A, a co-factor for HNF4A (Iordanidou et al, 2005;Bell and Michalopoulos, 2006) that plays a crucial role in liver metabolism through the control of Phase I and Phase II drug-metabolizing enzyme activities, glucose metabolism and lipid production (Parviz et al, 2003;Rhee et al, 2003;Odom et al, 2004). Expression of PGC1A is also dramatically upregulated in mouse liver immediately after birth (Lin et al, 2003) possibly to promote maturation of the neonatal hepatocytes.…”

“…To further mature the cells, we investigated the role of cAMP signalling, as studies using hepatic cell lines have shown that activation of this pathway induces hepatic gene expression, in part through the induction of the peroxisome proliferator-activated receptor γ co-activator 1α (PGC1A; PPARGC1A -Human Gene Nomenclature Database), a co-activator that functions together with HNF4A to regulate the expression of many genes involved in hepatocyte function (Bell and Michalopoulos, 2006;Arpiainen et al, 2008;Benet et al, 2010;Dankel et al, 2010). Treatment of the aggregates with 8-bromoadenosine-3Ј,5Ј-cyclic monophosphate (8-Br-cAMP), a cell-permeable analogue of cAMP, from days 32 to 44 of culture significantly enhanced the expression of PGC1A (15-fold), G6P (25-fold) and TAT (33-fold) but not that of HNF4A (Fig.…”