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J Biol Chem, Vol. 274, Issue 5, 3199-3206, January 29, 1999

Sp1 and Egr-1 Have Opposing Effects on the Regulation of the Rat Pgp2/mdr1b Gene

Jaideep V. ThottasseryDagger , Daxi SunDagger , Gerard P. Zambetti§, Amber TroutmanDagger , Vikas P. Sukhatme, Erin G. SchuetzDagger , and John D. SchuetzDagger

From the Departments of Dagger  Pharmaceutical Sciences and § Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 and the  Beth Israel Hospital and Harvard School of Medicine, Boston, Massachusetts 02215

The promoter of the rat pgp2/mdr1b gene has a GC-rich region (pgp2GC) that is highly conserved in mdr genes and contains an consensus Sp1 site. Sp1's role in transactivation of the pgp2/mdr1b promoter was tested in Drosophila Schneider cells. The pgp2/mdr1b promoter was strongly activated by co-transfected wild type Sp1 but not mutant Sp1 and mutation of the Sp1 site abrogated Sp1-dependent transactivation. In gel shift assays, the same mutations abolished Sp1-DNA complex formation. Moreover, basal activity of the pgp2/mdr1b Sp1 mutant promoter was dramatically lower. Enforced ectopic overexpression of Sp1 in H35 rat hepatoma cells revealed that cell lines overexpressing Sp1 had increased endogenous pgp2/mdr1b mRNA, demonstrating that Sp1 activates the endogenous pgp2/mdr1b gene. Pgp2GC oligonucleotide also bound Egr-1 in gel shift assays and Egr-1 competitively displaced bound Sp1. In transient transfections of H35 cells (and human LS180 and HepG2 cells) Egr-1 potently and specifically suppressed pgp2/mdr1b promoter activity and mutations in the Egr-1 site decreased Egr-1 binding and correlated with pgp2/mdr1b up-regulation. Ectopic overexpression of Egr-1 in H35 cells decreased Pgp expression and selectively increased vinblastine sensitivity. In conclusion, Sp1 positively regulates while Egr-1 negatively regulates the rat pgp2/mdr1b gene. Moreover, competitive interactions between Sp1 and Egr-1 in all likelihood determine the constitutive expression of the pgp2/mdr1b gene in H35 cells.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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