Effects of prostaglandin E 2 on gene expression in primary osteoblastic cells from prostaglandin receptor knockout mice

Abstract

Recent studies have shown that stimulation of osteoclastogenesis in cocultures of osteoblasts and spleen cells in response to prostaglandin E 2 (PGE 2) is markedly decreased when the osteoblasts are derived from cells lacking either the EP 2 or the EP 4 receptor. Induction of osteoclast formation requires upregulation of receptor activator of nuclear factor-κB ligand (RANKL) on cells of the osteoblastic lineage, which then binds to the RANK receptor on cells of the osteoclast lineage. Osteoprotegerin (OPG) is a decoy receptor for RANKL that can block its interaction with RANK. In addition, macrophage-colony stimulating factor (M-CSF) is essential for osteoclast formation. Finally, PGE 2 can increase interleukin-6 (IL-6), which may further enhance osteoclastogenesis. To study the relative influence of the EP 2 and EP 4 receptors on response of these factors to PGE 2, we examined mRNA levels for RANKL, OPG, M-CSF, and IL-6 in primary osteoblastic cell cultures derived from two lines of EP 2 knockout mice (EP 2−/−) and one line of EP 4 knockout mice (EP 4−/−) and the relevant wild-type controls (EP 2+/+ and EP 4+/+). The responses of cells from wild-type animals of all three lines were similar. After PGE 2 treatment, RANKL mRNA levels were increased at 2 h, and this was sustained over 72 h. Basal RANKL expression was moderately reduced in EP 2−/− cells and markedly reduced in EP 4−/− cells. PGE 2 increased RANKL mRNA in EP 2−/− cells and EP 4−/− cells, but the levels were significantly reduced compared with wild-type cells. There were no consistent changes in expression of M-CSF or OPG in the different genotypes or with PGE 2 treatment. IL-6 mRNA was variably increased by PGE 2 in both wild-type and knockout cells, although the absolute levels were somewhat lower in both EP 2−/− and EP 4 −/− cultures. Parathyroid hormone (PTH) increased RANKL and IL-6 and decreased OPG mRNA levels similarly in both wild-type and EP 2−/− or EP 4−/− cells. The major defect in the response to PGE 2 in animals lacking either EP 2 or EP 4 receptors is a reduction in basal and stimulated RANKL levels. Loss of EP 4 receptor appears to have a greater effect on basal RANKL expression than EP 2.