Estrogen Receptor - Spl Complexes Mediate Estrogen - induced Cathepsin D Gene Expression in MCF - 7 Human Breast Cancer Cells

Cathepsin D is an estrogen (17P-estradio1, E,)-inducible lysosomal protease. A putative estrogen receptor (ER)-Spl-like sequence (GGGCGG(n),&CGGG) has been identified in the,non-coding strand of the cathepsin D promoter (-199 to -165), and electromobility shift assays of nuclear extracts from MCF-7 and HeLa cells confirm that both the ER and Spl protein bind to ”P-labeled EWSpl oligo. For example, nuclear extracts from MCF-7 cells bind to the 32P-labeled EWSpl oligo; however, EW Spl binding can be decreased by selective competition with excess unlabeled estrogen responsive element and Spl oligos, immunodepletion with ER or Spl antibodies, and by treating cells with IC1 164,384, an antiestrogen which inhibits formation of ER homodimer. Moreover, E,-induced chloramphenicol acetyltransferase (CAT) activity in MCF-7 cells cotransfected with a human estrogen receptor expression plasmid and a plasmid containing an EWSpl sequence cloned upstream to a thymidine kinase promoter and a CAT reporter. In cotreatment studies, IC1 164,384 inhibited E,-induced CAT activity. In contrast, E, did not induce CAT activity in MCF-7 cells transfected with plasmids containing mutations in the ER or Spl segments of the EWSpl oligo, thus confirming that both cognate binding sites are required for estrogen responsiveness.