JBC Ideal method for primary cell transfection

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Originally published In Press as doi:10.1074/jbc.M002462200 on October 10, 2000

J. Biol. Chem., Vol. 276, Issue 5, 3348-3352, February 2, 2001
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Sp3 Is a Transcriptional Repressor of Transforming Growth Factor-beta Receptors*

Sudhakar Ammanamanchi and Michael G. BrattainDagger

From the Departments of Surgery and Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229

MCF-7E breast cancer cells express transforming growth factor-beta (TGF-beta ) receptors RI and RII in comparison to MCF-7L cells. We present data showing that Sp3 acts as a transcriptional repressor of RI and RII in MCF-7L cells and GEO colon cancer cells. MCF-7L and GEO cells express high levels of Sp3 protein. Gel shift analysis indicated enhanced binding of Sp3 from MCF-7L cells to a consensus Sp1 oligonucleotide. Southwestern data indicated increased binding of Sp3 to RI and RII promoters in MCF-7L cells, suggesting a correlation between Sp3 binding and reduced expression of TGF-beta receptors in MCF-7L cells. Cotransfection of CMV-Sp3 cDNA with RI and RII promoter-luciferase reporter constructs decreased RI and RII promoter activities by 70% in MCF-7E and GEO cells. Southwestern analysis detected the binding of transiently expressed Sp3 to RI and RII promoters in MCF-7E cells. Significantly, ectopic Sp3 expression led to repression of RI and RII transcripts in MCF-7E cells. This report demonstrates that inappropriate overexpression of Sp3 is a mechanism that contributes to repression of TGF-beta receptors.

* This work was supported by National Institutes of Health Grants CA 38173 and CA 72001.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.

Dagger To whom correspondence should be addressed: Dept. of Surgery, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229. Tel.: 210-567-4524; Fax: 210-567-4664; E-mail: brattainm@uthscsa.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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