Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas, USA.
Biol Reprod. 2011 Dec;85(6):1203-15. doi: 10.1095/biolreprod.111.093484. Epub 2011 Aug 10.
Our previous studies showed that the prototypical testicular toxic phthalate monoester, mono-(2-ethylhexyl) phthalate (MEHP), suppresses Sertoli cell TIMP2 levels and allows for the activation of MMP2 in seminiferous epithelium. Activation of MMP2 is important for triggering germ cell apoptosis and instigating germ cell detachment from Sertoli cells. These novel findings led us to examine the transcriptional regulation of the Timp2 gene that accounts for the decrease in Sertoli cell TIMP2 levels following MEHP exposure. Sequential deletion of the Timp2 5'-upstream activating sequence (1200 bp) was used to survey transcriptional activation in the Timp2 promoter region in response to MEHP. Results indicate that under control conditions in rat Sertoli cells, CCAAT enhancer-binding protein alpha (CEBPA) acts as a transactivator to initiate Timp2 gene transcription, and its action is deactivated by exposure to MEHP. By contrast, MYC protein acts as an inhibitor of Timp2 gene transcription, and its activity is increased after MEHP treatment. Addition of follicle-stimulating hormone (FSH) to cells causes translocation of CEBPA into the Sertoli cell nucleus and rescues MEHP-suppressed TIMP2 levels. Down-regulation of TIMP2 expression by MEHP exposure is blocked by forskolin (a cAMP-elevating agent), suggesting that the decrease in Sertoli cell TIMP2 expression following MEHP exposure is cAMP-dependent. Taken together, these data indicate that MEHP both disrupts the FSH-stimulated cAMP signaling pathway and activates the inhibitory signaling mediated by MYC protein, to ultimately account for the cellular mechanism underlying the decreased expression of TIMP2 in Sertoli cells.
我们之前的研究表明，典型的睾丸毒性邻苯二甲酸单酯，邻苯二甲酸二（2-乙基己基）酯（MEHP），抑制支持细胞 TIMP2 水平，并允许激活精子上皮中的 MMP2。MMP2 的激活对于触发生殖细胞凋亡和促使生殖细胞从支持细胞上脱离至关重要。这些新发现促使我们检查 Timp2 基因的转录调控，该基因解释了 MEHP 暴露后支持细胞 TIMP2 水平的降低。使用 Timp2 启动子区域的转录激活来连续删除 Timp2 5'-上游激活序列（1200bp）。结果表明，在大鼠支持细胞的对照条件下，CCAAT 增强子结合蛋白α（CEBPA）作为转录激活物，启动 Timp2 基因转录，其作用被 MEHP 暴露所失活。相比之下，MYC 蛋白作为 Timp2 基因转录的抑制剂，其活性在 MEHP 处理后增加。向细胞中添加卵泡刺激素（FSH）会导致 CEBPA 易位到支持细胞核中，并挽救 MEHP 抑制的 TIMP2 水平。MEHP 暴露下调 TIMP2 表达被 forskolin（一种 cAMP 升高剂）阻断，表明 MEHP 暴露后支持细胞 TIMP2 表达的降低是 cAMP 依赖性的。综上所述，这些数据表明，MEHP 既破坏了 FSH 刺激的 cAMP 信号通路，又激活了由 MYC 蛋白介导的抑制信号，最终解释了支持细胞中 TIMP2 表达降低的细胞机制。
