REGULATION BY ESTROGEN THROUGH THE 5'-FLANKING REGION OF THE MOUSE CALBINDIN-D28K GENE

Mouse calbindin-D-28k expression is regulated in vivo by estradiol in ovaries, uterus, and oviduct. To determine whether estrogen can have a n effect on the transcription of the calbindin-D-28k gene, the human b reast cancer cells T47D were transiently transfected with a plasmid co ntaining a 1.1 kilobase (kb) PstI/SacII fragment (-1075/+34) of the mo use calbindin gene ligated to the chloramphenicol acetyltransferase (C AT) gene and cotransfected with human estrogen receptor expression vec tor. 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 c himeric gene construct alone also resulted in an estradiol-dependent i nduction in CAT activity. Deletion mutant analysis demonstrated that t here are two regions of the mouse calbindin-D-28k promoter (between -1 075/-702 and between -175/-78) that contribute to the induction by est radiol. 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 region s there are multiple imperfect half-palindromic estrogen-responsive el ements. Gel retardation assays demonstrated weak protein-DNA interacti ons that were competed with cold oligonucleotide containing the vitell ogenin estrogen-response element. These findings indicate that the mou se calbindin-D-28k promoter is capable of conferring estrogen responsi veness, which may be mediated by several imperfect half-palindromic es trogen-responsive elements, and suggest, in light of previous studies concerning 1,25-dihydroxyvitamin D regulation, multiple steroid regula tion of the calbindin-D-28k gene.