Supporting data for “Elucidating the Roles and Regulation of Coxsackievirus and Adenovirus Receptor in the Testis”

Spermatogenesis is a highly complex, coordinated cellular process during which highly specialized haploid male gametes developed from spermatogonial stem cells. Timely restructuring of the cell junctions between Sertoli cells and Sertoli cells, as well as junctions between Sertoli cells and germ cells are required throughout spermatogenesis. Although those cell junctions share common features with cell junctions in other tissues, they have their own unique characteristics. However, the exact components, architecture, and the regulatory mechanism for the unique characteristics of testicular cell junctions remain to be elucidated. Coxsackievirus and adenovirus receptor (CXADR) is a membrane protein that is expressed by germ cells and Sertoli cells. The canonical membrane-bound CXADR can mediate Sertoli-Sertoli and Sertoli-germ cell interactions in either a homophilic or heterophilic manner. Our previous studies have revealed that Sertoli cell (SC)-specific CXADR knockout exhibits impaired spermatogenesis, suggesting that SC-CXADR is indispensable for spermatogenesis. Cytokines such as transforming growth factor-β3 (TGF-β3) have been demonstrated to play essential roles in modulating the disassembly and reassembly of those cell junctions at stage VIII of the cycle of the seminiferous epithelium. This dissertation focuses on the the molecular regulation of CXADR by TGF-β3 and the structure role of CXADR in testicular cells. The expression patterns of TGF-β3 and CXADR are highly correlated. Our studies showed that TGF-β3 downregulates the expression level of Cxadr mRNA, as well as CXADR protein. The disappearance of CXADR at the site of the Sertoli-Sertoli cell interface was observed following TGF-β3 treatment. Inhibitor treatment and siRNA knockdown assays demonstrated that clathirin-mediated endocytosis is involved in TGF-β3-induced CXADR downregulation and mis-localization. TGF-β3 promotes CXADR protein degradation via ubiquitin-proteasome pathway. Besides, TGF-β3 induces the degradation of Cxadr mRNA partially via p38 mitogen-activated protein kinase signaling pathway. By combining the proximity-dependent biotin identification assay and glutathione S-transferase pull down assay, an interactome of CXADR was plotted. Several important structural components of cell junctions are identified as potential interacting partners of CXADR, including desmoplakin, cofilin, and filamin-A. Gene ontology enrichment analysis revealed that CXADR is linked those proteins to biological processes related to the intermediate filament and actin filament organization. This thesis study revealed the regulatory mechanism of TGF-β3 on CXADR expression and an interactome of CXADR. Data obtained in this thesis study sheds new insights on the molecular structure of cell junctions as well as the regulation of cell junction dynamics in spematogenesis.精子发生（Spermatogenesis）是一类高度复杂且协同有序的细胞过程，在此过程中，精原干细胞可分化为高度特化的单倍体雄性配子。在整个精子发生进程中，支持细胞之间、支持细胞与生殖细胞之间的细胞连接需适时进行重构。尽管这类细胞连接与其他组织中的细胞连接具有共性，但它们具备独特的自身特征。然而，睾丸细胞连接的具体组成、结构架构以及赋予其独特性的调控机制，仍有待阐明。柯萨奇病毒与腺病毒受体（Coxsackievirus and adenovirus receptor, CXADR）是一种膜蛋白，由生殖细胞与支持细胞表达。经典的膜结合型CXADR可通过同嗜性或异嗜性方式介导支持细胞-支持细胞以及支持细胞-生殖细胞间的相互作用。我们既往的研究显示，支持细胞特异性CXADR敲除会导致精子发生受损，这表明支持细胞来源的CXADR对精子发生不可或缺。诸如转化生长因子-β3（transforming growth factor-β3, TGF-β3）这类细胞因子，已被证实可在生精上皮周期第Ⅷ期调控上述细胞连接的解离与重建过程。本学位论文聚焦于TGF-β3对CXADR的分子调控机制，以及CXADR在睾丸细胞中的结构功能。TGF-β3与CXADR的表达模式具有高度相关性。我们的研究发现，TGF-β3可下调Cxadr mRNA以及CXADR蛋白的表达水平。经TGF-β3处理后，支持细胞-支持细胞连接界面处的CXADR信号消失。抑制剂处理与小干扰RNA（siRNA）敲低实验证实，网格蛋白介导的内吞作用参与了TGF-β3诱导的CXADR下调与定位异常。TGF-β3可通过泛素-蛋白酶体通路促进CXADR蛋白降解。此外，TGF-β3还可部分通过p38丝裂原活化蛋白激酶信号通路降解Cxadr mRNA。通过结合邻近依赖生物素标记鉴定实验与谷胱甘肽S-转移酶下拉实验，我们绘制了CXADR的相互作用组。若干重要的细胞连接结构组分被鉴定为CXADR的潜在相互作用伴侣，包括桥粒斑蛋白、丝切蛋白以及细丝蛋白A。基因本体富集分析显示，CXADR与这些蛋白所参与的生物学过程涉及中间丝与肌动蛋白丝的组织调控。本学位论文研究揭示了TGF-β3对CXADR表达的调控机制，以及CXADR的相互作用组。本研究所得数据为细胞连接的分子结构以及精子发生过程中细胞连接动态变化的调控机制提供了新的见解。