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Biochemistry |
Ludwig Institute for Cancer Research [D. W., H-J. S. H., W. K. C.], Department of Medicine [H-J. S. H., W. K. C.], Center for Molecular Genetics [W. K. C.], and Cancer Center [W. K. C.], University of California-San Diego, California 92093-0660, and Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114 [A. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSImmunoprecipitation and Western...In Vitro PI3K Activity...RESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSImmunoprecipitation and Western...In Vitro PI3K Activity...RESULTSDISCUSSIONREFERENCES |
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, transforming growth factor ß, epidermal growth factor, PDGF, and tissue hypoxia (17, 18, 19, 20, 21, 22, 23, 24)
. In addition, inactivation of the p53 or von Hippel-Lindau tumor suppressor genes (25
, 26)
, as well as activation of oncogenes such as ras, raf, or src enhance VEGF expression (27, 28, 29)
. Expression of ßPDGFR is increased on neovascular endothelial cells in several pathological conditions including wound healing, inflammation, and glioma tumorigenesis (30, 31, 32, 33) . Although this induction has been suggested to play a role in neovascularization, the downstream mechanisms by which PDGF transduces signals via ßPDGFR expression in endothelial cells to mediate neovascularization remain obscure. In NIH fibroblasts and vascular smooth muscle cells, PDGF induces VEGF expression (19 , 20) , and in fibroblasts, this induction appears to be PKC dependent (19) . Here, we investigated whether PDGF might induce VEGF mRNA expression and protein production in endothelial cells expressing ßPDGFR and whether this causes an increased VEGF secretion that could stimulate endothelial cell proliferation. We observed that expression of ßPDGFR in endothelial cells created an autocrine PDGF regulation of VEGF expression, and further extracellular exposure of cells to PDGF markedly increased VEGF induction and secretion. Such VEGF in the conditioned media from PDGF-treated ßPDGFR-expressing endothelial cells stimulated endothelial cell mitogenesis and migration. These results suggest a potential autocrine VEGF action pathway that may enhance the paracrine effects of VEGF in stimulating angiogenesis.
Studies on ßPDGFR signaling have revealed that the binding of PDGF to its cell surface receptor activates its intrinsic tyrosine kinase activity and leads to phosphorylation of multiple tyrosine residues. The activated receptor initiates activation of intracellular signaling pathways by recruiting and activating a set of SH2-containing proteins including PLC
, the GAP, the phosphotyrosine phosphatase SHP-2 (Syp), the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), and other proteins (34
, 35)
. Although these early multiple signaling events downstream of ßPDGFR have been analyzed in various cell types, little is known about the pathways by which PDGF causes up-regulation of VEGF expression. Here, we present evidence showing that, in contrast to fibroblasts, PKC may not be required for VEGF induction in endothelial cells. Using chemical inhibitors, mutant ßPDGF receptors and a dominant-negative form of PI3K, we were able to show that PDGF-activated PI3K pathways may play an essential role in mediating VEGF expression in endothelial cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSImmunoprecipitation and Western...In Vitro PI3K Activity...RESULTSDISCUSSIONREFERENCES |
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Generation of Stable Cell Lines Expressing Dominant-Negative PI3K.
The p85 based-dominant negative mutant of PI3K (p85 
iSH2-N, deletion of amino acids 494–790) in pSG5 (38
, 39)
was kindly provided by Drs. M. Waterfield and J. Downward (Ludwig Institute for Cancer Research, London, and Imperial Cancer Research Fund, London, respectively). The EcoRI fragment of the p85 iSH2-N excised from pSG5 was subcloned into pcDNA3.1 Zerocin (Invitrogen). The plasmids were transfected into PAE/ßPDGFR cells using Lipofectin reagent (Life Technologies, Inc.). Individual zerocin-resistant clones were selected and analyzed by Western blotting for the expression of both the mutant PI3K and ßPDGFR.
