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Protein Phosphatase 3 Differentially Modulates Vascular Endothelial Growth Factor- and Fibroblast Growth Factor 2-Stimulated Cell Proliferation and Signaling in Ovine Fetoplacental Artery Endothelial Cells¹

Perinatal Research Laboratories,³ Department of Obstetrics and Gynecology, University of Wisconsin, Madison, Wisconsin 53715 Department of Biochemistry and Molecular Biology,⁴ Dalian Medical University, Dalian 116044, Liaoning, People's Republic of China Department of Reproductive Medicine,⁵ University of California, San Diego, California 92093

ABSTRACT

A critical process for vascular endothelial growth factor (VEGF)- and fibroblast growth factor 2 (FGF2)-regulated cellular function is reversible protein phosphorylation, which is tightly controlled by a balance of protein kinases and phosphatases. We have reported that in ovine fetoplacental artery endothelial (OFPAE) cells, VEGF and FGF2 stimulate cell proliferation in part via activation of mitogen-activated protein kinase kinase 1/2 (MAP2K1/2)/mitogen-activated protein kinase 3/1 (MAPK3/1) and phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog 1 (AKT1) pathways. In the present study, we examined if protein phosphatase 3 (PPP3) mediated VEGF- and FGF2-stimulated OFPAE cell proliferation via modulating activation of MAPK3/1 and AKT1. Small interfering RNA (siRNA) targeting human PPP3 catalytic subunit alpha (PPP3CA) was used to suppress PPP3CA protein expression in OFPAE cells. Compared with the scrambled siRNA, PPP3CA siRNA decreased PPP3CA protein levels by approximately 97% without altering protein levels of protein phosphatase 2 catalytic subunit alpha, total MAPK3/1, total AKT1, or glyceraldehyde-3-phosphate dehydrogenase. Knockdown of PPP3CA protein expression enhanced VEGF-stimulated, but not FGF2-stimulated, cell proliferation. Knockdown of PPP3CA protein expression did not significantly affect VEGF-induced MAPK3/1 and AKT1 phosphorylation but attenuated FGF2-induced MAPK3/1 and AKT1 phosphorylation. Thus, to our knowledge, the present study is the first to demonstrate successful knockdown of PPP3CA protein expression in any cell model using a single pair of double-strained siRNA. Moreover, specific knockdown of PPP3CA protein expression enhances VEGF-stimulated, but not FGF2-stimulated, OFPAE cell proliferation and attenuates FGF2-induced, but not VEGF-induced, MAPK3/1 and AKT1 activation. Thus, PPP3CA differentially modulates the VEGF- and FGF2-stimulated cell proliferation and signaling cascades in OFPAE cells. These data also suggest that signaling molecules other than MAPK3/1 and AKT1 play an important role in VEGF- and FGF2-stimulated cell proliferation after knockdown of PPP3CA in OFPAE cells.

AKT1, cell proliferation, FGF2, growth factors, kinases, MAPK3/1, phosphatases, placenta, placental endothelial cells, PPP3, pregnancy, VEGF

¹Supported in part by NIH grants HL64703 and HD38843 (J.Z.) and HL74947 and HL70562 (D.-B.C.).

Correspondence: ²Jing Zheng, Department of Obstetrics and Gynecology, Perinatal Research Laboratories, University of Wisconsin, PAB1 Meriter Hospital, 202 S. Park St., Madison, WI 53715. FAX: 608 257 1304; e-mail: jzheng{at}wisc.edu