Department of Medicine, Baylor College of Medicine , Houston, Texas 77030, United States.
J Proteome Res. 2017 Aug 4;16(8):2709-2728. doi: 10.1021/acs.jproteome.6b00981. Epub 2017 Jul 18.
Osteoblasts communicate both with normal cells in the bone marrow and with tumor cells that metastasized to bone. Here we show that osteoblasts release exosomes, we termed osteosomes, which may be a novel mechanism by which osteoblasts communicate with cells in their environment. We have isolated exosomes from undifferentiated/proliferating (D0 osteosomes) and differentiated/mineralizing (D24 osteosomes) primary mouse calvarial osteoblasts. The D0 and D24 osteosomes were found to be vesicles of 130-140 nm by dynamic light scattering analysis. Proteomics profiling using tandem mass spectrometry (LC-MS/MS) identified 206 proteins in D0 osteosomes and 336 in D24 osteosomes. The proteins in osteosomes are mainly derived from the cytoplasm (∼47%) and plasma membrane (∼31%). About 69% of proteins in osteosomes are also found in Vesiclepedia, and these canonical exosomal proteins include tetraspanins and Rab family proteins. We found that there are differences in both protein content and levels in exosomes isolated from undifferentiated and differentiated osteoblasts. Among the proteins that are unique to osteosomes, 169 proteins are present in both D0 and D24 osteosomes, 37 are unique to D0, and 167 are unique to D24. Among those 169 proteins present in both D0 and D24 osteosomes, 10 proteins are likely present at higher levels in D24 than D0 osteosomes based on emPAI ratios of >5. These results suggest that osteosomes released from different cellular state of osteoblasts may mediate distinct functions. Using live-cell imaging, we measured the uptake of PKH26-labeled osteosomes into C4-2B4 and PC3-mm2 prostate cancer cells. In addition, we showed that cadherin-11, a cell adhesion molecule, plays a role in the uptake of osteosomes into PC3-mm2 cells as osteosome uptake was delayed by neutralizing antibody against cadherin-11. Together, our studies suggest that osteosomes could have a unique role in the bone microenvironment under both physiological and pathological conditions.
成骨细胞不仅与骨髓中的正常细胞相互交流，还与转移到骨骼中的肿瘤细胞相互交流。在这里，我们表明成骨细胞释放外泌体，我们称之为骨外泌体，这可能是成骨细胞与周围环境中细胞进行交流的一种新机制。我们已经从未分化/增殖（D0 骨外泌体）和分化/矿化（D24 骨外泌体）的原代小鼠颅骨成骨细胞中分离出外泌体。动态光散射分析发现，D0 和 D24 骨外泌体为 130-140nm 的囊泡。使用串联质谱（LC-MS/MS）的蛋白质组学分析鉴定了 D0 骨外泌体中的 206 种蛋白质和 D24 骨外泌体中的 336 种蛋白质。骨外泌体中的蛋白质主要来源于细胞质（约 47%）和质膜（约 31%）。骨外泌体中的约 69%的蛋白质也存在于 Vesiclepedia 中，这些经典的外泌体蛋白包括四跨膜蛋白和 Rab 家族蛋白。我们发现，从未分化和分化的成骨细胞中分离出的外泌体在蛋白质含量和水平上存在差异。在仅存在于骨外泌体中的蛋白质中，169 种蛋白质在 D0 和 D24 骨外泌体中都存在，37 种蛋白质仅存在于 D0 中，167 种蛋白质仅存在于 D24 中。在 D0 和 D24 骨外泌体中都存在的 169 种蛋白质中，有 10 种蛋白质的 emPAI 比值>5，提示其在 D24 骨外泌体中的含量可能高于 D0 骨外泌体。这些结果表明，来自不同成骨细胞状态的骨外泌体可能介导不同的功能。通过活细胞成像，我们测量了 PKH26 标记的骨外泌体被 C4-2B4 和 PC3-mm2 前列腺癌细胞摄取的情况。此外，我们还表明，细胞黏附分子钙黏蛋白 11 在外泌体被 PC3-mm2 细胞摄取中起作用，因为中和钙黏蛋白 11 的抗体可延迟骨外泌体的摄取。总之，我们的研究表明，在生理和病理条件下，骨外泌体在骨微环境中可能具有独特的作用。
