Osteoporosis is one of the most widespread and destructive bone diseases in our modern world. There is a great need for anabolic agents for bone which could reverse this disease, but few are available for clinical use. Prostaglandin E receptor (EP4) agonist (EP4A) is one of the very few anabolic agents for bone in rat, but its systemic efficacy against bone loss at sub-optimal dose is limited in mice. As osteoblasts are regulated by extracellular matrix proteins, we tested whether deficiency of osteopontin (OPN), a secreted phosphorylated protein, could modulate the effects of EP4A (ONO-AE1-329) treatment at 30 μg/kg body weight, a sub-optimal dose, for 5 days/week for 4 weeks. OPN deficiency enhanced the anabolic effects of EP4A on bone volume. Histomorphometric analysis indicated that EP4A increased mineral apposition rate as well as bone formation rate in OPN-deficient but not in wild-type mice. Neither OPN deficiency nor EP4A altered osteoclast parameters. Importantly, OPN deficiency enhanced the direct anabolic action of EP4A locally injected onto the parietal bone in inducing new bone formation. Combination of OPN deficiency and EP4A treatment caused an increase in mineralized nodule formation in the cultures of bone marrow cells. Finally, OPN deficiency enhanced anabolic action of EP4A in the mice subjected to ovariectomy. These data indicate that OPN deficiency enhances the actions of EP4A at sub-optimal dose.
Norihiko Kato, Keiichiro Kitahara, Susan R Rittling, Kazuhisa Nakashima, David T Denhardt, Hisashi Kurosawa, Yoichi Ezura and Masaki Noda
Koichiro Komatsu, Akemi Shimada, Tatsuya Shibata, Satoshi Wada, Hisashi Ideno, Kazuhisa Nakashima, Norio Amizuka, Masaki Noda and Akira Nifuji
Bisphosphonates (BPs) are a major class of antiresorptive drug, and their molecular mechanisms of antiresorptive action have been extensively studied. Recent studies have suggested that BPs target bone-forming cells as well as bone-resorbing cells. We previously demonstrated that local application of a nitrogen-containing BP (N-BP), alendronate (ALN), for a short period of time increased bone tissue in a rat tooth replantation model. Here, we investigated cellular mechanisms of bone formation by ALN. Bone histomorphometry confirmed that bone formation was increased by local application of ALN. ALN increased proliferation of bone-forming cells residing on the bone surface, whereas it suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in vivo. Moreover, ALN treatment induced more alkaline phosphatase-positive and osteocalcin-positive cells on the bone surface than PBS treatment. In vitro studies revealed that pulse treatment with ALN promoted osteocalcin expression. To track the target cells of N-BPs, we applied fluorescence-labeled ALN (F-ALN) in vivo and in vitro. F-ALN was taken into bone-forming cells both in vivo and in vitro. This intracellular uptake was inhibited by endocytosis inhibitors. Furthermore, the endocytosis inhibitor dansylcadaverine (DC) suppressed ALN-stimulated osteoblastic differentiation in vitro and it suppressed the increase in alkaline phosphatase-positive bone-forming cells and subsequent bone formation in vivo. DC also blocked the inhibition of Rap1A prenylation by ALN in the osteoblastic cells. These data suggest that local application of ALN promotes bone formation by stimulating proliferation and differentiation of bone-forming cells as well as inhibiting osteoclast function. These effects may occur through endocytic incorporation of ALN and subsequent inhibition of protein prenylation.
Muneaki Ishijima, Kunikazu Tsuji, Susan R Rittling, Teruhito Yamashita, Hisashi Kurosawa, David T Denhardt, Akira Nifuji, Yoichi Ezura and Masaki Noda
Mechanical stress to bone plays a crucial role in the maintenance of bone homeostasis. It causes the deformation of bone matrix and generates strain force, which could initiate the mechano-transduction pathway. The presence of osteopontin (OPN), which is one of the abundant proteins in bone matrix, is required for the effects of mechanical stress on bone, as we have reported that OPN-null (OPN−/−) mice showed resistance to unloading-induced bone loss. However, cellular mechanisms underlying the phenomenon have not been completely elucidated. To obtain further insight into the role of OPN in mediating mechanical stress effect on bone, we examined in vitro mineralization and osteoclast-like cell formation in bone marrow cells obtained from hind limb bones of OPN−/− mice after tail suspension. The levels of mineralized nodule formation of bone marrow cells derived from the femora subjected to unloading were decreased compared with that from loaded control in wild-type mice. However, these were not decreased in cells from OPN−/− mice after tail suspension compared with that from loaded OPN−/− mice. Moreover, while spreading of osteoclast-like cells derived from bone marrow cells of the femora subjected to unloading was enhanced compared with that from loaded control in wild-type mice, this enhancement of spreading of these cells derived from the femora subjected to unloading was not recognized compared with those from loaded control in OPN−/− mice. These data provided cellular bases for the effect of the OPN deficiency on in vitro reduced mineralized nodule formation by osteoblasts and on enhancement of osteoclast spreading in vitro induced by the absence of mechanical stress. These in vitro results correlate well with the resistance to unloading-induced bone loss in OPN−/− mice in vivo, suggesting that OPN has an important role in the effects of unloading-induced alterations of differentiation of bone marrow into osteoblasts and osteoclasts.