Novel osteogenetic therapy using BMP and small molecular compound (ROCK inhibitor)

使用 BMP 和小分子化合物（ROCK 抑制剂）的新型成骨疗法

• 批准号: 14370478
• 负责人: ITOH Kazuyuki
• 金额: $ 9.15万
• 依托单位: Osaka Medical Center for Cancer and Cardiovascular Diseases
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370478/
• 关键词: bone formation; bone morphogenetic protein (BMP); cytoskeletoton; Rho; Rho associated coiled -coil kinase (ROCK); Mitogen-activated kinase (MAP kinase); T0614; actin; BMP; Rho kinase; MEK inhibitor; T-614; Osterix

There are multiple steps for osteogenesis from undifferentiated mesenchymal stem cells to osteoblast. These steps require longer period in the adult mammals (especially in monkey or human), and it becomes a big clinical problem. There is no available drug to stimulate the osteogenesis among the clinically approved medicines for the treatment of osteoporosis. One growth factor, named bone morphogenetic protein (BMP), is currently undertaken for the clinical trial for regeneration after surgery, however, the amount required for reasonable results in human is huge. In order to overcome these problems, our group has been screened small molecular compound, which stimulate osteogenesis by themselves or combination with BMP. We found three candidate compounds as follwing.1.Small GTP-binding protein-Rho-associated coiled-coil kinase (ROCK) inhibitor (Y-27632)2.Mitogen-activated protein kinase (MAP kinase) inhibitor (PD98059, U0126)3.Newly synthesized anti-rheumatic drug (T0614)We found that T0 … More 614 stimulated the expression of "osterix", key factor for ontogenesis, without affecting the expression of cbfa-1. In order to examine the mechanism for ROCK and MAP kinase inhibitor, we focused on the morphology of mesenchymal cells either treated with these inhibitors or transfected with various cDNAs of active/dominant negative forms of each kinase. We found that the actin cytoskeleton dynamics was dramatically changed by these treatments. Next we stimulated the preosteoblasts (MC3T3-E1 cells) with cytochalacin D or latrunculin B, which directly change the actin polymerization dynamics. Surprisingly, it revealed that most profound osteogenetic effect by BMP was observed when the cells were stimulated during the re-formation of focal adhesion after disappeared by the treatment with cytochalasin D or latrunculin B. These results suggest that regulation of actin dynamics itself effectively enhance the BMP-induced osteogenesis both in vitro and in vivo. We are currently studying the profound mechanism, and try to apply these results for clinical therapy. Less

从未分化间充质干细胞到成骨细胞的成骨过程有多个步骤。这些步骤在成年哺乳动物(尤其是猴子或人类)中需要较长的时间，成为一大临床难题。在临床批准的治疗骨质疏松症的药物中，没有可用于刺激成骨的药物。一种名为骨形态发生蛋白(BMP)的生长因子目前正在进行手术后再生的临床试验，然而，在人类身上获得合理结果所需的量是巨大的。为了克服这些问题，我们课题组已经筛选出了单独或与BMP联合刺激成骨的小分子化合物。我们发现了如下三个候选化合物：1.小分子GTP结合蛋白-Rho相关卷曲卷曲蛋白(ROCK)抑制剂(Y-27632)2.丝裂原活化蛋白激酶(MAP)抑制剂(PD98059，U0126)3.新合成的抗风湿药物(T0614)我们发现T0-…614刺激了个体发育关键因子“Osterix”的表达，但不影响CBFA-1的表达。为了探讨ROCK和MAP激酶抑制剂的作用机制，我们重点研究了经这两种抑制剂处理的间充质细胞和不同活性/显性负性形式的不同激酶的cDNA对间充质细胞形态的影响。我们发现，这些处理显著改变了肌动蛋白细胞骨架的动力学。接下来，我们用细胞松弛素D或乳杆菌凝集素B刺激前成骨细胞(MC3T3-E1细胞)，这直接改变了肌动蛋白聚合动力学。令人惊讶的是，BMP的成骨作用在细胞松质素D或Latrunculin B处理后消失的局灶性黏附的形成过程中被刺激时被观察到。这些结果表明，在体外和体内，肌动蛋白动力学的调节本身都有效地促进了BMP诱导的成骨。我们目前正在研究其深刻的机制，并试图将这些结果应用于临床治疗。较少

项目成果

Kuriyama, K., Higuchi, C., Tanaka, K., Yoshikawa, H., Itoh, K.: "A novel anti-rheumatic drug, T-614, stimulates osteoblastic differentiation in vitro and bone morphogenetic protein-2 induced bone formation in vivo"Biochem.Biophys.Res.Commun.. 299. 903-909

Kuriyama, K.、Higuchi, C.、Tanaka, K.、Yoshikawa, H.、Itoh, K.：“一种新型抗风湿药 T-614 可刺激体外成骨细胞分化和骨形态发生蛋白 2 诱导的骨

Itoh, K., Yoshioka, K., Nakase, T., Yoshikawa H.: "Stimulation of Bone Formation by Rho-associated Kinase Inhibitor"J.Bone Miner.Res.. 16. 235 (2001)

Itoh, K.、Yoshioka, K.、Nakase, T.、Yoshikawa H.：“Rho 相关激酶抑制剂刺激骨形成”J.Bone Miner.Res.. 16. 235 (2001)

Kuriyama, K. et al.: "A novel anti-rheumatic drug, T-614, stimulates osteoblastic differentiation in vitro and bone morphogenetic protein-2 induced bone formation in vivo."Biochem.Biophys.Res.Commun.. 299. 903-909 (2002)

Kuriyama, K. 等人：“一种新型抗风湿药 T-614 可在体外刺激成骨细胞分化，并在体内刺激骨形态发生蛋白 2 诱导骨形成。”Biochem.Biophys.Res.Commun. 299. 903

Higuchi, C. et al.: "Continuous inhibition of MAPK signaling promotes the early osteoblastic differentiation and mineralization of extracellular matrix."J.Bone Miner.Res.. 17. 1785-1794 (2002)

Higuchi, C. 等人：“持续抑制 MAPK 信号传导可促进早期成骨细胞分化和细胞外基质矿化。”J.Bone Miner.Res.. 17. 1785-1794 (2002)

Nishimura, Y. et al.: "Evidence for a role in the regulation of intracellular vesicle trafficking of lysosomes and endosomes in human breast cancer cells."Eur.J.Cell Biol.. (in press). (2004)

Nishimura, Y. 等人：“人乳腺癌细胞中溶酶体和内体的细胞内囊泡运输调节作用的证据。”Eur.J.Cell Biol..（出版中）。