Synergistic Effect of Interferon-Gamma and Tumor Necrosis Factor-Alpha on Coxsackievirus and Adenovirus Receptor Expression: An Explanation of Cell Sloughing During Testicular Inflammation in Mice
Synergistic Effect of Interferon-Gamma and Tumor Necrosis Factor-Alpha on Coxsackievirus and Adenovirus Receptor Expression: An Explanation of Cell Sloughing During Testicular Inflammation in Mice
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DOI：
10.1095/biolreprod.113.113407
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发表时间：
2014-03-01
影响因子：
3.6
通讯作者：
Lui, Wing-Yee
中科院分区：
文献类型：
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作者：
Gao, Ying;Lui, Wing-Yee
柯萨奇病毒和腺病毒受体(CAR)是一种表达在支持细胞和生殖细胞上的连接分子。它介导Sertoli-Germ细胞的黏附，促进前/细线期精母细胞跨越血-睾丸屏障的迁移，提示基于CAR的细胞黏附和迁移在精子发生中是至关重要的。干扰素-γ(IFNG)和肿瘤坏死因子-α(TNF)是两种主要的细胞因子，在睾丸炎症过程中会升高，导致生育力下降。我们研究了干扰素和肿瘤坏死因子对睾丸细胞黏附的破坏作用机制。我们已经证明，IFNG和肿瘤坏死因子(IFNG+TNF)联合治疗通过下调CAR的mRNA和蛋白水平而发挥协同作用。免疫荧光染色显示，IFNG+肿瘤坏死因子处理能有效地去除细胞接触部位的CAR。利用抑制剂和免疫共沉淀，我们证实了IFNG+肿瘤坏死因子通过泛素-蛋白酶体和NFKB途径介导CAR蛋白的降解。阻断泛素-蛋白酶体途径显著抑制CAR的降解，CAR在细胞-细胞接触部位的重现表明。此外，IFNG+肿瘤坏死因子通过转录调控降低CAR基因的表达。突变研究表明，IFNG+肿瘤坏死因子诱导的CAR抑制是通过抑制基础转录实现的。凝胶迁移率改变分析和染色质免疫沉淀分析进一步证实，IFNG+TNF处理不仅抑制了基础转录因子的结合，而且促进了NFKB亚基和Sp1(负调控因子)与CAR启动子区域的结合。综上所述，IFNG+肿瘤坏死因子治疗显著下调CAR的表达，这为解释在睾丸炎症过程中，上皮细胞的脱落是如何通过丧失基于CAR的细胞黏附而介导的。
Coxsackievirus and adenovirus receptor (CAR) is a junction molecule that expresses on Sertoli and germ cells. It mediates Sertoli-germ cell adhesion and facilitates migration of preleptotene/leptotene spermatocytes across the blood-testis barrier, suggesting that CAR-based cell adhesion and migration are crucial for spermatogenesis. Interferon-gamma (IFNG) and tumor necrosis factor alpha (TNF) are two major cytokines that are elevated during testicular inflammation and cause reduced fertility. We investigated the mechanism by which IFNG and TNF exert their disruptive effects on testicular cell adhesion. We have demonstrated that combined treatment with IFNG and TNF (IFNG+TNF) exerts a synergistic effect by downregulating CAR mRNA and protein levels. Immunofluorescence staining revealed that IFNG+TNF treatment effectively removes CAR from the site of cell-cell contact. Using inhibitor and co-immunoprecipitation, we confirmed that IFNG+TNF mediates CAR protein degradation via ubiquitin-proteasome and NFKB pathways. Blockage of ubiquitin-proteasome pathway significantly inhibits CAR degradation, as indicated by the reappearance of CAR at the site of cell-cell contact. Additionally, IFNG+TNF reduces CAR mRNA via transcriptional regulation. Mutational studies have shown that IFNG+TNF-induced CAR repression is achieved by suppression of the basal transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays further confirmed that IFNG+TNF treament not only inhibits binding of the basal transcription factors but also promotes binding of NFKB subunits and Sp1 (negative regulators) to the CAR promoter region. Taken together, IFNG+TNF treatment significantly downregulates CAR expression, which provides an explanation of how cell sloughing in the epithelium mediates, by loss of CAR-based cell adhesion, during testicular inflammation.