Comparison of the signaling mechanisms by which VEGF, H2O2, and phosphatase inhibitors activate endothelial cell ERK1/2 MAP-kinase

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Abstract

VEGF-induced ERK1/2 activation is mediated by a signaling mechanism involving the sequential activation of PLCγ-PKC-Raf1-MEK-ERK1/2. This signaling pathway is necessary, but not sufficient for ERK1/2 activation, as VEGF-induced generation of reactive oxygen species (ROS) is also required. The molecular interaction by which VEGF-induced ROS generation is coordinated with the PLCγ plus PKC-dependent pathway is not certain, and the goal of this study was to clarify this issue. Prior investigations examining ROS-induced signaling have focused on the cellular protein tyrosine phosphatases (PTPs), and we asked whether a PTP participates in ERK1/2 activation in endothelial cells. We show that both the general PTP inhibitor vanadate, and a dominant negative inhibitor of SHP-1, mimics the effects of VEGF in activating ERK1/2. The phosphatase inhibitors induce ERK1/2 activation in endothelial cells lacking VEGF receptors, indicating that the inhibitors target a downstream effector. As is the case after VEGF treatment, the phosphatase inhibitors do lead to the activation of PLCγ, and a pharmacological inhibitor of the Src kinases blocks this. These results lead to the conclusion that inhibition of a protein tyrosine phosphatase activates endothelial cell ERK1/2 by a signaling mechanism involving the sequential activation of Src-PLCγ-PKC-Raf1-MEK-ERK1/2. VEGF treatment most likely activates this pathway by inhibiting SHP-1 through a ROS-dependent mechanism.

Introduction

Vascular endothelial growth factor (VEGF) is a key activator of endothelial cell functions (Dvorak, 2002, Ferrara, 2002), and the utility of VEGF antagonist for blocking tumor angiogenesis and tumor growth is under clinical evaluation (Saaristo et al., 2000). VEGF binds to two high affinity receptors, known as fms-like tyrosine kinase 1 (FLT1) and kinase insert domain receptor tyrosine kinase (KDR), that are predominantly specific to endothelial cells (de Vries et al., 1992, Terman et al., 1992). Both receptors contain insert sequences within their catalytic domains, seven extracellular immunoglobulin-like domains, and are closely related to the PDGF family of receptor tyrosine kinases. Although expression of both VEGF receptors occurs in adult endothelial cells, recent findings demonstrate that KDR, and not FLT1, is able to mediate the mitogenic, chemotactic, and survival responses to VEGF (Keyt et al., 1996).

Like many other growth factors, VEGF stimulates DNA synthesis via a signaling mechanism that involves the extracellular signal-regulated kinase (ERK1/2) MAP kinases (Takahashi et al., 1999). The signal transduction utilized by VEGF is somewhat unique compared to other growth factors in that neither Ras guanine nucleotide-releasing protein (Grb2) nor Ras is involved. Evidence supporting this conclusion includes the observations that expression of a dominant-negative Ras does not block VEGF-induced ERK1/2 activity (Takahashi et al., 1999), and VEGF treatment does not lead to the recruitment of Grb2 to activated receptor. Instead, the VEGF-induced signaling pathway involves the sequential activation of PLCγ-PKC-Raf-MEK-ERK1/2. VEGF-induced PLCγ activation is dependent upon the recruitment of PLCγ to tyrosine 1175 to activated receptor (Takahashi et al., 2001).

It has more recently become apparent that the PLCγ-PKC-Raf-MEK-ERK1/2 signaling pathway is necessary, but not sufficient, for ERK1/2 activation. For example, pharmacological inhibition of the Src-family of tyrosine kinases blocks VEGF-induced ERK1/2 activation (Pedram et al., 2002). While the molecular interactions by which Src mediates ERK1/2 activation have not been clarified, a role of Src in PLCγ activation has been documented (He et al., 1999). VEGF-induced MAP kinase activation is also dependent upon the Rac-mediated generation of reactive oxygen species (ROS) (Colavitti et al., 2002). Experimental evidence supporting this conclusion includes the observation that treatment of cells with VEGF results in a transient increase in ROS, and inhibition of this response inhibits the enhanced ERK1/2 activation.

