RESUMEN
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.
Asunto(s)
Linfocitos B/metabolismo , Regulación de la Expresión Génica/inmunología , Glicoproteínas de Membrana Plaquetaria/genética , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Transcripción Genética/inmunología , Factor de Crecimiento Transformador beta/farmacología , Linfocitos B/inmunología , Sitios de Unión/genética , Sitios de Unión/inmunología , Línea Celular Transformada , Núcleo Celular/genética , Núcleo Celular/metabolismo , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Glicoproteínas de Membrana Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , ARN Mensajero/biosíntesis , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
To understand the molecular mechanisms that direct the expression of the gene encoding the platelet-activating factor (PAF) receptor, the 5'-flanking region of the human PAF receptor gene was cloned, and its promoter activity in myeloid cell lines was characterized. By the 5'-rapid amplification of cDNA ends method and primer extension, the transcription initiation site was mapped to an adenosine residue 137 bases upstream of the ATG translation initiation codon. The promoter region lacks a typical TATA or CCAAT box. However, the sequence encompassing the transcription initiation site shows high homology to the initiator (Inr) sequence. Transfection of promonocytic U937 cells with recombinant plasmids containing a series of truncated segments of the 5'-flanking region linked to the luciferase reporter gene revealed that the sequence from nucleotides -44 to +27 relative to the transcription initiation site was sufficient to promote a high level of gene expression. The promoter activity was much lower in nonexpressing HeLa cells and promyelocytic HL-60 cells, which express relatively low levels of the PAF receptor. Gel mobility shift analysis demonstrated the binding of nuclear factors extracted from myelocytic cells to the -16/+18 sequence containing the Inr element. No binding activity was detected using the nuclear extracts from the nonmyelocytic HeLa cells. The DNA-protein complexes were sequence-specific since the binding was not significantly affected by the mutated Inr sequences or the Inr sequence of the terminal deoxynucleotidyltransferase gene. Furthermore, point mutations in the Inr element significantly reduced promoter activity in both U937 and THP-1 cell lines. When Me2SO or retinoic acid was used to induce granulocytic differentiation of HL-60 cells, a distinct Inr-protein complex was induced concurrently, but the complex was not observed in 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiated HL-60 cells or Me2SO-induced differentiated U937 cells, indicating that the inducible Inr binding activity is granulocyte-specific. These results suggest that distinct nuclear factors interact with the unique Inr element and play a role in the transcriptional regulation of the PAF receptor in various myeloid cells.