Northern Blot Analysis.
Cells were grown to 90% confluency and starved in SFM containing 0.1% BSA (SFM/BSA) for 18–20 h. After stimulation with PDGF-BB (R&D System) at indicated concentrations, cells were harvested, and total cellular RNA was isolated using the Trizol reagent (Life Technologies, Inc.). For drug treatment experiments, cells were preincubated either with the vehicle, DMSO (final concentration, 0.01%) alone, or the following drugs dissolved in DMSO for 30 min before the addition of PDGF or TPA (Sigma): 5 µg/ml actinomycin D (Sigma), 5 µg/ml cycloheximide (Sigma), 5 µM bisindolylmaleimide GF 109203X (BIM; Calbiochem), and wortmannin or LY24009 (Calbiochem) at the indicated concentrations. A 15-µg total cellular RNA sample was then fractionated through 1% agarose containing 2.2 M formaldehyde and transferred to Hybond+ nylon membranes (Amersham). A VEGF165 cDNA probe (0.6 kb) was 32P- radiolabeled by the random primer method and then hybridized to filters in QuickHyb solution (Stratagene) following the manufacturer’s protocol. To monitor RNA loading, different regions of the membranes were hybridized with a GAPDH probe (1.25 kb). Washed filters were analyzed using a PhosphorImager (Molecular Dynamics).
CM, VEGF ELISA, and Chemotaxis Assay.
Cells were seeded at equal numbers and grown to 90% confluency in 15-cm dishes. After washing twice with SFM/BSA, cells were starved in 10 ml SFM/BSA for 16 h. PDGF-BB was then added to a final concentration of 50 ng/ml. Twenty-four h after PDGF addition, the CM was collected, cleared by centrifugation (10,000 x g for 30 min), and stored in aliquots at -80°C until use. VEGF ELISA was performed using a human VEGF Quantikine kit (R&D System) according to the manufacturer’s protocol. An endothelial cell migration assay was carried out exactly as described previously (16)
, except that PAE cells expressing VEGF receptor KDR/VEGFR-2 (PAE/KDR) were used instead of human umbilical vascular endothelial cells. The PAE/KDR cells responded to VEGF but not to PDGF or basic fibroblast growth factor, as described previously (36)
.
BrdUrd Labeling and Immunofluorescence.
PAE/KDR cells were seeded on glass coverslips in a 24-well tissue culture dishes in the presence of 10% serum. After 24-h incubation, cells were washed one time with SFM/BSA and starved in SFM/BSA for 48 h. Quiescent cells were stimulated with CM. Cells were also stimulated with serum or recombinant VEGF (positive controls) or recombinant PDGF (negative control). BrdUrd was added at 8 h after stimulation, and cells were labeled for an additional 14 h. After fixation in 3.7% formaldehyde in PBS for 15 min, cells were permeabilized in 0.3% Triton-PBS for 15 min and then incubated with a rat monoclonal anti-BrdUrd antibody (Accurate Scientific), followed by incubation with fluorescently labeled secondary antibody. BrdUrd incorporation was examined using a Zeiss Axiophot epifluorescence microscope and photographed using a x40 objective.
