Prostaglandin E2-mediated anabolic effect of a novel inhibitor of phosphodiesterase 4, XT-611, in the in vitro bone marrow culture.
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.


BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Miyamoto
Ken-ichi
K
Department of Hospital Pharmacy, School of Medicine, Kanazawa University, Kanazawa, Japan. miyaken@pharmacy.m.kanazawa-u.ac.jp
Suzuki
Hirokazu
H
Yamamoto
Shinya
S
Saitoh
Yukie
Y
Ochiai
Eiji
E
Moritani
Shuzo
S
Yokogawa
Koichi
K
Waki
Yoshihiro
Y
Kasugai
Shohei
S
Sawanishi
Hiroyuki
H
Yamagami
Hideomi
H
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D045930:Anabolic Agents / Q000494:pharmacology*; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D015232:Dinoprostone / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D011687:Purines / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar
C461440:3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo(1,2-i)purin-5-one; D045930:Anabolic Agents; D010726:Phosphodiesterase Inhibitors; D011687:Purines; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D015232:Dinoprostone