Role for mitogen-activated protein kinases in phenobarbital-induced expression of cytochrome P450 2B in primary cultures of rat hepatocytes

doi:10.1016/j.toxlet.2005.08.006

Toxicology Letters

Volume 161, Issue 1, 8 February 2006, Pages 61-72

https://doi.org/10.1016/j.toxlet.2005.08.006 Get rights and content

Abstract

Phenobarbital (PB) alters expression of numerous hepatic genes, including genes of cytochrome P450 2B1 and 2B2 (CYP2B). However, the intracellular mechanisms remain to be fully elucidated. The present study investigated the involvement of mitogen-activated protein kinases (MAPKs) in rat hepatocytes in primary culture. We showed that PB induced an early, dose-dependent activation of ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 MAPKs. Regarding the PB (1 mM) induction of CYP2B mRNA expression, while chemically inhibiting JNK had no effect, specific inhibitors of the ERK (U0-126) and p38 (SB-203580) pathways up- and down-regulated this expression, respectively. However, although such a regulation was confirmed when testing the effect of a dominant negative mutant of the ERK pathway on the CYP2B2 enhancer-promoter activity, no such transcriptional role was found with the p38 pathway. Moreover, upon arrest of transcription, the stability of CYP2B mRNA remained unaffected by SB-203580. In conclusion, we show that the ERK pathway negatively regulates CYP2B2 enhancer-promoter activity and that, despite p38 activation upon PB exposure, the sensitivity of CYP2B mRNA expression to SB-203580 appears to be unrelated to this kinase.

Introduction

Cytochrome P450 (CYP) superfamily monooxygenases are important for the oxidative biotransformation of drugs and toxins. Although they are primarily detoxifying enzymes, CYPs can bioactivate some of their substrates, leading in some cases to the production of highly toxic metabolites. As, in many cases, CYP gene expression is known to be controlled by CYP substrates, toxic effects might consequently be favoured. In this context, a better knowledge of CYP regulation seems to be essential.

Among the known classes of CYP inducers, one comprises the anti-epileptic drug phenobarbital (PB) and several PB-related or -unrelated lipophilic chemicals termed “PB-like” inducers (such as several barbiturates, the halogen pesticide dichlordiphenyltrichloroethane (DDT), and the CYP2B1 inhibitor clotrimazole) (Lubet et al., 1992). PB and PB-like inducers have been shown to enhance, mainly in the liver, expression of various CYP genes, including those for CYP2B1 and CYP2B2 (CYP2B) (Luc et al., 1996, Bauer et al., 2004). Despite many studies on the mode of action of PB on CYP expression, this remains as yet to be fully elucidated. Although PB induction has long been known to involve a transcriptional activation of PB-responsive CYP2B genes (Adesnik et al., 1981, Hardwick et al., 1983), the key cis- and trans-acting factors of this activation have been identified only quite recently: a 51 bp response element termed PBREM (PB-responsive enhancer module), identified in a distal region of the mouse Cyp2b10 (Honkakoski et al., 1998), rat CYP2B1/2 (Smirlis et al., 2001, Trottier et al., 1995) and human CYP2B6 (Sueyoshi et al., 1999) 5′-flanking regions, and the orphan nuclear receptor constitutive active receptor or constitutive androstane receptor (CAR) (Choi et al., 1997). In response to PB and PB-like inducers, CAR is translocated to the nucleus where it heterodimerizes with the retinoid X receptor (RXR) in order to transactivate the PBREM (Kawamoto et al., 1999, Sueyoshi and Negishi, 2001). However, as PB is not an agonist ligand of CAR, how it elicits CAR translocation and activation is not fully understood. Several groups, including ours, have shown the involvement of protein kinases and phosphatases in the mechanism whereby PB induces CYP genes (Corcos and Lagadic-Gossmann, 2001, Sueyoshi and Negishi, 2001). Interestingly, some of the phosphorylation/dephosphorylation events demonstrated are directly involved in the nuclear translocation and activation of CAR (Kawamoto et al., 1999). Nevertheless, none of these studies clearly shows activation of kinases or phosphatases upon PB exposure, although Negishi's group recently reported recruitment of protein phosphatase 2A to the cytoplasmic CAR–HSP90 complex in mouse primary hepatocytes (Yoshinari et al., 2003). Also, the AMP-activated protein kinase (AMPK) might be involved in this process (Rencurel et al., 2005).