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Immunoprecipitation and Western Blot Analyses. |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSImmunoprecipitation and Western...In Vitro PI3K Activity...RESULTSDISCUSSIONREFERENCES |
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In Vitro PI3K Activity Assay. |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSImmunoprecipitation and Western...In Vitro PI3K Activity...RESULTSDISCUSSIONREFERENCES |
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-32P] ATP]. The reactions were stopped by the addition of 80 µl of 1 M HCl. The phospholipids were extracted with 160 µl of chloroform:methanol (1:1), and the organic phase was dried and stored at -80°C. Radiolabeled samples were redissolved in chloroform and chromatographed on silica TLC plates (J. T. Baker), using a borate buffer developing system (42)
. Signals were detected by autoradiography. |
RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSImmunoprecipitation and Western...In Vitro PI3K Activity...RESULTSDISCUSSIONREFERENCES |
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. Although VEGF mRNA was not detectable in parental PAE cells (not shown) or PAE cells expressing the empty vector (Fig. 1C)
, expression of ßPDGFR in PAE (PAE/ßPDGFR) cells caused production of a significant amount of VEGF mRNA (Fig. 1, B and C, first lane)
and protein (Fig. 5B, first lane)
without stimulation. This was probably due to an autocrine induction of VEGF by a small amount of PDGF expressed by the PAE cells because PDGF-BB neutralizing antibody (R&D Systems) blocked such induction (data not shown). Exposure of PAE/ßPDGFR cells to exogenous PDGF-BB caused a further and substantial induction of VEGF mRNA (Fig. 1, B and C)
. Similar results were observed in human umbilical vascular endothelial cells that express exogenous ßPDGFR (data not shown). Basal levels of VEGF mRNA were increased by 1 ng/ml PDGF-BB and reached a peak level by 20 ng/ml in a concentration-dependent fashion (Fig. 1C)
. Kinetic studies using 50 ng/ml PDGF-BB revealed that maximal induction was achieved by 2 h and then started to decrease at 4 h, gradually returning to basal level by 12 h (Fig. 1B)
. Taken together, these results showed that introduction of ßPDGFR into endothelial cells created an autocrine loop with endogenous PDGF, resulting in VEGF induction by the cells. Exposure of these cells to exogenous PDGF further augmented VEGF induction in a concentration- and time-dependent manner.
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, three protein bands were recognized by anti-VEGF antibody: (a) a major band with a molecular mass of 23 kDa was similar to that VEGF165 (Fig. 2A, middle band)
; (b) an upper band that was most likely VEGF189; and (c) a lower band that appears to be a proteolytic product rather than VEGF121 that lacks heparin-binding domain and was removed from the partially purified samples. For better quantitation, the original nonpurified CM was used for VEGF ELISA. As shown in Fig. 2B
, PDGF stimulation of PAE/ßPDGFR cells caused a 5-fold increase in VEGF protein accumulation, which correlated with the VEGF mRNA levels in these cells as shown in Fig. 1
. Although vector-transfected cells expressed little, if any, VEGF protein, untreated PAE/ßPDGFR cells expressed an elevated VEGF protein, also consistent with the low VEGF mRNA levels in these cells (Fig. 1)
.
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). However, the CM from PDGF-induced cells greatly increased levels of BrdUrd incorporation as did recombinant VEGF protein (Fig. 3)
. Anti-VEGF neutralizing antibody completely blocked the elevated BrdUrd signal in either case, whereas an IgG control antibody had no effect, indicating that VEGF protein in the CM was responsible for the increased BrdUrd incorporation. Thus, the PDGF-induced VEGF protein was functional and able to stimulate endothelial cell mitogenesis.
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. Analysis of VEGF mRNA levels at various time points after actinomycin D addition in PDGF-treated and -untreated cells revealed that the half life of VEGF mRNA was 
35 min (data not shown), similar to that in other systems (43)
. To determine whether de novo protein synthesis was required for PDGF-induced VEGF transcription, the PAE/ßPDGFR cells were exposed to PDGF in the presence or absence of cycloheximide, an inhibitor of translational elongation. Cycloheximide alone and cycloheximide together with PDGF increased both the basal and induced levels of VEGF mRNA, suggesting that: (a) PDGF-induced VEGF transcription did not require de novo protein synthesis; and (b) VEGF mRNA degradation required the synthesis of a labile protein(s). As a control, both the reagents showed similar effects on TPA-induced VEGF transcription, as has been shown previously in other cell systems (19
, 20)
. Thus, PDGF-driven VEGF expression in PAE/ßPDGFR cells was regulated at the transcriptional level and did not require de novo protein synthesis.
