Transcriptional control of the human MCP-2 gene promoter by IFN-g and IL-1b in connective tissue cells

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-1b or IFN-g. To understand the transcriptional regulation of the gene, we have analyzed the structure and function of the promoter region. The sequence of the 59-flanking region was determined and the transcription start site was found to be located at 68 nucleotides upstream of the ATG translation start codon. 59-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-1b, IFN-g, 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-1b alone failed to affect expression of any of the constructs both in diploid fibroblasts and in osteosarcoma cells. In both cell lines IFN-g increased the activity of all mutants that possessed the region between -340 and -301. In MG-63 cells, stimulation with the combination of IL-1b and IFN-g 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-g, 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. J. Leukoc. Biol. 66: 502–511; 1999.