Dieckol, a Component of Ecklonia cava, Suppresses the Production of MDC/CCL22 via Down-Regulating STAT1 Pathway in Interferon-? Stimulated HaCaT Human Keratinocytes

Kang, Na-Jin; Koo, Dong-Hwan; Kang, Gyeoung-Jin; Han, Sang-Chul; Lee, Bang-Won; Koh, Young-Sang; Hyun, Jin-Won; Lee, Nam-Ho; Ko, Mi-Hee; Kang, Hee-Kyoung; Yoo, Eun-Sook

HaCaT cells (5.0×10 5 cells/mL) were pretreated with epigallocatechingallate (EGCG; 10 mM) or dieckol (2.5, 5, 10 mM) for 30 min. The phosphorylation of STAT1 was determined in cells stimulated with IFN-γ (10 ng/mL) for 15 min. The phosphorylation or level of each protein in whole cell lysates was determined by Western blotting with the indicated antibodies. (B) HaCaT cells were pre-treated with EGCG (10 mM) or dieckol (2.5, 5, 10 mM) for 2 h. The nuclear translocation of the STAT1 protein was determined in cells stimulated with IFN-γ for 20 min. Immunofluorescence staining for STAT1 was performed by using a primary antibody against STAT1, followed by a DyLight488-conjugated secondary antibody. The fluorescence was then identified by using a confocal microscope (FV500, Olympus Corp.), and the images were acquired at constant two-photon excitation microscopy (PMT), gain, offset, magnification (40× oil immersion objective with a zoom factor of 1.5), and resolution. associated MAPKs depending on the cell type (Pearson et al., 2001; Madonna et al., 2008) . We thus investigated the involvement of these signaling kinases in IFN-γ induced MDC production in HaCaT cells. We first determined time dependent activation of three MAPKs (ERK, JNK, p38) after IFN-γ treatment. As illustrated in Fig. 4A . IFN-γ induced the phosphorylation of ERK at 5 min, while there was no effect on the phosphorylation of JNK and p38. Then we examined the inhibitory effect of dieckol on the ERK activation in IFN-γ-stimulated HaCaT cells. PD98059 (a specific ERK inhibitor, 10 mM), strongly suppressed ERK phosphorylation, but, dieckol did not inhibit ERK phosphorylation (Fig. 4B) . To confirm whether MAPKs pathway associate with MDC production, we determined the MDC level after the treatment of known MAPKs inhibitors in IFNγ-stimulated HaCaT cells. SP600125 (a specific JNK inhibitor) and PD98059 (a specific ERK inhibitor), and SB203580 (a specific p38 inhibitor) did not affect on the MDC production induced by IFN-γ (Fig. 4C) . These results suggest that MAPKs pathway does not contribute on the production of MDC in IFN-γ stimulated HaCaT cells.