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, two specific PI3K inhibitors with different modes of action, wortmannin and LY24009, blocked PDGF-induced VEGF expression in a concentration-dependent manner, suggesting that this induction requires PI3K. Because PKC has been shown previously to be essential for PDGF-dependent VEGF induction in NIH3T3 mouse fibroblasts (19)
, we tested the effects of the specific PKC inhibitor, bisindolylmaleimide GF 109203X (BIM), on VEGF induction in PAE/ßPDGFR cells. BIM inhibited TPA-induced VEGF expression (Fig. 5C)
but had no inhibitory effect on PDGF-induced VEGF expression; rather, potentiation of the PDGF-induced VEGF up-regulation was apparent (Fig. 5B)
. Although the mechanism by which this inhibitor potentiated VEGF induction by PDGF is not known, these data demonstrated that the induction was independent of PKC in endothelial cells. The selective effects of both the PKC and PI3K inhibitors on PDGF- or TPA-induced VEGF expression established their specificity. These results strongly suggested that PI3K, but not PKC, was necessary for PDGF-induced VEGF transcription in PAE/ßPDGFR cells, whereas the opposite was the case for TPA-induced VEGF expression (Fig. 5C)
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Maximal VEGF Induction Requires the Ability of ßPDGFR to Activate PI3K.
To further test the involvement of PI3K in PDGF-induced VEGF expression, we used mutant ßPDGFR forms that had been shown previously to be useful in a dissection of the relative role of each of the ß receptor-associated proteins in PDGFR-mediated mitogenic signaling (37)
. These receptors are shown schematically in Fig. 6A
. The F5 ßPDGFR mutant lacks five tyrosine phosphorylation sites and consequently does not associate with PLC, GAP, SHP-2, or PI3K. The Y40/51 receptor was made by restoring the two tyrosine residues required for docking PI3K back to the F5 receptor, resulting in binding and activation of PI3K but not the other three proteins. The F40/51 receptor lacks the docking sites for PI3K alone and therefore binds all effectors but PI3K (37)
. R634 is a kinase-defective receptor. Each receptor species was introduced into PAE cells; populations containing similar amounts of cell surface receptors (Fig. 6B, top panel)
were isolated by fluorescence-activated cell sorting, and their levels of expression were comparable with that reported in NIH3T3 cells as determined by Western blotting (data not shown).
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, and that PDGF stimulation resulted in tyrosine phosphorylation of each mutant receptor except the R634 kinase-defective mutant (Fig. 6B, middle panel)
. The coprecipitation of p85, a regulatory subunit of PI3K, was consistent with this activation, as was the small amount of p85 protein coprecipitated with the F5, F40/51, or R634 receptors (Fig. 6B, bottom panel)
. As expected, PI3K activity was detected in the immunoprecipitates from activated wild-type and Y40/51 receptors but not from the other mutant receptors (Fig. 6C)
.
Next we investigated the ability of the mutant receptors to mediate PDGF-driven VEGF transcription. Cells expressing the wild-type and mutant receptors were serum starved and treated with PDGF-BB for 2 h. Steady-state VEGF mRNA levels were analyzed by Northern blotting (Fig. 7A)
. PDGF-dependent VEGF expression was absent in cells expressing the R634 kinase-defective receptors, indicating that the intrinsic kinase activity of the receptor was a requirement for PDGF-driven VEGF induction. Mutating the tyrosine phosphorylation sites for PI3K binding (F40/51) caused a decrease in VEGF transcription by 3-fold in comparison to the wild-type receptors. Cells expressing the F5 mutant receptors, which lack five tyrosine phosphorylation sites and fail to associate with PI3K as well as other effectors (PLC, SHP-2, and GAP; Ref. 37
), expressed slightly less VEGF mRNA than the cells expressing the F40/51 receptors, suggesting that receptor-mediated signaling pathway(s) other than PI3K, PLC-, SHP-2, or GAP may also contribute somewhat to PDGF-dependent VEGF induction. Consistent with this, restoration of the PI3K docking sites to the F5 receptor (Y40/51) resulted in a 2-fold increase in VEGF expression compared with the F5 receptors. It should be pointed out that, as shown in Figs. 1
and 2
, the basal level of VEGF mRNA in receptor transfectants increased largely due to an autocrine PDGF induction of VEGF expression. Therefore, the magnitude of VEGF increase upon exogenous PDGF stimulation appears to represent an increased response to doses of PDGF. In this light, the effects of mutant receptors on VEGF induction are best assessed by comparing levels of VEGF mRNA among PDGF-stimulated cells expressing those receptors rather than comparing the magnitude of VEGF stimulation upon exogenous PDGF stimulation among mutant receptors.
