Microtubules-interfering agents restrict aryl hydrocarbon receptor-mediated CYP1A2 induction in primary cultures of human hepatocytes via c-jun-N-terminal kinase and glucocorticoid receptor

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Abstract

Disruption of microtubules has been shown to cause suppression of inducibility of major cytochromes P450 (CYP) through several nuclear receptors. Here we tested the effects of structurally different clinically used microtubules-interfering agents (MIAs), such as colchicine, vincristine, vinblastine, nocodazole and taxol on aryl hydrocarbon receptor signaling pathway in human hepatocytes. We show that tested MIAs inhibit 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible expression of CYP1A2 mRNA and restrict TCDD-dependent nuclear translocation of aryl hydrocarbon receptor. On the other hand, these MIAs increased the content of aryl hydrocarbon receptor protein and mRNA by transcriptional mechanism. We show that the MIAs activate c-Jun -N-terminal kinase (JNK), partly p38 but not extracellular-regulated protein kinase (ERK). Consistently, sorbitol, a model activator of JNK, inhibited TCDD-mediated induction of CYP1A2 mRNA and down-regulated tyrosine aminotransferase mRNA — a target gene of glucocorticoid receptor. Dexamethasone had the opposite effect on aryl hydrocarbon receptor signaling and decreased aryl hydrocarbon receptor mRNA and augmented the inducibility of CYP1A2 by TCDD. We conclude that the effects of tested MIAs on aryl hydrocarbon receptor-CYP1A2 signaling pathway are dual, i.e. they inhibit transcriptional activity and nuclear translocation of aryl hydrocarbon receptor but in parallel increase aryl hydrocarbon receptor protein and mRNA level. Microtubules destabilizers have the same effects as stabilizer taxol. This implies that aryl hydrocarbon receptor functions depend on microtubules dynamics but not integrity. Perturbation of aryl hydrocarbon receptor-CYP1A2 signaling by MIAs comprises glucocorticoid receptor-JNK and probably aryl hydrocarbon receptor-JNK/glucocorticoid receptor interactions. We also demonstrate that the effects of MIAs in human hepatocytes do not proceed via arresting cell cycle as confirmed by flow cytometry (FACS) analyses.

Introduction

Cytochrome P450 (CYP) CYP1A2 is an important member of the P-450 superfamily of xenobiotics-metabolizing enzymes. CYP1A2 is involved in the process of chemically induced carcinogenesis. This enzyme is hepatospecific and it is induced by polychlorinated biphenyls, polyaromatic hydrocarbons, aromatic amines etc. (Maurel, 1996). Induction of CYP1A2 is mediated by aryl hydrocarbon receptor. Aryl hydrocarbon receptor resides in cytosol in a multiprotein complex with chaperone proteins hsp90 and XAP2. Upon binding a ligand, aryl hydrocarbon receptor translocates to the nucleus, where it forms heterodimer with aryl hydrocarbon receptor nuclear translocator (ARNT) and triggers gene transcription. Thereafter, aryl hydrocarbon receptor is exported from nucleus and is subjected to degradation by proteasome (Pollenz et al., 2006).

The functions of aryl hydrocarbon receptor are affected by various physiological and patho-physiological factors. We observed that microtubules disruption by colchicine or nocodazole inhibit 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent induction of CYP1A1 in human hepatoma cells (HepG2) and in rat hepatocytes (Dvorak et al., 2006). Other authors described that synchronization of murine hepatoma cells in G2/M phase of the cell cycle by nocodazole produced the inhibition of the aryl hydrocarbon receptor dependent CYP1A1 expression (Scholler et al., 1994). Aryl hydrocarbon receptor transcriptional activity in 1c1c7 cells and in human monocytic cells (U937) is oscillating during the transition between individual phases of the cell cycle (Santini et al., 2001). In contrast, cell cycle independent inhibition of aryl hydrocarbon receptor transcriptional activity by arsenite was reported in primary mouse hepatocytes (Bonzo et al., 2005). This is in accordance with our recent study where we reported CYP1A1 down-regulation by colchicine and nocodazole in quiescent cells, i.e. rat hepatocytes (Dvorak et al., 2006).

In this work, we examined the role of microtubules in the function of aryl hydrocarbon receptor -CYP1A2 signaling pathway in human hepatocytes — i.e. quiescent, non-transformed, human, primary cells. We hypothesized that microtubule interfering agents (MIAs) could affect aryl hydrocarbon receptor-mediated transcriptional up-regulation of CYP1A2 and that these effects may proceed through glucocorticoid receptor or c-Jun-N-terminal kinase (JNK).

