Expression and regulation of the antimicrobial peptides, human beta-defensin-1 and 2, in gingival epithelial cells.

摘要

Gingival epithelia are continually exposed to bacterial challenge that may initially be resisted by antimicrobial peptides. The goals of this study were to determine mRNA and peptide expression of human β-defensin-1 (hBD-1) and β-defensin-2 (hBD-2) in gingival epithelial cells (HGE) and tissue, and to study the signaling pathways of hBD-2 up-regulation in response to challenge from Fusobacterium nucleatum (F.n.). HBD-1 mRNA was detected in unstimulated and stimulated cultures of HGE, and was not significantly modulated by any stimulants. HBD-2 mRNA was up-regulated by PMA, F.n. cell wall and TNFα, but not by P.g. cell wall. Unstimulated and stimulated fibroblasts did not express HBD-l1 and hBD-2 mRNA. Kinetic analysis of hBD-2 regulation showed rapid activation by TNFα and F.n., and slower activation by PMA, indicating involvement of multiple signaling pathways. E. coli and F.n. LPSs, even in the presence of human serum, were poor stimulants of hBD-2. HBD-1 and hBD-2 mRNA was expressed in all tissue samples, and their expression varied between individuals. In gingival tissue, hBD-1 and hBD-2 mRNAs were predominately localized in suprabasal stratified epithelium and the peptides were detected in upper epithelial layers. In cultured epithelial cells, hBD-1 and hBD-2 peptides were detected in differentiating, involucrin-positive cells, although hBD-2 expression required stimulation by F.n., PMA or TNFα.; We investigated the signaling pathways in hBD-2 regulation. Results from gel shift analysis and immunofluorescence indicated involvement of NF-κB in hBD-2 regulation by F.n. However, hBD-2 induction by F.n. was not inhibited by pretreatment with two NF-κB inhibitors, pyrrolidine dithiocarbamate and peptide-aldehyde MG132. These two inhibitors blocked the NF-κB pathway as shown by several analyses. To investigate the role of MAP kinase pathways in hBD-2 induction, we tested the ability of SB203580 and tyrphostin AG126 to block hBD-2 mRNA induction by F.n. SB203580 and AG126 partially blocked hBD-2 induction, and combination of these two inhibitors completely blocked hBD-2 induction, indicating involvement of p38 and JNK MAP kinase pathways. Expression of β-defensins may play a role as part of the innate host defenses in maintaining normal gingival health; understanding the molecular mechanisms of β-defensin regulation may be applicable for clinical uses.