Liou Shu-Fen, Hsu Jong-Hau, Chu Hsin-Chieh, Lin Hung-Hong, Chen Ing-Jun, Yeh Jwu-Lai
Department of Pharmacy, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan.
Department of Paediatrics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
J Cell Physiol. 2015 Sep;230(9):2038-48. doi: 10.1002/jcp.24904.
Phosphodiesterase (PDE) inhibitors have been suggested as a possible candidate for the treatment of osteopenia, including osteoporosis. KMUP-1 is a novel xanthine derivative with inhibitory activities on the PDE 3, 4, and 5 iso-enzymes to suppress the degradation of cAMP and cGMP. This study aimed to investigate the effect of KMUP-1 on osteoblast differentiation and the underlying cellular and molecular mechanisms. Primary osteoblasts and osteoblastic MC3T3-E1 cells were examined. KMUP-1 enhanced alkaline phosphatase (ALP) activity and mineralization compared to untreated controls in primary osteoblasts and MC3T3-E1 cells. KMUP-1 also increased the mRNA expression of the osteoblastic differentiation markers, including collagen type 1a, ALP, osteocalcin, osteoprotegerin, BMP-2, and Runx2, a key transcription regulator for osteoblastic differentiation. The osteogenic effect of KMUP-1 was abolished by BMP signaling inhibitor, noggin. Furthermore, we found that KMUP-1 upregulated Smad1/5/8 phosphorylations with subsequent BRE-Luc activation confirmed by transient transfection assay. In addition, KMUP-1 inactivated glycogen synthase kinase-3β (GSK-3β), with associated nuclear translocation of β-catenin. Co-treatment with H89 and KT5823, cAMP and cGMP pathway inhibitors, respectively, reversed the KMUP-1-induced activations of Smad1/5/8, β-catenin, and Runx2. The findings demonstrate for the first time that KMUP-1 can promote osteoblast maturation and differentiation in vitro via BMP-2/Smad1/5/8 and Wnt/β-catenin pathways. These effects are mediated, in part, by the cAMP and cGMP signaling. Thus, KMUP-1 may be a novel osteoblast activator and a potential new therapy for osteoporosis.
磷酸二酯酶（PDE）抑制剂已被认为是治疗骨质减少（包括骨质疏松症）的一种可能候选药物。KMUP-1是一种新型黄嘌呤衍生物，对PDE 3、4和5同工酶具有抑制活性，可抑制cAMP和cGMP的降解。本研究旨在探讨KMUP-1对成骨细胞分化的影响及其潜在的细胞和分子机制。对原代成骨细胞和成骨样MC3T3-E1细胞进行了检测。与未处理的原代成骨细胞和MC3T3-E1细胞对照相比，KMUP-1增强了碱性磷酸酶（ALP）活性和矿化作用。KMUP-1还增加了成骨细胞分化标志物的mRNA表达，包括1a型胶原蛋白、ALP、骨钙素、骨保护素、骨形态发生蛋白-2（BMP-2）和Runx2，Runx2是成骨细胞分化的关键转录调节因子。BMP信号抑制剂头蛋白可消除KMUP-1的成骨作用。此外，我们发现KMUP-1上调了Smad1/5/8的磷酸化，随后通过瞬时转染试验证实了BRE-Luc的激活。此外，KMUP-1使糖原合酶激酶-3β（GSK-3β）失活，并伴有β-连环蛋白的核转位。分别与cAMP和cGMP途径抑制剂H89和KT5823共同处理，可逆转KMUP-1诱导的Smad1/5/8、β-连环蛋白和Runx2的激活。这些发现首次证明，KMUP-1可通过BMP-2/Smad1/5/8和Wnt/β-连环蛋白途径在体外促进成骨细胞成熟和分化。这些作用部分由cAMP和cGMP信号介导。因此，KMUP-1可能是一种新型的成骨细胞激活剂和治疗骨质疏松症的潜在新疗法。
