IMPLANTATION AND PREGNANCY - A
Monday, August 2, 2004
10:30 AM–12:30 PM
(273) EXOGENOUS NITRIC OXIDE-STIMULATED CELL PROLIFERATION VIA ACTIVATION OF MEK/ERK1/2 PATHWAY IN OVINE FETO-PLACENTAL ARTERY ENDOTHELIAL (OFPAE) CELLS.
Zheng, Jing1, Wen, YunXia1, Chung, JinYoung1, Magness, Ronald1, 2, 3, 1 Dept, of Ob/Gyn2 Dept of, Pediatrics3 Dept. of Animal Sci, Madison, WI
ABSTRACT- Nitric Oxide (NO), a local vasodilator, is increased dramatically during pregnancy and plays a role in regulating placental vasodilation. We have shown that bFGF dose-dependently increases endothelial NO synthase (eNOS) expression via activation of MEK/ERK1/2, and also increases cell proliferation in OFPAE cells. Hypothesis: Exogenous NO stimulates cell proliferation via activation of the MEK/ERK1/2 cascade. Methods: For cell proliferation assays, OFPAE cells were treated with sodium nitroprusside (SNP; 1nM-100uM), an NO donor. After 72 hr the number of cells was determined. Additional cells were treated with 1 uM SNP in the absence or presence of PD98059 (a specific MEK inhibitor; 2.5-40 uM) in a similar fashion. To determine whether SNP activates the ERK1/2 cascade, cells were treated with 1 uM SNP for 0, 1, 5, 10, 30, or 60 minutes. Cells were then analyzed for ERK1/2 phosphorylation using immunocytochemistry and Western analysis. Results: SNP dose-dependently stimulated (p<0.05) cell proliferation with a maximal effect at 1 uM SNP (∼4.5 fold). PD98059 dose-dependently inhibited (p <0.05) SNP-induced cell proliferation, beginning at 5 uM and reaching a maximum at 20 uM. SNP at 1 uM also time-dependently phosphorylated ERK1/2. The ERK1/2 phosphorylation first appeared after 10 min of treatment, reached a maximum at 30 min, and then remained elevated through 60 min. Conclusions: These data indicate that exogenous NO stimulated cell proliferation is mediated via activation of the MEK/ERK1/2 signaling pathway. These data also suggest that bFGF-induced cell proliferation may in part be mediated through an NO/MEK cascade. Supported by NIH grants HL64703, HL57653, HL38843.
KEY WORDS: placenta, kinase, nitric oxide, endothelium