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

Human monocyte chemotactic protein-2 (MCP-2) is a member of the CC chemokin e family, It is produced by mononuclear leukocytes, diploid fibroblasts, an d tumor cells after induction with IL-1 beta or IFN-gamma. To understand th e transcriptional regulation of the gene, we have analyzed the structure an d 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 ATC translation start codon, 5'-Deletion mut ants were generated and transfected into E6SM diploid fibroblasts and MG-63 osteosarcoma cells, Expression was measured by luciferase assay in transfe cted unstimulated cells and after stimulation with IL-1 beta, IFN-gamma, or a combination, The region between nucleotides -143 and -73 (relative to th e transcription initiation site), containing putative cis-elements for GATA -1, H-APF1, AP-1, and GAS, is important for basal transcription levels in b oth cell lines, Stimulation for 18 h with IL-1 beta alone failed to affect expression of any of the constructs both in diploid fibroblasts and in oste osarcoma 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, st imulation with the combination of IL-1 beta and IFN-gamma caused an additio nal increase in expression of the constructs from -340 onward. Finally, the presence of transcription factors in nuclear extracts of MG-63 cells and t heir specificity to hind 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 tran scription of the MCP-2 gene in fibroblasts and thereby can indirectly contr ibute to recruitment of various leukocyte cell types to inflammatory sites.