Alendronate Affects Osteoblast Differentiation by Crosstalk through EphrinB1-EphB Interaction

Objective: Bisphosphonates are therapeutic agents for the treatment of postmenopausal osteoporosis. Although they have been associated with delayed healing in injured tissues, resulting in inappropriate femoral fractures and osteonecrosis of the jaw (ONJ), the pathophysiological mechanisms involved are not clear. Our hypothesis is that alendronate, a member of the N-containing bisphosphonates, indirectly inhibits osteoblast function through the coupling of osteoclasts to osteoblasts by ephrinB-EphB interaction.

Method: Two-month old mice were assigned to two different groups with 15 mice per group and each received either saline or alendronate (10 μg/100 g/week) subcutaneously for 8 weeks. We performed real time RT-PCR and Western blot assay using spongiosa of femurs and bone marrow cells from femurs of mice. Moreover, we performed immunohistochemistry of ephrinB1, EphB1 and B3 using femurs of mice. 

Result: We found that alendronate increased gene and protein expression of ephrinB1 and EphB1, as well as B3, in femurs of mice injected with alendronate. Alendronate stimulated ephrinB1 protein level in monocytes or pre-osteoclasts of bone marrow whereas it enhanced EphB1 and EphB3 protein in osteoblasts of trabecular bone. Alendronate suppressed the expression of bone sialoprotein (BSP) and osteonectin in both femurs and bone marrow osteoblastic cells of mice. The drug inhibited osteoblast marker genes in bone marrow osteoblasts which are a mixed culture. After elimination of monocytes or pre-osteoclasts from bone marrow cells, alendronate did not affect osteoblast differentiation indicating the need for pre-osteoclasts for alendronate’s effects. Alendronate stimulated EphB1 and EphB3 protein expression in osteoblasts whereas it enhanced ephrinB1 protein in monocytes or pre-osteoclasts. In addition, a reverse signal by ephrinB1 inhibited osteoblast differentiation and suppressed BSP gene expression.

Conclusion: Alendronate, through its direct effects on the pre-osteoclast, appears to regulate expression of ephrinB1 which regulates and acts through the EphB1, B3 receptors on the osteoblast to suppress osteoblast differentiation.