Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan; Department of Glocal Healthcare Science, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
J Biol Chem. 2021 Jan-Jun;296:100601. doi: 10.1016/j.jbc.2021.100601. Epub 2021 Mar 26.
The expression and function of some xenobiotic transporters vary according to the time of the day, causing the dosing time-dependent changes in drug disposition and toxicity. P-glycoprotein (P-gp), encoded by the ABCB1 gene, is highly expressed in the kidneys and functions in the renal elimination of various drugs. The elimination of several P-gp substrates was demonstrated to vary depending on administration time, but the underlying mechanism remains unclear. We found that adenosine deaminase acting on RNA (ADAR1) was involved in the circadian regulation of P-gp expression in human renal proximal tubular epithelial cells (RPTECs). After synchronization of the cellular circadian clock by dexamethasone treatment, the expression of P-gp exhibited a significant 24-h oscillation in RPTECs, but this oscillation was disrupted by the downregulation of ADAR1. Although ADAR1 catalyzes adenosine-to-inosine (A-to-I) RNA editing in double-stranded RNA substrates, no significant ADAR1-regulated editing sites were detected in the human ABCB1 transcripts in RPTECs. On the other hand, downregulation of ADAR1 induced alternative splicing in intron 27 of the human ABCB1 gene, resulting in the production of retained intron transcripts. The aberrant spliced transcript was sensitive to nonsense-mediated mRNA decay, leading to the decreased stability of ABCB1 mRNA and prevention of the 24-h oscillation of P-gp expression. These findings support the notion that ADAR1-mediated regulation of alternative splicing of the ABCB1 gene is a key mechanism of circadian expression of P-gp in RPTECs, and the regulatory mechanism may underlie the dosing time-dependent variations in the renal elimination of P-gp substrates.
一些外源性转运蛋白的表达和功能随时间变化而变化，导致药物处置和毒性的剂量依赖性变化。由 ABCB1 基因编码的 P-糖蛋白（P-gp）在肾脏中高度表达，在各种药物的肾脏消除中起作用。已经证明几种 P-gp 底物的消除取决于给药时间，但潜在机制尚不清楚。我们发现，作用于 RNA 的腺苷脱氨酶（ADAR1）参与了人肾近端小管上皮细胞（RPTEC）中 P-gp 表达的昼夜节律调节。用地塞米松处理同步细胞生物钟后，RPTEC 中 P-gp 的表达表现出明显的 24 小时振荡，但 ADAR1 的下调破坏了这种振荡。虽然 ADAR1 在双链 RNA 底物中催化腺苷到肌苷（A-to-I）的 RNA 编辑，但在 RPTEC 中未检测到人类 ABCB1 转录本中明显受 ADAR1 调控的编辑位点。另一方面，ADAR1 的下调诱导了人类 ABCB1 基因 27 号内含子的选择性剪接，导致产生保留内含子的转录本。异常剪接的转录本对无意义介导的 mRNA 降解敏感，导致 ABCB1 mRNA 的稳定性降低，并防止 P-gp 表达的 24 小时振荡。这些发现支持 ADAR1 介导的 ABCB1 基因选择性剪接调节是 RPTEC 中 P-gp 昼夜表达的关键机制的观点，并且调节机制可能是 P-gp 底物肾脏消除中剂量依赖性变化的基础。
