Regulation of VEGF-induced endothelial cell PAF synthesis: role of p42/44 MAPK, p38 MAPK and PI3K pathways

Abstract

1. Vascular endothelial growth factor (VEGF) is a potent angiogenic and inflammatory mediator. We have recently shown that this latter effect requires the activation of Flk-1 receptor and subsequent endothelial cell (EC) PAF synthesis. However, the intracellular events that regulate EC PAF synthesis upon Flk-1 stimulation by VEGF remain to be elucidated. 2. Using specific inhibitors and Western blot analysis, we herein report that in bovine aortic endothelial cells (BAEC), VEGF induces the synthesis of PAF through the cascade activation of Flk-1 receptor, phospholipase Cgamma (PLCgamma), protein kinase C (PKC) and p42/44 mitogen-activated protein kinases (MAPK). 3. Moreover, we demonstrate that VEGF-mediated PAF synthesis requires the activation of p38 MAPK, likely by directing the conversion of lyso-PAF to PAF. 4. Interestingly, we observed that VEGF also promoted the activation of the phosphatidyl inositol-3-phosphate kinase (PI3K) pathway, and that its blockade potentiated PAF synthesis following a VEGF treatment. Consequently, it appears that the PI3K pathway acts as a negative regulator of EC PAF synthesis. 5. Taken together, these results allow a better understanding of the intracellular events activated upon EC stimulation by VEGF, and shed a new light on the mechanisms by which VEGF induces PAF synthesis.

Figure 1

Inhibitors effect on VEGF-induced EC PAF synthesis. BAEC were grown to confluence in six-well tissue culture plate. The media was replaced by 1 ml of HBSS – HEPES (10 mM)+CaCl₂ (10 mM), and cells were pretreated with tyrosine kinase inhibitor genistein (Gen, 10 μM), PLC inhibitor U73122 (10 μM), PKC inhibitor GF109203X (GFX, 1 μM), p42/44 MAPK pathway inhibitor PD98059 (PD, 10 μM) or p38 MAPK inhibitor SB203580 (SB, 10 μM). Cells were then incubated with [³H]-acetate, and stimulated with VEGF (1 nM) for 15 min. The values are means of at least eight experiments±s.e.mean. ^*P<0.05, ^***P<0.001 as compared with control buffer (PBS). †††P<0.001 as compared with VEGF (1 nM), as determined by analysis of variance followed by Bonferoni's t-test.

Figure 2

VEGF-induced activation of PLCγ. (A) Western blot analysis of VEGF time-dependent effect on PLCγ activation. Near-confluent BAEC were serum-starved in DMEM for 16 h and stimulated with VEGF (1 nM) for various periods of time. Cells were lysed, proteins were immunoprecipitated with an anti-PLCγ antibody and separated with a 10% SDS – PAGE. Western blot analysis was performed with an anti-PY20 antibody. (B) and (C) effect of U73122 (10 μM) on VEGF- and PBS-induced PLCγ phosphorylation respectively. BAEC were pretreated 10 min with U73122 and U73343 prior to a 5 min VEGF treatment.

Figure 3

VEGF effect on p42/44 MAPK phosphorylation. (A) Western blot analysis of VEGF time-dependent effect on p42/44 MAPK activation. Confluent BAEC were stimulated with VEGF (1 nM) for various periods of time. Cells were lysed, proteins were separated on a 10% SDS – PAGE. Western blot analysis was performed with an anti-phospho-p42/44 MAPK antibody. (B) BAEC were pretreated with genistein (Gen, 10 μM), U73122 (10 μM), GF109203X (GFX, 1 μM), PD98059 (PD, 10 μM), SB203580 (SB, 10 μM), then the cells were treated as in (A).

Figure 4

Effect of VEGF on p38 MAPK phoshorylation. (A) Western blot analysis of the phosphorylated form of p38 MAPK was conducted as described in Figure 3A with the use of an anti-phospho-p38 MAPK antibody. (B) Cells were pretreated as described in Figure 3B, and processed as described in A.

