TNF-alpha-induced Increase of Human beta-defensin-2 Expression in HaCaT Cell Lines

Park, Young Do; Lee, Kyu Suk

Abstract

BACKGROUND: Several kinds of epithelial cells and focal lymph nodes are known to be involved in the skin's immune reaction. Especially, internal antimicrobial peptide play an important role in protecting microbial agents. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which is produced by epithelial cells after stimulation with microorganisms or inflammatory mediators. hBD-2 participates in the increase of the cell-mediated immune reaction. It also affects the proliferation and differentiation of epithelial cells and fibroblasts, resulting in enhancement of wound healing. However, little is known as to whether the TNF-alpha induces the expression of hBD-2 in HaCaT cells through the NF-kappaB or MAPKs pathways.
OBJECTIVE
Research was undertaken to investigate the roles of NF-kappaB and MAPKs transcription factors in the molecular pathway of TNF-alpha-induced hBD-2 expression in HaCaT cell lines.
METHODS
The expression of hBD-2 in TNF-alpha-treated HaCaT cells was analyzed by immunofluorescence staining and reverse transcription polymerase chain reaction (RT-PCR). The expression of NF-kappaB was analyzed by Western blot analysis and electrophoretic mobility shift assay (EMSA).
RESULTS
Strong positive hBD-2 immunofluorescence staining in TNF-alpha-treated HaCaT cells was observed. According to RT-PCR analysis, the expression of hBD-2 increased TNF-alpha-treated HaCaT cells by dose-dependent and time-dependent manners. In addition, according to Western blot analysis and EMSA, NF-kappaB was also activated in TNF-alpha-treated HaCaT cells. Interestingly, the expression of hBD-2 in TNF-alpha-treated HaCaT cells was attenuated in the presence of NF-kappaB inhibitors, PDTC or MG132. Furthermore, MAPKs inhibitors, especially SB (p38 inhibitor), partially attenuated the TNF-alpha-induced hBD-2 expession, but not PD (ERK inhibitor) and SP (JNK inhibitor).
CONCLUSION
These results collectively suggest that hBD-2 is up-regulated in TNF-alpha-treated HaCaT cells through activation of NF-kappaB and p38 MAPKs pathway. Our data regarding the up-regulation of hBD-2 may help us to understand the antimicrobial mechanism in normal skin or in skin diseases.