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 intracellular levels of immunoreactive protein in MCF-7 human breast cancer cells. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alone does not affect cathepsin D gene expression in this cell line; however, in cells cotreated 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 estrogen receptor (hER) and the pBC12/S1/pac plasmid, which contains the 5' promoter region (-296/+57) of the cathepsin D gene and an alkaline phosphatase reporter gene. The E2-responsive ER/Sp1 sequence (-199 to -165) in the cathepsin D 5' region contains an imperfect GTGCGTG (-175/-181) xenobiotic responsive element (XRE); the role of this sequence in Ah responsiveness was investigated in gel electrophoretic mobility shift assays and with plasmid constructs containing a wild-type ER/Sp1 oligonucleotide or a mutant ER/Sp1-"XRE" oligonucleotide containing two C-->A mutations in the XRE sequence (antisense strand). In plasmid constructs which contained a chloramphenicol acetyltransferase reporter gene and the wild-type ER/Sp1 promoter sequence, E2-induced chloramphenicol acetyltransferase activity and mRNA levels were inhibited by TCDD whereas no inhibition was observed with the mutant ER/Sp1-"XRE" plasmids. Electrophoretic mobility shift assays showed that the nuclear or transformed cytosolic Ah receptor complex blocked formation of the ER-Sp1 complex with the wild-type but not the ER/Sp1 mutant oligonucleotide. Moreover, incubation of the wild-type bromodeoxyuridine-substituted ER/Sp1 oligonucleotide with the nuclear Ah receptor complex gave a specifically bound cross-linked 200-kDa band. These data demonstrate 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.
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station 77843-4466, USA.
V
V
Krishnan
W
W
Porter
M
M
Santostefano
X
X
Wang
S
S
Safe
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D001943:Breast Neoplasms; D002402:Cathepsin D / Q000096:biosynthesis*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003001:Cloning, Molecular; D004958:Estradiol / Q000494:pharmacology*; D004965:Estrogen Antagonists / Q000494:pharmacology*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D018336:Receptors, Aryl Hydrocarbon / Q000096:biosynthesis / Q000502:physiology; D011960:Receptors, Estrogen / Q000096:biosynthesis / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D015262:Xenobiotics / Q000494:pharmacology
D004965:Estrogen Antagonists; D016376:Oligonucleotides, Antisense; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D015262:Xenobiotics; D004958:Estradiol; D015500:Chloramphenicol O-Acetyltransferase; D000469:Alkaline Phosphatase; D002402:Cathepsin D