Programming Airway Epithelial Differentiation by Rad

描述（由申请人提供）：鳞状细胞分化是一种重要的组织重塑过程，涉及许多细胞事件，从促进屏障功能到导致气道中的细胞转化和致癌。维生素A缺乏是已知的气道鳞状细胞分化的原因之一。我们进行了一项研究，以确定是否Rad，其表达是由维生素A缺乏诱导，调节气道鳞状细胞分化，如一个小的富含脯氨酸的蛋白质，SPRR 1B，鳞状细胞分化的标志，在人气管支气管上皮（TBE）细胞的表达。人气管、支气管和食管组织的免疫组化研究表明，Rad蛋白在纤毛细胞和鳞状细胞中大量表达，而在粘液细胞中很少表达。北方杂交和真实的时间RT-PCR分析表明，TBE细胞中Rad/热尔恩/Kir（RGK）Ras-like亚家族成员Rad的表达与SPRR 1 B的诱导表达在时间和空间上相关，而与MUC 5 B/MUC 5AC的表达水平呈负相关。利用Tet-on Rad和突变型Rad（S66 N）在HBE 1细胞中诱导的细胞系统，我们证明了Rad，而不是突变型Rad（S66 N）表达是一个关键的信号分子，参与协调气道上皮细胞分化的鳞状细胞和粘液细胞表型。初步研究表明，Rad通过与微管的相互作用，发挥蛋白激酶C（PKC）介导的信号通路诱导SPRR 1B的表达。一个类似的PKC介导的信号传导显然参与了MUC 5 B/MUC 5AC表达的衰减。这些初步结果强烈表明，拉德可能作为一个“开关”的因素，是能够编程气道上皮细胞分化鳞状细胞和粘液细胞表型。本研究假设Rad蛋白通过蛋白激酶C（PKC）/MAPK/AP-1途径调控气道上皮细胞分化。为了验证这一假设，目的1是确定Rad与微管相互作用的结构-功能关系，以及这种相互作用在SPRR 1B表达调控中的后果。目的二是研究镭-微管复合物如何激活PKC同工酶以及激活哪种同工酶。目的3是阐明Rad-microtubule/ PKC激活后SPRR 1B表达的下游信号传导。目的4是阐明Rad转录激活SPRR 1B表达的分子本质。我们将阐明这一过程中涉及的顺式元件和反式作用因子。目的5是为原代人气道上皮细胞/组织开发一种生物学范式，以研究Rad在减弱气道上皮细胞分化中的功能。这些将获得的结果将不仅是感兴趣的一般生物科学，但也有助于我们开发一个潜在的有效的治疗工具，用于控制的可塑性的上皮细胞，经常看到在不同的生理和病理阶段的气道。总之，我们提出了一种综合的方法，利用组合的细胞系，原代细胞，和离体组织来测试这一假设。

DESCRIPTION (provided by applicant): Squamous cell differentiation, an important tissue remodeling process, has been implicated in many cellular events from promoting barrier function to rendering cell transformation and carcinogenesis in the airway. Vitamin A deficiency is one of known causes for airway squamous cell differentiation. We undertook a study to determine whether Rad, whose expression is induced by vitamin A deficiency, modulates airway squamous cell differentiation, such as the expression of a small proline-rich protein, SPRR1B, a hallmark of squamous cell differentiation in human tracheobronchial epithelial (TBE) cells. Immunohistochemical studies of human tracheobronchial and esophageal tissues indicated that Rad protein is significant presence in ciliated and squamous cells, but scarce in mucous cells. Both Northern blot and Real Time RT-PCR analysis demonstrated that the expression of Rad, a member of Rad/Gern/Kir (RGK) Ras-like subfamily, is temporally and spatially correlated with the induction of SPRR1B, but inversely correlated with the level of MUC5B/MUC5AC expression in human TBE cells. Utilizing Tet-on Rad and mutant Rad(S66N) inducible cell systems in HBE1 cells, we demonstrated that Rad, but not the mutant Rad(S66N) expression is a crucial signaling molecule involved in coordinating airway epithelial cell differentiation between the squamous and mucous cell phenotypes. Initial studies suggested that Rad through the interaction with microtubule exerts a protein kinase C (PKC)-mediated signaling pathway in the induction of SPRR1B expression. A similar PKC-mediated signaling is apparently involved in the attenuation of MUC5B/MUC5AC expression by Rad. These preliminary results strongly suggest that Rad may serve as a "switch" factor that is able to programming airway epithelial cell differentiation between squamous and mucous cell phenotypes. In this proposal, we hypothesize that Rad protein regulates airway epithelial call differentiation through a protein kinase C (PKC)/MAPK/AP-1 pathways. In order to test the hypothesis, Aim 1 is to determine the structure-functional relationship of Rad to interact with microtubule and the consequence of the interaction in the regulation of SPRR1B expression. Aim 2 is to see how and which PKC isozyme is activated by Rad-microtubule complex. Aim 3 is to elucidate down stream signaling after Rad-microtubule/ PKC activation for SPRR1B expression. Aim 4 is to elucidate the molecular nature of the transcriptional activation of SPRR1B expression by Rad. We will elucidate both cis-elements and trans-acting factors involved in the process. Aim 5 is to develop a biological paradigm for primary human airway epithelial cells/tissues of the function of Rad in the attenuation of airway epithelial cell differentiation. These to-be-obtained results will not only be of interest to general biological science, but also assist us to develop a potential effective therapeutic tool for the control of the plasticity of epithelial cells that are frequently seen in airways under various physiological and pathological stages. In summary, we have proposed a comprehensive approach utilizing combined cell line, primary cells, and ex vivo tissues to test this hypothesis.