Sirianni Rosa, Chimento Adele, De Luca Arianna, Zolea Fabiana, Carpino Amalia, Rago Vittoria, Maggiolini Marcello, Andò Sebastiano, Pezzi Vincenzo
Department of Pharmaco-Biology, University of Calabria, 87036 Arcavacata di Rende (CS), Italy.
J Biol Chem. 2009 Oct 16;284(42):28905-16. doi: 10.1074/jbc.M109.041020. Epub 2009 Aug 13.
Our recent studies have revealed that estrogens stimulate an autocrine mechanism determining Leydig tumor cell proliferation. Estrogen overproduction is due to an elevated steroidogenic factor-1 (SF-1) expression and cAMP-response element-binding protein (CREB) phosphorylation, both inducing aromatase overexpression. Although we have shown that increased SF-1 expression depends mainly on higher local insulin-like growth factor I production, the mechanisms and factors determining increased CREB activation in Leydig tumor cells are not completely understood. In this study, we investigated the role of cyclooxygenase-2 (COX-2) in CREB dependent-aromatase expression in Leydig tumor cells. We found that COX-2 is expressed in rat and human Leydigiomas as well as in the rat Leydig tumor cell line R2C, but not in normal testis. Our data indicate that in R2C cells the COX-2-derived prostaglandin E2 (PGE2) binds the PGE2 receptor EP4 and activates protein kinase A (PKA) and ultimately CREB. Inhibitors for COX-2 (NS398), EP4 (AH23848), and PKA (H89) decreased aromatase expression and activity as a consequence of a decreased phosphorylated CREB recruitment to the PII promoter of the aromatase gene. The COX-2/PGE2/PKA pathway also seems to be involved in aromatase post-translational activation, an observation that requires further studies. The reduction in aromatase activity was responsible for a drop in estrogen production and subsequent reduction in cyclin E expression resulting in a decrease in tumor Leydig cell proliferation. Furthermore, COX-2 silencing caused a significant decrease in CREB phosphorylation, aromatase expression, and R2C cell proliferation. These novel findings clarify the mechanisms involved in the growth of Leydig cell tumors and should be taken into account in determining new therapeutic approaches.
我们最近的研究表明，雌激素会刺激一种自分泌机制，该机制决定了睾丸间质细胞瘤细胞的增殖。雌激素过量产生是由于类固醇生成因子-1（SF-1）表达升高以及环磷酸腺苷反应元件结合蛋白（CREB）磷酸化，二者均诱导芳香化酶过度表达。尽管我们已经表明SF-1表达增加主要取决于局部胰岛素样生长因子I产量的提高，但决定睾丸间质细胞瘤细胞中CREB激活增加的机制和因素尚未完全明确。在本研究中，我们调查了环氧合酶-2（COX-2）在睾丸间质细胞瘤细胞中CREB依赖性芳香化酶表达中的作用。我们发现COX-2在大鼠和人类睾丸间质细胞瘤以及大鼠睾丸间质细胞瘤细胞系R2C中表达，但在正常睾丸中不表达。我们的数据表明，在R2C细胞中，COX-2衍生的前列腺素E2（PGE2）与PGE2受体EP4结合并激活蛋白激酶A（PKA），最终激活CREB。COX-2抑制剂（NS398）、EP4抑制剂（AH23848）和PKA抑制剂（H89）会降低芳香化酶的表达和活性，这是因为磷酸化CREB募集到芳香化酶基因的PII启动子的数量减少。COX-2/PGE2/PKA途径似乎也参与了芳香化酶的翻译后激活，这一观察结果需要进一步研究。芳香化酶活性的降低导致雌激素产生减少，随后细胞周期蛋白E表达降低，从而导致肿瘤睾丸间质细胞增殖减少。此外，COX-2沉默导致CREB磷酸化、芳香化酶表达和R2C细胞增殖显著降低。这些新发现阐明了睾丸间质细胞瘤生长所涉及的机制，在确定新的治疗方法时应予以考虑。
