Molecular Programming of Salivary Gland Gene Expression

描述(申请人提供)：适当的唾液腺功能对口腔健康至关重要。头颈癌的放射治疗经常会引起显著的副作用，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，这仍然是治疗的主要挑战。我们研究的长期目标是阐明参与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。自噬是动态平衡控制机制之一，是一种结构性的细胞分解代谢过程，细胞蛋白质和细胞器被吞噬，通过溶酶体机械消化和循环。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，急性低氧应激利用JNK1/Beclin 1依赖的途径诱导自噬，对低氧应激诱导的唾液细胞死亡提供短暂的保护。此外，我们还建立了AQP5-Cre转基因小鼠，其中Cre重组酶通过敲除水通道蛋白-5(AQP5)基因的外显子1在唾液腺泡细胞中表达，因为AQP5蛋白在唾液腺泡细胞中优先表达。利用这些知识和这些工具，我们建议研究自噬在调节唾液腺泡细胞在各种类型和/或严重的应激或损伤后的自我平衡控制、再生和适应性反应中的作用。我们的中心假设是：1)ATG5功能的丧失损害了唾液腺泡细胞对应激保持动态平衡控制的能力(目标1)，2)自噬在损伤过程中起到了短暂的细胞保护作用(目标2)，以及3)自噬、凋亡和死亡通路之间的串扰决定了应激或受损的唾液腺泡细胞的命运(目标3)。我们推测，自噬通过促进腺泡细胞的存活和再生来保护唾液腺免受应激和病理侮辱，作为一种应激适应反应。我们的目标将通过下列手段进行：(1)鉴定AQP5-Cre小鼠与Atg5f/f小鼠杂交后唾液腺泡细胞靶向ATG5失活的特征，这是我们实验计划所基于的独特来源；(2)使用慢性异丙肾上腺素注射模型确定自噬对动态平衡控制的贡献，并使用下颌下导管结扎/去结扎模型分别确定唾液腺泡细胞死亡和再生的贡献，以及(3)研究自噬与其他细胞死亡途径在潜在的唾液适应性反应中的串扰。这些研究将极大地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，它们将提供一个独特的机会来评估自噬靶向治疗的可行性，以改善或恢复人类损伤后的唾液腺(Dys)功能。 公共卫生相关性：自噬是一个与细胞死亡相关的生物过程，在正常生理和许多病理条件下具有重要意义，包括急慢性疾病状态和无数癌症。了解这些自噬过程如何调节对细胞应激的适应，对于开发针对患病唾液组织的有效疗法以及唾液腺组织替代的组织工程至关重要。拟议的研究不仅将开辟唾液研究领域，还将有助于制定一种策略，以防止头颈癌患者在接受放射治疗时因疾病或细胞死亡而导致唾液腺功能丧失。

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.