Structural characterization of the human interleukin-13 receptor alpha1 gene promoter.
Human cancer cells have been found to express a large number of IL-13 receptors. We have previously shown that mRNA encoding one of these receptors, IL-13Ralpha1, is increased in cisplatin-resistant cells and is upregulated in tumor cells cultured with cisplatin. To understand the molecular mechanism of IL-13Ralpha1 gene expression, we cloned approximately 52 kbp of the IL-13Ralpha1 gene and sequenced the first exon and about 1 kbp of the upstream DNA. The first exon is 211 bp and contains 88 bp of coding sequence, while the first intron is about 13 kbp in length. The promoter region, which is GC rich, was found to lack both TATA and CCAAT boxes. Transient expression assays revealed that transcription of the IL-13Ralpha1 gene was significantly higher in cisplatin-resistant cells than in parental, cisplatin-sensitive cells. Deletion analysis of the IL-13Ralpha1 promoter identified a 70-bp core promoter region upstream of the transcription initiation site. Electrophoretic gel mobility shift assays showed that a synthetic IL-13Ralpha1 oligonucleotide (nt -40 to nt -15) bound a nuclear factor from cisplatin-resistant cells to a significantly greater degree than the equivalent factor from parental cells. This oligonucleotide was found to contain a palindromic sequence with a BstEII recognition site at its center. This palindromic sequence functions to mediate upregulation of IL-13Ralpha1 promoter in cisplatin-resistant cells and deletion or disruption of this sequence also resulted in severe reduction of the promoter activity. These findings suggest that IL-13Ralpha1 expression is upregulated at the transcriptional level in cisplatin-resistant cells. The characterization of both the IL-13Ralpha1 promoter and the transcription factors binding to it may contribute to our understanding of IL-13Ralpha1 regulation in cancer cells.
Ise
T
T
Department of Molecular Biology, University of Occupational and Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, 807-8555, Japan.
Izumi
H
H
Nagatani
G
G
Takano
H
H
Wada
M
M
Kuwano
M
M
Kohno
K
K
D000970:Antineoplastic Agents / Q000494:pharmacology; D001483:Base Sequence; D002945:Cisplatin / Q000494:pharmacology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D053661:Interleukin-13 Receptor alpha1 Subunit; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D018123:Receptors, Interleukin / Q000235:genetics*; D053660:Receptors, Interleukin-13; D015183:Restriction Mapping; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D000970:Antineoplastic Agents; D017931:DNA Primers; D018076:DNA, Complementary; C508181:IL13RA1 protein, human; D053661:Interleukin-13 Receptor alpha1 Subunit; D012333:RNA, Messenger; D018123:Receptors, Interleukin; D053660:Receptors, Interleukin-13; D014157:Transcription Factors; D008156:Luciferases; D002945:Cisplatin