2004;72:5041C5051. of proinflammatory cytokine expression by LPS and IL-1. Taken together, this study showed that LPS and IL-1 coordinate a synergy on cytokine production by upregulating MyD88 expression in HGFs. (InvivoGen, San Diego, CA) (was calculated by subtracting for GAPDH from for genes of interest and the was RTC-5 calculated by subtracting the for control cells from for treated cells. The fold change was calculated as 2?LPS, IL-1 or both for 24 h. After the treatment, the cells were rinsed with cold PBS and lysed with the buffer from Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Both firefly and renilla luciferase levels were measured in a luminometer according to the instruction from the manufacturer. The firefly luciferase levels were normalized to the renilla luciferase levels. 2.6. Immunoblotting of MyD88 and MAPK For immunoblotting of MyD88, cell lysate containing 25C50 g protein was electrophoresed in a 10% polyacrylamide gel. After transferring proteins to RTC-5 a PVDF membrane, MyD88 was immunoblotted with goat anti-human MyD88 primary antibody (R&D Systems) and horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA). MyD88 were visualized by incubating the membrane with chemiluminescence reagent (NEN Life Science Products) for 1 min and exposing it to x-ray film for 1C10 min. The X-ray films were scanned using an Epson scanner (Perfection 1200U). For mitogen-activaetd protein kinase (MAPK) immunoblotting, anti-total or anti-phosphorylated extracellular regulated kinase (ERK), c-Jun N-terminal kinases (JNK) or p38 MAPK primary antibodies and HRPconjugated secondary antibody were used. The density of bands on the images was quantified using Adobe Photoshop version 10.0.1. The results were presented as the ratios of phosphorylated ERK, JNK or p38 vs. total ERK, JNK or p38. 2.7. Treatment of cells with the inhibitors of signaling pathways Human gingival fibroblasts were treated with LPS, Rabbit Polyclonal to MRPL46 IL-1 or the combination of LPS and IL-1 in the absence or presence of 5 or 10 M of ERK pathway inhibitor PD98059, JNK pathway inhibitor SP600125, or p38 MAPK pathway inhibitor SB203580, or 2.5 or 5 M of NFB pathway inhibitor Bay117085 (Calbiochem/EMD Biosciences, Inc., San Diego, CA) for 24 h. After the treatment, IL-6 in medium was quantified using ELISA. 2.8. RNA interference Human gingival fibroblasts were transiently transfected with 200 nM of MyD88 siRNA (Thermo-Fisher Scientific, Waltham, MA) or the scrambled control siRNA (Santa Cruz Biotechnology, Inc., Dallas, TX) using RTC-5 Lipofectamine RNAi MAX reagent (Thermo-Fisher Scientific) for 24 h by following the manufacturer’s instructions. After the transfection, fibroblasts were treated with LPS, IL-1 or both LPS and IL-1 for 24 h. 2.9. Statistic analysis Data were presented as mean SD. Student tests were performed to determine the statistical significance of gene expression or luciferase activity among different experimental groups. A value of LPS alone stimulated expression of several cytokines and chemokines such as IL-6, chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte-chemotactic protein (MCP)-1, and IL-8, but had no significant effect on molecules involved in the signaling transduction and transcription factors. In contrast, IL-1 is a stronger stimulator as it stimulated not only cytokines and chemokines, but also molecules involved in the signal transduction and transcription such as interferon regulatory transcription factor (IRF)1, NFKB1, NFKB1A and prostaglandin-endoperoxide synthase (PTGS) 2, also known as cyclooxygenase-2 (COX-2). Strikingly, the combination of LPS and IL-1 is much more potent than LPS or IL-1 alone in the stimulation of the proinflammatory molecules. For example, while LPS and IL-1 stimulated IL-6 expression by 2- and 74-fold, respectively, the combination of LPS and IL-1 led to a 201-fold increase. These data revealed a strongly synergy between LPS and IL-1 in the upregulation of proinflammatory gene expression. Table 1 Synergistic effect of LPS and IL-1 on expression of gene expression LPSLPSLPSLPSLPSLPSLPS and IL-1 on inflammatory cytokine secretion from gingival fibroblasts. Results showed that LPS and IL-1 synergistically increased secretion of IL-6 secretion (Fig. 1A), GM-CSF, also known as CSF2 (Fig. 1B), and IL-1 (Fig. 1C). All these results are consistent with the findings from the PCR array as shown in Table 1. Since the expression of 84 genes was RTC-5 amplified at the same time and under the same temperature by the PCR array, the fold changes of gene expression may not be same as those by quantitative realtime PCR. However, both the data from the PCR array and real-time PCR showed a.