Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture
Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture
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DOI：
10.1074/jbc.m115.698530
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发表时间：
2016-06-17
影响因子：
4.8
通讯作者：
Bhat, Suraj P.
中科院分区：
文献类型：
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作者：
Gangalum, Rajendra K.;Bhat, Ankur M.;Bhat, Suraj P.
外泌体携带细胞类型特异性分子货物到细胞外目的地，因此充当细胞间通信和遗传信息从一个细胞转移到另一个细胞的横向载体。我们之前已经证明，小热休克蛋白α B-结晶蛋白（α B）从包装在外泌体中的成人视网膜色素上皮细胞（ARPE19）中输出。在这里，我们证明通过shRNA抑制α B的表达可以抑制ARPE19细胞的外泌体分泌，这表明外泌体货物可能在外泌体生物发生（合成和/或分泌）中发挥作用。培养基和细胞提取物的蔗糖密度梯度分离表明外泌体继续合成，但外泌体分泌受到抑制。在α B表达被抑制的细胞中，外泌体标志物CD63 （LAMP3）的分布明显改变，从正常的分散模式转变为核周围的堆叠模式。有趣的是，天然细胞和α b抑制细胞的总抗cd63 （LAMP3）免疫荧光保持不变，这表明在外泌体分泌受损的情况下，外泌体的合成仍在继续。重要的是，抑制α B的表达导致RPE细胞的表型，其中包含液泡数量增加和增大（融合）的囊泡，显示CD63（LAMP3）和LAMP1的存在增加，表明内溶酶体腔室增强。Rab7标记的增加进一步证实了这一点（已知Rab7 - gtpase 7与内核体成熟晚有关）。这些数据共同指出α B在外泌体生物发生中的调节作用，可能是通过其参与促进外泌体分泌的内吞途径的分支点。
Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein alpha B-crystallin (alpha B) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of alpha B via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where alpha B expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and alpha B-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of alpha B results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (Rab-GTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for alpha B in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes.