Prosaposin knockdown in Caco-2 cells decreases cellular levels of coenzyme Q10 and ATP, and results in the loss of tight junction barriers.
Prosaposin knockdown in Caco-2 cells decreases cellular levels of coenzyme Q10 and ATP, and results in the loss of tight junction barriers.
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DOI：
10.3164/jcbn.16-32
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发表时间：
2017-03
影响因子：
2.4
通讯作者：
Yamamoto Y
中科院分区：
文献类型：
--
作者：
Kashiba M;Terashima M;Sagawa T;Yoshimura S;Yamamoto Y
辅酶Q10 （CoQ10）是线粒体电子传递链的重要组成部分，也是最重要的抗氧化剂之一。我们之前发现糖蛋白prosaposin （Psap）在人类细胞中结合CoQ10。虽然Psap在肠道中表达，但其在胃肠道中的作用尚不清楚。为了阐明Psap在肠道中的作用，我们建立了Psap敲低（KD） Caco-2细胞，这是一种肠上皮细胞系。与亲代Caco-2细胞相比，Psap KD Caco-2细胞的细胞CoQ10水平显著降低。与亲代Caco-2细胞相比，Psap KD Caco-2细胞的细胞ATP水平也显著降低。据报道，肠道中较低的ATP水平会导致紧密连接形成的失败。正如预期的那样，Psap KD Caco-2单层膜不产生经上皮电阻，而亲本Caco-2单层膜产生经上皮电阻。此外，荧光染料路西弗黄通过Psap KD Caco-2单层渗漏，而不通过亲本Caco-2单层渗漏。这些结果表明Psap对于维持细胞中辅酶q10和ATP的水平至关重要，因此在胃肠道中形成紧密连接。
Coenzyme Q10 (CoQ10) is a key component of the mitochondrial electron transfer chain and is one of the most important antioxidants. We previously found that glycoprotein prosaposin (Psap) binds CoQ10 in human cells. Although Psap is expressed in the intestines, its role in the gastrointestinal tract is not clear. To elucidate the role of Psap in the intestines, we established Psap knockdown (KD) Caco-2 cells, which are an intestinal epithelial cell line. Cellular CoQ10 levels decreased significantly in Psap KD Caco-2 cells as compared to parental Caco-2 cells. Cellular ATP levels also decreased significantly in Psap KD Caco-2 cells as compared to parental Caco-2 cells. Lower ATP levels in the intestines have been reported to result in the failure of tight junction formation. As expected, Psap KD Caco-2 monolayers did not produce transepithelial electrical resistance, while parental Caco-2 monolayers did. Moreover, a fluorescent dye, lucifer yellow, leaked out through Psap KD Caco-2 monolayers, whereas it did not through parental Caco-2 monolayers. These results indicate that Psap is essential to maintain cellular levels of CoQ10 and ATP, and consequently to form tight junctions in the gastrointestinal tract.