The goal of this study was to clarify the molecular interactions by which VEGF-induced ROS generation is coordinated with the PLCγ-PKC-Raf1-MEK pathway to allow for maximal ERK1/2 stimulation. Prior investigations examining ROS-induced signaling have, in part, focused on the cellular protein tyrosine phosphatases (Meng et al., 2002), and these studies demonstrated that ROS-induced inhibition of PTPs augments tyrosine phosphorylation of key signaling proteins and enhancement of their activities. We tested whether a PTP also participates in VEGF-induced ERK1/2 activation, focusing on Src homology 2 (SH2) domain phosphatase-1 (SHP-1) (also called PTP1C), a cytoplasmic protein tyrosine phosphatase predominantly expressed in hematopoietic cells (Zhang et al., 2000). SHP-1 recruitment to the receptors for many cytokines and growth factors plays a negative regulatory role in signal transduction pathways (Berg et al., 1999, Long, 1999, Wu et al., 2003), and a naturally occurring mouse mutant, motheaten, expressing an inactive SHP-1 displays multiple defects characterized by overgrowth of hematopoietic cells (Tsui et al., 1993, Yu et al., 1996). Two manuscripts (Guo et al., 2000, Nakagami et al., 2002) have described experimental results consistent with SHP-1 playing a negative role in VEGF-induced signaling; in particular MAP-kinase activation and cell proliferation. Both studies found that tumor necrosis factor (TNF) inhibits the VEGF-induced phosphorylation and activation of KDR. TNF treatment leads to the activation of SHP-1 and its recruitment to KDR. The role of SHP-1 in VEGF-induced signaling has not been determined, although VEGF treatment does lead to SHP-1 recruitment to activated receptor (Kroll and Waltenberger, 1997).

Our results indicate that inhibition of cellular receptor tyrosine phosphatases, using vanadate or inhibition of SHP-1 using a dominant negative strategy, mimics the effect of VEGF-induced ROS generation in the activation of ERK1/2. Significantly, the signaling mechanism mediating the cellular response to phosphatase inhibition is independent of VEGF receptors.

Section snippets

Cell culture

Human umbilical vein endothelial cells (HUVEC) were cultured on 0.2% gelatin-coated tissue culture plates in M199 containing 10% newborn calf serum and Endothelial Growth Factor (Sigma), 7.5 μg/ml. Passage one or two cells were used for each experiment. Porcine aortic endothelial cells (PAEC), human embryonic kidney epithelial (HEK) 293 cells were cultured in DMEM containing 10% newborn calf serum. The development and use of stably transfected KDR-expressing PAEC (KDR-PAEC) in our laboratory

Results

ROS generation is a necessary step in the signaling pathway that mediates VEGF-induced ERK1/2 activation (Colavitti et al., 2002); yet the cellular targets of this activity are not certain. Fig. 1A shows that bovine catalase inhibits VEGF-induced ERK1/2 phosphorylation in HUVEC; the protocol for this experiment involved Western blotting using phospho-specific antibodies to ERK1/2. The effect is not a nonspecific consequence of catalase activity because catalase had no effect on EGF-induced

Discussion

A goal of this study was to clarify how VEGF-induced ROS generation coordinates with the PLCγ-PKC-Raf1-MEK pathway to allow ERK1/2 activation. The described experimental results lead us to the hypothesis, shown schematically in Fig. 8, that in the absence of VEGF or H2O2, a protein tyrosine phosphatase (perhaps SHP-1) is active, and blocks the signaling pathways leading to ERK1/2 activation. The model predicts that an early event after both VEGF-induced ROS generation and H2O2 treatment is

Acknowledgments

This work was supported by grants CA86289 and HL067019 from the NIH.

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