There are several reports on the mutual interactions between aryl hydrocarbon receptor and glucocorticoid receptor (Linder et al., 1999). We found that colchicine and nocodazole down-regulate glucocorticoid receptor-controlled genes in human hepatocytes, such a CYP2B6, CYP2C8/9, CYP2C19, CYP3A4, constitutive androstane receptor (CAR), pregnane X receptor (PXR) and tyrosine aminotransferase (TAT). This was due to inhibition of nuclear translocation of glucocorticoid receptor (Dvorak et al., 2003, Pascussi et al., 2003). The importance of microtubules in proper glucocorticoid receptor function was reported by other authors as well (Dvorak et al., 2005, Galigniana et al., 1999, Galigniana et al., 2001, Galigniana et al., 1998, Harrell et al., 2004, Pratt et al., 2004, Whitesell and Cook, 1996).

The involvement of JNK in glucocorticoid receptor (Krstic et al., 1997, Rogatsky et al., 1998) and aryl hydrocarbon receptor (Tan et al., 2004, Tan et al., 2002) functions was described. However, the data come from cell lines. Since aryl hydrocarbon receptor, glucocorticoid receptor and JNK affect cell cycle and reciprocally the functions of aryl hydrocarbon receptor, glucocorticoid receptor and JNK are cell cycle-dependent, it is important that we performed this study in primary cultures of human hepatocytes; i.e. non-proliferating and non-transformed cells. We recently demonstrated in our laboratory that JNK is involved in inhibition of glucocorticoid receptor by colchicine and nocodazole in human hepatocytes (Dvorak et al., 2004).

The aim of the study was to examine the effects of MIAs acting through different mechanisms on aryl hydrocarbon receptor-CYP1A2 signaling in primary cultures of human hepatocytes. Both microtubule disruptors, i.e. colchicine, nocodazole, vincristine, vinblastine and stabilizer taxol were included. The study should confirm/refute the involvement of glucocorticoid receptor and JNK in aryl hydrocarbon receptor-CYP1A2 signaling. In addition, the MIAs examined in this study are widely used in clinical practice as anticancer drugs, therefore this study could have important toxicological implication in chemotherapy.

We show that MIAs affect the dynamics of microtubules which in turn leads to lowered transcriptional activity of aryl hydrocarbon receptor and to reduction of its nuclear translocation. The explanation for this phenomenon might be either aryl hydrocarbon receptor-JNK cross-talk or the involvement of glucocorticoid receptor in this process.

Section snippets

Materials

Collagen-coated culture dishes were purchased from BD Biosciences (Le Pont de Claix, France). Hyperfilm™ ECL and chemiluminescence-developing reagents were from Amersham-Pharmacia Biotech (Amersham, United Kingdom). 2,3,7,8-tetrachlorodibenzo-p-dioxin was purchased from Ultra Scientific (RI, USA). LightCycler FastStart DNA MasterPLUS SYBR Green I was purchased from Roche Diagnostic Corporation (Meylan, France). Oligonucleotide primers used in RT-PCR reactions were purchased from Invitrogen.

Effect of MIAs on integrity of microtubules in primary human hepatocytes

First, the integrity of microtubules network was monitored by immunofluorescence staining of tubulin. Microtubules network collapsed after 24 h of treatment with colchicine (1 μM), nocodazole (40 μM), vincristine (10, 40 μM), and/or vinblastine (10, 40 μM). Microtubules remained intact in the presence of taxol (1, 10, 40 μM) (Fig. 1A and B).

Effects of MIAs on TCDD-inducible expression of CYP1A2 mRNA

Human hepatocytes cultures obtained from five different patients were treated for 24 h with MIAs in the presence of TCDD, which is potent ligand of aryl

Discussion

In the current paper, we describe the effects of selected MIAs on aryl hydrocarbon receptor-CYP1A2 signaling pathway in primary cultures of human hepatocytes. We found that induction of CYP1A2 mRNA was inhibited by all MIAs tested, regardless of microtubules stabilization or disruption (Table 1). This implies that aryl hydrocarbon receptor signaling depends on microtubules dynamics but not on microtubules integrity.

The effects of MIAs on aryl hydrocarbon receptor- mediated transcriptional

Acknowledgements

This work was supported by the grant from the Ministry of Education, Youth and Sports of the Czech Republic MSM 6198959216 (to J.U.), by the grant from Grant Agency of the Czech Republic GACR 303/07/0128 (to Z.D.) and by EMBO Short Term Fellowship ASTF 275-2006 (to Z. D.).

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