OVARIAN TUMOR NECROSIS FACTOR

本应用程序的目的是描述肿瘤坏死因子α (TNF)和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，TNF抑制FSH和LH刺激小鼠和人颗粒细胞分泌雌二醇，但不改变cAMP水平；这一观察结果与大鼠颗粒细胞的观察结果不同，在大鼠颗粒细胞中，cAMP水平因TNF而急剧降低。因此，TNF在人和小鼠颗粒细胞中的作用位点似乎位于cAMP后位点，而在大鼠模型中，TNF在cAMP前和cAMP后位点均有抑制作用。第一个目的是确定TNF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性作用，这些作用将与雌二醇分泌相关。此外，TNF对转染PKA催化亚基的小鼠颗粒细胞中PKA活性的影响将被检测并与雌二醇分泌相关。第二个目标将确定TNF对芳香化酶依赖性转录因子、甾体生成因子-1 （SF-1）和cAMP反应元件结合蛋白（CREB）的影响，包括它们的表达、磷酸化和与芳香化酶启动子的结合。TNF对SF-1和CREB的表达和磷酸化的影响将通过磷酸化特异性抗体和Western blots来确定。利用电移动性转移试验，将评估TNF对SF-1和CREB结合芳香化酶启动子能力的影响。第三个目标是确定TNF对芳香化酶基因表达和分泌的影响是否通过TNF 1型和/或2型受体介导。这两种受体之间是否存在协同作用以抑制TNF的作用将被确定。野生型颗粒细胞（TNF受体I型和II型为+/+）将被反义TNF RI或TNF RII转染，并评估TNF对芳香化酶基因表达和雌二醇分泌的影响。此外，RI（-/-）和RII（-/-）细胞将分别转染RI和RII受体，TNF对FAN通路包括raf-1、ERK1和2的影响及其在抑制雌二醇分泌中的作用将被确定，因为这些激酶是TNFRI通路信号传导的核心。总的来说，这些研究将为TNF在调节卵巢芳香化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.