Molecular Programming of Salivary Gland Gene Expression

描述（申请人提供）：正常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明显的副作用，影响正常的唾液腺功能，最常见的是口干症。目前的治疗方法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。自噬是一种稳态控制机制，是一种基本的细胞分解代谢降解过程，细胞蛋白质和细胞器被吞噬，通过溶酶体消化并循环利用。自噬相关基因Atg5在自噬过程中起着不可或缺的作用。我们的初步数据表明，急性缺氧应激利用JNK1/Beclin 1依赖性途径诱导自噬，为唾液细胞提供短暂的保护，防止缺氧应激引起的细胞死亡。此外，我们构建了Aqp5-Cre转基因小鼠，由于Aqp5蛋白在唾液腺泡细胞中优先表达，通过敲除Aqp5基因的外显子1，将Cre重组酶靶向在唾液腺泡细胞中表达。利用这些知识和工具，我们建议研究自噬在不同类型和/或严重程度的唾液腺泡细胞应激或损伤后调节稳态控制、再生和适应性反应中的作用。我们的主要假设是：1)atg5功能的丧失削弱了唾液腺泡细胞对应激维持稳态控制的能力（Aim 1）； 2)自噬在损伤过程中起着短暂的细胞保护作用（Aim 2）； 3)自噬、凋亡和坏死途径之间的串扰决定了应激或损伤唾液腺泡细胞的命运（Aim 3）。我们假设自噬通过促进腺泡细胞存活和再生作为应激适应反应来保护唾液腺免受应激和病理损伤。我们将通过以下途径实现我们的目标：(1)通过Aqp5-Cre小鼠与Atg5f/f小鼠杂交，对唾液腺泡靶向Atg5失活小鼠进行表征，这代表了我们实验计划所依据的独特来源；(2)使用慢性异丙肾上腺素注射模型确定自噬对稳态控制的贡献，并使用下颌下导管结扎/去结扎模型分别确定自噬对唾液腺泡细胞死亡和再生的贡献。(3)研究自噬与其他细胞死亡途径在潜在唾液适应性反应中的串扰。这些研究将大大提高我们对有害环境中唾液腺稳态控制和/或再生的理解。此外，它们将提供一个独特的机会来评估自噬靶向治疗改善或恢复人类损伤后唾液腺功能的可行性。

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.