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 regulation of Cx26, we identified a protected DNase I footprint region (-140 to -113) in the rat Cx26 promoter. This rCx26 Promoter Footprinting Region, or CPFR, contains an Sp binding site (CCGCCC) overlapping with an AP-2 binding site (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 shift assay (EMSA), and these complexes were markedly enhanced during pregnancy and lactation. Antibody supershift analysis further identified the presence of Sp1, Sp3, and AP-2 in these binding complexes. Human mammary epithelial MCF-10A and MCF-12A cells were transiently transfected with chimeric mutant rCx26 promoter/luciferase reporter constructs, and luciferase activities measured. Mutations along the CPFR fragment drastically reduced the promoter activity, specially at the Sp/AP-2 overlapping site. Cotransfection of AP-2 with rCx26 promoter/reporter constructs into MCF-10 cells markedly induced the reporter activity. These data infer that AP-2, along with previously reported Sp transcription factors, is involved in the up-regulation of Cx26 gene during pregnancy and lactation.
Tu
Z J
ZJ
Division of Hematology, Oncology, and Transplantation, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Pan
W
W
Gong
Z
Z
Kiang
D T
DT
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007774:Lactation / Q000502:physiology; D008321:Mammary Glands, Animal / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D017630:Connexins; D004268:DNA-Binding Proteins; C000607042:GJB2 protein, human; C000607044:Gjb2 protein, rat; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C083115:activator protein-2 binding element; D000072259:Connexin 26