FUNCTIONAL-ROLE OF THE CIS-ACTING ELEMENTS IN HUMAN MONOCYTE CHEMOTACTIC PROTEIN-1 GENE IN THE REGULATION OF ITS EXPRESSION BY PHORBOL ESTER IN HUMAN GLIOBLASTOMA CELLS

The monocyte chemotactic protein-1 (MCP-1) is a 76 amino acid protein that specifically attracts monocytes. The expression of MCP-1 gene can be induced by lipopolysaccharides (LPS), phorbol esters (TPA) and sev eral cytokines. However, how they regulate MCP-1 gene expression is no t known. We tested whether the two putative TPA-responsive elements (T REs) and one kappa B enhancer-like region found in the MCP-1 promoter region, are involved in this regulation of MCP-1 gene expression. The 5' untranslated region of MCP-1 gene was linked to chloramphenicol ace tyl transferase (CAT) reporter gene and transfected into human gliobla stoma cells in which endogenous MCP gene expression was found to be st imulated by TPA and tumor necrosis factor-alpha (TNF-alpha). The 128 b p 5'-flanking region containing one TRE was adequate for basal promote r activity but the presence of both TREs in the MCP-1 promoter region were needed to give TPA responsive enhancement (2.5 fold) of expressio n of the marker gene. Mutations in either of the TRE's could abolish t he TPA induction of CAT expression. Replacement of the kappa B enhance r-like element with a TRE-like sequence caused a 10-fold enhancement o f CAT expression by TPA treatment. Random mutation of kappa B enhancer -like element did not affect CAT expression or its TPA induction. None of the MCP promoter constructs showed significant increase in CAT exp ression by treatment with tumor necrosis factor-alpha (TNF-alpha). Thi s result suggested that the TNF regulation of MCP-1 gene involves othe r parts of the gene besides the proximal 5' flanking region.