Abundant expression of Kallikrein 1 gene in human keratinocytes was mediated by GATA3.
Among Tissue kallikrein genes (KLKs), KLK1 is abundantly expressed in human skin. Although its putative promoter is known to have various cis-elements, they have not been functionally tested. In the present study, the regulation mechanism of KLK1 promoter supporting such abundant expression was examined. Luciferase assay targeting the KLK1 promoter (nucleotide -1153/+40 from the major transcriptional start site) was performed on NHEK human keratinocyte. -954/-855, -428/-236, and -100/+40 had the induction activity. The motif search program failed to find unique binding motifs in -428/-236, whereas both -954/-855 and -100/+40 had a unique GATAs binding motif. Electrophoretic mobility shift assay (EMSA) and DNA footprinting confirmed the binding of NHEK nuclear protein to these motifs that were supershifted by anti-GATA3 antibody. Among GATA isoforms, GATA3 alone could be amplified in RNA obtained from NHEK. Moreover, introduction of GATA3 into fibroblastic NIH3T3 cells enhanced the activity of KLK1 promoter containing -954/+40, while that of GATA3 dominant negative mutant to NHEK cells impaired the same promoter's activity. Thus, GATA3 was found to bind the site located at -954/-855 and to be a key regulator of abundant KLK1 expression in human keratinocyte.
Son
Do Ngoc
DN
Department of Hygiene, Kanazawa University School of Medicine, Kanazawa, Japan.
Li
LiHua
L
Katsuyama
Hironobu
H
Komatsu
Nahoko
N
Saito
Masayuki
M
Tanii
Hideji
H
Saijoh
Kiyofumi
K
D000704:Analysis of Variance; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay; D050990:GATA3 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020840:Tissue Kallikreins / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D050990:GATA3 Transcription Factor; C494749:GATA3 protein, human; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D049409:Luciferases, Firefly; D020840:Tissue Kallikreins