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。目前的治疗方法 策略只能提供部分缓解，许多患者仍然对治疗无效，并存在以下风险 重大不良事件，包括严重感染和癌症。因此，更好地理解 炎症信号通路和新的临床前机制的确定是临床治疗的关键挑战 IBD研究。促炎症细胞因子肿瘤坏死因子和Toll样蛋白下游信号传导 受体(TLR)通路在IBD的发病机制中起重要作用。在之前的工作中，我们发现RNA 结合蛋白Sam68是肿瘤坏死因子和肿瘤坏死因子受体诱导的转录因子NF-κB激活所必需的， 炎症的主要调节因子，表明Sam68参与了NF-κB依赖的炎症。 我们的新的初步结果表明，Sam68在人和小鼠的肠道中显著表达 上皮细胞(IEC)；Sam68基因敲除(KO)小鼠对葡聚糖硫酸钠有显著的保护作用 (DSS)和恶唑酮诱导的结肠炎；在UC的炎症结肠中Sam68蛋白显著升高 病人。在此基础上，我们假设Sam68是IECS炎症的关键介质，并且 靶向IEC Sam68将通过双重抑制肿瘤坏死因子-1和肿瘤坏死因子-2为UC的治疗提供新的策略 TLR介导的炎症途径。我们建议研究IEC特异性的分子机制。 使用3D结肠镜等尖端工具在人和实验性小鼠结肠炎中的Sam68信号 来源于UC患者的结肠细胞和新型IEC特异性条件KO(CKO)小鼠。目标1将 IEC特异性Sam68信号在肿瘤坏死因子和TLR诱导的炎症中的分子机制 并确定其所需的Sam68的结构域和翻译后修饰 炎症功能。目的2将研究sAM68在体内的作用(S)在原代培养的IEC中的作用 炎症条件下用DSS和恶唑酮激发的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 两只KO小鼠。这一目标将利用从野生型和Sam68-KO小鼠中制备的3D结肠素来研究其作用 研究Sam68在IEC增殖、通透性和炎症信号转导中的作用。 目的3描述Sam68在UC患者中的促炎作用。这一目标将研究IEC特有的生长， IEC和IEC的增殖、凋亡和TJ蛋白的表达以及促炎信号的激活 用对照和患者来源的3D肠道器官和 免疫细胞。这项研究的成功完成将填补理解小说的关键知识空白 UC中依赖肿瘤坏死因子和肿瘤坏死因子受体的炎症信号的机制，并为 以Sam68为靶点治疗UC的新疗法的开发此外，这项研究也将作为 探讨Sam68在其他依赖肿瘤坏死因子和TLR的慢性炎症性疾病中的作用。

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.