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

<p> </p> <p>Spermatogenesis is a highly complex, coordinated cellular process during which highly specialized haploid male gametes developed from spermatogonial stem cells.<br> 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.<br> Although those cell junctions share common features with cell junctions in other tissues, they have their own unique characteristics.<br> However, the exact components, architecture, and the regulatory mechanism for the unique characteristics of testicular cell junctions remain to be elucidated.<br> <br> Coxsackievirus and adenovirus receptor (CXADR) is a membrane protein that is expressed by germ cells and Sertoli cells.<br> The canonical membrane-bound CXADR can mediate Sertoli-Sertoli and Sertoli-germ cell interactions in either a homophilic or heterophilic manner.<br> Our previous studies have revealed that Sertoli cell (SC)-specific CXADR knockout exhibits impaired spermatogenesis, suggesting that SC-CXADR is indispensable for spermatogenesis.<br> 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.<br> <br> This dissertation focuses on the the molecular regulation of CXADR by TGF-β3 and the structure role of CXADR in testicular cells.<br> <br> The expression patterns of TGF-β3 and CXADR are highly correlated.<br> Our studies showed that TGF-β3 downregulates the expression level of Cxadr mRNA, as well as CXADR protein.<br> The disappearance of CXADR at the site of the Sertoli-Sertoli cell interface was observed following TGF-β3 treatment.<br> Inhibitor treatment and siRNA knockdown assays demonstrated that clathirin-mediated endocytosis is involved in TGF-β3-induced CXADR downregulation and mis-localization.<br> TGF-β3 promotes CXADR protein degradation via ubiquitin-proteasome pathway.<br> Besides, TGF-β3 induces the degradation of Cxadr mRNA partially via p38 mitogen-activated protein kinase signaling pathway.<br> <br> By combining the proximity-dependent biotin identification assay and glutathione S-transferase pull down assay, an interactome of CXADR was plotted.<br> Several important structural components of cell junctions are identified as potential interacting partners of CXADR, including desmoplakin, cofilin, and filamin-A.<br> Gene ontology enrichment analysis revealed that CXADR is linked those proteins to biological processes related to the intermediate filament and actin filament organization.<br> <br> </p> <p>This thesis study revealed the regulatory mechanism of TGF-β3 on CXADR expression and an interactome of CXADR.<br> 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.<br> </p>