Epidermal Growth Factor Receptor Is an Obligatory Intermediate for Oxytocin-Induced Cyclooxygenase 2 Expression and Prostaglandin F2α Production in Bovine Endometrial Epithelial Cells

Abstract

Oxytocin (OT) triggers the luteolytic pulses of prostaglandin F(2 alpha) (PGF(2 alpha)) from the endometrial epithelial cells in ruminants. We have proposed that the embryonic signal interferon-tau exerts its antiluteolytic effect by disrupting the OT signaling axis. Accordingly, we have attempted to define the signaling pathway of OT-induced PGF(2 alpha) production in the bovine endometrium using our newly characterized epithelial cell line (bEEL). OT receptor was coupled to the classical G alpha(q) pathway as evidenced by calcium release and activation of phospholipase C. Similarly, OT-induced PGF(2 alpha) production was mediated through the canonical ERK1/2 pathway. Because of the importance of receptor and nonreceptor tyrosine kinases in G protein-coupled receptor signaling, we studied the role of epidermal growth factor receptor (EGFR), c-Src, and phosphoinositide 3-kinase (PI3K) on OT-induced PGF(2 alpha) production in association with cyclooxygenase 2 (COX2) expression and ERK1/2 and Akt phosphorylation. The EGFR inhibitor AG1478 (10 microm) nearly abolished basal and OT-induced PGF(2 alpha) production and down-regulated COX2 expression and ERK1/2 phosphorylation. Because the transactivated EGFR can serve as a ligand for the signaling proteins with Src homology 2 (SH2) domain, we hypothesized a role for c-Src and PI3K in OT-induced PGF(2 alpha) production. Inhibitors of c-Src (PP2, 10 microm) and PI3K (LY294002, 25 microm) produced a significant decrease in OT-induced PGF(2 alpha) production and reduced COX2 expression. Also, PP2, but not LY294002, decreased OT-induced ERK1/2 phosphorylation. Because LY294002 did not affect ERK1/2 phosphorylation, but inhibited PGF(2 alpha) production and down-regulated COX2 expression, it is likely that the Akt pathway is also involved in PGF(2 alpha) production. Thus, EGFR may simultaneously activate c-Src and PI3K to amplify the OT signaling to increase the output of PGF(2 alpha) in bEEL cells.

The Multidrug Resistance-Associated Protein 4 (MRP4) Appears as a Functional Carrier of Prostaglandins Regulated by Oxytocin in the Bovine Endometrium

“…5A). Consistent with our previous characterization of OT response in bEEL cells (27), the effect of OT could be mimicked by the protein kinase C (PKC) activator PMA (10 nM) but not after activation of the PKA pathway by 8-bromo-cAMP (500 M) (Fig. 5B).…”

“…However, in the present study, we have concentrated on possible regulation by OT and IFN known to regulate PG biosynthesis and action at the time of recognition of pregnancy (5,10,38). We have used our newly developed bEEL cell line (6,27) to investigate the modulation of MRP4 and PGT in response to OT or IFN. We have used IFN at 10 ng/ml, a concentration in the low physiological range shown to inhibit OT stimulation of PG production and induce Stat phosphorylation, without direct action on COX-2 or PG output when added alone (6) (Fig.…”

“…The bovine endometrial epithelial cell line (bEEL) and bovine caruncular stromal cells (CSC) were cultured as reported previously (27). Briefly, a frozen aliquot was grown in RPMI 1640 medium without phenol red [Life Technologies, Inc.-BRL (Invitrogen), Mississauga, Ontario, Canada] supplemented with 10% fetal bovine serum and 50 IU penicillin-streptomycin (Wisent Inc., Québec, Québec, Canada); confluent cultures were trypsinized and seeded at 4 ϫ 10 4 cells/ml.…”

“…The potential regulation of PGT/SLCO2A1 and MRP4/ABCC4 genes was studied in response to OT and IFN in our recently developed bovine endometrial epithelial cell line identified as bEEL (6,27) (Fig. 2).…”

Comparison of the Effects of Early Pregnancy with Human Interferon, Alpha 2 (IFNA2), on Gene Expression in Bovine Endometrium1

“…For parathyroid hormone-like hormone (PTHLH), a role in early pregnancy in the rat has been shown by antagonization of PTHLH action leading to fetoplacental growth restriction [82]. Trafficking protein, kinesin-binding 2 (TRAK2, alias GRIF1) is a further potentially interesting gene, because a regulatory role in epidermal growth factor receptor (EGFR) degradation has been shown [83] and EGFR is involved in oxytocin-induced PGF2a production [84].…”