E2 suppressed RANKL expression via inhibition of upstream inflammatory stimulators

Objectives: Systemic bone loss accompanying with estrogen deficiency has been proposed as a risk factor for periodontal disease in postmenopausal women. Previously, we discovered that estrogen (17â-estradiol, E2) exhibited a significant inhibitory effect on the bone-resorbing cytokines such as TNF-á, IL-1â, IL-6 and RANKL induced by E. coli lipopolysaccharide (LPS) in periodontal ligament (PDL) cells. A recent study by Wada et al. suggested that E. coli LPS stimulated RANKL expression in hPDL cells through the prior induction of TNF-á, IL-1â and IL-6. Therefore, we speculated that E2 suppressed RANKL expression in hPDL cells through the prior inhibition of TNF-á, IL-1â. Methods: To verify whether the modulatory effect of estrogen on RANKL expression was mediated by upstream cytokines TNF-á, IL-1â and IL-6, neutralizing antibodies for these cytokines were added in cultured human PDL cells. Gene expression of RANKL was investigated by quantitative Real-time polymerase chain reaction (PCR) analysis. The amount of soluble RANKL(sRANKL) in conditioned culture media and membrane-bound RANKL in the cell lysates were measured using enzyme-linked immunosorbent assays (ELISA). Results: In our study, we found that it took 48 h to detect a significant change of LPS-induced RANKL expression, whereas TNF-á, IL-1â and IL-6 expression was significantly enhanced within 6 h of LPS treatment. E2 greatly attenuated the inducing effects of LPS on TNF-á, IL-1â and IL-6 expression in PDL cells. E2 suppressed LPS-induced RANKL expression at both mRNA and protein level in PDL cells as well. Admination of neutralizeing antibody for these cytokines supressed the upregulation effects of LPS on RANKL expression in a dose-dependent manner. Conclusions: Our results suggested that estrogen may play significant roles in modulating the periodontal tissue responses to LPS, and may modulate RANKL expression in a manner dependent on upstream inflammatory stimulators.