Role of Sam68 in Proinflammatory Signaling

摘要 溃疡性结肠炎（UC）是一种慢性炎症性肠病（IBD），无法治愈。当前治疗 策略只能提供部分缓解，许多患者仍然难以治疗，并存在 严重不良事件，包括严重感染和癌症。因此，更好地了解 炎症信号通路和新的临床前机制的鉴定是 IBD研究促炎细胞因子肿瘤坏死因子（TNF）和Toll样因子的下游信号传导 TLR通路在IBD发病机制中起主要作用。在之前的工作中，我们发现RNA 结合蛋白Sam 68是TNF和TLR诱导的转录因子NF-κB活化所必需的， 这表明Sam 68参与了NF-κ B依赖性炎症。 我们的新的初步结果表明，Sam 68在人和小鼠的肠道中显著表达， 上皮细胞（IEC）; Sam 68敲除（KO）小鼠显著免受葡聚糖硫酸钠的影响 （DSS）和恶唑酮诱导的结肠炎;并且在UC的发炎结肠中Sam 68蛋白显著升高 患者基于此，我们假设Sam 68是IEC中炎症的关键介质， 靶向IEC Sam 68将通过双重抑制TNF-α和 TLR介导的炎症通路。我们建议研究IEC特异性的分子机制， 使用尖端工具（如3D colonoids）在人类和实验鼠结肠炎中进行Sam 68信号传导 来源于UC患者的结肠细胞和新的IEC特异性Sam 68条件性KO（cKO）小鼠。目标1将 描述IEC特异性Sam 68信号传导在TNF和TLR诱导的炎症中的分子机制 信号传导和鉴定其所需的Sam 68的结构域和翻译后修饰。 炎症功能。目的2将研究Sam 68在稳态和稳态条件下在原代IEC中的体内作用。 使用用DSS和恶唑酮攻击的Sam 68-cKO小鼠作为实验性结肠炎， 模型，并使用自发性结肠炎模型，Sam 68-cKO / Wiscott Aldrich综合征蛋白（WASP）KO 双KO小鼠。该目的将利用从野生型和Sam 68-KO小鼠制备的3D类结肠来研究作用。 的Sam 68在IEC增殖，渗透性和炎症信号传导中的作用，以研究Sam 68的稳态作用。 目的3将描述Sam 68在UC患者中的促炎作用。这一目标将研究IEC特定的增长， 增殖、凋亡和TJ蛋白的表达，以及IEC中促炎信号的激活， 使用对照和患者来源的3D肠类器官的共培养测定的粘膜免疫细胞， 免疫细胞成功完成这项研究将填补理解小说的关键知识空白 UC中TNF-和TLR依赖性炎症信号传导的潜在机制，并为研究UC中TNF-和TLR依赖性炎症信号传导的机制铺平道路。 开发靶向Sam 68的新型治疗剂以治疗UC。此外，这项研究还将作为 为探索Sam 68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。

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.