Rac GTPase signaling during embryonic salivary gland branching morphogenesis

描述(申请人提供)：严重的唾液腺功能减退和口干(口干)是越来越常见的情况，通常是由以下原因引起的：1)干燥综合征，一种以唾液分泌腺泡组织为靶点的自身免疫性疾病，2)头颈部癌症的放疗和化疗方案，3)数千种药物的副作用。目前基于口干症的治疗是不充分和暂时的，因此产生了对长期解决方案的重大临床需求，包括用在人工工程生物相容支架上生长的功能性组织来替换不可逆转的受损或丢失的唾液组织。然而，由于缺乏对控制腺体结构和功能的精确分子信号机制的了解，在人工工程组织中刺激和/或维持唾液上皮细胞分化的相当大的挑战一直受到阻碍。这项拟议的研究将通过探索GTPase RAC信号在调节唾液腺分支形态发生和组织极化中的作用来解决我们对组织形成的理解中的这些空白，这些过程对功能器官的发育至关重要。从这些研究中获得的知识将进一步用于研究RAC在人工工程3D纳米支架上促进唾液上皮细胞组织和极化方面的作用，朝着未来产生功能性人工唾液腺结构的目标迈进。其具体目的是：(1)确定Rac1 GTPase是否是唾液腺分支形态发生和根尖-基础组织极性建立所必需的；(2)确定Rac激活是否能够促进人工构建的生物相容性PLGA纳米纤维支架上的唾液上皮细胞极化。我们将使用体外整体器官培养系统来检测小鼠胚胎颌下腺中rac1的功能，并使用实时延时或固定共聚焦显微镜来成像分支、形态发生和尖基极的动态变化。数据还将使用生化免疫印迹和QRT-PCR技术进行分析，并使用成像软件、图像分割和计算细胞图方法进行严格量化。唾液腺疾病，如Sjvgren综合征、涎腺癌和口干症，都以唾液腺功能低下为原因，这给受影响的个人、他们的家庭和整个医疗系统带来了巨大的负担。该项目所获得的早期唾液腺器官发生的信号机制的知识将对组织工程和再生医学领域以及未来研究RAC信号在唾液腺疾病中的功能和可能的解除调控具有重要意义。提案中使用的缩写：2D，二维；3D，三维；BM，基底膜；ECM，细胞外基质；GTP酶，鸟苷核苷酸交换因子；IB，免疫印迹；IF，免疫荧光；IP，免疫沉淀；PAR，分割缺陷蛋白；PLGA，聚乳酸-羟基乙酸共聚物；rac1，RAS相关的C3肉毒底物1；SMG，下颌下唾液腺；Tiam1，T细胞淋巴瘤侵袭和转移诱导蛋白1。 与公共卫生相关：严重的唾液腺功能低下是大多数接受唾液腺疾病治疗的患者的共同特征，如干燥综合征(SS)、唾液肿瘤、正在接受头颈癌治疗的成年人以及那些服用抗唾液酸药物的患者。目前以口干症为基础的治疗是不充分的，是暂时的，包括药物和味觉刺激剂。更长期的解决方案包括组织替代和再生治疗，然而，在工程化组织中维持和刺激上皮细胞组织和分化的重大挑战仍然存在。由于上皮细胞的分泌功能对器官功能至关重要，了解调节和维持组织结构和分化的细胞机制对于再生或工程功能组织至关重要。从这项资助中获得的数据将促进关于小GTP酶RAC在控制唾液腺分支形态发生中的新角色的基本科学知识。利用三维体外整体器官培养系统，我们将研究RAC GTPase介导的信号通路如何控制主要的发育过程，如唾液腺分支形态发生和组织极性的形成，这是唾液单向和受控流动所必需的。更重要的是，将研究RAC在生物兼容3D纳米纤维支架上促进唾液上皮细胞组织和极化方面的作用，朝着在生物兼容支架上创建用于再生医学的人工唾液腺结构的未来目标。

DESCRIPTION (provided by applicant): Profound salivary gland hypofunction and xerostomia (dry mouth) are increasingly common occurrences and are often the consequences of: 1) Sjogren's Syndrome, an autoimmune disease that targets saliva-secreting acinar tissue, 2) radiation and chemotherapeutic regimens for head and neck cancers and 3) adverse side effects from thousands of medications. Current xerostomia-based treatments are inadequate and temporary thus creating a significant clinical need for long-term solutions that include replacing irreversibly damaged or lost salivary tissue, with functional tissue grown on artificially engineered biocompatible scaffolds. However, the considerable challenge of stimulating and/or maintaining salivary epithelial cell differentiation in artificially engineered tissues has been hindered by the lack of knowledge of precise molecular signaling mechanisms that control glandular structure and function. The proposed study will address these gaps in our understanding of tissue formation by exploring the role of GTPase Rac signaling in the regulation of salivary gland branching morphogenesis and tissue polarization, processes that are crucial for development of a functional organ. The knowledge gained from these studies will further be used to investigate the role of Rac in promoting salivary epithelial cell organization and polarization on artificially engineered 3D nanoscale scaffolds, towards the future goal of generating functional artificial salivary gland constructs. The specific aims are to: (1) determine whether Rac1 GTPase is required for salivary gland branching morphogenesis and the establishment of apico-basal tissue polarity, and (2) determine whether Rac activation can promote salivary epithelial cell polarization on artificially