OVARIAN TUMOR NECROSIS FACTOR

本申请的目的是使用小鼠模型描述肿瘤坏死因子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刺激的小鼠和人类颗粒细胞的雌二醇分泌，而不改变cAMP的水平;这一观察结果与大鼠颗粒细胞中的cAMP水平急剧降低，以响应TNF不同。因此，TNF在人和小鼠颗粒细胞中的作用位点似乎是在cAMP后位点，而在大鼠模型中，TNF在cAMP前位点以及cAMP后位点抑制。第一个目标是确定TNF对小鼠卵巢颗粒细胞中促性腺激素刺激的蛋白激酶A活性的时间和剂量依赖性效应，这些效应将与雌二醇分泌相关。此外，TNF对PKA催化亚基转染的小鼠颗粒细胞中PKA活性的影响将被检查，并与雌二醇分泌相关。第二个目标是确定TNF对芳香化酶依赖性转录因子、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使用磷酸特异性抗体和蛋白质印迹来确定。使用电迁移率变动分析，将评估TNF对SF-1和CREB结合芳香酶启动子的能力的影响。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。这两种受体对TNF的抑制作用是否存在协同作用有待进一步研究。将用反义TNF RI或TNF RII转染野生型颗粒细胞（I型和II型TNF受体+/+），并评估TNF对芳香酶基因表达和雌二醇分泌的影响。 此外，RI（-/-）和RII（-/-）细胞将分别用RI和RII受体转染，并且将确定TNF对FAN途径（包括raf-1和ERK 1和2）的影响及其在抑制雌二醇分泌中的作用，因为这些激酶是TNFRI途径中信号传导的核心。总的来说，这些研究将提供新的见解TNF的行动，在调节卵巢芳香化酶的表达。

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.