Lipopolysaccharide increases IL-6 secretion via activation of the ERK1/2 signaling pathway to up-regulate RANKL gene expression in MLO-Y4 cells

Lipopolysaccharide (LPS) plays an important role in bone resorption, which involves numerous cytokines through various signaling pathways. RANKL and interleukin (IL)-6 are two important cytokines that are involved in bone remodeling. The aim of this study was to evaluate the effect of LPS on RANKL and IL-6 gene expression, the relationship of RANKL and IL-6, and the role of extracellular signal-regulated kinases 1/2 (ERK1/2) on IL-6 secretion induced by LPS in MLO-Y4 cells. The cells were stimulated by LPS at different concentrations (1, 10, 100, 500, and 1000ng/mL) for different durations (0.5, 1, 2, 4, and 8h and 0.5, 1, 1.5, 2, and 4h), and the mRNA expressions of RANKL and IL-6 were determined by PCR. In the presence of 100ng/mL LPS at different time points (0.5, 1, 1.5, 2, and 4h), IL-6 secretion and ERK1/2 phosphorylation in the cells were determined by ELISA and western blotting, respectively. STAT3 phosphorylation in cells simulated by 100ng/mL LPS at different time points (0.5, 1, 2, 4, and 8h) was assessed by western blotting. We found that LPS significantly up-regulated RANKL expression and activated the ERK1/2 pathway to induce IL-6 mRNA expression and protein synthesis in MLO-Y4 cells. However, the increased IL-6 was blocked by pre-treatment of MLO-Y4 cells with the ERK1/2 inhibitor U0126 (10 mu M), and the enhanced RANKL was blocked by the STAT3 inhibitor S3I-201 (100 mu M). Our results indicate that LPS up-regulates osteocyte expression of RANKL and IL-6, and the increased RANKL is associated with the up-regulation of IL-6, which involves the ERK1/2 pathway.