engineered, biocompatible PLGA nanofibrous scaffolds. We will use an ex vivo whole organ culture system to examine Rac1 function in the mouse embryonic submandibular salivary gland and live time-lapse or fixed confocal microscopy to image the dynamics of branching morphogenesis and apico-basal polarity. Data will also be analyzed using biochemical immunoblotting and QRT-PCR techniques and rigorously quantified using imaging software, image segmentation and computational Cell graph methods. Salivary gland diseases like Sjvgren's syndrome, salivary adenocarcinomas and xerostomia, all feature salivary gland hypofunction as a cause, which poses an enormous burden to affected individuals, their families and the health care system as a whole. The knowledge gained from this project on the signaling mechanisms underlying early salivary gland organogenesis will be of considerable significance to the fields of tissue engineering and regenerative medicine and to future studies examining the function and possible deregulation of Rac signaling in salivary gland diseases. Abbreviations used in proposal: 2D, two-dimensional; 3D, three-dimensional; BM, basement membrane; ECM, extracellular matrix; GEF, guanine nucleotide exchange factor; GTPase, guanosine triphosphate hydroxylase; IB, immunoblotting; IF, immunofluorescence; IP, immunoprecipitation; Par, partitioning-defective proteins; PLGA, polylactic-co-glycolic acid polymer; Rac1, Ras-related C3 botulinum substrate 1; SMG, submandibular salivary gland; Tiam1, T-cell lymphoma invasion and metastasis-inducing protein 1. PUBLIC HEALTH RELEVANCE: Profound salivary gland hypofunction is a common feature in a majority of patients treated for salivary gland diseases such as Sjvgren's syndrome (SS), salivary neoplasms, adults being treated for head and neck cancer and those taking medications with anti-sialogogue sequelae. Current xerostomia-based treatments are inadequate, temporary and include pharmacological and gustatory stimulants. More long term solutions include tissue replacement and regeneration therapies, however, the significant challenge of maintaining and stimulating epithelial cell organization and differentiation in engineered tissues, remains. Since epithelial cell secretory function is crucial to organ function, understanding the cellular mechanisms regulating and maintaining tissue structure and differentiation is critical to regenerating or engineering functional tissues. The data obtained from this grant will advance basic scientific knowledge regarding novel roles for the small GTPase Rac in the control of salivary gland branching morphogenesis. Utilizing three-dimensional ex vivo whole organ culture systems, we will examine how Rac GTPase-mediated signaling pathways can control major developmental processes such as salivary gland branching morphogenesis and the formation of tissue polarity, an indispensable requirement for unidirectional and controlled flow of saliva. More significantly, the role of Rac in promoting salivary epithelial cell organization and polarization on biocompatible 3D nanofibrous scaffolds will be examined, towards the future goal of creating an artificial salivary gland construct on biocompatible scaffolds for use in regenerative medicine.