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Originally published In Press as doi:10.1074/jbc.M207841200 on September 20, 2002
J. Biol. Chem., Vol. 277, Issue 48, 45904-45910, November 29, 2002
Identification of a 16-Nucleotide Sequence That Mediates
Post-transcriptional Regulation of Rat CYP2E1 by Insulin*
Arlette
Moncion,
Nhu Traï
Truong,
Alessio
Garrone,
Philippe
Beaune,
Robert
Barouki, and
Isabelle
de Waziers
From the INSERM U490, Laboratoire de Toxicologie Moléculaire,
Faculté de Médecine, 45 Rue des Saints Pères 75270, Paris Cedex 06, France
Insulin directly down-regulates the gene
expression of the rat CYP2E1 by altering its mRNA stability (De
Waziers, I., Garlatti, M., Bouguet, J., Beaune, P. H., and
Barouki, R. (1995) Mol. Pharmacol. 47, 474-479).
Because the regulation of CYP mRNA stability was poorly understood,
the molecular mechanisms involved in this regulation in the rat
hepatoma H4IIEC3 cell line were studied. By using RNase T1 protection
methods, the formation of a major CYP2E1 RNA-protein complex was
observed. By competition experiments, the binding site of this complex
was located on a 16-nucleotide sequence in the 5'-proximal region of
the CYP2E1-coding sequence. Insulin did not modify the binding pattern
of proteins to this sequence. and transfections of expression vectors
or antisense oligonucleotides were undertaken to demonstrate the actual
functionality of the 16-mer sequence. The insertion of this sequence in
a luciferase gene was sufficient to render the chimeric mRNA
sensitive to insulin. Furthermore, transfection of H4IIEC3 cells with
antisense oligonucleotide complementary to this sequence blocked the
insulin effect on the CYP2E1 mRNA expression, i.e. its
rapid degradation. All these results demonstrate that this
16-nucleotide sequence is implicated in the CYP2E1 post-transcriptional
regulation by insulin.
*
This work was supported by the INSERM, the Université
Paris V René Descartes, the Region Ile de France (SESAME), the
Ligue Contre le Cancer, the Association de Recherches Contre le Cancer, and the Fondation pour la Recherche Médicale.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 33-1-42-86-21- 49; Fax: 33-1-42-86-20-72; E-mail:
Isabelle.Waziers@biomedicale.univ-paris5.fr.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
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