Expression of Human beta Defensin-2 in HaCaT Cell by Lipoteichoic Acid and Peptidoglycan

Kim, Ji Eun; Jeong, Mi Sook; Kim, Myeung Nam; Hong, Chang Kwun; Ro, Byung In; Seo, Seong Jun

Abstract

BACKGROUND
Defensin, a major family of antimicrobial peptides, is small cationic, cysteine rich peptides with wide range of antimicrobial activity against Gram negative and Gram positive bacteria, fungi, yeast, and virus. Expression of human defensin-2 is upregulated by bacteria, virus, fungus and pro-inflammatory cytokines. However, this peptide was found to be only bacteriostatic, but not bactericidal, against the Gram positive bacteria. OBJECTIVE: To evaluate human defensin-2 (hBD-2) expression after exposure of human skin keratinocytes to the cell wall component of Gram positive bacteria such as lipoteichoic acid (LTA) and peptidoglycan(PEN), and to compare quantitatively the amount of expression with that after their exposure to the cell wall component of Gram negative bacteria. METHODS: Expression of hBD-2 was measured by reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry(IHC). RESULTS: 1. In RT-PCR results, the amount of hBD-2 expression after exposure to LPS was larger than those of PEN and LTA at 6 and 12 hours (p=0.02). At 24 hours, hBD-2 expression showed a peak in PEN stimulated group (p=0.09). 2. In Western blot analysis, hBD-2 expressions, when treated with PEN and LTA, were stronger than that treated with LPS at 6 and 12 hours. 3. In IHC, hBD-2 was stained much stronger in LPS stimulated group than PEN or LTA stimulated groups at 12 hours. CONCLUSION: Our study demonstrated that exposure of human skin keratinocytes to the cell wall components of Gram positive bacteria such as LTA and PEN triggered production of hBD-2 in addition to the cell wall component of Gram negative bacteria such as LPS, however, the amounts of expression were relatively stronger in LPS treated group.