This proposal tests the hypothesis that the TCDD-induced decreased in hCG bioactivity in human trophoblast cells is mediated by the AhR and involves a TCDD-dependent alteration in one or more steps in the normal biosynthetic pathway of hCG production. We will characterize the AhR- and TCDD-signaling pathways in syncytiotrophoblast cells and to determine the mechanism(s) by which TCDD affects the bioactivity of hCG produced by these cells. Our working hypothesis has evolved from the result of a series of experiments has evolved from the results of a series of experiments in the previous project period. These results show that TCDD induces a decreases in the ratio of bioactive (B) to immunoreactive (I) CG that is observed with human syncytiotrophoblast cells are treated in vitro and when pregnant macaques are treated in vivo. Given that production of bioactive hCG is a complex process involving numerous integrated and regulated steps and the exact site of action at which TCDD exerts this effect is currently unknown, we will carry out a series of integrated studies designed to examine the effect of TCDD on each step of this pathway. This progression of experimental aims will allow us to define the specific mechanistic event(s) responsible for the TCDD-induced decrease in hCG bioactivity. Experiments described in the first specific aim will identify and characterize the AhR signaling system in syncytiotrophoblast cells, they will examine the role of the AhR in the TCDD-dependent decrease in bioactive hCG secretion and determine the effect of TCDD on hCG gene expression.
The second aim will determine whether the changes in AP-1 activity induced by TCDD are AhR-dependent Whether or not the TCDD effects on AP-1 activity are AhR-dependent, we will conduct experiments to address the regulation of autocrine/paracrine actions of EGF/EGFR by AP-1 in TCDD- treated syncytiotrophoblast cells. The objective of third specific aim is to investigate the biochemical changes in the hCG molecules which are produced by TCDD-treated syncytiotrophoblast cells. We will determine if the conformation alterations in hCG that result in decreased bioactivity are consistent with the mechanisms of TCDD toxicity determined in Specific Aims 1 and 2. In the fourth specific aim, we will investigate the intracellular localization of hCG subunits in syncytiotrophoblast cells following TCDD treatment, and test the hypotheses that the decrease in hCG bioactivity induced by TCDD is associated with altered intracellular distribution of the hCG subunits.
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