Opposing roles of activator protein-1 and CCAAT/enhancer binding protein beta in the regulation of inducible granulysin gene expression in a human monocytic cell line, THP-1.

We previously reported that inducible granulysin gene expression in a human monocytic cell line, THP-1 is dominantly dependent on transcription factor activator protein-1 (AP-1). Here, we further examined the precise regulatory mechanisms underlying granulysin gene expression using THP-1 cells treated with Acholeplasma laidlawii. Transfection of reporter gene constructs into THP-1 cells indicated that the presence of a positive regulatory element(s) is located from -329 to -85 base pairs, containing two distinct AP-1 binding sites and one nuclear factor-kappaB (NF-kappaB) binding site. Deletion or mutation of the NF-kappaB binding site failed to affect inducible promoter activity, whereas deletion or mutation of both the AP-1 binding sites abrogated the promoter activity. Interestingly, deletion of the putative CCAAT/enhancer binding protein beta (C/EBPbeta) binding site upstream of the positive regulatory element induced the augmentation of granulysin promoter activity. Electrophoretic mobility shift assays demonstrated that nuclear extract prepared from A. laidlawii-treated THP-1 cells generated a specific binding to oligonucleotides, including AP-1, C/EBPbeta, and NF-kappaB element. Furthermore, over-expression of liver-enriched transcriptional activator protein, a subunit of C/EBPbeta, augmented A. laidlawii-induced granulysin promoter activity, whereas over-expression of liver-enriched transcriptional inhibitory protein inhibited the promoter activity. NF-kappaB p50 homodimer had no transactivation property, although it bound to the NF-kappaB site. These results indicate that AP-1 and C/EBPbeta, but not NF-kappaB participate in the regulation of inducible granulysin gene expression in THP-1 cells. Moreover, the Toll-like receptor 2-dependent signalling pathway may be involved in A. laidlawii-induced transactivation of the granulysin promoter. Thus, these results suggest that the gene expression of granulysin in macrophages would be exquisitely regulated by positive and negative transcription factors when microbial invasion occurs.