Programming Airway Epithelial Differentiation by Rad

描述(申请人提供)：鳞状细胞分化是一种重要的组织重塑过程，参与了许多细胞事件，从促进屏障功能到在呼吸道中实现细胞转化和癌变。维生素A缺乏是呼吸道鳞状细胞分化的已知原因之一。我们开展了一项研究，以确定由维生素A缺乏诱导的Rad是否调节呼吸道鳞状细胞分化，例如富含脯氨酸的小蛋白SPRR1B的表达，这是人类气管支气管上皮(TBE)细胞鳞状细胞分化的标志。免疫组织化学研究表明，Rad蛋白主要存在于纤毛细胞和鳞状细胞中，而在粘液细胞中很少。Northern印迹和实时定量RT-PCR分析表明，Rad/Gern/Kir(RGK)Ras样亚家族成员Rad的表达与SPRR1B的诱导存在时空相关性，而与MUC5B/MUC5AC的表达水平呈负相关。利用Tet-on Rad和突变型Rad(S66N)诱导的HBE1细胞系统，我们证明了Rad而不是突变型Rad(S66N)的表达是调节鳞状细胞和粘液细胞表型之间的呼吸道上皮细胞分化的关键信号分子。初步研究表明，Rad通过与微管的相互作用，通过蛋白激酶C(PKC)介导的信号通路诱导SPRR1B的表达。Rad对MUC5B/MUC5AC表达的抑制作用与类似的PKC介导的信号转导有关。这些初步结果有力地表明，Rad可能是一个“开关”因子，能够编程在鳞状细胞和粘液细胞表型之间的气道上皮细胞分化。在这个提议中，我们假设Rad蛋白通过蛋白激酶C(PKC)/MAPK/AP-1通路调节呼吸道上皮细胞的分化。为了验证这一假说，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SPRR1B表达调控中的结果。目的2是观察Rad-微管复合体是如何激活PKC同工酶的，以及激活的是哪一种同工酶。目的3阐明Rad-微管/PKC激活后SPRR1B表达的下游信号转导。目的4阐明Rad对SPRR1B转录激活的分子本质。我们将阐明参与这一过程的顺式元件和反式作用因子。目的5是为原代人呼吸道上皮细胞/组织建立Rad在减弱呼吸道上皮细胞分化中的作用的生物学范式。这些即将获得的结果不仅对一般生物科学感兴趣，而且有助于我们开发一种潜在的有效治疗工具来控制呼吸道上皮细胞的可塑性，这些细胞在不同的生理和病理阶段经常出现在呼吸道中。综上所述，我们提出了一种综合的方法，利用组合的细胞系、原代细胞和体外组织来验证这一假设。

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.