Miyamoto Ken-ichi, Suzuki Hirokazu, Yamamoto Shinya, Saitoh Yukie, Ochiai Eiji, Moritani Shuzo, Yokogawa Koichi, Waki Yoshihiro, Kasugai Shohei, Sawanishi Hiroyuki, Yamagami Hideomi
Department of Hospital Pharmacy, School of Medicine, Kanazawa University, Kanazawa, Japan.
J Bone Miner Res. 2003 Aug;18(8):1471-7. doi: 10.1359/jbmr.2003.18.8.1471.
The mechanism of osteoblast formation by a novel PDE4 inhibitor, XT-611, was studied in the in vitro bone marrow culture system. The compound potentiated the osteoblast differentiation through accumulation of cyclic AMP after autocrine stimulation of EP4 receptor by PGE2 in pro-osteoblastic cells.
We previously reported that inhibitors of phosphodiesterase (PDE)4 isoenzyme increase osteoblast formation in an in vitro bone marrow culture system and inhibit bone loss in animal osteoporosis models. Here we investigated the mechanism of the effect of a novel PDE4 inhibitor, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (XT-611), on osteoblast formation in the in vitro bone marrow culture system.
Rodent bone marrow cells were cultured in the presence of 0.2 mM ascorbic acid phosphate ester, 1 mM beta-glycerophosphate, and 10 nM dexamethasone for 10 days. Drug treatments were done for 24 h on day 3 of culture.
PDE4 inhibitors, including XT-611, but not PDE3 and PDE5 inhibitors, increased mineralized nodule formation in rat and mouse bone marrow cell cultures. During culture of the bone marrow cells, prostaglandin E2 (PGE2) production increased with a peak on day 4, but the increase was completely inhibited by indomethacin, an unselective cyclo-oxygenase (COX) inhibitor. Spontaneous and XT-611-induced mineralized-nodule formation was also inhibited by indomethacin and COX-2 inhibitors, in a similar potential. Alkaline phosphatase-positive nodule formation in the absence or presence of XT-611 was inhibited by an antagonist of EP4 receptor, AH23848B, and synergistically potentiated by 11-deoxy-PGE1, but it was not influenced by other EP antagonists and agonists examined. The expression of PDE4 and EP4 mRNAs was observed in bone marrow cells. The effect of XT-611 was also confirmed to involve an increase of cyclic AMP and the cyclic AMP-dependent protein kinase pathway.
These results suggest that PGE2 stimulates differentiation of osteoblast progenitor cells through the EP4 receptor in an autocrine manner, and the PDE4 inhibitor potentiates the differentiation by inhibiting hydrolysis of cyclic AMP in the cells.
在体外骨髓培养系统中研究了新型磷酸二酯酶4（PDE4）抑制剂XT-611促进成骨细胞形成的机制。该化合物通过前列腺素E2（PGE2）在成骨前体细胞中自分泌刺激EP4受体后使环磷酸腺苷（cAMP）蓄积，从而增强成骨细胞分化。
我们之前报道过，磷酸二酯酶（PDE）4同工酶抑制剂在体外骨髓培养系统中可增加成骨细胞形成，并在动物骨质疏松模型中抑制骨质流失。在此，我们研究了新型PDE4抑制剂3,4-二丙基-4,5,7,8-四氢-3H-咪唑并[1,2-i]嘌呤-5-酮（XT-611）在体外骨髓培养系统中对成骨细胞形成的作用机制。
将啮齿动物骨髓细胞在含有0.2 mM抗坏血酸磷酸酯、1 mMβ-甘油磷酸酯和10 nM地塞米松的条件下培养10天。在培养第3天进行24小时的药物处理。
包括XT-611在内的PDE4抑制剂可增加大鼠和小鼠骨髓细胞培养物中矿化结节的形成，而PDE3和PDE5抑制剂则无此作用。在骨髓细胞培养过程中，前列腺素E2（PGE2）的产生在第4天达到峰值，但非选择性环氧化酶（COX）抑制剂吲哚美辛可完全抑制其增加。吲哚美辛和COX-2抑制剂以相似的效力抑制自发的以及XT-611诱导的矿化结节形成。EP4受体拮抗剂AH23848B可抑制在有无XT-611情况下碱性磷酸酶阳性结节的形成，而11-脱氧-PGE1可协同增强其形成，但不受其他检测的EP拮抗剂和激动剂的影响。在骨髓细胞中观察到了PDE4和EP4 mRNA的表达。XT-611的作用还被证实涉及cAMP增加以及cAMP依赖性蛋白激酶途径。
这些结果表明，PGE2以自分泌方式通过EP4受体刺激成骨祖细胞的分化，而PDE4抑制剂通过抑制细胞内cAMP的水解来增强这种分化。
