Lipopolysaccharide and High Concentrations of Glucose Enhances Zoledronate-induced Increase in RANKL/OPG Ratio by Upregulating PGE2 Production in Osteoblasts

Abstract

: Preexisting diseases, such as diabetes and chronic inflammation in periodontal tissue, are risk factors associated with bisphosphonate-related osteonecrosis of the jaw. Osteoblasts produce prostaglandin (PG)E 2 via cyclooxygenases (COX), and the autocrine action of PGE 2 impacts the function of osteoblasts, including receptor activator of NF-kappa B li gand (RANKL) and osteoprotegerin (OPG) production. This study assessed the effects of the stimulation of zoledronate in the presence of lipopolysaccharide (LPS) and high concentrations of glucose on the expression of COX-2, RANKL, and OPG, in addition to PGE 2 production in osteoblasts. MG-63 cells were cultured in medium containing 1 µg/ml LPS, 25 mM glucose (high glucose), and/or zoledronate (1×10 -8 , 1×10 -7 , 1×10 -6 , 5×10 -6 , or 1×10 -5 M). The mRNA expression of COX-2, RANKL, and OPG genes was determined by real-time polymerase chain reaction. The concentrations of RANKL and OPG protein and PGE 2 in the culture supernatant were examined by enzyme-linked immunosorbent assay. Zoledronate at a con centration of 5×10 -6 M overwhelmingly increased COX-2 mRNA expression. The expression levels of RANKL and OPG as well as PGE 2 production was significantly increased in cells stimulated with 5×10 -6 M zoledronate in the presence of LPS and high glucose than in the unstimulated cells (control). NS398, a specific inhibitor of COX-2, blocked the stimulatory ef fects of zoledronate (in the presence of LPS and high glucose) on PGE 2 production and the protein expression levels of RANKL and OPG. The ratio of RANKL/OPG was also increased following zolendronate stimulation. In addition, a signifi cant difference was observed not in the stimulation with zoledronate alone, but by the stimulation of zoledronate in the pres ence of LPS and high glucose as compared that in controls. These results suggest that LPS and high concentrations of glu cose enhances zoledronate-induced increase in RANKL/OPG ratio via the autocrine action of NS398-blocked PGE 2 in osteoblasts. assessed the effects of three types of bisphosphonate, zoledronate, alen dronate, and clodronate, on mRNA expression involved in the osteoblast differentiation and function in MG-63 cells and human primary osteo -blasts. In their experiments, treatment with zoledronate at concentrations ranging from 1×10 -5 to 1×10 -9 M decreased the expression of osteoblast differentiation-related transcription factors, collagenous, and non-colla genous protein, bone morphogenic protein, and ALPase, whereas the expression levels of transforming growth factor β and vascular endothe lial growth factor were increased. They also revealed that the effects of zoledronate on these mRNA expression levels was found to be generally dose-dependent. In the present study, the amount of total RNA eluted from cells was less obvious in the stimulation of 1×10 -5 M zoledronate than in the case of a stimulation under or equal to 5×10 -6 M (data not shown), and COX-2 expression levels were lower in 1×10 -5 M than 5×10 -6 M cases. Although we did not assess the other type of osteoblastic cell line, except for MG-63 cells, it is possible that zoledronate, at a concentration exceeding 5×10 -6 M, might have cytotoxic effects in oste oblasts.