MOLECULAR MECHANISM OF INHIBITION OF ESTROGEN-INDUCED CATHEPSIN-D GENE-EXPRESSION BY 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN MCF-7 CELLS

17 beta-Estradiol (E2) induces cathepsin D mRNA levels and intracellul ar levels of immunoreactive protein in MCF-7 human breast cancer cells , 2,3,7,8-Tetrachlbrodibenzo-p-dioxin (TCDD) alone does not affect cat hepsin D gene expression in this cell line; however, in cells cotreate d with TCDD and E2, TCDD inhibited E2-induced cathepsin D mRNA levels, the rate of gene transcription, and levels of immunoreactive protein. The inhibitory responses were observed within 30 to 120 min after the cells were treated with TCDD, TCDD also inhibited E2-induced secreted alkaline phosphatase activity in aryl hydrocarbon (Ah)-responsive MCF -7 and wild-type mouse Hepa 1c1c7 cells cotransfected with the human e strogen receptor (hER) and the pBC12/S1/pac plasmid, which contains th e 5' promoter region (-296/+57) of the cathepsin D gene and an alkalin e phosphatase reporter gene. The E2-responsive ER/Sp1 sequence (-199 t o -165) in the cathepsin D 5' region contains an imperfect GTGCGTG (-1 751-181) xenobiotic responsive element (XRE); the role of this sequenc e in Ah responsiveness was investigated in gel electrophoretic mobilit y shift assays and with plasmid constructs containing a wild-type ER/S p1 oligonucleotide or a mutant ER/Sp1-''XRE'' oligonucleotide containi ng two C-->A mutations in the XRE sequence (antisense strand). In plas mid constructs which contained a chloramphenicol acetyltransferase rep orter gene and the wild-type ER/Sp1 promoter sequence, E2-induced chlo ramphenicol acetyltransferase activity and mRNA levels were inhibited by TCDD whereas no inhibition was observed with the mutant ER/Sp1-''XR E'' plasmids. Electrophoretic mobility shift assays showed that the nu clear or transformed cytosolic Ah receptor complex blocked formation o f the ER-Spl complex with the wild-type but not the ER/Sp1 mutant olig onucleotide. Moreover, incubation of the wild type bromodeoxyuridine-s ubstituted ER/Sp1 oligonucleotide with the nuclear Ah receptor complex gave a specifically bound cross-linked 200-kDa band. These data demon strate that Ah receptor-mediated inhibition of E2-induced cathepsin D gene expression is due to disruption of the ER-Sp1 complex by targeted interaction with an overlapping XRE.