Involving AP-2 transcription factor in connexin 26 up-regulation during pregnancy and lactation

Gap junction connexin 26(Cx26) is up-regulated in mammary epithelial cells during pregnancy and lactation. To understand the transcriptional regulatio n of Cx26, we identified a protected DNase I footprint region (-140 to -113 ) in the rat Cx26 promoter. This rCx26 Promoter Footprinting Region, or CPF R, contains an Sp binding site (CCGCCC) overlapping with an AP-2 binding si te (GCCCGCGGC), and is evolutionarily conserved. Nuclear extracts from rat mammary glands and human MCF-10 mammary epithelial cells formed protein-DNA complexes with the labeled CPFR probe in the electrophoretic mobility shif t assay (EMSA), and these complexes were markedly enhanced during pregnancy and lactation. Antibody supershift analysis further identified the presenc e of Sp1, Sp3, and AP-2 in these binding complexes. Human mammary epithelia l MCF 10A and MCF-12A cells were transiently transfected with chimeric muta nt rCx26 promoter/luciferase reporter constructs, and luciferase activities measured. Mutations along the CPFR fragment drastically reduced the promot er activity, specially at the Sp/AP-2 overlapping site. Cotransfection of A P-2 with rCx26 promoter/reporter constructs into MCF-10 cells markedly indu ced the reporter activity. These data infer that AP-2, along with previousl y reported Sp transcription factors, is involved in the up-regulation of Cx 26 gene during pregnancy and lactation. Mol. Reprod. Dev. 59:17-24, 2001. ( C) 2001 Wiley-Liss, Inc.