Departments of Pharmacology Orthodontics, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan Transcriptome Research Group, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Hokkaido University, Kita 13, Nishi 7, Kita-ku, Sapporo 060-8586, Japan Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Chiyoda-ku, Tokyo 113-8510, Japan.
J Endocrinol. 2013 Oct 4;219(2):145-58. doi: 10.1530/JOE-13-0040. Print 2013 Nov.
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.
双膦酸盐（BPs）是一类主要的抗吸收药物，其抗吸收作用的分子机制已得到广泛研究。最近的研究表明，BPs 不仅靶向骨吸收细胞，还靶向成骨细胞。我们之前的研究表明，局部应用含氮双膦酸盐（N-BP）阿仑膦酸钠（ALN）短时间内可增加大鼠牙再植模型中的骨组织。在这里，我们研究了 ALN 促进骨形成的细胞机制。骨组织形态计量学证实，局部应用 ALN 可增加骨形成。ALN 增加了位于骨表面的成骨细胞的增殖，而体内抑制了抗酒石酸酸性磷酸酶（TRAP）阳性破骨细胞的数量。此外，ALN 处理诱导了更多的骨表面碱性磷酸酶阳性和骨钙素阳性细胞，而 PBS 处理则诱导了更少的细胞。体外研究表明，脉冲处理 ALN 可促进骨钙素表达。为了追踪 N-BP 的靶细胞，我们在体内和体外应用荧光标记的 ALN（F-ALN）。F-ALN 被摄取到体内和体外的成骨细胞中。这种细胞内摄取被内吞作用抑制剂所抑制。此外，内吞作用抑制剂丹磺酰尸胺（DC）抑制了 ALN 刺激的体外成骨细胞分化，并抑制了体内碱性磷酸酶阳性成骨细胞的增加和随后的骨形成。DC 还阻断了 ALN 在成骨细胞中对 Rap1A 异戊烯化的抑制。这些数据表明，局部应用 ALN 通过刺激成骨细胞的增殖和分化以及抑制破骨细胞功能来促进骨形成。这些作用可能通过 ALN 的内吞作用和随后的蛋白异戊烯化抑制来发生。
