Role of CD98 Glycoprotein in Intestinal Inflammation

描述(申请人提供)：CD98在协调肠上皮细胞事件中发挥重要作用，如黏附/极性、氨基酸运输和与细胞表面分子的直接结合。CD98在胞外C末端带有PDZ结合区的独特分子定位表明，胞外信号在其多功能中发挥了作用。例如，CD98的胞外磷酸化增强了异型细胞与细胞之间的相互作用，而具有丝氨酸磷酸化位点的胞外区域对这种作用至关重要。CD98的表达在炎症性肠病(IBD)等炎症性炎症条件下增加。早期的研究支持肠上皮细胞特异性CD98诱导屏障功能障碍和刺激促炎介质的产生的能力。本课题组的研究共同表明，CD98的表达在人类肠病(IBD)的发病机制中起着重要作用。我们的总体假设是，结肠CD98在启动和持续肠结肠炎中起着关键作用。本研究的初步目的是探讨CD98/细菌相互作用在肠道屏障功能丧失中的作用。此外，我们还将重点研究CD98在自发性结肠炎中的作用。随后将使用有针对性的纳米技术方法来评估下调炎症结肠中CD98表达的效果。该项目将涉及各种生化、化学、纳米技术、分子、体外和体内方法。预计拟议的实验将有助于确定CD98在肠道炎症中的功能作用的分子机制，并允许开发针对肠道炎症状况的治疗策略，包括IBD。我的第一个目标将集中在研究肠上皮细胞CD98和CD98/细菌的相互作用是否参与肠屏障功能的丧失。本课题组获得的初步数据显示，结肠CD98的过表达增加了肠道通透性、细菌易位和促炎细胞因子的表达，如IL-1和TNF。我们最初的目标是调查细菌是否启动了特异性过表达结肠CD98的小鼠的肠道屏障功能。具体地说，Villin-CD98+/+转基因小鼠将接受抗生素治疗，并评估这些动物的肠道屏障功能和细胞因子表达与未经处理的Villin-CD98+/+转基因小鼠的关系。其次，CD98与细菌之间的直接相互作用及其信号传递过程将通过EX和体内方法进行研究。这些实验应该阐明CD98表达和CD98/细菌相互作用在肠道屏障功能丧失中的顺序作用。因此，我的第二个目的是研究结肠CD98表达对结肠炎的调节作用。我们已经证明，与野生型动物相比，在Villin-CD98转基因小鼠(其中CD98在结肠细胞中表达)DSS诱导的结肠炎更严重。相比之下，CD98低表达(CD98fl/+Villin-Cre)的小鼠比CD98高表达(CD98fl/+)的小鼠对DSS更具抵抗力。我们建议通过评估IL-10-/-/CD98fl/+Villin-CRE、IL-10-/-/Villin-CD98+/+和IL-10-/-/CD98fl/+动物的自发炎症反应来确定CD98在慢性结肠炎中的作用。最后，我的第三个目标将研究使用包裹在纳米颗粒(NPs)中的非病毒载体作为载体来定点递送ant-CD98 siRNA，以有效抑制诱导的结肠炎。最近，我们描述了一种以结肠为靶点的独创技术，将抗炎分子加载到纳米颗粒中，并将其包裹在海藻酸盐-壳聚糖水凝胶中。利用这项技术，我们获得了初步数据，表明负载肿瘤坏死因子？siRNA的纳米粒可以抑制肠道炎症。我们进一步建议特异性地靶向CD98 siRNA并将其传递到诱导结肠炎小鼠中过表达CD98的炎症结肠(上皮和免疫)细胞。

DESCRIPTION (provided by applicant): CD98 plays an important role in coordinating intestinal epithelial events, such as adhesion/polarity, amino acid transport, and direct binding o cell surface molecules. The unique molecular orientation of CD98 with a PDZ-binding domain in the extracellular C-terminal tail suggests that extracellular signaling plays a role in its multipl functions. For example, ecto-phosphorylation of CD98 enhances heterotypic cell-cell interactions, and the extracellular domain possessing serine phosphorylation sites is crucial for this effect. CD98 expression is increased in inflammatory inflammation conditions, such as inflammatory bowel disease (IBD). Earlier research supports the ability of intestinal epithelial cell-specific CD98 to induce barrier dysfunction and stimulate the production of pro-inflammatory mediators. Studies by our group have collectively shown that CD98 expression plays a role in the pathogenesis of intestinal bowel disease (IBD) in humans. Our overall hypothesis is that colonic CD98 plays a critical role in initiating and perpetuating intestinal coltis. The initial aim of this proposal is to investigate the role of CD98/bacteria interactions in the los of intestinal barrier function. We will additionally focus on establishing the role of CD98 in spontaneous colitis. The efficacy of knockdown of CD98 expression in inflamed colon will subsequently be assessed using a targeted nanotechnology approach. The project will involve a variety of biochemical, chemical, nanotechnological, molecular, in vitro, and in vivo approaches. It is envisaged that the proposed experiments will facilitate identification of the molecular mechanisms underlying the functional role of CD98 in intestinal inflammation and allow the development of therapeutic strategies targeting intestinal inflammatory conditions, including IBD. My first aim will focus on investigating if intestinal epithelial CD98 and CD98/bacteria interactions participate in loss of intestinal barrier function. Preliminary data obtained by our group showed that overexpression of colonic CD98 increases intestinal permeability, bacterial translocation and expression of pro-inflammatory cytokines, such as IL-1¿ and TNF¿. Our initial aim is to investigate whether bacteria initiate intestinal barrier function in mice that specificaly overexpress colonic CD98. Specifically, Villin-CD98+/+ transgenic mice will be treated with antibiotic, and the intestinal barrier function and cytokine expression in these animals assessed in relation to untreated Villin-CD98+/+ transgenic mice. Secondly, direct interactions between CD98 and bacteria and their signaling processes will be investigated via ex and in vivo approaches. These experiments should clarify the sequential role of CD98 expression and CD98/bacteria interactions in loss of intestinal barrier function. Thus, my second aim will study the colonic CD98 expression effect on the modulation of colitis. We have demonstrated that DSS-induced colitis is aggravated in villin-CD98 transgenic mice (in which CD98 is expressed in colonocytes) than wild-type animals. In contrast, mice with low colonic CD98 expression (CD98fl/+Villin-Cre) are more resistant to DSS, compared to those with higher colonic CD98 expression (CD98fl/+). We propose to establish the role of CD98 expression in chronic colitis by assessing spontaneous inflammation in IL-10-/-/CD98fl/+Villin-Cre, IL-10-/-/Villin-CD98+/+ and IL-10-/-/CD98fl/+ animals. Finally, my third aim will investigate the site-directed delivery of ant-CD98 siRNA using non-viral vectors encapsulated in nanoparticles (NPs) as carriers effectively inhibits induced colitis. Recently, we described an original technique targeting the colon with anti-inflammatory molecules loaded in NPs and encapsulated in an alginate-chitosan hydrogel. Using this technology, we obtained preliminary data showing that TNF¿ siRNA-loaded NPs suppress intestinal inflammation. We further propose to specifically target and deliver CD98 siRNA to inflamed colonic (epithelial and immune) cells that overexpress CD98 in mice with induced colitis.