Kirii Mami, Yoshida Yui, Takashima Shigeo, Uchio-Yamada Kozue, Oh-Hashi Kentaro
Graduate School of Natural Science and Technology, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
Mol Biol Rep. 2025 Jun 3;52(1):540. doi: 10.1007/s11033-025-10591-3.
Since the endoplasmic reticulum (ER) stress response is found in almost all tissues, its regulation and downstream factors have been vigorously explored.
In this study, we performed a comprehensive microarray analysis of genes induced by tunicamycin (Tm) stimulation in the rat cardiac fibroblast cell line H9c2. Tm treatment induced a marked increase in solute carrier family 6 member 9, Slc6a9, mRNA as well as typical ER stress-inducible factors GADD153 and GRP78 mRNA. Slc6a9 mRNA in H9c2 cells was also increased by ER stress inducers thapsigargin (Tg) and brefeldin A (BFA) other than Tm. The effects of inhibitors on the ER-localized stress sensors IRE1, ATF6, and PERK were examined, and the PERK inhibitor GSK2606414 significantly suppressed Tm-induced Slc6a9 mRNA expression. Analysis of Slc6a9 mRNA expression in PERK and ATF4-deficient HEK293 cells established by genome editing showed that Slc6a9 mRNA expression by Tm was suppressed in these cells. Furthermore, the long variant of Slc6a9 mRNA was increased in Tm-treated HEK293, while the short variant was negligible. Analysis of the nucleotide sequence of the 5'-flanking region of the Slc6a9 gene revealed an ATF4-binding sequence in exon1 is highly conserved among many species. As expected, our luciferase reporter assay containing the ATF4 binding sequence near the transcription start site showed a marked increase in promoter activity upon Tm stimulation and ATF4 co-expression.
The ATF4-binding sequence found near the transcription start site of the Slc6a9 gene is highly conserved among species and the element is functional in the PERK-ATF4 system. This study will help elucidate Slc6a9-mediated regulation of intracellular and extracellular amino acid homeostasis under pathophysiological conditions.
由于内质网（ER）应激反应几乎在所有组织中都存在，因此对其调控及下游因子进行了深入研究。
在本研究中，我们对衣霉素（Tm）刺激大鼠心脏成纤维细胞系H9c2后诱导的基因进行了全面的微阵列分析。Tm处理导致溶质载体家族6成员9（Slc6a9）的mRNA以及典型的内质网应激诱导因子GADD153和GRP78的mRNA显著增加。除Tm外，内质网应激诱导剂毒胡萝卜素（Tg）和布雷菲德菌素A（BFA）也使H9c2细胞中的Slc6a9 mRNA增加。研究了抑制剂对内质网定位的应激传感器IRE1、ATF6和PERK的影响，PERK抑制剂GSK2606414显著抑制了Tm诱导的Slc6a9 mRNA表达。对通过基因组编辑建立的PERK和ATF4缺陷型HEK293细胞中Slc6a9 mRNA表达的分析表明，这些细胞中Tm诱导的Slc6a9 mRNA表达受到抑制。此外，在Tm处理的HEK293细胞中，Slc6a9 mRNA的长变体增加，而短变体可忽略不计。对Slc6a9基因5'侧翼区域核苷酸序列的分析显示，外显子1中的ATF4结合序列在许多物种中高度保守。正如预期的那样，我们的荧光素酶报告基因检测在转录起始位点附近包含ATF4结合序列，结果显示在Tm刺激和ATF4共表达时启动子活性显著增加。
在Slc6a9基因转录起始位点附近发现的ATF4结合序列在物种间高度保守，且该元件在PERK-ATF4系统中具有功能。本研究将有助于阐明病理生理条件下Slc6a9介导的细胞内和细胞外氨基酸稳态调控。
