Transcriptional control of the human MCP-2 gene promoter by IFN-γ and IL-1β 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-1 beta 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 ATC translation start codon, 5'-Deletion mutants were generated and transfected into E(6)SM diploid fibroblasts and MG-63 osteosarcoma cells, Expression was measured by luciferase assay in transfected unstimulated cells and after stimulation with IL-1 beta, 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-1 beta 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-1 beta 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 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 transcription of the MCP-2 gene in fibroblasts and thereby can indirectly contribute to recruitment of various leukocyte cell types to inflammatory sites.