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）：深刻的唾液腺功能低下和静态（口干）越来越常见，通常是：1）Sjogren综合征，一种靶向唾液腺泡的自身免疫性疾病，靶向分泌唾液的腺泡，2）辐射和化学治疗方案对头部和3次不良范围造成不利的治疗方案和3）不良侧面的副作用。当前基于静脉的治疗是不足和临时的，因此对长期解决方案产生了巨大的临床需求，包括替换不可逆损害或丢失的唾液组织，并且在人工工程的生物相容性脚手架上生长的功能性组织。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，刺激和/或维持人为工程组织中唾液上皮细胞分化的挑战已阻碍。拟议的研究将通过探索GTPase RAC信号传导在调节唾液腺分支形态发生和组织极化的调节中的作用，这是我们对组织形成的理解中的这些差距，这对于功能器官的发展至关重要。从这些研究中获得的知识将进一步用于研究RAC在促进唾液上皮细胞组织中的作用，并在人工设计的3D纳米级支架上进行两极分化，以实现产生功能性人工唾液腺结构的未来目标。具体目的是：（1）确定Rac1 GTPase是否需要唾液腺分支形态发生和建立Apico-Basal组织极性，并且（2）（2）确定RAC激活是否可以促进唾液上皮细胞在人工设计的，生物相容性的PLGA Nananobibrous Scaffolds上促进唾液上皮细胞极化。我们将使用体内整个器官培养系统来检查小鼠胚胎下颌唾液腺中的RAC1功能，并实时延时或固定的共聚焦显微镜，以模拟分支形态发生和apico-basal极性的动力学。还将使用生化免疫印迹和QRT-PCR技术对数据进行分析，并使用成像软件，图像分割和计算细胞图方法严格量化数据。 唾液腺疾病，例如Sjvgren综合征，唾液腺癌和静脉疾病，所有这些疾病都以唾液腺功能低下为原因，这为受影响的个人，家庭和整个医疗保健系统造成了巨大的负担。从该项目获得的关于早期唾液腺器官发生的信号传导机制所获得的知识将对组织工程和再生医学领域具有相当大的意义，以及未来研究研究功能和可能放松RAC Signal Decrumation唾液腺内疾病的功能和可能放松管制的研究。提案中使用的缩写：2D，二维； 3D，三维； BM，地下膜； ECM，细胞外基质； GEF，鸟嘌呤核苷酸交换因子； GTPase，三磷酸三磷酸羟化酶； IB，免疫印迹；如果，免疫荧光； IP，免疫沉淀； PAR，分配缺陷蛋白； PLGA，聚乳糖 - 糖酸聚合物； Rac1，与RAS相关的C3肉毒杆菌底物1； SMG，下颌下唾液腺； TIAM1，T细胞淋巴瘤侵袭和诱导转移的蛋白1。 公共卫生相关性：大多数接受唾液腺疾病治疗的患者，例如SJVGREN综合征（SS），唾液性肿瘤，成年人接受过头颈癌和患有抗Sialogogue Secelae的药物治疗的大多数患者，唾液腺功能低下是一个共同特征。当前基于静脉的治疗不足，临时性，包括药理学和味觉刺激剂。但是，更多的长期解决方案包括组织置换和再生疗法，但是，仍然存在维持和刺激上皮细胞组织的重大挑战以及工程组织中的分化。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工程功能组织至关重要。从该赠款中获得的数据将促进有关小型GTPase RAC在控制唾液腺分支形态发生中的新作用的基本科学知识。利用三维的全体器官培养系统，我们将研究RAC GTPase介导的信号传导途径如何控制主要的发育过程，例如唾液腺分支分支形态发生以及组织极性的形成，这是对唾液流动和控制流动流的不可或缺的要求。更重要的是，将研究RAC在促进唾液上皮细胞组织中的作用以及在生物相容性的3D纳米纤维支架上的极化，以实现未来的目标，即在生物相容性脚手架上创建人工唾液腺构造，以用于再生药物。