Effects of phosphodiesterase 7 inhibition by RNA interference on the gene expression and differentiation of human mesenchymal stem cell-derived osteoblasts
Effects of phosphodiesterase 7 inhibition by RNA interference on the gene expression and differentiation of human mesenchymal stem cell-derived osteoblasts
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DOI：
10.1016/j.bone.2008.02.021
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发表时间：
2008-07-01
期刊：
影响因子：
4.1
通讯作者：
Lamberg-Allardt, Christel J. E.
中科院分区：
文献类型：
--
作者：
Pekkinen, Minna;Ahlstrom, Mikael E. B.;Lamberg-Allardt, Christel J. E.
第二信使分子环磷酸腺苷（CAMP）在骨代谢的激素调节中发挥着重要作用。cAMP被环核苷酸磷酸二酯酶（PDE）灭活，PDE是一个酶超家族，分为11个已知家族，命名为PDE 1-11。本研究的目的是研究PDE 7和PDEB抑制对人成骨细胞基因表达和分化的影响。培养从人类间充质干细胞（hMSC）分化的成骨细胞，并用POP和PDE 8 PCR产物产生的短干扰RNA（SiRNA）处理。从细胞中提取总RNA，用cDNA微阵列和定量实时PCR检测基因表达。在分化过程中测定bALP测量值，并通过定量茜素红S染色测定矿化。通过RNA干扰抑制PDE 7和PDE 8使PDE 7A的基因表达降低60- 70%，PDE 7 B降低40- 50%，PDE 8A降低30%。PDE 7沉默增加了β-连环蛋白、骨钙素、半胱天冬酶-8和cAMP反应元件结合蛋白5（CREB-5）基因的表达，并降低了1，25-二羟维生素D3受体基因的表达。PDE 8A沉默增加了抗凋亡基因的表达，但降低了骨甘氨酸（骨诱导因子）和骨形态发生蛋白1（BMP-1）的表达。与对照相比，PDE 7沉默使bALP和矿化增加多达三倍。用PDE 7选择性PDE抑制剂BRL-50481处理对矿化具有与基因沉默相似的作用。PDE 7沉默也增加了毛喉素刺激的cAMP反应，但对增殖率没有影响。此外，PDE 7沉默通过依赖于蛋白激酶A的机制增加骨钙素表达。我们的研究结果表明，特定的基因沉默与RNAi方法是一个有用的工具，抑制特定的PDE的基因表达和PDE 7沉默上调几个成骨基因，增加矿化。PDE 7可能在成骨细胞分化的调节中发挥重要作用。(C)2008年爱思唯尔公司All rights reserved.
The second messenger molecule cyclic adenosine monophosphate (CAMP) plays an important role in the hormonal regulation of bone metabolism. cAMP is inactivated by the cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 known families designated PDE 1-11. The aim of this study was to investigate the effect of PDE7 and PDEB inhibition on the gene expression and differentiation of human osteoblasts. Osteoblasts differentiated from human mesenchymal stem cells (hMSC) were cultured and treated with short interfering RNAs (siRNAs) generated from POP and PDE8 PCR products. Total RNA was isolated from the cells, and gene expression was assayed with cDNA microarray and quantitative real-time PCR. bALP measurements were assayed during differentiation, and mineralization was determined by quantitative Alizarin red S staining. PDE7 and PDE8 inhibition by RNA interference decreased the gene expression of PDE7A by 60-70%, PDE7B by 40-50%, and PDE8A by 30%. PDE7 silencing increased the expression of beta-catenin, osteocalcin, caspase-8, and cAMP responsive element-binding protein 5 (CREB-5) genes and decreased the expression of the 1, 25-dihydroxyvitamin D3 receptor gene. PDE8A silencing increased the expression of anti-apoptotic genes, but decreased the expression of osteoglycin (osteoinductive factor) and bone morphogenetic protein 1(BMP-1). PDE7 silencing increased bALP and mineralization up to three-fold compared to controls. Treatment with the PDE7-selective PDE inhibitor BRL-50481 had similar effects on mineralization as the gene silencing. The PDE7 silencing also increased forskolin stimulated cAMP response, but had no effect on the proliferation rate. Furthermore, osteocalcin expression was increased by PDE7 silencing by a mechanism dependent on protein kinase A. Our results show that specific gene silencing with the RNAi method is a useful tool for inhibiting the gene expression of specific PDEs and that PDE7 silencing upregulates several osteogenic genes and increases mineralization. PDE7 may play an important role in the regulation of osteoblastic differentiation. (C) 2008 Elsevier Inc. All rights reserved.