Rac GTPase signaling during embryonic salivary gland branching morphogenesis

胚胎唾液腺分支形态发生过程中的 Rac GTPase 信号传导

• 批准号: 8125444
• 负责人: Sharon Sequeira
• 金额: $ 4.92万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8125444
• 关键词: Abbreviations; Acinar Cell; Address; Adenocarcinoma; Adhesions; Adult; Adverse effects; Affect; Apical; Autoimmune Diseases; Basal Cell; Basement membrane; Basic Science; Biochemical; Biocompatible; Cell Communication; Cell Culture Techniques; Cell Differentiation process; Cell Polarity; Cell physiology; Cells; Clinical; Complex; Computer software; Confocal Microscopy; Data; Development; Developmental Process; Disease; Drosophila genus; Embryo; Engineering; Epithelial Cells; Extracellular Matrix; Family; Family health status; Future; Glycolates; Goals; Grant; Graph; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Head and Neck Cancer; Healthcare Systems; Image; Immunoblotting; Immunofluorescence Immunologic; Immunoprecipitation; Individual; Intercellular Junctions; Knowledge; Life; Maintenance; Mediating; Membrane Proteins; Methods; Mixed Function Oxygenases; Molecular; Monomeric GTP-Binding Proteins; Morphogenesis; Mus; Natural regeneration; Neoplasm Metastasis; Neoplasms; Organ; Organ Culture Techniques; Organogenesis; Pathology; Patients; Pharmaceutical Preparations; Polymers; Process; Production; Proteins; Radiation; Regenerative Medicine; Regimen; Regulation; Role; Saliva; Salivary; Salivary Gland Diseases; Salivary Gland Neoplasms; Salivary Gland Tissue; Salivary Glands; Signal Pathway; Signal Transduction; Sjogren' s Syndrome; Solutions; Structure; Syndrome; System; T-Cell Lymphoma; Techniques; Therapeutic; Tight Junctions; Time; Tissue Engineering; Tissues; Xerostomia; base; biocompatible polymer; botulinum; cell motility; gland development; imaging Segmentation; in vivo; inhibitor/antagonist; interest; member; nanofiber; nanoscale; novel; research study; rho; saliva secretion; scaffold; therapeutic development; three dimensional structure; two-dimensional

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.

描述（由申请人提供）：严重的唾液腺功能减退和口干症（口干）越来越常见，通常是以下原因造成的：1) 干燥综合征，一种针对唾液分泌腺泡组织的自身免疫性疾病，2) 放射和化疗方案头颈癌和 3) 数千种药物的不良副作用。目前基于口干症的治疗方法是不充分且暂时的，因此临床上迫切需要长期解决方案，包括用在人工设计的生物相容性支架上生长的功能组织来替代不可逆受损或丢失的唾液组织。然而，由于缺乏控制腺体结构和功能的精确分子信号传导机制的知识，在人工工程组织中刺激和/或维持唾液上皮细胞分化的巨大挑战受到阻碍。拟议的研究将通过探索 GTPase Rac 信号在唾液腺分支形态发生和组织极化调节中的作用来解决我们对组织形成理解中的这些空白，这些过程对于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 纳米级支架上促进唾液上皮细胞组织和极化中的作用，以实现生成功能性人工唾液腺结构的未来目标。具体目标是：(1) 确定 Rac1 GTPase 是否是唾液腺分支形态发生和顶端基底组织极性建立所必需的，以及 (2) 确定 Rac 激活是否可以促进人工工程、生物相容性 PLGA 上的唾液上皮细胞极化纳米纤维支架。我们将使用离体全器官培养系统来检查小鼠胚胎下颌下唾液腺中的 Rac1 功能，并使用实时延时或固定共聚焦显微镜来对分支形态发生和顶端基底极性的动态进行成像。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。 干燥综合征、唾液腺癌和口干症等唾液腺疾病均以唾液腺功能减退为病因，这给受影响的个人、其家庭和整个医疗保健系统带来了巨大的负担。从该项目中获得的有关早期唾液腺器官发生的信号传导机制的知识对于组织工程和再生医学领域以及未来研究唾液腺疾病中 Rac 信号传导的功能和可能的失调具有重要意义。提案中使用的缩写：2D，二维； 3D，三维； BM，基底膜； ECM，细胞外基质； GEF，鸟嘌呤核苷酸交换因子； GTP酶、三磷酸鸟苷羟化酶； IB，免疫印迹； IF，免疫荧光； IP，免疫沉淀； Par，分配缺陷蛋白； PLGA，聚乳酸-乙醇酸聚合物； Rac1，Ras相关的C3肉毒杆菌底物1； SMG，颌下唾液腺； Tiam1，T细胞淋巴瘤侵袭和转移诱导蛋白1。 公共卫生相关性：严重的唾液腺功能减退是大多数接受唾液腺疾病治疗的患者的一个共同特征，例如干燥综合征 (SS)、唾液肿瘤、接受头颈癌治疗的成人以及服用具有抗唾液酸后遗症药物的患者。目前基于口干症的治疗方法是不充分的、暂时的，并且包括药物和味觉兴奋剂。更长期的解决方案包括组织替代和再生疗法，然而，维持和刺激工程组织中的上皮细胞组织和分化的重大挑战仍然存在。由于上皮细胞分泌功能对器官功能至关重要，因此了解调节和维持组织结构和分化的细胞机制对于再生或改造功能组织至关重要。从这项资助中获得的数据将推进关于小 GTPase Rac 在控制唾液腺分支形态发生中的新作用的基础科学知识。利用三维离体全器官培养系统，我们将研究 Rac GTPase 介导的信号通路如何控制主要发育过程，例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不可或缺的要求。更重要的是，我们将研究 Rac 在生物相容性 3D 纳米纤维支架上促进唾液上皮细胞组织和极化的作用，以实现在生物相容性支架上创建人工唾液腺结构用于再生医学的未来目标。