The role of Sp1 in the differential expression of transforming growth factor-beta receptor type II in human breast adenocarcinoma MCF-7 cells

Progression of MCF-7 cells from early passage (MCF-7E, <200 passage) to lat e passage (MCF-7L, >500 passage) correlates with a loss of sensitivity to e xogenous TGF beta 1, We have previously shown that loss of TGF beta sensiti vity is due to decreased expression of the transforming growth factor recep tor type II (T beta RII) and is associated with increased tumorigenicity in nude mice. Reduced T beta RII expression in MCF-7L cells is caused by decr eased T beta RII promoter activity in this cell line. Our previous studies using 5' deletion constructs of this promoter revealed that MCF-7L cells we re unable to support transcription of the minimal promoter (-47 to +2) to t he same levels as the MCF-7E cells. This region of the promoter contains an Sp1 element at position -25 from the major transcription start site. In th is study, we investigated the role of Sp1 in T beta RII transcription. Muta tion of the Sp1 site resulted in decreased transcription of T beta RII in M CF-7E and MCF-7L cells, indicating that this site played a role in transcri ption of this promoter. Gel shift assays using the proximal Spl site from t he T beta RII promoter showed enhanced DNA:protein complex formation with n uclear proteins isolated from MCF-7E cells compared with MCF-7L cells. Supe rshift analysis identified this binding activity as Sp1, Western blot analy sis of Sp1 levels demonstrated that MCF-7E cells contain increased Sp1 prot ein compared with MCF-7L cells, paralleling the increased binding activity. Differential Sp1 activity was also demonstrated by higher levels of transc ription of an Sp1-dependent insulin-like growth factor II promoter construc t in MCF-7E cells compared with MCF-7L cells. Co-transfection of an Sp1 exp ression vector with a T beta RII promoter construct in MCF-7L cells induced the expression from the promoter-CAT constructs and resulted in an increas e of endogenous T beta RII protein levels. These results demonstrate that t he transcriptional repression of T beta RII in MCF-7L cells is caused, in p art, by lower Sp1 levels.