Role of Sam68 in Proinflammatory Signaling

Sam68 在促炎信号传导中的作用

基本信息

批准号：
10598098
负责人：
Parameswaran Ramakrishnan
金额：
$ 56.37万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2026-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10598098
关键词：
3-Dimensional Acute Address Adverse event Affect Apoptosis Binding Biochemical Biological Assay Biological Response Modifier Therapy Bone Marrow Transplantation CRISPR/Cas technology Caspase Cell Line Cells Chronic Coculture Techniques Colitis Colon Colonic inflammation Complex Cytoplasm DNA-Protein Interaction Deletion Mutation Development Digestive System Disorders Disease remission Environmental Risk Factor Epithelial Cell Proliferation Epithelial Cells Future Gel Gene Expression Genes Genetic Genetic Transcription Growth Hematopoietic Human Immune Incidence Infection Inflammation Inflammation Mediators Inflammatory Inflammatory Bowel Diseases Intestinal permeability Intestines Knock-out Knockout Mice Knowledge Malignant Neoplasms Mediating Mediator Membrane Modeling Molecular Mucositis Mucous Membrane Mus Organoids Oxazolone Partial Remission Pathogenesis Pathogenicity Pathologic Pathway interactions Patients Permeability Persons Play Point Mutation Post-Translational Protein Processing Proctitis Proliferating Proteins RNA Binding RNA-Binding Proteins Receptor Signaling Refractory Relapse Research Risk Role SRC-associated p68 protein Signal Pathway Signal Transduction Signaling Protein Sodium Dextran Sulfate Spontaneous colitis Structure-Activity Relationship System TLR2 gene TNF gene Testing Therapeutic Tissues Toll-Like Receptor Pathway Toll-like receptors Transcriptional Activation Tumor Necrosis Factor Receptor Ulcerative Colitis United States Wiskott-Aldrich Syndrome Work chemically induced colitis chromatin immunoprecipitation chronic inflammatory disease cohort conditional knockout cytokine design experimental study gut inflammation improved in vivo intestinal epithelium intestinal homeostasis murine colitis new therapeutic target novel novel therapeutic intervention pre-clinical protein protein interaction targeted treatment tool transcription factor transcriptome sequencing treatment strategy tumor necrosis factor receptor binding vector

项目摘要

Abstract Ulcerative colitis (UC) is a chronic form of inflammatory bowel disease (IBD) with no cure. Current treatment strategies offer only partial remission, with many patients remaining refractory to treatment, and carry risks of significant adverse events including serious infections and cancer. Thus, an improved understanding of inflammatory signaling pathways and identification of novel preclinical mechanisms are critical challenges in IBD research. Signaling downstream of the proinflammatory cytokine tumor necrosis factor (TNF) and toll like receptor (TLR) pathways play major roles in IBD pathogenesis. In previous work, we discovered that the RNA binding protein Sam68 is required for both TNF- and TLR-induced activation of the transcription factor NF-κB, a master regulator of inflammation, suggesting that Sam68 contributes to NF-κB-dependent inflammation. Our new preliminary results show that Sam68 is prominently expressed in human and murine intestinal epithelial cells (IEC); Sam68 knockout (KO) mice are significantly protected from dextran sulfate sodium (DSS)- and oxazolone-induced colitis; and Sam68 protein is significantly elevated in inflamed colons of UC patients. Based on this, we hypothesize that Sam68 is a critical mediator of inflammation in IECs, and targeting IEC Sam68 will provide a novel therapeutic strategy in UC via dual inhibition of TNF- and TLR-mediated inflammatory pathways. We propose to study the molecular mechanisms of IEC-specific Sam68 signaling in human and experimental murine colitis using cutting edge tools such as 3D colonoids derived from UC patient colonocytes and novel IEC-specific Sam68 conditional KO (cKO) mice. Aim 1 will delineate molecular mechanisms of IEC-specific Sam68 signaling in TNF- and TLR-induced inflammatory signaling and identify the structural domains and posttranslational modifications of Sam68 required for its inflammatory functions. Aim 2 will study the in vivo role(s) of Sam68 in primary IECs under homeostatic and inflammatory conditions, using Sam68-cKO mice challenged with DSS and oxazolone as experimental colitis models, and using a spontaneous colitis model, Sam68-cKO / Wiscott Aldrich Syndrome Protein (WASP) KO double KO mice. This aim will utilize 3D colonoids prepared from wild type and Sam68-KO mice to study role of Sam68 in IEC proliferation, permeability and inflammatory signaling to study the homeostatic role of Sam68. Aim 3 will delineate the proinflammatory role of Sam68 in UC patients. This aim will study IEC-specific growth, proliferation, apoptosis, and expression of TJ proteins, and activation of proinflammatory signaling in IEC's and mucosal immune cells using co-culture assays of control and patient derived 3D intestinal organoids and immune cells. Successful completion of this study will fill critical knowledge gaps in understanding novel mechanisms underlying TNF- and TLR-dependent inflammatory signaling in UC and pave the way for the development of novel therapeutics targeting Sam68 to treat UC. Moreover, this study will also serve as the basis to explore the role of Sam68 in other TNF- and TLR-dependent chronic inflammatory diseases as well.

