Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro.
Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro.
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DOI：
10.1016/j.matbio.2016.02.003
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发表时间：
2016-05
期刊：
影响因子：
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通讯作者：
Napierala D
中科院分区：
文献类型：
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作者：
Chaudhary SC;Kuzynski M;Bottini M;Beniash E;Dokland T;Mobley CG;Yadav MC;Poliard A;Kellermann O;Millán JL;Napierala D
矿化是磷酸钙晶体在纤维状细胞外基质(ECM)中沉积的过程。在矿化组织中，如牙本质、骨和肥大的软骨，这一过程是由一种特殊的细胞外小泡(EV)启动的，称为基质小泡(MV)。虽然已经提出MV是由质膜脱落形成的，但调控具有矿化活性的MV形成的细胞和分子机制还没有完全阐明。在这些研究中，17IIA11、ST2和MC3T3-E1成骨细胞系被用来确定MV的形成在矿化过程的启动过程中是如何调节的。此外，还比较了血液和B16-F10黑色素瘤细胞系的17IIA11细胞和外切体分泌的MV的分子组成，以确定MV区别于其他EV的分子特征。Western印迹分析表明，17IIA11细胞释放的MV具有高水平的钙磷调节蛋白，但不表达外体标记CD81和HSP70。此外，我们发现17IIA11细胞释放的MV的分子组成在暴露于经典的成骨分化诱导剂，即抗坏血酸和磷酸盐时发生变化。溶酶体蛋白LAMP1和LAMP2A仅在成骨因子刺激的细胞分泌的MV中检测到。对成骨细胞分泌MV的纳米颗粒定量跟踪分析表明，标准的成骨因子刺激MV的分泌，而磷酸盐是其分泌的主要驱动力。在分子水平上，磷酸盐诱导的MV分泌是通过激活细胞外信号调节蛋白ERK1/2来实现的，并伴随着丝状肌动蛋白的重组。综上所述，我们确定了具有矿化能力的MV不同于外切体，并确定了磷酸盐在ECM矿化过程中的新作用。这些数据为矿化过程启动过程中MV的形成机制提供了新的见解。
Mineralization is a process of deposition of calcium phosphate crystals within a fibrous extracellular matrix (ECM). In mineralizing tissues, such as dentin, bone and hypertrophic cartilage, this process is initiated by a specific population of extracellular vesicles (EV), called matrix vesicles (MV). Although it has been proposed that MV are formed by shedding of the plasma membrane, the cellular and molecular mechanisms regulating formation of mineralization-competent MV are not fully elucidated. In these studies, 17IIA11, ST2, and MC3T3-E1 osteogenic cell lines were used to determine how formation of MV is regulated during initiation of the mineralization process. In addition, the molecular composition of MV secreted by 17IIA11 cells and exosomes from blood and B16-F10 melanoma cell line was compared to identify the molecular characteristics distinguishing MV from other EV. Western blot analyses demonstrated that MV released from 17IIA11 cells are characterized by high levels of proteins engaged in calcium and phosphate regulation, but do not express the exosomal markers CD81 and HSP70. Furthermore, we uncovered that the molecular composition of MV released by 17IIA11 cells changes upon exposure to the classical inducers of osteogenic differentiation, namely ascorbic acid and phosphate. Specifically, lysosomal proteins Lamp1 and Lamp2a were only detected in MV secreted by cells stimulated with osteogenic factors. Quantitative nanoparticle tracking analyses of MV secreted by osteogenic cells determined that standard osteogenic factors stimulate MV secretion and that phosphate is the main driver of their secretion. On the molecular level, phosphate-induced MV secretion is mediated through activation of extracellular signal-regulated kinases Erk1/2 and is accompanied by re-organization of filamentous actin. In summary, we determined that mineralization-competent MV are distinct from exosomes, and we identified a new role of phosphate in the process of ECM mineralization. These data provide novel insights into the mechanisms of MV formation during initiation of the mineralization process.