Threonine Phosphorylation of STAT1 Safeguards Gut Epithelial Integrity and Restricts Interferon-mediated Cytotoxicity [RNA-seq]

Uncontrolled inflammation drives tissue damage, highlighting the need for tightly regulated immune responses and tissue integrity, particularly in barrier tissues like the intestine. To maintain this exquisite balance, intestinal epithelial cells (IECs) employ molecular circuits that preserve tissue integrity following inflammation. STAT1 is traditionally viewed as a pro-inflammatory driver in the intestine, acting as a central signaling mediator downstream of interferons (IFN). Here, we identify threonine 748 (Thr748) phosphorylation of Stat1 as an evolutionarily conserved adaptation that reciprocally regulates IEC integrity and IFN responsiveness. Mice expressing a phospho-deficient T748A Stat1 mutant exhibit severe pathology similar to Stat1 deficient littermates, underscoring Thr748's critical role in Stat1-driven protection following intestinal inflammation. Bone marrow transfer experiments further demonstrate that this protective effect is non-hematopoietic. Integrated genomic and transcriptomic analyses reveal that Thr748 phosphorylation modulates Stat1 DNA binding, directly activating the Itgb4 promoter and enhancing integrin ß4 expression in IECs following inflamamtion. In vitro intestinal organoid models, combined with gain- and loss-of-function experiments, show that Stat1 promotes integrin ß4 expression via Thr748 phosphorylation following damage, boosting epithelial resilience independently of IFN-induced Tyrosine 701 (Tyr701) phosphorylation. In contrast, IFN stimulation triggers Tyr701 phosphorylation of Stat1, upregulating Zbp1—a sensor of cytotoxic damage-associated nucleic acids—while suppressing integrin ß4, leading to epithelial cytotoxicity, which is mitigated by Thr748 phosphorylation. Our findings uncover a modular architecture of Stat1 signaling that enables epithelial adaptation to damage, with Thr748 phosphorylation acting as a rheostat to preserve tissue integrity while restricting IFN-mediated cytotoxicity. Overall design: To elucidate the protective epithelial transcriptional programs driven by the Thr748 phosphorylation of Stat1 following colitis-induced damage, we performed RNA sequencing (RNA-seq) on IECs isolated from Wt and Stat1T748A mice under both basal and DSS-treated conditions. A total of 2–5 × 105 cells per condition were lysed in 0.5 mL of TRIzol Reagent (Thermo Fisher) following the indicated treatments. RNA-seq analysis was performed by the Genome Information Research Center (GIRC) at Osaka University. Briefly, total RNA was extracted, and cDNA libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, Cat. No. 20020594) according to the manufacturer's instructions. Sequencing was conducted on an Illumina HiSeq 2500 platform using 75-base single-end reads. Base calling was performed with Illumina Casava v1.8.2 software. Sequenced reads were aligned to the mouse reference genome (mm10/GRCm38) using TopHat v2.0.13 with Bowtie2 v2.2.3 and SAMtools v0.1.19. Gene expression levels were quantified as fragments per kilobase of exon per million mapped reads (FPKM) using Cuffnorm v2.2.1. Further data processing and analysis were conducted on the Galaxy platform (https://usegalaxy.org/). Read quality was assessed using FastQC (v0.73), and adaptor sequences were trimmed prior to alignment with HISAT2 (v2.2.1) using default parameters. Gene counts were generated with featureCounts (v2.0.3), and differential expression analysis was performed using DESeq2 (v2.11.40.7). Visualization of results, including volcano plots and heatmaps, was performed using Volcano Plot (v0.0.5) and heatmap2 (v3.1.3), respectively. Gene ontology enrichment and pathway analysis were conducted using the PANTHER GO-Slim Biological Process database (v17.0, released 2022/02/22).失控的炎症会引发组织损伤，凸显了严格调控免疫应答与维持组织完整性的必要性，在肠道这类屏障组织中尤为如此。为维持这一精妙平衡，肠上皮细胞（intestinal epithelial cells, IECs）会激活分子通路，在炎症发生后维系组织完整性。传统观点认为，信号转导与转录激活因子1（STAT1）是肠道内的促炎调控因子，作为干扰素（IFN）下游的核心信号介导蛋白发挥作用。本研究发现，STAT1的苏氨酸748（Thr748）位点磷酸化是一种进化保守的适应性机制，可双向调控IEC的完整性与IFN应答能力。表达磷酸化缺陷型T748A STAT1突变体的小鼠，其病理表型与STAT1敲除同窝小鼠相似且更为严重，这进一步证实了Thr748位点在肠道炎症后STAT1介导的保护作用中的关键功能。骨髓移植实验进一步证实，该保护效应并非由造血细胞介导。整合基因组与转录组的分析结果显示，Thr748位点磷酸化可调控STAT1的DNA结合能力，在炎症发生后直接激活整合素β4（Itgb4）的启动子，并上调IEC中整合素β4的表达。体外肠道类器官模型结合功能获得与功能缺失实验显示，在组织损伤后，STAT1可通过Thr748位点磷酸化促进整合素β4的表达，不依赖于IFN诱导的酪氨酸701（Tyr701）位点磷酸化，从而增强上皮细胞的抗损伤能力。与之相反，IFN刺激会触发STAT1的Tyr701位点磷酸化，上调细胞毒性损伤相关核酸的感知蛋白ZBP1，同时抑制整合素β4的表达，最终引发上皮细胞毒性，而Thr748位点磷酸化可缓解这一过程。本研究的发现揭示了STAT1信号通路的模块化架构，该架构可使上皮细胞适应损伤，其中Thr748位点磷酸化充当调节开关，在维持组织完整性的同时限制IFN介导的细胞毒性。实验整体设计：为阐明结肠炎诱导的组织损伤后，STAT1的Thr748位点磷酸化所驱动的上皮保护性转录程序，我们对野生型（Wt）与Stat1T748A小鼠在基础状态及葡聚糖硫酸钠（DSS）处理后的分离IEC进行了RNA测序（RNA-seq）。每个实验组取2~5×10^5个细胞，在按指定方案处理后，于0.5 mL TRIzol试剂（赛默飞世尔科技）中裂解。RNA-seq分析由大阪大学基因组信息研究中心（Genome Information Research Center, GIRC）完成。简要流程如下：提取总RNA后，按照试剂盒说明书，使用TruSeq链特异性mRNA样本制备试剂盒（Illumina，货号：20020594）构建cDNA文库。测序在Illumina HiSeq 2500平台上完成，采用75bp单端测序读长。碱基识别由Illumina Casava v1.8.2软件完成。测序读段通过TopHat v2.0.13结合Bowtie2 v2.2.3与SAMtools v0.1.19，比对至小鼠参考基因组mm10/GRCm38。使用Cuffnorm v2.2.1将基因表达量量化为每百万比对读段的外显子每千碱基片段数（FPKM）。后续的数据处理与分析在Galaxy平台（https://usegalaxy.org/）上完成。使用FastQC（v0.73）评估测序读段质量，并使用默认参数通过HISAT2（v2.2.1）比对前，去除接头序列。使用featureCounts（v2.0.3）生成基因计数矩阵，并通过DESeq2（v2.11.40.7）进行差异表达分析。结果可视化（包括火山图与热图）分别通过Volcano Plot（v0.0.5）与heatmap2（v3.1.3）完成。基因本体富集分析与通路分析使用PANTHER GO-Slim生物过程数据库（v17.0，发布于2022/02/22）完成。