抽象的溃疡性结肠炎（UC）是一种慢性炎症性肠病（IBD），目前尚无治愈方法。策略只能提供部分缓解，许多患者仍然难以治疗，并且存在以下风险：重大不良事件，包括严重感染和癌症，从而提高了对这些不良事件的认识。炎症信号通路和新的临床前机制的鉴定是关键挑战 IBD 研究。促炎细胞因子肿瘤坏死因子 (TNF) 和 Toll 样下游信号传导。受体（TLR 途径）在 IBD 发病机制中发挥重要作用在之前的工作中，我们发现 RNA。结合蛋白 Sam68 是 TNF 和 TLR 诱导的转录因子 NF-κB 激活所必需的，炎症的主要调节因子，表明 Sam68 有助于 NF-κB 依赖性炎症。我们的新初步结果表明 Sam68 在人类和小鼠肠道中显着表达上皮细胞 (IEC)；Sam68 敲除 (KO) 小鼠受到葡聚糖硫酸钠的显着保护 (DSS) 和恶唑酮诱导的结肠炎；Sam68 蛋白在 UC 发炎结肠中显着升高基于此，我们勇敢地承认 Sam68 是 IEC 炎症的关键介质，并且靶向 IEC Sam68 将通过双重抑制 TNF-α 和 TNF-α 为 UC 提供一种新的治疗策略我们建议研究 IEC 特异性的分子机制。使用 3D 结肠等尖端工具在人类和实验性小鼠结肠炎中进行 Sam68 信号传导源自 UC 患者结肠细胞和新型 IEC 特异性 Sam68 条件 KO (cKO) 小鼠。描绘 TNF 和 TLR 诱导炎症中 IEC 特异性 Sam68 信号传导的分子机制信号传导并识别 Sam68 所需的结构域和翻译后修饰目标 2 将研究 Sam68 在体内稳态和原代 IEC 中的作用。炎症状况，使用接受 DSS 和恶唑酮攻击的 Sam68-cKO 小鼠作为实验性结肠炎模型，并使用自发性结肠炎模型 Sam68-cKO / Wiscott Aldrich 综合征蛋白 (WASP) KO 该目标将利用野生型和 Sam68-KO 小鼠制备的 3D 克隆体来研究其作用。 Sam68 在 IEC 增殖、通透性和炎症信号传导中的作用，以研究 Sam68 的稳态作用。目标 3 将描述 Sam68 在 UC 患者中的促炎作用该目标将研究 IEC 特异性生长， IEC 和 TJ 蛋白的增殖、凋亡和表达以及促炎信号的激活使用对照和患者衍生的 3D 肠道类器官的共培养测定来检测粘膜免疫细胞和免疫细胞的成功完成将填补理解新奇的关键知识空白。 UC 中 TNF 和 TLR 依赖性炎症信号传导的潜在机制，并为此外，这项研究也将作为针对 Sam68 治疗 UC 的治疗小说的开发。为探讨 Sam68 在其他 TNF 和 TLR 依赖性慢性炎症性疾病中的作用奠定了基础。