title: Transcriptional regulation of platelet-activating factor receptor gene in B lymphoblastoid Ramos cells by TGF-beta.
abstract: 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-beta 2. The TGF-beta 2-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-beta-responsive element (T beta RE) lies between the sequence from -44 to -17 relative to the transcriptional start site. Insertion of the T beta RE upstream of the unresponsive minimal thymidine kinase promoter conferred the TGF-beta-inducibility. Gel mobility shift assay demonstrated the specific binding of nuclear factors to the T beta RE. The T beta RE 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-beta 2-treatment. Concomitant treatment of cells with cycloheximide abolished the increases in both T beta RE-binding activity and expression of PAF receptor mRNA, indicating that de novo protein synthesis is required to exert TGF-beta 2 effect. Methylation interference analysis revealed that the T beta RE-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-beta-induced promoter activity. Collectively, these results clearly demonstrate that a T beta RE 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-beta 2.
authors.affiliation: Division of Cardiovascular Research, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.
authors.forename: H H
authors.initials: HH
authors.lastname: Yang
authors.forename: J H
authors.initials: JH
authors.lastname: Pang
authors.forename: R Y
authors.initials: RY
authors.lastname: Hung
authors.forename: L Y
authors.initials: LY
authors.lastname: Chau
mesh_terms: D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D010980:Platelet Membrane Glycoproteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D014158:Transcription, Genetic / Q000187:drug effects / Q000276:immunology*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics / Q000276:immunology
chemical_list: D004268:DNA-Binding Proteins; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D016212:Transforming Growth Factor beta; C039351:platelet activating factor receptor