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.
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