Transcriptional Regulation of Platelet-Activating Factor Receptor Gene in B Lymphoblastoid Ramos Cells by TGF-β

The expression of platelet-activating factor (PAF) receptor gene was up-regulated in a time- and dose-dependent manner in a B cell line (Ramos) following exposure to TGF-β2. The TGF-β2-induced increment of PAF receptor mRNA was at least partly due to an increase in transcriptional rate as demonstrated by nuclear run-off experiments. Transient transfection of cells with PAF receptor transcript I gene promoter fused to a luciferase reporter gene revealed that the TGF-β-responsive element (TβRE) lies between the sequence from -44 to -17 relative to the transcriptional start site. Insertion of the TβRE upstream of the unresponsive minimal thymidine kinase promoter conferred the TGF-β-inducibility. Gel mobility shift assay demonstrated the specific binding of nuclear factors to the TβRE. The TβRE binding activity was gradually increased and reached a maximum at 3 h and subsequently returned to basal level at 5 h in cells following TGF-β2-treatment. Concomitant treatment of cells with cycloheximide abolished the increases in both TβRE-binding activity and expression of PAF receptor mRNA, indicating that de novo protein synthesis is required to exert TGF-β2 effect. Methylation interference analysis revealed that the TβRE-binding protein recognized a purine-rich sequence, 5′-GGGGTG-3′. Point mutations of the consecutive guanine nucleotides significantly reduced the DNA-binding activity and the TGF-β-induced promoter activity. Collectively, these results clearly demonstrate that a TβRE proximal to the transcriptional initiation site of the human PAF receptor transcript I gene mediates the up-regulation of PAF receptor gene expression in Ramos cells by TGF-β2.