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

Abstract

SUMMARYWe 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 À329 to À85 base pairs, containing two distinct AP-1 binding sites and one nuclear factor-kB (NF-kB) binding site. Deletion or mutation of the NF-kB 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 b (C/EBPb) 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 speci®c binding to oligonucleotides, including AP-1, C/EBPb, and NF-kB element. Furthermore, over-expression of liver-enriched transcriptional activator protein, a subunit of C/EBPb, augmented A. laidlawii-induced granulysin promoter activity, whereas over-expression of liver-enriched transcriptional inhibitory protein inhibited the promoter activity. NF-kB p50 homodimer had no transactivation property, although it bound to the NF-kB site. These results indicate that AP-1 and C/EBPb, but not NF-kB 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.

Expression and Secretion of Cathelicidin LL-37 in Human Epithelial Cells after Infection byMycobacterium bovisBacillus Calmette-Guérin

“…In addition, it has been reported that activation of AP-1 is regulated via MAPK signaling pathways (11). Thus, we could not exclude the participation of AP-1 in M. bovis BCG-induced LL-37 expression in A549 cells.…”

Serratia marcescensSerralysin Induces Inflammatory Responses through Protease-Activated Receptor 2

“…Although the C/EBP molecules other than C/EBP␤ can also bind to the C/EBP binding site, C/EBP␤ is especially involved in the inducible expression of several genes that are important for inflammatory and immune responses (31,32,49,50). Therefore, to identify the C/EBP␤ protein, the nuclear extract from the stimulated EBC-1 cells was preincubated with C/EBP␤-specific antibody before the addition of a radiolabeled probe.…”

Granulysin activates antigen-presenting cells through TLR4 and acts as an immune alarmin

“…[14][15][16][17] Consequently both forms of GNLY can be induced in the course of inflammation and have also been shown to be up-regulated by stimulation of lymphoid cells by proinflammatory mediators and pathogenassociated molecular patterns. 18,19 GNLY exhibits lytic activity against a variety of microorganisms and tumors. 3,14,[20][21][22] It binds to a variety of lipids, including cholesterol and sphingolipids, and damages microbial cell membranes by binding to negatively charged molecules.…”