Protein phosphatase 3 differentially modulates vascular endothelial growth factor- and fibroblast growth factor 2-stimulated cell proliferation and signaling in ovine fetoplacental artery endothelial cells

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.

FIG. 1.

Transfection of OFPAE cells with the scrambled siRNA conjugated with Cy3. Cells were cultured in 60-mm culture dishes in DMEM containing 5% fetal bovine serum, 5% calf serum, and 1% penicillin-streptomycin. After reaching 50–60% of confluence, cells were transfected with the scrambled siRNA (20 nM). After 5 and 48 h of transfection, cells were examined. Top) 5 h of transfection. Middle and Bottom) 48 h of transfection. Reddish fluorescence indicates presence of Cy3 inside cells. Bar = 50 μm.

FIG. 2.

Effects of PPP3CA siRNA on protein expression of PPP3CA, PPP2CA, and GAPDH in OFPAE cells. Cells at 50–60% confluence were transfected with the scrambled or PPP3CA siRNA (20 and 40 nM) for 48 h. Proteins (20 μg) were used for Western blot analysis for PPP3CA, PPP2CA, and GAPDH. Lanes 1 and 2: scrambled siRNA at 20 and 40 nM, respectively; lanes 3 and 4: PPP3CA siRNA at 20 and 40 nM, respectively. Data normalized to GAPDH protein levels are expressed as the fold-value (mean ± SEM) of the scrambled siRNA from three independent experiments. Means with an asterisk differ significantly (P < 0.05) from the scrambled siRNA control. ssiRNA, scrambled siRNA; siRNA, PPP3CA siRNA.

FIG. 3.

Effects of PPP3CA siRNA on VEGF- and FGF2-stimulated OFPAE cell proliferation. Cells after transfection with the scrambled or PPP3CA siRNA were seeded in 96-well plates (4000 cells/well). After serum starvation, cells were treated without (control) or with VEGF or FGF2 for 48 h. Data are expressed as a percentage (mean ± SEM) of the control from eight independent experiments. Numbers of cells per well in the control were 6036 ± 544. Asterisks indicate significant (P < 0.05) differences from the control. Number symbols denote significant (P < 0.05) differences from the scrambled siRNA at the corresponding dose of growth factor.

FIG. 4.

Effects of PPP3CA siRNA on VEGF-induced MAPK3/1 phosphorylation in OFPAE cells. Cells were transfected with the scrambled or PPP3CA siRNA for 48 h. After serum starvation, cells were treated with 10 ng/ml of VEGF. Proteins (15 μg/lane) were subjected to Western blot analysis. Data normalized to total MAPK3/1 are expressed as the fold-value (mean ± SEM) of the controls (time zero) from seven independent experiments. Asterisks indicate significant (P < 0.05) differences from the corresponding time-zero control.

FIG. 5.

Effects of PPP3CA siRNA on FGF2-induced MAPK3/1 phosphorylation in OFPAE cells. Cells were transfected with the scrambled or PPP3CA siRNA for 48 h. After serum starvation, cells were treated with 10 ng/ml of FGF2. Proteins (15 μg/lane) were subjected to Western blot analysis. Data normalized to total MAPK3/1 are expressed as the fold-value (mean ± SEM) of the controls (time zero) from seven independent experiments. Asterisks indicate significant (P < 0.05) differences from the corresponding time-zero control. Number symbols denote significant (P < 0.05) differences from the scrambled siRNA.

FIG. 6.

Effects of PPP3CA siRNA on VEGF-induced AKT1 phosphorylation in OFPAE cells. Cells were transfected with the scrambled or PPP3CA siRNA for 48 h. After serum starvation, cells were treated with 10 ng/ml of VEGF. Proteins (15 μg/lane) were subjected to Western blot analysis. Data normalized to total AKT1 are expressed as the fold-value (mean ± SEM) of the controls (time zero) from five independent experiments. Asterisks indicate significant (P < 0.05) differences from the corresponding time-zero control.

FIG. 7.

Effects of PPP3CA siRNA on FGF2-induced AKT1 phosphorylation in OFPAE cells. Cells were transfected with the scrambled or PPP3CA siRNA for 48 h. After serum starvation, cells were treated with 10 ng/ml of FGF2. Proteins (15 μg/lane) were subjected to Western blot analysis. Data normalized to total AKT1 are expressed as the fold-value (mean ± SEM) of the controls (time zero) from five independent experiments. Asterisks indicate significant (P < 0.05) differences from the corresponding time-zero control. Number symbols denote significant (P < 0.05) differences from the scrambled siRNA.