A study on diurnal mRNA expression of CYP1A1, AHR, ARNT, and PER2 in rat pituitary and liver

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Abstract

The ligand activated basic-helix-loop-helix (bHLH)-PAS transcription factor, the aryl hydrocarbon receptor (AHR) protein, heterodimerizes with its partner protein the aryl hydrocarbon receptor nuclear translocator (ARNT). The heterodimer activates transcription via xenobiotic responsive elements to regulate the transcription of a battery of biotransformation genes as well as genes involved in growth, differentiation, and cellular homeostasis. In this study we have investigated the diurnal expression of cytochrome P450 1A1, one of the genes in the AHR target gene battery, in rat pituitary and liver. The mRNA expression patterns of AHR, ARNT, and the periodic gene (PER2) were also analyzed. PER2 belongs to another group of bHLH-PAS transcription factor complexes, which are involved in the control of circadian rhythms. Diurnal variation of cytochrome P450 1A1 (CYP1A1) mRNA expression was observed in the anterior and posterior pituitary and in the liver. The accumulation of CYP1A1 mRNA occurred during different times of the day and exhibited an opposite expression in anterior and posterior pituitary, respectively. A daily upregulation of CYP1A1 and PER2 mRNAs that was in antiphase to the AHR and ARNT mRNAs was seen in the liver. The AHR/ARNT system is considered a defense system against toxic chemicals. The high inducibility of CYP1A1 in the pituitary, shown in an earlier study, as well as the tissue specific expression patterns shown here, suggest that AHR and CYP1A1 may play a physiological role in controlling neuroendocrine functions.

Introduction

The aryl hydrocarbon receptor (AHR), the aryl hydrocarbon receptor nuclear translocator (ARNT), and the periodic protein (PER) belong to a subclass of transcription factors named basic-helix-loop-helix (bHLH)-PAS proteins. The functions of bHLH-PAS proteins seem to have their origin in early photoreceptor proteins (Pellequer et al., 1998). bHLH-PAS proteins regulate fundamental biological processes involving the maintenance of homeostasis, adaptation to environmental stimuli, circadian rhythmicity, and development (Wenger and Gassmann, 1997, Schmidt and Bradfield, 1996, Dunlap, 1999, Crews, 1998).

The AHR and ARNT proteins mediate the aryl hydrocarbon-dependent induction of cytochrome P450 1A1 (CYP1A1) (for reviews see Nebert et al., 2000, Gu et al., 2000). The most potent inducer of CYP1A1, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), elicits a wide range of biological effects through the activation of AHR. The AHR is located in the cytoplasm as a complex with other proteins. After the binding of a ligand, the AHR translocates to the nucleus where it heterodimerizes with ARNT. This heterodimer binds to the xenobiotic responsive element (XRE) and activates the transcription of a large number of genes, the AHR battery, leading to the synthesis of the CYP1A1 protein and several other proteins involved in xenobiotic metabolism, growth, differentiation, and cellular homeostasis (Nebert, 1994, Nebert et al., 2000). A feedback process regulating the expression of the CYP1A1 gene and other genes in the AHR battery by a CYP1A1-dependent oxidation of an endogenous substrate/AHR ligand has been suggested by the studies of CYP1A1 deficient mouse hepatoma cells (Hankinson et al., 1985, RayChaudhuri et al., 1990, Chang and Puga, 1998).

The normal cellular functions of the AHR receptor and the AHR/ARNT heterodimer are not yet known. A photoproduct derived from tryptophan (6-formylindolo[3,2-b]carbazole, FICZ), which binds to the AHR with very high affinity, however, has been described and suggested to be an endogenous ligand for the AHR (Rannug et al., 1987, Rannug et al., 1995). The photoproduct, FICZ, is metabolized by CYP1A1, and in contrast to TCDD, and other slowly metabolized AHR ligands, causes a transient rise in CYP1A1 gene transcripts (Wei et al., 1998, Wei et al., 1999, Wei et al., 2000). Thus, the formation of the CYP1A1 protein seems to autoregulate CYP1A1 gene expression after exposure to the photoproduct FICZ. Based on the very high affinity of the photoproduct to the AHR and the rapid, transient expression of a battery of response genes, we have suggested that oxidation products of tryptophan are mediators of light via binding to the AHR. In this regard, they may have a role in light-regulated biological rhythms (Rannug et al., 1998 Wei et al., 1999).

The bHLH-PAS protein PER2 is involved in the control of circadian rhythms. Cellular oscillators or clock genes are expressed after binding of the bHLH-PAS heterodimer of the two proteins CLOCK and BMAL to the recognition sequence (E-box) in DNA, giving rise to transiently elevated levels of several mRNAs and proteins. The proteins feed back, after a lag phase, to depress the level of their own transcripts and thereby sustain a circadian rhythmicity (Dunlap, 1999). PER2, together with another PER protein (PER1) and cryptochrome proteins (CRY1, CRY2), is an essential component of negative regulation of the two clock proteins CLOCK and BMAL1 (Chang and Reppert, 2001).

We have previously observed a strong expression of CYP1A1 mRNA and protein in rat pituitary after treatment with the AHR ligand TCDD (Huang et al., 2000). The aim of this study was to characterize in more detail the expression of CYP1A1 in the anterior and posterior pituitary in relation to the expression of AHR and ARNT in the same tissues. An additional objective was to investigate the diurnal rhythms in the expression of the CYP1A1 gene and of AHR and ARNT. In parallel, we characterized the expression pattern of the PER2 gene. The mRNA levels in anterior pituitary, posterior pituitary, and liver of male Sprague–Dawley rats were analyzed at six different time points 08:00, 12:00, 16:00, 20:00, 00:00, and 04:00 hours by a semi-quantitative RT-PCR method.

Section snippets

Materials

We used the RNeasy™ Mini Kit and QIAshredder from QIAGEN GmbH (Germany). The RNase-free DNase I was purchased from Boehringer Mannheim. SuperScript™ II RNase H Reverse Transcriptase (RT), RNaseOUT™ Recombinant Ribonuclease Inhibitor, Taq DNA Polymerase (recombinant), and dNTP Set were purchased from GibcoBRL (Life Technology). Random hexamers were acquired from Pharmacia Biotech.

Experimental animals and sample preparation

Male Sprague–Dawley rats (B and K Stockholm, Sweden) with body weights of 200 g were housed in polypropylene cages

CYP1A1 expression

When the level of CYP1A1 expression was normalized to that of β-actin, a diurnal variation in CYP1A1 mRNA expression was observed in the anterior and posterior pituitary, as well as in the liver (Table 1, Fig. 1, Fig. 2). The diurnal accumulation of CYP1A1 occurred during different times of the day, and exhibited an opposite pattern of expression in anterior and posterior pituitary, respectively (Fig. 1A). At midnight (00:00 hours time point) CYP1A1 mRNA exhibited the highest level in anterior

Discussion

The results of this study show temporal patterns in the mRNA expression of CYP1A1, AHR, and ARNT in pituitary and liver from Sprague–Dawley rats. In these samples, the diurnal expression of the PER2 gene, coding for a PAS-protein, involved in the regulation of the mammalian clock genes CLOCK/BMAL1, was demonstrated. Furthermore, the data provide a detailed analysis and comparison of the expression of PER2, AHR, ARNT, and CYP1A1 in the anterior and posterior pituitary, which were earlier

Acknowledgements

This work was supported by the grant 12X-10815 from the Swedish Medical Research Council, and by Swedish Match. We thank Dr Eva Ahlbom for her assistance in tissue sample collection and Ms Margareta Sandström for technical assistance in total RNA extraction.

References (35)

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