Gastroenterology

Gastroenterology

Volume 135, Issue 5, November 2008, Pages 1636-1644.e3
Gastroenterology

Basic—Alimentary Tract
Circadian Clock-Controlled Intestinal Expression of the Multidrug-Resistance Gene mdr1a in Mice

https://doi.org/10.1053/j.gastro.2008.07.073Get rights and content

Background & Aims

P-glycoprotein, the product of the multidrug resistance (mdr) gene, functions as a xenobiotic transporter contributing to the intestinal barrier. Although intestinal expression of the mdr1a gene and its efflux pump function has been shown to exhibit 24-hour variation, the mechanism of the variations remains poorly understood. Here, we demonstrated that the molecular components of the circadian clock act as regulators to control 24-hour variation in the expression of the mdr1a gene.

Methods

Luciferase reporter assay and gel mobility shift assay were used to study the mechanism of transcriptional regulation of the mdr1a gene by clock gene products. The messenger RNA levels and protein abundances in colon 26 cells and mouse intestine were measured by quantitative real-time polymerase chain reaction and Western blotting, respectively.

Results

Hepatic leukemia factor (HLF) and E4 promoter binding protein-4 (E4BP4) regulated transcription of the mdr1a gene by competing with each other for the same DNA binding site. Molecular and biochemical analyses of HLF- and E4BP4-down-regulated colon 26 cells and the intestinal tract of Clock mutant mice suggested that these 2 proteins consisted of a reciprocating mechanism in which HLF activated the transcription of the mdr1a gene, whereas E4BP4 periodically suppressed transcription at the time of day when E4BP4 was abundant.

Conclusions

The intestinal expression of the mdr1a gene is influenced by the circadian organization of molecular clockwork. Our present findings provide a link between the circadian timekeeping system and xenobiotic detoxification.

Section snippets

Materials and Methods

A detailed methods section is provided in the Supplementary Materials (see Supplementary materials online at www.gastrojournal.org).

Transcriptional Regulation of mdr1a Gene by Clock Gene Products

To explore whether products of clock genes regulate expression of the mdr1a gene, we first looked for consensus sequences within the promoter region of the mdr1a gene. Two nucleotide sequences showing homology with the PAR bZIP protein response element (PARRE) RTTAYGYAAY (where R is a purine and Y is a pyrimidine) were found within 1.0 kilobase (kb) of the mdr1a gene 5′-flanking region (Figure 1A). Furthermore, an E-box element CACGTG, consensus-binding site for CLOCK/BMAL1, was also identified

Discussion

PAR bZIP transcription factors DBP, TEF, and HLF have been shown to function as mediators of circadian output physiology. They are expressed in robust circadian fashion in most organs, although the expression of HLF is more restricted to the brain, liver, kidney, and small intestine.30 Recent studies suggested that PAR bZIP proteins and E4BP4 participate in the control of the expression of genes responsible for xenobiotic detoxification, such as cytochrome P450 enzymes, carboxylesterases, and

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  • Cited by (0)

    Supported, in part, by a grant-in-aid for Scientific Research on Priority Areas “Cancer” from the Ministry of Education, Culture, Sport, Science and Technology (18014020 and 20014016, to S.O.); a grant-in-aid for Scientific Research (B) (18390050, to S.O.); and a grant-in-aid for Encouragement of Young Scientists (18790692, to S.K.) from the Japan Society for the Promotion of Science.

    Y.M., S.K., and S.O. equally contributed to this study.

    The authors disclose no conflicts.

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