Molecular Programming of Salivary Gland Gene Expression

描述（由申请人提供）：适当的唾液腺功能对口腔健康至关重要。头颈部癌症的放射治疗通常会引起影响正常唾液腺功能的显著副作用，最常见的是口干症。目前的治疗无法永久恢复唾液功能，这仍然是一个主要的治疗挑战。我们的长期研究目标是阐明唾液腺稳态控制和再生的分子和细胞机制，特别是信号网络。自噬是体内平衡控制机制之一，是一种组成性细胞分解代谢降解过程，其中细胞蛋白质和细胞器被吞噬，通过溶酶体机制消化并再循环。自噬相关基因Atg 5已被确定为自噬中不可或缺的参与者。我们的初步数据表明，急性缺氧应激利用JNK 1/Beclin 1依赖性途径诱导自噬，对缺氧应激引起的唾液细胞死亡提供短暂的保护。此外，我们产生了Aqp 5-Cre转基因小鼠，其中Cre重组酶通过敲除水通道蛋白-5（Aqp 5）基因的外显子1而靶向在唾液腺泡细胞中表达，因为Aqp 5蛋白优先在唾液腺泡细胞中表达。利用这些知识和这些工具，我们建议研究自噬在管理稳态控制，再生和适应性反应后的压力或损伤的各种类型和/或严重程度的唾液腺泡细胞的作用。我们的主要假设是：1）Atg 5功能的丧失损害了唾液腺泡细胞针对应激维持稳态控制的能力（目的1），2）自噬在损伤期间起短暂的细胞保护作用（目的2），以及3）自噬、凋亡和坏死途径之间的串扰决定应激或损伤的唾液腺泡细胞的命运（目的3）。我们推测自噬通过促进腺泡细胞的存活和再生作为一种应激适应反应来保护唾液腺免受应激和病理损伤。我们将通过以下方式实现我们的目标：（1）表征来自Aqp 5-Cre小鼠与Atg 5 f/f小鼠杂交的具有唾液腺泡靶向Atg 5失活的小鼠，其代表我们的实验计划所基于的独特来源，（2）使用慢性异丙肾上腺素-肾上腺素受体拮抗剂，测定自噬对体内平衡控制的贡献。注射模型和使用下颌下腺导管结扎/去结扎模型的唾液腺泡细胞死亡和再生，和（3）研究自噬与其他细胞死亡途径在唾液适应性反应中的相互干扰。这些研究将大大提高我们对唾液腺在有害环境中的自我平衡控制和/或再生的理解。此外，他们将提供一个独特的机会，以评估自噬靶向治疗的可行性，以改善或恢复唾液腺（dys）功能损伤后的人。 公共卫生相关性：自噬是一种与细胞死亡相关的生物学过程，在正常生理学和许多病理学状况中具有重要意义，包括急性和慢性疾病状态以及无数癌症。了解这些自噬过程如何调节细胞应激的适应，对于开发针对患病唾液组织的有效治疗方法以及用于组织替代的唾液腺组织工程至关重要。拟议的研究不仅将打开唾液研究的领域，而且还将有助于制定一项战略，以防止接受放射治疗的头颈癌患者因疾病或细胞死亡而丧失唾液腺功能。

DESCRIPTION (provided by applicant): Proper salivary gland function is critical for oral health. Radiation therapy for head and neck cancer often causes notable side effects that impact normal salivary gland function, most commonly xerostomia. Current therapies are unable to permanently restore salivary function, which remains a major therapeutic challenge. The long-term goal of our research is to elucidate the molecular and cellular mechanisms, in particular the signaling networks, involved in salivary gland homeostatic control and regeneration. One of the homeostatic control mechanisms, autophagy, is a constitutive cellular catabolic degradation process whereby cellular proteins and organelles are engulfed, digested through the lysosomal machinery and recycled. The autophagy-related 5 gene, Atg5, has been established as an indispensable player in autophagy. Our preliminary data suggest that acute hypoxic stress utilizes the JNK1/Beclin 1-dependent pathway to induce autophagy, providing transient protection against hypoxic stress-elicited cell death in salivary cells. Moreover, we generated Aqp5-Cre transgenic mice, in which the Cre recombinase was targeted to express in salivary acinar cells by being knocked in the exon 1 of Aquaporin-5 (Aqp5) gene, as Aqp5 protein is preferentially expressed in salivary acinar cells. Utilizing this knowledge and these tools, we propose to investigate the role of autophagy in governing homeostatic control, regeneration and adaptive responses following stress or injury of various types and/or severity to salivary acinar cells. Our central hypotheses are: 1) Loss-of-Atg5-function impairs the ability of salivary acinar cells to maintain homeostatic control against stress (Aim 1), 2) Autophagy plays a transient cytoprotective role during injury (Aim 2), and 3) Crosstalk among autophagic, apoptotic and necrotic pathways decides the fate of stressed or injured salivary acinar cells (Aim 3). We postulate that autophagy protects salivary glands from stress and pathologic insults by promoting acinar cell survival and regeneration as a stress adaptation response. Our objective will be pursued through the following means: (1) Characterize mice with salivary acinar-targeted Atg5 inactivation from crossing Aqp5-Cre mice with Atg5f/f mice, representing a unique source on which our experimental plan is based, (2) Determine the contribution of autophagy to homeostatic control using a chronic isoproterenol- injection model and to salivary acinar cell death and regeneration using a submandibular ductal ligation/de- ligation model, respectively, and (3) Investigate crosstalk of autophagy with other cell death pathways in underlying salivary adaptive responses. These studies will greatly improve our understanding of salivary gland homeostatic control and/or regeneration in a deleterious environment. In addition, they will provide a unique opportunity to evaluate the feasibility of autophagy-targeted therapies to ameliorate or restore salivary gland (dys)function following injury in human. PUBLIC HEALTH RELEVANCE: Autophagy is a biological process, associated with cell death, which has important implications in normal physiology and many pathological conditions, including acute and chronic disease states and a myriad of cancers. Understanding how these autophagic processes modulate adaptation to cellular stress is essential for developing effective therapeutics to target diseased salivary tissues, as well as tissue engineering of salivary glands for tissue replacement. The proposed studies will not only open the field of salivary research, but will also assist in developing a strategy to prevent the loss of salivary gland function resulting from disease or cell death in head and neck cancer patients undergoing radiation therapy.