Chemical inhibition of APE1 redox and DNA repair functions affects the inflammatory response via different but overlapping mechanisms.. Chemical inhibition of APE1 redox and DNA repair functions affects the inflammatory response via different but overlapping mechanisms.

The presence of oxidized DNA lesions, such as 7,8-dihydro-8-oxoguanine (8-oxoG) and apurinic/apyrimidinic sites (AP sites), has been described as epigenetic signals that are involved in gene expression control. In mammals, Apurinic-apyrimidinic endonuclease 1/Redox factor-1 (APE1/Ref-1) is the main AP endonuclease of the base excision repair (BER) pathway and is involved in active demethylation processes. In addition, APE1/Ref-1, through its redox function, regulates several transcriptional factors. However, the transcriptional control targets of each APE1 function are not completely known. In this study, a transcriptomic approach was used to investigate the effects of chemical inhibition of APE1/Ref-1 redox or DNA repair functions by E3330 or methoxyamine (MX) in an inflammatory cellular model. Under lipopolysaccharide (LPS) stimulation, both E3330 and MX reduced the expression of some cytokines and chemokines. Interestingly, E3330 treatment reduced cell viability after 48 h of the treatment. Genes related to inflammatory response and mitochondrial processes were downregulated in both treatments. In the E3330 treatment, RNA processing and ribosome biogenesis genes were downregulated, while they were upregulated in the MX treatment. Furthermore, in the E3330 treatment, the cellular stress response was the main upregulated process, while the cellular macromolecule metabolic process was observed in MX-upregulated genes. Nuclear respiratory factor 1 (NRF1) was predicted to be a master regulator of the downregulated genes in both treatments, while the ETS transcription factor ELK1 (ELK1) was predicted to be a master regulator only for E3330 treatment. Decreased expression of ELK1 and its target genes and a reduced 28S/18S ratio were observed, suggesting impaired rRNA processing. In addition, both redox and repair functions can affect the expression of NRF1 and GABPA target genes. The master regulators predicted for upregulated genes were YY1 and FLI1 for the E3330 and MX treatments, respectively. In summary, the chemical inhibition of APE1/Ref-1 affects gene expression regulated mainly by transcriptional factors of the ETS family, showing partial overlap of APE1 redox and DNA repair functions, suggesting that these activities are not entirely independent. This work provides a new perspective on the interaction between APE1 redox and DNA repair activity in inflammatory response modulation and transcription. Overall design: The cells were grouped as follows: LPS-stimulated (LPS), LPS + 100 µM E3330 (LPS+E3330), and LPS + 6 mM MX (LPS+MX). The LPS group was used as reference sample

氧化DNA损伤（如7,8-二氢-8-氧代鸟嘌呤（8-oxoG）和无嘌呤/无嘧啶位点（AP位点））的存在，已被描述为参与基因表达调控的表观遗传信号。在哺乳动物中，无嘌呤无嘧啶内切核酸酶1/氧化还原因子-1（Apurinic-apyrimidinic endonuclease 1/Redox factor-1，APE1/Ref-1）是碱基切除修复（base excision repair，BER）通路中的主要AP内切核酸酶，同时参与活性去甲基化过程。此外，APE1/Ref-1还可通过其氧化还原功能调控多种转录因子。然而，目前尚不明确APE1两种功能各自的转录调控靶点。本研究采用转录组学方法，探究了在炎症细胞模型中，E3330或甲氧胺（methoxyamine，MX）对APE1/Ref-1氧化还原功能或DNA修复功能的化学抑制效应。在脂多糖（lipopolysaccharide，LPS）刺激下，E3330与MX均可降低部分细胞因子及趋化因子的表达。值得注意的是，E3330处理48小时后可降低细胞活力。两种处理方式下，与炎症反应及线粒体过程相关的基因均出现下调。在E3330处理组中，RNA加工与核糖体生物发生相关基因表达下调，而在MX处理组中此类基因则出现上调。进一步而言，E3330处理组中上调的主要生物学过程为细胞应激反应，而MX处理组上调基因则富集于细胞大分子代谢过程。核呼吸因子1（nuclear respiratory factor 1，NRF1）被预测为两种处理组下调基因的核心调控因子，而ETS转录因子ELK1（ELK1）仅被预测为E3330处理组的核心调控因子。实验观察到ELK1及其靶基因的表达降低，且28S/18S核糖体RNA比值下降，提示rRNA加工过程受损。此外，APE1的氧化还原与修复功能均可影响NRF1及GABPA靶基因的表达。两种处理组上调基因的核心调控因子分别为YY1与FLI1，其中E3330处理组对应YY1，MX处理组对应FLI1。综上，对APE1/Ref-1的化学抑制会影响主要由ETS家族转录因子调控的基因表达，且APE1的氧化还原与DNA修复功能存在部分重叠，表明这两种活性并非完全独立。本研究为APE1氧化还原功能与DNA修复活性在炎症反应调控及转录过程中的相互作用提供了新视角。整体实验设计：细胞分为以下三组：脂多糖刺激组（LPS组）、LPS+100 µM E3330组（LPS+E3330组）以及LPS+6 mM 甲氧胺（MX）组（LPS+MX组），其中LPS组作为对照样本。