Morgan Larry, Antenos Monica, Kirby Gordon M
Department of Biomedical Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Department of Biomedical Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Toxicology. 2022 Apr 15;471:153162. doi: 10.1016/j.tox.2022.153162. Epub 2022 Mar 25.
Cytochrome P450 2a5 (Cyp2a5) is distinct from other P450 enzymes in that it is induced in the endoplasmic reticulum (ER) of mouse hepatocytes in conditions that are injurious to the liver. These conditions cause ER stress eventually resulting in apoptosis if not rectified. We previously showed that mouse hepatic Cyp2a5 is induced during reductive ER stress caused by the intramolecular disulfide form of dithiothreitol, trans-4,5-dihydroxy-1,2-dithiane (DTT), and that overexpression of Cyp2a5 provides partial protection against apoptosis due to bilirubin (BR), a compound known to cause ER stress. The purpose of this study was to investigate the mechanism of Cyp2a5 gene regulation by DTTox and to determine if Cyp2a5 plays a cytoprotective role during reductive ER stress. Exposure to DTT (10 mM) and another reductive ER stressor, 2-mercaptoethanol (1 mM), for 48 h markedly increased Cyp2a5 protein levels in primary mouse hepatocytes. In addition, DTT transactivated Cyp2a5 via a mechanism involving the transcription factor nuclear factor-(erythroid-derived 2)-like 2 (Nrf2). Expression of the BR-conjugating enzyme, UDP glucuronosyl transferase 1A1 (UGT1A1) was also increased after DTT treatment, however, this was reduced by Cyp2a5 overexpression. Hemin, a porphyrin inducer of Cyp2a5, induced mRNA splicing of X-box binding protein 1 (XBP-1), a transcription factor involved in the ER stress response, however, this was also reduced by Cyp2a5 overexpression. Finally, overexpression of Cyp2a5 partially blocked DTT-mediated caspase-3 cleavage in Hepa 1-6 cells suggesting a cytoprotective role during ER stress. These findings demonstrate that Nrf2-mediated induction of Cyp2a5 in a reducing ER environment provides partial protection against ER stress-induced apoptosis by decreasing XBP-1 mRNA splicing and caspase-3 cleavage.
细胞色素P450 2a5（Cyp2a5）与其他P450酶不同，因为它在对肝脏有损伤的条件下，在小鼠肝细胞的内质网（ER）中被诱导。这些条件会导致内质网应激，如果不纠正最终会导致细胞凋亡。我们之前表明，小鼠肝脏Cyp2a5在由二硫苏糖醇的分子内二硫键形式反式-4,5-二羟基-1,2-二硫烷（DTT）引起的还原性内质网应激期间被诱导，并且Cyp2a5的过表达提供了对胆红素（BR）诱导的细胞凋亡的部分保护，胆红素是一种已知会引起内质网应激的化合物。本研究的目的是研究DTTox对Cyp2a5基因调控的机制，并确定Cyp2a5在还原性内质网应激期间是否发挥细胞保护作用。将原代小鼠肝细胞暴露于DTT（10 mM）和另一种还原性内质网应激源2-巯基乙醇（1 mM）48小时，可显著增加Cyp2a5蛋白水平。此外，DTT通过涉及转录因子核因子（红系衍生2）样2（Nrf2）的机制反式激活Cyp2a5。DTT处理后，BR结合酶尿苷二磷酸葡萄糖醛酸基转移酶1A1（UGT1A1）的表达也增加，然而，这被Cyp2a5过表达所降低。血红素是Cyp2a5的卟啉诱导剂，可诱导参与内质网应激反应的转录因子X盒结合蛋白1（XBP-1）的mRNA剪接，然而，这也被Cyp2a5过表达所降低。最后，Cyp2a5的过表达部分阻断了Hepa 1-6细胞中DTT介导的半胱天冬酶-3切割，表明在内质网应激期间发挥细胞保护作用。这些发现表明，在还原性内质网环境中，Nrf2介导的Cyp2a5诱导通过减少XBP-1 mRNA剪接和半胱天冬酶-3切割，对内质网应激诱导的细胞凋亡提供部分保护。
