Abstract
Integrin-associated protein (IAP or CD47) is expressed in a variety of tissues, including the nervous system and immune system. To understand how cells control the expression of the IAP gene, we cloned the 5'-proximal region of the human IAP gene and investigated IAP promoter activity by transient transfection. RT-PCR confirmed the expression of IAP transcripts in human neuroblastoma IMR-32 and hepatoma HepG2 cells. Deletion analysis identified a core promoter of the human IAP gene located between nucleotide positions -232 and -12 relative to the translation initiation codon in these two cell lines. Site-directed mutagenesis and gel electrophoretic mobility shift assay identified a alpha-Pal/NRF-1 binding element within the IAP core promoter. Supershift assays using the alpha-Pal/NRF-1 antiserum confirmed the binding of this transcription factor on the alpha-Pal/NRF-1 site. Overexpression of the DNA binding domain of alpha-Pal/NRF-1 in cells enhanced DNA-alpha-Pal/NRF-1 binding in vitro. Furthermore, overexpression of full-length alpha-Pal/NRF-1 significantly enhanced IAP promoter activity while overexpression of dominant-negative mutant reduced promoter activity both in the cultured human cell lines and primary mouse cortical cells. These results revealed that alpha-Pal/NRF-1 is an essential transcription factor in the regulation of human IAP gene expression.
Highlights
Surface protein associated with integrin ␣v3 and it was involved in the enhancement of neutrophil adhesion, chemotaxis, and phagocytosis triggered by an extracellular matrix [1, 6, 7]
We investigated the regulation of Integrin-associated protein (IAP) gene promoter in human neuroblastoma and hepatoma cell lines by using luciferase reporter and gel electrophoretic mobility shift assays
We found that ␣-Pal/NRF-1 in the core promoter region is a positive regulator of the human IAP gene
Summary
Cell Culture—Human neuroblastoma IMR-32 (CCRC 60014) and hepatoma HepG2 (CCRC 60048) cell lines were purchased from Culture Collection and Research Center, Food Industry and Development Institute, Hsinchu, Taiwan. The DNA binding reaction was conducted at 4 °C for 30 min in a mixture containing 3 g of nuclear extract, 10 mM Tris-Cl (pH 7.5), 50 mM sodium chloride, 0.5 mM dithiothreitol, 0.5 mM EDTA, 1 mM magnesium chloride, 4% glycerol, 0.05 g poly(dI-dC)1⁄7poly(dI-dC) (Amersham Biosciences) and 2 ϫ 104 cpm of 32P-labeled double-stranded oligonucleotides. HepG2 cells (8 ϫ 105) were placed onto 6-cm dishes for overexpression of ␣-Pal/ NRF-1 using the same procedure for transient transfection of the reporter plasmids.
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