Regulation by estrogen through the 5'-flanking region of the mouse calbindin-D28k gene.
Mouse calbindin-D28k expression is regulated in vivo by estradiol in ovaries, uterus, and oviduct. To determine whether estrogen can have an effect on the transcription of the calbindin-D28k gene, the human breast cancer cells T47D were transiently transfected with a plasmid containing a 1.1 kilobase (kb) PstI/SacII fragment (-1075/+34) of the mouse calbindin gene ligated to the chloramphenicol acetyltransferase (CAT) gene and cotransfected with human estrogen receptor expression vector. T47D cells, transfected and treated with estradiol (10(-11) - 10(-7) M for 64-65 h), exhibited a dose-dependent increase in CAT activity (up to 6.2-fold). Transfection of MCF-7 breast cancer cells with the chimeric gene construct alone also resulted in an estradiol-dependent induction in CAT activity. Deletion mutant analysis demonstrated that there are two regions of the mouse calbindin-D28k promoter (between -1075/-702 and between -175/-78) that contribute to the induction by estradiol. These fragments, when linked to the thymidine kinase promoter to construct a heterologous promoter chimera, were able to convert the thymidine kinase promoter to estrogen responsiveness. In these regions there are multiple imperfect half-palindromic estrogen-responsive elements. Gel retardation assays demonstrated weak protein-DNA interactions that were competed with cold oligonucleotide containing the vitellogenin estrogen-response element. These findings indicate that the mouse calbindin-D28k promoter is capable of conferring estrogen responsiveness, which may be mediated by several imperfect half-palindromic estrogen-responsive elements, and suggest, in light of previous studies concerning 1,25-dihydroxyvitamin D regulation, multiple steroid regulation of the calbindin-D28k gene.
Gill
R K
RK
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103-2714, USA.
Christakos
S
S
D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms; D064092:Calbindin 1; D064026:Calbindins; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D012091:Repetitive Sequences, Nucleic Acid; D064030:S100 Calcium Binding Protein G / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured
C576498:CALB1 protein, human; C576499:Calb1 protein, mouse; D064092:Calbindin 1; D064026:Calbindins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D064030:S100 Calcium Binding Protein G; D004958:Estradiol; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase