Apico-basal Polarity Signaling Controls Expression of Epithelial Cyrokines Through NF-kB

摘要 慢性炎症广泛存在于肾、肝、肠等有上皮性实质的器官中。 健康问题。人们普遍认为它涉及上皮细胞和细胞间的相互作用 免疫细胞。我们也知道上皮屏障在维持慢性炎症中起作用。 由于紧密连接(TJ)对管腔抗原和细菌的渗透性增加。接着，组装TJ 并受参与尖基极性的信号通路控制，如Par-Poly复合体，它 包括PAR6、非典型PKC(ι和ζ异构体，APKC)和PAR3。最近的研究表明，此外， 上皮细胞可通过促炎或抗炎细胞因子增强或抑制炎症。使用PKCι 在条件性基因敲除小鼠模型中，我们已经证明了aPKC拮抗上皮天然免疫因子-kB 并促进抗炎IL-10的分泌。此外，我们已经证明了这一机制是 对于防止轻微的促炎损伤引发致命的急性炎症来说是必要的。重要的是 在旁分泌肿瘤坏死因子α刺激下，小分子伴侣BAG-1M抑制正常的蛋白激酶C重折叠并增加其活性。 泛素化。APKC由Hsp70陪伴，发生在中间丝(角蛋白)支架上。这两个 角蛋白为基础的aPKC重折叠和对核因子-kB的下游作用是上皮细胞特有的。 这个项目的首要目标是确定特定于上皮细胞的分子机制，从而导致 在抗炎的细胞间相互作用中。长期的想法是利用这些机制来发现新的， 肠、肝或肾慢性炎症的替代治疗方法。在出版的基础上 和初步证据，我们假设，(1)Bag-1M与角蛋白-HSC/Hsp70复合体在 在低微摩尔浓度范围内，抑制蛋白激酶C的折叠和随后的自动磷酸化。 (2)生理水平的aPKC通过间接调控上皮细胞的抗炎作用。 抑制核因子-kB、细胞因子转录和TJ功能。在具体目标1中，我们将分析这些机制 参与由旁分泌和微生物区系的促炎信号引起的PKC不稳定。在……里面 具体目标2我们将研究aPKC下游的信号通路，TJ的相对贡献 通透性与细胞因子分泌对局部炎症以及导致核因子-2的可能间接途径的影响 通过aPKC抑制和控制细胞因子的转录。我们将使用人类上皮细胞的组合 在培养物(CACO-2)中，未转化的原代培养物和有条件的PKCιFLOX/FLOX与肠细胞或 肝细胞特异性Cre表达小鼠进行机制和功能实验以测试 假设。所有这些生物系统都显示出aPKC下调。 炎症和aPKC依赖的核因子-kB抑制在我们实验室的初步数据或出版物中，因此 确保所有拟议研究的可行性。据我们所知，我们是第一个调查 先天免疫中的尖-基极性信号。

Summary Chronic inflammation in organs with epithelial parenchyma, such as kidney, liver and intestine is a widespread health problem. It is generally accepted that it involves intercellular interactions among epithelial cells and immune cells. It is also known that the epithelial barrier has a role in the maintenance of a chronic inflammation by due increased permeability of tight junctions (TJ) to luminal antigens and bacteria. In turn, TJ are assembled and controlled by signaling pathways involved in apico-basal polarity, such as the Par-polarity complex, which includes Par6, atypical PKC (ι and ζ isoforms,aPKC), and Par3. Recent work has shown that, in addition, epithelial cells can enhance or limit inflammation by means of pro- or anti-inflammatory cytokines. Using a PKCι conditional knockout mouse model, we have shown that aPKC antagonizes epithelial innate immunity NF-kB and promotes anti-inflammatory IL-10 secretion. Furthermore, we have demonstrated that this mechanism is necessary to prevent a mild pro-inflammatory injury from triggering a deadly acute inflammation. Importantly, under paracrine TNFα stimulation, a small chaperone Bag-1M inhibits normal aPKC refolding and increases its ubiquitination. aPKC chaperoning by Hsp70 occurs on an intermediate filament (keratin) scaffold. Both the keratin-based aPKC refolding and the downstream effects on NF-kB are specific to epithelial cells. The overarching goal of this project is to identify molecular mechanisms, specific to epithelial cells, which result in anti-inflammatory intercellular interactions. The long-term idea is to harness these mechanisms to find novel, alternative therapeutic approaches to chronic inflammation in intestine, liver, or kidney. On the basis of published and preliminary evidence, we hypothesize that, (1) that Bag-1M interacts with keratin-Hsc/Hsp70 complexes at concentrations in the low micromolar range, inhibiting aPKC refolding and subsequent auto-phosphorylation. And (2) aPKC at physiological levels, controls the anti-inflammatory role of the epithelium through indirect inhibition of NF-kB, cytokine transcription, and TJ function. In specific aim 1, we will analyze the mechanisms involved in aPKC destabilization caused by pro-inflammatory signals both paracrine and from microbiota. In specific aim 2 we will study the signaling pathways downstream of aPKC, the relative contribution of TJ permeabilization versus cytokine secretion to local inflammation and possible indirect pathways leading to NF- kB inhibition and control of cytokine transcription by aPKC. We will use a combination of human epithelial cells in culture (Caco-2), non-transformed primary cultures, and conditional PKCιflox/flox crossed with enterocyte or hepatocyte-specific CRE expresser mice to conduct mechanistic and functional experiments to test the hypotheses. All these biological systems have been shown to display the aPKC downregulation upon inflammation, and aPKC-dependent NF-kB inhibition in preliminary data or publications from our lab, thus ensuring the feasibility of all the proposed studies. To our knowledge, we are the first to investigate the role of apico-basal polarity signaling in innate immunity.