Transcriptional control of the human MCP-2 gene promoter by IFN-gamma and IL-1beta 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-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.
Van Coillie
E
E
Rega Institute for Medical Research, Laboratory of Molecular Immunology, University of Leuven, Belgium.
Van Aelst
I
I
Fiten
P
P
Billiau
A
A
Van Damme
J
J
Opdenakker
G
G
D001483:Base Sequence; D001859:Bone Neoplasms / Q000473:pathology; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002474:Cell-Free System; D002478:Cells, Cultured; D054412:Chemokine CCL8; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D003062:Codon / Q000235:genetics; D004357:Drug Synergism; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D007375:Interleukin-1 / Q000494:pharmacology*; D008969:Molecular Sequence Data; D018945:Monocyte Chemoattractant Proteins / Q000096:biosynthesis / Q000235:genetics*; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D015345:Oligonucleotide Probes; D012516:Osteosarcoma / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012333:RNA, Messenger / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000096:biosynthesis; D011994:Recombinant Proteins; D015183:Restriction Mapping; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
C515453:CCL8 protein, human; D054412:Chemokine CCL8; D003062:Codon; D007375:Interleukin-1; D018945:Monocyte Chemoattractant Proteins; D009363:Neoplasm Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011994:Recombinant Proteins; D007371:Interferon-gamma