OVARIAN TUMOR NECROSIS FACTOR

本研究的目的是利用小鼠模型研究肿瘤坏死因子α及其受体系统抑制促性腺激素刺激的卵巢颗粒细胞雌二醇分泌的机制。初步数据表明，在小鼠和人的颗粒细胞中，肿瘤坏死因子抑制FSH和黄体生成素刺激的雌二醇的分泌，而不改变cAMP的水平，这一观察结果不同于大鼠颗粒细胞的cAMP水平因肿瘤坏死因子的反应而显著降低。因此，在人类和小鼠颗粒细胞中，肿瘤坏死因子的作用部位似乎在夏令营后，而在大鼠模型中，肿瘤坏死因子抑制在夏令营前和夏令营后部位。第一个目的是确定肿瘤坏死因子对促性腺激素刺激的小鼠卵巢颗粒细胞蛋白激酶A活性的时间和剂量依赖效应，这些效应将与雌二醇的分泌有关。此外，还将检测肿瘤坏死因子对转染PKA催化亚基的小鼠颗粒细胞中PKA活性的影响，并将其与雌二醇分泌相关联。第二个目的是确定肿瘤坏死因子对芳香酶依赖的转录因子、类固醇生成因子-1(SF-1)和cAMP反应元件结合蛋白(CREB)的影响，包括它们的表达、磷酸化和与芳香酶启动子的结合。肿瘤坏死因子对SF-1和CREB表达和磷酸化的影响将通过磷酸化特异性抗体和Western blotts来确定。利用电子迁移率位移分析，将评估肿瘤坏死因子对SF-1和CREB结合芳香酶启动子能力的影响。第三个目的将确定肿瘤坏死因子对芳香酶基因表达和分泌的影响是否通过肿瘤坏死因子1型和/或2型受体介导。这两种受体之间对肿瘤坏死因子的抑制作用是否存在协同作用将被确定。将反义肿瘤坏死因子RI或肿瘤坏死因子受体II导入野生型颗粒细胞，观察肿瘤坏死因子对芳香酶基因表达和雌二醇分泌的影响。此外，RI(-/-)和RII(-/-)细胞将分别导入RI和RII受体，而肿瘤坏死因子对包括RAF-1、ERK1和2在内的FAN通路的影响以及它们在抑制雌二醇分泌中的作用将被确定，因为这些通路是TNFRI通路中的信号中心。总体而言，这些研究将为肿瘤坏死因子在调节卵巢芳香酶表达中的作用提供新的见解。

The objective of this application is to delineate the mechanisms by which tumor necrosis factor alpha (TNF) and in receptor system inhibit gonadotropin-stimulated ovarian granulosa cell estradiol secretion using a mouse model. Preliminary data indicate that TNF inhibits FSH and LH stimulated estradiol secretion by granulosa cells in the mouse and human without altering the level of cAMP; this observation is different than that in rat granulosa cells in which cAMP levels are drastically reduced in response to TNF. Thus, the site of action of TNF in humans and mouse granulosa cells appears to be at post cAMP sites whereas in the rat model TNF inhibits at sites prior to cAMP as well as at post cAMP sites. The first aim will determine the time and dose dependent effects of TNF on gonadotropin stimulated protein kinase A activity in mouse ovarian granulosa cells, and those effects will be correlated with estradiol secretion. In addition, the effects of TNF on PKA activity in mouse granulosa cells transfected with a PKA catalytic subunit will be examined and correlated with estradiol secretion. The second aim will determine the effects of TNF on aromatase dependent transcription factors, steroidogenic factor-1 (SF-1) and cAMP response element binding protein (CREB) including their expression, phosphorylation and binding to the aromatase promoter. The effects of TNF on the expression and phosphorylation of SF-1 and CREB will be determined using phospho- specific antibodies and Western blots. Using electromobility shift assays, the effects of TNF on the ability of SF-1 and CREB to bind the aromatase promoter will be assessed. The third aim will determine if the effects of TNF on aromatase gene expression and secretion are mediated via TNF type 1 and/or type 2 receptors. Whether cooperativity exists between these two receptors for TNF's inhibitory action will be determined. Wild type granulosa cells (+/+ for TNF receptors type I and II) will be transfected with antisense TNF RI or TNF RII) and effects of TNF on aromatase gene expression and estradiol secretion will be assessed. In addition, RI(-/-) and RII(-/-) cells will be transfected with RI and RII receptor, respectively, and the effects of TNF on the FAN pathway including raf-1, and ERK1 and 2 and their role in inhibition estradiol secretion will be determined since these kinases are a centerpiece for signaling in the TNFRI pathway. Overall, these studies will provide novel insights into TNF action in regulating aromatase expression in the ovary.