Several studies have demonstrated a role for mitogen-activated protein kinases (MAPKs) in the control of diverse biotransformation enzymes. For example, whereas ERK2 positively regulates the induction of antioxidant responsive element (ARE)-dependent phase II detoxifying enzymes (Yu et al., 1999), p38 kinase pathway functions as a negative regulator towards these enzymes (Yu et al., 2000). The c-Jun N-terminal kinase pathway has also been shown to play such a negative role on the effects of bile acids on the CYP7A1 gene (Gupta et al., 2001). With respect to PB, it has previously been shown that treatment of hepatocytes with this xenobiotic induced activation of the ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) pathway (Hodges et al., 2000). However, a role for such an activation in the mechanism of PB induction of CYP2B remains to be thoroughly studied, especially as previous data from our group have suggested the involvement of the ERK pathway in the regulation of CYP2B and CYP3A1/2 mRNA induction in primary rat hepatocytes (Joannard et al., 2000). Nothing is known about the role of the other MAPKs in this induction, but it is worth noting that PB elicits cell proliferation in liver, a process known to involve different MAPKs (Zhang and Liu, 2002).

The aim of the present work was to further analyse the involvement of MAPK in the transduction pathways involved in PB induction of CYP2B expression in rat hepatocytes in primary culture. By the use of a chemical inhibitor and of a transiently transfected dominant negative mutant of the MAPK pathway, we demonstrated that ERK1/2 negatively regulates PB induction of CYP2B2, via an effect on the enhancer-promoter. Concerning the other MAPK pathways, we found that exposure of rat hepatocytes to PB resulted in an early dose-dependent activation of the p38α isoform. Furthermore, the use of a known inhibitor of this kinase, SB-203580, led to a dose-dependent inhibition of PB induction of CYP2B mRNA expression, probably via a p38-independent effect.

Section snippets

Chemicals

Liberase was obtained from Boehringer Mannheim (Mannheim, Germany). Cell culture medium (Williams’ E) was purchased from Gibco BRL (Life Technologies, Paisley, Scotland) and fetal calf serum from Seromed (Biochrom KG, Berlin, Germany) and from Dominique Dutscher (Brumath, France). PB was obtained from Coopération Pharmaceutique Française (Melun, France). Dexamethasone, ITS (insulin from bovine pancreas, human transferrin, sodium selenite), and 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole

Early activation of MAPKs upon PB application in primary cultures of rat hepatocytes

In order to assess the involvement of MAPKs in the intracellular pathways underlying PB induction of CYP2B, we first tested whether PB was capable of eliciting an early activation of MAPKs. This was carried out using specific antibodies raised against the phosphorylated and total forms of MAPKs.

Concerning the ERK pathway, we observed that phosphorylation of ERK1 and 2 was significantly increased only at concentrations of 3 and 5 mM PB following a 15 min treatment (Fig. 1A). Furthermore, regarding

Discussion

Although several recent studies have clearly demonstrated the involvement of MAPKs in the regulation of the induction of phase II drug metabolizing enzymes (DME) by several xenobiotics (Yu et al., 1999, Yu et al., 2000), less is known about such a role in the control of phase I DME. In the present study, we have found that exposure of hepatocytes to PB results in an early dose-dependent activation of the MAPKs ERK, JNK and p38. Regarding the CYP2B1/2 induction by PB, to our knowledge, this

Acknowledgements

We wish to thank Laurent Corcos for valuable discussion, and Sophie Langouët and Marc Le Vee for their technical advice on Q-PCR. We are very grateful to Angel Nebreda for providing p38α antiserum. F.J. was the recipient of a D.Phil. scholarship from the Ligue contre le Cancer, Comité des Côtes d’Armor.

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Role for mitogen-activated protein kinases in phenobarbital-induced expression of cytochrome P450 2B in primary cultures of rat hepatocytes

Abstract

Introduction

Section snippets

Chemicals

Early activation of MAPKs upon PB application in primary cultures of rat hepatocytes

Discussion

Acknowledgements

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J. Biol. Chem.