Zhao Yulan, Jiang Yidan, Feng Yue, Zhao Ruqian
MOE Joint International Research Laboratory of Animal Health and Food Safety, Nanjing Agricultural University, Nanjing, Jiangsu, PR China; Key Laboratory of Animal Physiology & Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, PR China.
MOE Joint International Research Laboratory of Animal Health and Food Safety, Nanjing Agricultural University, Nanjing, Jiangsu, PR China; Key Laboratory of Animal Physiology & Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, PR China.
Poult Sci. 2025 Jan;104(1):104393. doi: 10.1016/j.psj.2024.104393. Epub 2024 Oct 9.
Glucocorticoid receptors are distributed in various cells of the body and participate in the regulation of metabolism and immunity in response to glucocorticoids. RNA mA methylation participates in various metabolic and inflammatory responses, yet it remains elusive whether mA is involved in GR regulation during immune activation. Here, we observed uncoupled GR responses with increased mRNA yet suppressed protein levels in the LPS-challenged broilers chicken liver, in association with global elevation of RNA mA methylation, especially in the expression of METTL3 and YTHDF2. Further analysis using isolated primary hepatocytes and Kupffer cells revealed that such uncoupled GR responses and mA hypermethylation occurred specifically in KCs. The same GR and mA responses were reproduced in LPS-activated KC cell line, implying a possible role of mA in the post-transcriptional suppression of GR in KC. Indeed, mA inhibitor cycloleucine alleviated LPS-induced GR protein suppression, whilst GR antagonist RU486 had no effect on global mA methylation in KC. We observed that YTHDF2 siRNA can alleviate LPS-induced GR mRNA stability decrease. Subsequently, specific mA sites on GR were predicted and verified. Mutation mA sites and luciferase reporter assay was also applied to validate these findings. Mechanistically, mA methylation on the transcripts of GR impairs its mRNA stability in a YTHDF2-dependent manner, which leads to the decrease of its protein. Our study indicates successive roles of RNA mA modification in the down regulation of GR expression, which provides new drug targets for epigenetic therapy of liver inflammation.
糖皮质激素受体分布于机体的各种细胞中，参与糖皮质激素对代谢和免疫的调节。RNA mA甲基化参与多种代谢和炎症反应，但在免疫激活过程中，mA是否参与糖皮质激素受体（GR）的调节仍不清楚。在这里，我们观察到在受到脂多糖（LPS）攻击的肉鸡肝脏中，GR反应与mRNA增加但蛋白水平受到抑制的情况不相关，这与RNA mA甲基化的整体升高有关，特别是在甲基转移酶样蛋白3（METTL3）和YTHDF2的表达方面。使用分离的原代肝细胞和库普弗细胞进行的进一步分析表明，这种不相关的GR反应和mA高甲基化特异性地发生在库普弗细胞中。在LPS激活的库普弗细胞系中重现了相同的GR和mA反应，这意味着mA在库普弗细胞中对GR的转录后抑制可能发挥作用。事实上，mA抑制剂环亮氨酸减轻了LPS诱导的GR蛋白抑制，而GR拮抗剂RU486对库普弗细胞中的整体mA甲基化没有影响。我们观察到YTHDF2小干扰RNA（siRNA）可以减轻LPS诱导的GR mRNA稳定性降低。随后，对GR上的特定mA位点进行了预测和验证。还应用了突变mA位点和荧光素酶报告基因检测来验证这些发现。从机制上讲，GR转录本上的mA甲基化以YTHDF2依赖的方式损害其mRNA稳定性，从而导致其蛋白减少。我们的研究表明RNA mA修饰在GR表达下调中具有连续作用，这为肝脏炎症的表观遗传治疗提供了新的药物靶点。
