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Integrins are heterodimeric cell surface receptors that mediate cell-cell and cell-matrix interaction. The vitronectin and osteopontin receptor αvβ3 integrin has increased expression levels and is implicated in the adhesion, activation, and migration of osteoclasts on the bone surface as well as osteoclast polarization. αvβ3 integrin plays an important role in osteoclast differentiation and resorption. In addition, Arg-Gly-Asp (RGD)-containing peptides, small molecular inhibitors, and antibodies to αvβ3 integrin have been shown to inhibit bone resorption in vitro and in vivo. Here we examined the effects of a disintegrin HSA-ARLDDL a genetically modified mutant of rhodostomin conjugated with human serum albumin, which is highly selective of αvβ3, on RANKL-induced osteoclastogenesis and ovariectomy (OVX)-induced osteoporosis. In RANKL-induced osteoclastogenesis, HSA-ARLDDL significantly inhibited osteoclast formation, and IC50 was at nM range. Post-treatment HSA-ARLDDL also inhibits osteoclast formation. Furthermore, weekly administration of HSA-ARLDDL significantly inhibits the increase in serum bone resorption marker levels and decrease in cancellous bone loss in tibia and femur induced by OVX. On the other hand, HSA-ARLDDL did not affect the differentiation and calcium deposition of osteoblasts. These results indicate that the highly selective and long-acting αvβ3 integrin antagonists could be developed as effective drugs for postmenopausal osteoporosis.

Metabolites from arachidonic acids play the pivotal roles in inflammatory arthritis. Arachidonic acid could be metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) to produce the bioactive eicosanoids. Although the down-stream products of COX including prostaglandin E2 are well-known inflammatory stimulators, the role of LOX products in inflammatory arthritis is still unclear. Here we found that the downstream product of 15-LOX, 15-S-hydroxyeicosatetraenoic acid (15-(S)-HETE), can enhance the expression of placenta growth factor (PLGF), which is recently considered to play an important role in rheumatoid arthritis. 15-(S)-HETE increased the expression of PLGF in human rheumatoid arthritis synovial fibroblasts in a time-dependent and concentration-dependent manner. PI3K-Akt, NF-κB signaling pathways were involved in the potentiation effects of 15-(S)-HETE. In addition, COX-2 was up-regulated by the treatment of 15-(S)-HETE and the increase of COX-2 expression participated in 15-(S)-HETE-induced PLGF expression, which was confirmed by COX-2 shRNA or pharmacological COX-2 inhibitor. Moreover, it was found that treatment of prostaglandin E2 (PGE2), which was the main down-stream metabolite of COX-2, increased the expression of PLGF. EP1, EP2, EP3 and EP4 agonists could up-regulate PLGF as well. In animal studies, we found that the adjuvant-induced expression of PLGF and COX-2 was inhibited in 15-LOX knockout mice. These results indicated that PLGF up-regulation by 15-LOX downstream product may be involved in inflammatory arthritis.

Glutamate, an important central excitatory neurotransmitter, is also secreted by osteoblasts and may be involved in the regulation of bone metabolism. Glutamate receptors for N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) are demonstrated in bone cells. Here we investigated the in vivo effects of glutamate by local injection of AMPA, NMDA, and their antagonists into tibia as well as their in vitro effects on the maturation of osteoblasts and formation of osteoclasts. AMPA receptor antagonist CNQX and NMDA receptor antagonist MK-801 significantly inhibited the maturation and mineralization of osteoblasts in high-glutamate alpha-MEM. On the other hand, AMPA and NMDA up-regulated the mineralized deposition and osteocalcin mRNA expression of primary osteoblasts cultured in glutamate-free DMEM. AMPA and NMDA induced the phosphorylation of extracellular signal-related kinases (ERK) in osteoblasts within 15 min. In addition, NMDA but not AMPA up-regulated the number of osteoclasts while MK801 antagonized this potentiating effect. To explore the action of glutamate agonists on bone formation in animal model, AMPA was locally injected into tibia and it was found that the bone volume in secondary spongiosa significantly increased and co-treatment of CNQX antagonized the enhancing effect of AMPA. These results suggest that glutamate may play a physiological role in regulating the maturation of osteoblasts and osteoclastogenesis. Activation of both AMPA and NMDA receptors regulates the maturation of osteoblasts. NMDA but not AMPA affects receptor for activation of NF-kappaB ligand (RANKL)-induced osteoclastogenesis.