Estrogen receptor-Sp1 complexes mediate estrogen-induced cathepsin D gene expression in MCF-7 human breast cancer cells.
Cathepsin D is an estrogen (17 beta-estradiol, E2)-inducible lysosomal protease. A putative estrogen receptor (ER)-Sp1-like sequence (GGGCGG(n)23ACGGG) 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 Sp1 protein bind to 32P-labeled ER/Sp1 oligo. For example, nuclear extracts from MCF-7 cells bind to the 32P-labeled ER/Sp1 oligo; however, ER/Sp1 binding can be decreased by selective competition with excess unlabeled estrogen responsive element and Sp1 oligos, immunodepletion with ER or Sp1 antibodies, and by treating cells with ICI 164,384, an antiestrogen which inhibits formation of ER homodimer. Moreover, E2-induced chloramphenicol acetyltransferase (CAT) activity in MCF-7 cells cotransfected with a human estrogen receptor expression plasmid and a plasmid containing an ER/Sp1 sequence cloned upstream to a thymidine kinase promoter and a CAT reporter. In cotreatment studies, ICI 164,384 inhibited E2-induced CAT activity. In contrast, E2 did not induce CAT activity in MCF-7 cells transfected with plasmids containing mutations in the ER or Sp1 segments of the ER/Sp1 oligo, thus confirming that both cognate binding sites are required for estrogen responsiveness.
Krishnan
V
V
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station 77843-4466.
Wang
X
X
Safe
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001943:Breast Neoplasms; D002402:Cathepsin D / Q000096:biosynthesis*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology*; D004965:Estrogen Antagonists / Q000494:pharmacology; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D016376:Oligonucleotides, Antisense; D010761:Phosphorus Radioisotopes; D053284:Polyunsaturated Alkamides; D011960:Receptors, Estrogen / Q000096:biosynthesis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000096:biosynthesis / Q000378:metabolism*; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D004965:Estrogen Antagonists; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D010761:Phosphorus Radioisotopes; D053284:Polyunsaturated Alkamides; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D013463:Sulfuric Acid Esters; D004958:Estradiol; C051781:ICI 164384; D000255:Adenosine Triphosphate; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase; D002402:Cathepsin D; C007482:dimethyl sulfate