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Promoter Function and the Role of Cytokines in the Transcriptional Regulation of Rabbit CYP2E1 and CYP2E2☆

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Abstract

Rabbit CYP2E1 and CYP2E2 show considerable similarity in the 5′ flanking region, but a 32-base-pair element (32-BPE) that is repeated in 2E1 is present only as a single inexact copy in 2E2. In the present investigation, footprinting disclosed two specific binding sites for liver nuclear proteins, and the DNase I sensitivity profiles of the two genes were found to be different. Several positive and negative regulatory elements were identified by transfection with a series of constructs of upstream CYP2E sequences fused to the luciferase gene. Both genes have an HNF-1 consensus motif with one nucleotide mismatch, which affects binding affinity and promoter activity. Investigation of DNA–protein interactions revealed that Sp1 and NFκB bind exclusively to the 32-BPE of 2E1 and 2E2, respectively, suggesting a possible regulatory role for the 32-BPE. Interleukin-1α (IL-1α) gave rise to a 2.5-fold increase in the promoter activity of 2E1 in HepG2 cells, and the IL-1α-mediated induction of reporter gene expression was almost completely prevented when the 32-BPE was deleted. Increased DNA binding and Sp1 protein content as a result of IL-1α treatment, as well as cotransfection experiments with pPacSp1, suggest that Sp1 is a transcription activator for the induction of 2E1 by IL-1α in HepG2 cells.

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    ☆

    This research was supported by Grant AA-06221 from the National Institutes of Health (to M.J.C.).

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    To whom correspondence should be addressed. Fax: (734) 763-4581. E-mail: [email protected].

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