Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity
Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity
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DOI：
10.1074/jbc.m701116200
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发表时间：
2007-05-25
影响因子：
4.8
通讯作者：
McKee, Marc D.
中科院分区：
文献类型：
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作者：
Addison, William N.;Azari, Fereshteh;McKee, Marc D.
细胞产生的无机焦磷酸盐（PPi）通过与晶体结合来抑制矿化。它的普遍存在被认为可以防止“软”组织矿化，而它在骨骼和牙齿中通过组织非特异性碱性磷酸酶（Tnap，Tnsalp，Alp 1，Akp 2）降解为P-i可能有助于晶体生长。虽然PPi的晶体结合特性在很大程度上被理解，但对其对成骨细胞活性的影响知之甚少。我们使用MC 3 T3-E1成骨细胞培养来研究PPi对成骨细胞功能和基质矿化的影响。PPi剂量依赖性地抑制培养物中的矿化。PPi还可通过Erk 1/2和p38 MAPK信号通路诱导骨桥蛋白（OPN）水平升高。PPi的Opn调节对膦甲酸（磷酸盐摄取抑制剂）和左旋咪唑（Tnap酶活性抑制剂）也不敏感，这表明Opn水平的增加不是由于磷酸盐的变化。外源性OPN抑制矿化，但通过Tnap的去磷酸化逆转了这种作用，表明OPN通过其带负电荷的磷酸残基抑制矿化，并且与PPi一样，通过Tnap的水解降低了其矿物质抑制效力。使用酶动力学研究，我们已经表明，PPi抑制Tnap介导的β-甘油磷酸（一种常用的有机磷酸盐来源培养矿化研究）通过混合类型的抑制P-i释放。总之，PPi通过至少三种不同的机制防止MC 3 T3-E1成骨细胞培养物中的矿化，包括直接结合生长晶体、诱导Opn表达和抑制Tnap活性。
Inorganic pyrophosphate (PPi) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent "soft" tissues from mineralizing, whereas its degradation to P-i in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alp1, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PPi are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PPi on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PPi. This inhibition could be reversed by Tnap, but not if PPi was bound to mineral. PPi also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PPi was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PPi, hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PPi inhibits Tnap-mediated P-i release from beta-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PPi prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity.