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

总结 肾、肝、肠等上皮实质器官的慢性炎症是一种广泛存在的疾病。 健康问题。人们普遍认为，它涉及上皮细胞和上皮细胞之间的细胞间相互作用。 免疫细胞。还已知上皮屏障在维持慢性炎症中发挥作用 由于紧密连接（TJ）对管腔抗原和细菌的渗透性增加。依次组装TJ 并由参与顶端-基底极性的信号通路控制，例如 Par-极性复合体， 包括 Par6、非典型 PKC（ι 和 ζ 同工型，aPKC）和 Par3。最近的工作表明，此外， 上皮细胞可以通过促炎或抗炎细胞因子增强或限制炎症。使用 PKCι 条件敲除小鼠模型中，我们发现aPKC拮抗上皮先天免疫NF-kB 并促进抗炎IL-10的分泌。此外，我们还证明了该机制 对于防止轻微的促炎性损伤引发致命的急性炎症是必要的。重要的是， 在旁分泌 TNFα 刺激下，小伴侣 Bag-1M 抑制正常 aPKC 重折叠并增加其 泛素化。 Hsp70 的 aPKC 陪伴发生在中间丝（角蛋白）支架上。两者都 基于角蛋白的 aPKC 重折叠和对 NF-kB 的下游影响是上皮细胞特有的。 该项目的总体目标是确定上皮细胞特有的分子机制，从而导致 在抗炎细胞间相互作用中。长期的想法是利用这些机制来寻找新颖的、 针对肠道、肝脏或肾脏慢性炎症的替代治疗方法。在已发表的基础上 和初步证据，我们假设，(1) Bag-1M 与角蛋白-Hsc/Hsp70 复合物相互作用 低微摩尔浓度范围内，抑制 aPKC 重折叠和随后的自磷酸化。 (2)aPKC在生理水平上，通过间接的方式控制上皮的抗炎作用。 抑制 NF-kB、细胞因子转录和 TJ 功能。在具体目标1中，我们将分析其机制 参与旁分泌和微生物群的促炎信号引起的 aPKC 不稳定。在 具体目标2 我们将研究aPKC下游的信号通路，TJ的相对贡献 透化作用与细胞因子分泌对局部炎症的影响以及导致 NF-的可能间接途径 aPKC 对细胞因子转录的 kB 抑制和控制。我们将使用人类上皮细胞的组合 培养物 (Caco-2)、未转化的原代培养物以及与肠上皮细胞杂交的条件 PKCιflox/flox 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。所有这些生物系统均已显示出 aPKC 下调 我们实验室的初步数据或出版物中存在炎症和 aPKC 依赖性 NF-kB 抑制，因此 确保所有拟议研究的可行性。据我们所知，我们是第一个研究 先天免疫中的顶端-基底极性信号传导。

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.