Figure 5

Effect of VEGF on MKK-3/6 phoshorylation. Western blot analysis of the phosphorylated form of MKK-3/6 was conducted as described in Figure 3A with the use of an anti-phospho-MKK-3/6 antibody.

Figure 6

Effect of Wortmannin and LY294002 on VEGF-induced EC PAF synthesis. Experiments were conducted as described in Figure 1, with the exception that BAEC were pretreated for 10 min without or with the PI3K pathway inhibitors Wortmannin (Wort, 10 – 100 nM) or LY294002 (1 – 10 μM). The values are means of at least eight experiments. ^***P<0.001 as compared with control buffer (PBS). †P<0.05 as compared with VEGF (1 nM) as determined by analysis of variance followed by Bonferoni's t-test.

Figure 7

Effect of VEGF on PI3K pathway activation. (A) Confluent BAEC were stimulated with VEGF (1 nM) for various periods of time. Cells were lysed and separated on a 10% SDS – PAGE. Western blot analysis was performed with an anti-phospho-Akt antibody. (B) Western blot determination of the phosphorylated form of Akt reveals the effect of VEGF-induced Akt phosphorylation. Experiments were carried out as in Figure 6A, except that BAEC were pretreated 5 min with genistein (Gen, 10 μM), U73122 (10 μM), GF109203X (GFX, 1 μM), PD98059 (PD, 10 μM), SB203580 (SB, 10 μM), Wortmannin (Wort, 100 nM) or LY294002 (10 μM) prior to a 7.5 min treatment with VEGF (1 nM).

Figure 8

Effect of Wortmannin and LY294002 on VEGF-induced activation of the p42/44 and p38 MAPK. (A) Confluent BAEC were pretreated with or without Wortmannin (Wort; 100 nM) or LY294002 (LY; 10 μM), and were stimulated and processed as described in Figure 3A. Western blot analysis was performed with an anti-phospho-p42/44 MAPK antibody. (B) Confluent BAEC were pretreated with or without Wortmannin (Wort; 100 nM) or LY294002 (LY; 10 μM), and were stimulated and processed as described in Figure 3B. Western blot analysis was performed with an anti-phospho-p38 MAPK antibody.

Figure 9

VEGF effect on IP production. (A) Confluent BAEC were labelled with [³H]-myoinositol, and stimulated with or without VEGF (1 nM) for various periods of time. The cells were scraped, total IP were isolated by extraction with 1,1,2-trichlorotrifluoroethane and tri-n-octylamine, purified by chromatography with a Dowex AG1X8 column (200 – 400 mesh) and quantified by scintillation counting. The values are means of at least four experiments±s.e.mean. (B) Confluent BAEC were pretreated either with U73122 (10 μM), U73343 (10 μM), Wortmannin (Wort; 100 nM) or LY294002 (LY; 10 μM), and stimulated with VEGF (1 nM) for 5 min. IP production was determined as in A. ^***P<0.001 as compared with control buffer (PBS). †††P<0.001 as compared with VEGF (1 nM) as determined by analysis of variance followed by a Bonferoni t-test.

Figure 10

Proposed pathway for the induction of EC PAF synthesis by VEGF. The synthesis of PAF upon Flk-1 autophosphorylation by VEGF is predominantly mediated through the activation of a Ras-independent pathway (PLCγ and PKC) (solid line), promoting the downstream activation of the Raf and p42/44 MAPK pathway. In addition, VEGF activates the MKK-3/6 which induces the activation of p38 MAPK. Both p42/44 and p38 MAPK are known to activate the phospholipid remodelling pathway which leads to PAF synthesis. Meanwhile, Flk-1 autophosphorylation also activates PI3K, which appears to act as a negative regulator of PAF synthesis in BAEC (dashed line). Since VEGF-induced p42/44 and p38 MAPK phosphorylation were not affected by Wortmannin or LY294002, PI3K likely exerts its inhibitory effect downstream of p42/44 and p38 MAPK. Hence, the PI3K pathway might act on PAF synthesis in BAEC by downregulating the activity of the phospholipid remodelling pathway.