Structural and functional analysis of the promoter region of the human MCP-3 gene: Transactivation of expression by novel recognition sequences adjacent to the transcription initiation site

Human monocyte chemotactic factor-3 (MCP-3) belongs to the C-C chemokines, which are cytokines involved in cell recruitment in inflammation and cancer, Northern blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that the MCP-3 gene is expressed in many human tissues and tumor cell lines and that the expression level is increased by various stimuli, Measles virus and phorbol 12-myristate 13-acetate (PMA) induced MCP-3 mRNA after 6 hr of stimulation, Interferon-beta (IFN-beta) induced MCP-3 mRNA after 16 hr, a time point when the PMA-induced mRNA had the tendency to level off, No significant increase in MCP-3 mRNA levels was observed in MG-63 cells after stimulation with interleukin-1 beta (IL-1 beta), To elucidate the regulation of MCP-3 gene expression, we determined the sequence of 5 kb of the MCP-3 promoter, This sequence contained a microsatellite that was shown to be polymorphic in various cell lines, Next 5'-deletion mutants of the promoter were generated and transfected into MG-63 cells, demonstrating the presence of several positive and negative transcriptional regulatory elements, One of the positive elements was located at -37, only 21 bp upstream from the TATAA box, This. element was similar to an AP-I element and also to a homeodomain protein Pbx1 binding site, A deletion mutant from -110 to +52 possessed the highest promoter activity, and the longer deletion mutants had relatively low activities, The region between -190 and -172 contained an Ets-like element and inhibited promoter activity, Stimulation with PMA dramatically increased promoter activity through activation of a positive element present between -172 and -100, The same 5'-deletion mutants were transfected into HeLa and Jurkat cells, None of the deletion mutants had any significant activity in Jurkat cells. In HeLa cells, low levels of MCP-3 mRNA were detected by RT-PCR, but the profile of the promoter activities of the deletion mutants was different hom that seen in MG-63 cells.