Van Coillie E, Van Aelst I, Fiten P, Billiau A, Van Damme J, Opdenakker G
Rega Institute for Medical Research, Laboratory of Molecular Immunology, University of Leuven, Belgium.
J Leukoc Biol. 1999 Sep;66(3):502-11. doi: 10.1002/jlb.66.3.502.
Human monocyte chemotactic protein-2 (MCP-2) is a member of the CC chemokine family. It is produced by mononuclear leukocytes, diploid fibroblasts, and tumor cells after induction with IL-1beta or IFN-gamma. To understand the transcriptional regulation of the gene, we have analyzed the structure and function of the promoter region. The sequence of the 5'-flanking region was determined and the transcription start site was found to be located at 68 nucleotides upstream of the ATG translation start codon. 5'-Deletion mutants were generated and transfected into E6SM diploid fibroblasts and MG-63 osteosarcoma cells. Expression was measured by luciferase assay in transfected unstimulated cells and after stimulation with IL-1beta, IFN-gamma, or a combination. The region between nucleotides -143 and -73 (relative to the transcription initiation site), containing putative cis-elements for GATA-1, H-APF1, AP-1, and GAS, is important for basal transcription levels in both cell lines. Stimulation for 18 h with IL-1beta alone failed to affect expression of any of the constructs both in diploid fibroblasts and in osteosarcoma cells. In both cell lines IFN-gamma increased the activity of all mutants that possessed the region between -340 and -301. In MG-63 cells, stimulation with the combination of IL-1beta and IFN-gamma caused an additional increase in expression of the constructs from -340 onward. Finally, the presence of transcription factors in nuclear extracts of MG-63 cells and their specificity to bind to various oligonucleotide probes in this [-340; -301] region were evidenced by electromobility shift assays. These results show that IFN-gamma, produced by lymphocytes and NK cells, induces the transcription of the MCP-2 gene in fibroblasts and thereby can indirectly contribute to recruitment of various leukocyte cell types to inflammatory sites.
人单核细胞趋化蛋白-2（MCP-2）是CC趋化因子家族的成员。它由单核白细胞、二倍体成纤维细胞和经白细胞介素-1β或干扰素-γ诱导后的肿瘤细胞产生。为了解该基因的转录调控，我们分析了其启动子区域的结构和功能。确定了5'侧翼区域的序列，发现转录起始位点位于ATG翻译起始密码子上游68个核苷酸处。构建了5'缺失突变体并转染到E6SM二倍体成纤维细胞和MG-63骨肉瘤细胞中。通过荧光素酶测定法检测转染的未刺激细胞以及经白细胞介素-1β、干扰素-γ或二者联合刺激后的表达情况。核苷酸-143至-73之间的区域（相对于转录起始位点），包含GATA-1、H-APF1、AP-1和GAS的假定顺式元件，对两种细胞系中的基础转录水平都很重要。单独用白细胞介素-1β刺激18小时未能影响二倍体成纤维细胞和骨肉瘤细胞中任何构建体的表达。在两种细胞系中，干扰素-γ均增加了所有含有-340至-301区域突变体的活性。在MG-63细胞中，白细胞介素-1β和干扰素-γ联合刺激使从-340起构建体的表达进一步增加。最后，通过电泳迁移率变动分析证明了MG-63细胞核提取物中转录因子的存在及其与该[-340；-301]区域各种寡核苷酸探针结合的特异性。这些结果表明，淋巴细胞和自然杀伤细胞产生的干扰素-γ可诱导成纤维细胞中MCP-2基因的转录，从而可间接促进各种白细胞类型募集到炎症部位。
