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

总结 慢性炎症在具有上皮实质的器官，如肾、肝和肠中是广泛存在的。 健康问题。一般认为，它涉及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慢性炎症的维持中起作用 由于紧密连接（TJ）对管腔抗原和细菌的渗透性增加。反过来，TJ组装 并由参与顶端-基底极性的信号传导途径控制，如Par-极性复合物， 包括Par 6、非典型PKC（I和II亚型，aPKC）和Par 3。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限制炎症。使用PKCι 在条件性基因敲除小鼠模型中，我们已经证明aPKC拮抗上皮天然免疫NF-κ B， 并促进抗炎IL-10分泌。此外，我们已经证明，这种机制是 以防止轻微的促炎性损伤引发致命的急性炎症。重要的是， 在旁分泌TNFα刺激下，小分子伴侣Bag-1 M抑制正常aPKC重折叠，并增加其 泛素化由Hsp 70的aPKC陪伴发生在中间丝（角蛋白）支架上。两者 基于角蛋白的aPKC重折叠和对NF-kB的下游作用对上皮细胞是特异性的。 该项目的首要目标是确定上皮细胞特异性的分子机制， 在抗炎细胞间的相互作用。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。根据公布的 和初步证据，我们假设，（1）Bag-1 M与角蛋白-Hsc/Hsp 70复合物相互作用， 在低微摩尔浓度范围内，抑制aPKC再折叠和随后的自磷酸化。 （2）生理水平的aPKC，通过间接调控上皮细胞的抗炎作用 抑制NF-κ B、细胞因子转录和TJ功能。在具体目标1中，我们将分析 参与由旁分泌和来自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， 透化与细胞因子分泌对局部炎症的影响以及导致NF-κ B的可能间接途径。 通过aPKC抑制kB和控制细胞因子转录。我们将使用人类上皮细胞 在培养物（Caco-2）、未转化的原代培养物和与肠上皮细胞或肠上皮细胞杂交的条件性PKCιflox/flox中， 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NF-κ B抑制，因此 确保所有拟议研究的可行性。据我们所知，我们是第一个调查 先天免疫中的顶-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.