PARENT SESSION
IMPLANTATION AND PREGNANCY - B

Tuesday, August 3, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(494) Erk1/2 REGULATES MMP PRODUCTION IN JEG-3 CELL CULTURE.

Andrassy, Julie ¹, Leco, Kevin ², Yang, Kaiping³, Watson, Andrew³, ¹ University of Western Ontario, London, ON, Canada² University of Western Ontario, London, ON, Canada³ University of Western Ontario, London, ON, Canada

ABSTRACT- Trophoblast cells, the embryonic lineage contributing to the placenta, invade into the uterine endometrium during implantation. Matrix metalloproteinases (MMPs) are implicated as critical mediators of trophoblast invasion. In addition, the mitogen activated protein kinase (MAPK) signaling family, is required for placental development as Mek 1 and p38 MAPK murine null lines both display a mid-gestation lethality that is attributable to placental defects. The objective of our study was to examine the role of Erk1/2 MAPK signaling in regulating MMP release using a choriocarcinoma trophoblast cell model, the JEG-3 cell line. JEG-3 cells were pre-treated for one hour with a 10M concentration of pharmacological inhibitors of the upstream activators of Erk1/2, PD98059, U0126 and U0124 (inactive analogue) in serum-free minimal essential medium media. After pre-treatment, cells were stimulated with either 10% fetal bovine serum or 10 ng/ml of epidermal growth factor (EGF) in the presence or absence of PD98059, U0126 or U0124 (inactive analogue), for 15 and 30 minutes. Cells were lysed and the samples analyzed by Western blot methods. In addition, conditioned media was collected from EGF treatment groups following 24 hours of culture to detect MMPs by gelatin zymography. Serum and EGF stimulation of JEG-3 cells for 15 and 30 minutes resulted in an increase in phosphorylated Erk1/2 protein. The presence of PD98059 and U0126 negated this stimulation of Erk1/2 phosphorylation and resulted in a decrease in phosphorylated Erk1/2 protein. This effect was greater in the U0126-treated cells, reflecting this inhibitor's greater potency. Treatment of EGF-stimulated cells with PD98059 and U0126 decreased the gelatinolytic activity of MMP-2 as seen by zymography. Our results indicate that Erk1/2 regulates MMP production in JEG-3 cells and that MAPK signaling may mediate trophoblast invasion during implantation. Research supported by the Canadian Institute of Health Research (CIHR).

KEY WORDS: Implantation, Fertility, Trophoblast, Growth Factors