Supplementary MaterialsSupplemental data include four figures and can be found with this article online at http://e-emm. semi-quantitative RT-PCR. (D) 12-HHT was added to the culture media (0, 25, 75 or 150 nM). After 24 h, the culture media were harvested, and IL-6 was measured by ELISA. Data indicate the means S.D. of three impartial experiments (** 0.01). 12-HHT reduces UVB-induced IL-6 synthesis inhibition of the p38 MAPK/NF-B pathway We next investigated the signaling mechanism by which 12-HHT down-regulates IL-6 synthesis upon UVB irradiation. MAPK has been implicated in the synthesis of the inflammatory cytokines induced by UV irradiation (Peus et al., 1999; Pfundt et al., 2001; Bode and Dong, 2003). Thus, we hypothesized that the ability of 12-HHT to modulate IL-6 synthesis would be mediated by MAPK and, subsequently, the pro-inflammatory activities of transcription factors, such as NF-B. In accordance with previous reports, the activation of MAPK reached a maximum at 60 min post-irradiation (Physique 3A). Furthermore, IL-6 synthesis upon UVB irradiation was attenuated when the HaCaT cells were treated with MAPK inhibitors [e.g., p38 kinase inhibitor SB203580, ERK inhibitor PD98059 or JNK Telaprevir cost inhibitor SP600125], and, among these, SB203580 showed the most Telaprevir cost marked attenuation effect (Physique 3B). These results suggest that p38 MAPK is usually a major regulator of IL-6 synthesis. Telaprevir cost Next, the measurement of MAPK phosphorylation using western blotting revealed that this UVB-induced phosphorylation of p38 MAPK was significantly suppressed by 12-HHT treatment (Physique 3C); in contrast, no notably changes were detected in the phosphorylation of ERK or JNK (Supplemental Data Physique S1), indicating that 12-HHT down-regulates UVB-induced IL-6 synthesis in a largely p38 MAPK-dependent manner. It is well known that Telaprevir cost NF-B acts down-stream of p38 MAPK in UV-induced signal transduction, thus regulating the expression of a variety of inflammatory cytokines. Therefore, we investigated the effect of 12-HHT on UVB-induced NF-B activation, using a luciferase reporter gene assay. We detected an inhibitory effect of 12-HHT around the UVB-induced activation of NF-B, showing that pretreatment with Bay11-7082, a specific inhibitor of NF-B, markedly prevented UVB-induced IL-6 synthesis (Physique 3B) and that treatment with 12-HHT substantially reduced the transcriptional activity of NF-B in HaCaT cells after UVB irradiation (Physique 3D). Taken together, these results suggest that 12-HHT inhibits the p38 MAPK/NF-B pathway which is usually activated by UVB irradiation, thus leading to the reduction of IL-6 synthesis. Open in a separate window Physique 3 12-HHT reduces UVB-induced IL-6 synthesis inhibition of the p38 MAPK/NF-B pathway. (A) HaCaT cells were starved for 12 h and irradiated with UVB (5 mJ/cm2) for various occasions (0, 30, 60 and 120 min). The cell lysates were examined by western blotting to analyze p-p38, p38, p-JNK, JNK, p-ERK and ERK. (B) Starved HaCaT cells were pretreated with SB203580 (20 M), PD98059 (20 M), SP600125 (20 M) or Bay11-7082 (20 M) for 60 min and then irradiated with UVB (5 mJ/cm2) for 24 h. The level of IL-6 synthesis was measured by MMP3 ELISA. (C) Upon UVB irradiation (5 mJ/cm2), the HaCaT cells were immediately incubated with ethanol (control) or 12-HHT (150 nM) for 60 min. The cell lysates were prepared and examined by western blotting using antibodies specifically recognizing p38 MAPK and p-p38 MAPK. The p-p38 MAPK signals are presented as the fold induction relative to the control samples and are shown with densitometry values expressed as the means S.D. of three impartial experiments. (D) HaCaT cells were co-transfected with both NF-B-dependent luciferase construct and pSV40–galactosidase construct for 24 h and serum-starved for an additional 6 h. These transfected cells were either sham-irradiated or stimulated with UVB (5 mJ/cm2) and treated with ethanol (control) or 12-HHT (150 nM) for Telaprevir cost 1 h. The relative fold increase of luciferase activity was calculated, as described in the Methods. Data indicate the means S.D. of three impartial experiments (* 0.05 and ** 0.01). 12-HHT inhibits UVB-induced IL-6 synthesis through the induction of MKP-1 MKP-1 is usually a dual-specificity phosphatase that directly dephosphorylates p38 MAPK and, thus, attenuates UV-induced inflammation (Keyse,.