Investigation of the effects of urea cycle amino acids on the expression of ALB and CEBPB in the human hepatocellular carcinoma cell line FLC-4
Investigation of the effects of urea cycle amino acids on the expression of ALB and CEBPB in the human hepatocellular carcinoma cell line FLC-4
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DOI：
10.1007/s13577-020-00383-1
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发表时间：
2020-05
期刊：
影响因子：
4.3
通讯作者：
T. J. Fujimi;Y. Mezaki;T. Masaki;Ayasa Tajima;Mariko Nakamura;A. Yoshikawa;N. Murai;M. Aizawa
中科院分区：
文献类型：
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作者：
T. J. Fujimi;Y. Mezaki;T. Masaki;Ayasa Tajima;Mariko Nakamura;A. Yoshikawa;N. Murai;M. Aizawa
细胞系是在分子水平上研究肝功能的有力工具。然而，它们通常不适合严格评估氨基酸组成的影响，因为许多品系需要含血清的培养基来维持。在这里，我们的目的是研究鸟氨酸和精氨酸，这是包括在肝细胞的特征代谢过程中，对人肝癌细胞系（FLC-4），可以在无血清培养基中培养的影响。在以下三种条件下培养FLC-4细胞：+鸟氨酸/ -精氨酸、-鸟氨酸/ -精氨酸和-鸟氨酸/ +精氨酸。通过定量聚合酶链反应和酶联免疫吸附试验评价白蛋白表达，并显示基于鸟氨酸或精氨酸的存在无明显差异。然而，两个肝脏富集的转录因子（CEBPB和HNF 1A），这是参与调节白蛋白的表达，在含精氨酸的培养基中生长的细胞中的mRNA水平显着高于在含鸟氨酸的培养基中生长的细胞。Western blotting结果显示，在精氨酸培养基中培养的细胞中，激活型和抑制型C/EBPβ亚型的水平均显著增加。此外，我们还发现，培养基中多胺来源的鸟氨酸和精氨酸的耗尽不会导致多胺缺乏。当鸟氨酸和精氨酸耗尽时，白蛋白的产生在mRNA水平上显著降低，CEBPB mRNA水平升高，C/EBPβ活化形式的水平升高。本研究的结果表明，在肝细胞中，这两种氨基酸可能具有不同的功能，因此它们引起不同的细胞反应。
Cell lines are powerful tools for research into liver function at the molecular level. However, they are generally unsuitable for rigorously assessing the effects of amino acid composition, because many lines require serum-containing medium for their maintenance. Here, we aimed to investigate the effects of ornithine and arginine, which are included in the characteristic metabolic process in hepatocyte, on a human hepatoma-derived cell line (FLC-4) that can be cultured in serum-free medium. FLC-4 cells were cultured under the following three conditions: + ornithine/ – arginine, – ornithine/ – arginine, and –ornithine/ + arginine. Albumin expression evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay and showed no obvious differences based on the presence of ornithine or arginine. However, the mRNA levels of two liver-enriched transcription factors (CEBPBandHNF1A), which are involved in regulating albumin expression, were significantly higher in cells grown in medium-containing arginine than that in cells grown in ornithine-containing medium. Western blotting showed that the levels both activating and inhibitory C/EBPβ isoforms were significantly increased in cells grown in arginine medium. Furthermore, we have found that depletion of both ornithine and arginine, the polyamine sources, in the medium did not cause polyamine deficiency. When ornithine and arginine were depleted, albumin production was significantly reduced at the mRNA level,CEBPBmRNA levels were increased, and the level of activating form of C/EBPβ was increased. The results of this study suggest that in hepatocyte, these two amino acids might have different functions, and because of which they elicit disparate cellular responses.