Effect Of PPP3CA SiRNA On VEGF-and FGF2-Stimulated Cell Proliferationin Ovine Fetoplacental Artery Endothelial Cells

During pregnancy, angiogenesis and vasodilatation are two key mechanisms essential for increasing placental blood flows, which are directly correlated with fetal growth and survival as well as neonatal weights and survivability. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF2) are two of the most potent angiogenic factors. Expression of FGF2 and VEGF in placentas is positively associated with the placental vascular growth and blood flows during pregnancy. It is well established that the biological actions of VEGF and FGF2 are initiated by binding to their specific receptors thereby activating the cytoplasmic tyrosine kinase domains. Upon activation, these receptor-tyrosine kinases initiate multiple downstream cellular protein kinase signaling pathways, including 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 oncogen homolog 1 (AKT1) pathways. MAPK3/1 and AKT1, as major threo- nine/tyrosine protein kinases, are involved in regulating cell survival, proliferation, and migration by phosphorylation and activation their downstream substrates. Recently, we have reported that the VEGF- and FGF2-stimulated OFPAE cell proliferation is mediated at least partly via the MAP2K1/2//MAPK3/1 and PI3K/AKT1 signaling pathways.Protein dephosphorylation is the same important process with phosphorylation. This process is tightly controlled by protein phosphatases. Protein phosphatase 3 (PPP3, also term as PP2B or calcineurin) is the only Ca²⁺/calmodulin dependent member of PPP, which is composed of catalytic and regulatory subunits. Three PPP3 catalytic (α,β,γ, also named as PPP3CA, PPP3CB, PPP3CC) and two regulatory (B1 and B2, also named as PPP3R1 and PPP3R2) subunits have been identified. More and more people focused on PP2B due to its wide distribution and multiply functions. Recent studies showed PP2B plays important role in the vascular development as well as cardiac hypertrophy and vascular smooth musclar differentiation. It also indicated that suppression of PPP3 activity by its pharmacological inhibitor cyclosporine A (CsA, a widely used immunosuppressant) inhibits VEGF-, but not FGF2-induced angiogenesis in human umbilical vein endothelial and intestinal microvascular endothelial cells. This inhibition can be mediated via blocking or attenuating endothelial MAPK3/1 activation, implicating a reciprocal relationship between PPP3 and MAPK3/1 activations in endothelial cells similar to that observed in cardiomyocytes and B cells. In contrast, CsA is also capable of promoting MAPK3/1 activation in human first-trimester trophoblasts and canine kidney epithelial cells. Little is known about the role of PPP3 in the PI3K/AKT1 pathway, except that one recent report showed that PPP3 inhibition by FK506 (another selective PPP3 inhibitor) had no effect on activation of the PI3K/AKT pathway in A549 cells. In this study, we determined that if suppression of PPP3CA inhibited FGF2- and VEGF- stimulated cell proliferation and attenuated FGF2- and VEGF- induced activation of MAPK3/1 and AKT1.In this study, small interfering RNA (siRNA) specifically targeting human PPP3CA was used to suppress PPP3CA protein expression in OFPAE cells. PPP3CA siRNA decreased PPP3CA protein levels by 97%, while failing to alter protein levels of PPP2 catalytic subunitα(PPP2CA), total MAPK3/1 and AKT1, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase), as compared with the scrambled siRNA. This inhibitory effect of PPP3CA siRNA on PPP3CA protein expression maintained at least for 3 days after the transfection. Violet crystal method was used to determined the effect of PPP3CA on VEGF- and FGF2- induced cell proliferation. Both VEGF and FGF2 dose-dependently stimulated (p < 0.05) cell proliferation. Western Blot was performed to further demonstrate effect of PPP3CA on VEGF- and FGF2- induced MAPK3/1 and AKT1 phosphorylation. Suppression of PPP3CA protein expression did not significantly affect VEGF-induced MAPK3/1 and AKT1 phosphorylation, whereas attenuating(p < 0.05) FGF2-induced MAPK3/1 and AKT1 phosphorylation. Thus, these data suggest that PPP3CA plays an important role in VEGF-, but not FGF2- stimulated OFPAE cell proliferation and in FGF2-, but not VEGF-induced MAPK3/1 and AKT1 activation.Our current observations that PPP3CA participates VEGF-, but not FGF2-stimulated endothelial proliferation are consistent with the previous reports using other endothelial cell types. Meanwhile, in contrast to these previous reports showing that inhibition of PPP3 activity attenuates VEGF-induced angiogenesis, knockdown of PPP3CA protein expression enhances OFPAE cell proliferation. It is noteworthy that in both of previous studies, inhibition of PPP3 activity is carried out by its pharmacological inhibitor CsA, which executes its action via binding to the interface of the catalytic and regulatory subunits of PPP3. Being generally considered to be a selective inhibitor of PPP3 and widely used in investigating roles of PPP3, this drug, however, can also function independent of PPP3. For example, it has been shown that CsA inhibition of the opening of mitochondrial inner membrane permeability transition pore is not mediated via PPP3. Thus, it is possible that PPP3 might not be the only major target in the CsA-inhibited VEGF-stimulated in vitro and in vivo angiogenic responses. On the other hand, OFPAE cells were exposed to VEGF at least 16 hr (serum starvation period) after knockdown of PPP3CA protein expression. This relatively chronic knockdown of PPP3CA might lead to extensive, different changes in signaling network as compared with the relatively acute ( 30 min to 1 hr) inhibition of PPP3 by CsA before VEGF stimulation. Moreover, in the current study, the siRNA designed targets only PPP3CA, which may contribute portion of total PPP3 activity in OFPAE cells. Our current study cannot exclude the possibility that the catalyticβsubunit plays a role in VEGF-stimulated endothelial proliferation.Although we have demonstrated that in OFPAE cells that activation of the MAP2K1/2/MAPK3/1 and PI3K/AKT1 pathways is critical for VEGF- and FGF2-stimulated cell proliferation. The increased VEGF-stimulated and unchanged FGF2-stimulated cell proliferation in OFPAE cells in this study suggest that after knockdown of PPP3CA signaling cascades other than the MAP2K1/2/MAPK3/1 and PI3K/AKT1 might emerge as major players in mediating the VEGF- and FGF2-stimulated cell proliferation. Indeed, we have also observed that MAPK11 (also termed as p38 MAPK) is a key signaling in mediating VEGF- and FGF2-induced OFPAE cell proliferation. Alternatively, differential regulation of other protein hosphatases (i.e. MAPK phosphatase [MAPK], and Phosphatase and TENsin Homolog [PTEN]) might contribute to differential activation of MAPK3/1 and AKT1 by VEGF and FGF2 in OFPAE cells transfected with PPP3CA siRNA.Taken together, our current data demonstrate that PPP3CA plays a critical role in VEGF-stimulated cell proliferation and FGF2-induced MAPK3/1 and AKT1 activation in fetoplacental endothelial cells. Our current findings further advance our understanding of the complex signaling mechanism controlling placental endothelial function. Future studies are needed to dissect these signaling networks, which may provide fundamental information for modulating placental vasculature and blood flows by altering activation of signaling cascades by angiogenic factors.