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and VEGF ELISA (Fig. 7B, lower panel)
. Only a small amount of VEGF proteins was detected by both methods in the CM collected from cells expressing the F5 or F40/51 receptors. In contrast, a significant increase in VEGF protein accumulation was observed in the CM from the cells expressing Y40/51 receptors, in agreement with the VEGF mRNA data shown in Fig. 7A
. Apparently, the effects of the F5 and F40/51 receptors on VEGF protein accumulation in the CM was more profound than on VEGF transcription, possibly due to decreases of VEGF protein translation, secretion, and/or protein stability.
Finally, we determined whether the levels of VEGF proteins in the CM collected from the cells expressing mutant receptors correlated with their activity to stimulate VEGF receptor (KDR)-mediated endothelial cell migration in a modified Boyden chamber in vitro (Fig. 8)
. The CM from the PDGF-treated wild-type and the Y40/51 cells both stimulated PAE/KDR cell migration more than 2-fold over that of the CM from unstimulated cells. Neutralizing anti-VEGF monoclonal antibody suppressed the chemotactic activity of recombinant human VEGF and the CM to background level, but control IgG had no effect, indicating that VEGF was a major component in the CM responsible for stimulating PAE/KDR cell migration. In contrast, the CM from the F5, F40/51, or R634 cells had little stimulatory activity. Taken together, these data show that the levels of PDGF induction of VEGF mRNA and protein were correlated with the ability of mutant receptors to associate and activate PI3K, indicating that PDGF-induced VEGF expression is largely mediated by PI3K activity. We also showed that the amount of VEGF protein expressed by these cells was functional in its ability to stimulate endothelial cell migration.
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iSH2-N; Refs. 38
and 39
). Two independent clones were selected for having similarly high levels of expression of the exogenous p85 iSH2-N protein and ßPDGFR compared with clones expressing the empty vector (Fig. 9A)
. As shown in Fig. 9B
, overexpression of the p85 iSH2-N significantly blocked PDGF-induced VEGF mRNA, whereas expression of the empty vector had no effect. Consistent with the data obtained with the mutant receptors (Fig. 7A)
, dominant-negative PI3K constructs had little effect on relative levels of VEGF increases in response to exogenous PDGF because they also inhibited basal levels of VEGF mRNA that is caused by autocrine PDGF induction of VEGF expression, which apparently also requires PI3K activity. The inhibitory effect of the p85 mutant on VEGF induction appeared somewhat greater than that obtained by preventing activation of receptor-associated PI3K from F40/51 mutant receptor (Fig. 5A)
, perhaps suggesting its ability to interfere with upstream signaling molecules needed for activation of PI3K both by the receptor and by Ras, because Ras has been shown to be required for efficient activation of PI3K by PDGF (45)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSImmunoprecipitation and Western...In Vitro PI3K Activity...RESULTSDISCUSSIONREFERENCES |
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The expression of ßPDGFR on endothelial cells is a prerequisite for PDGF to elicit a direct effect on neovascularization. A large number of fresh samples of human astrocytomas of all grades examined by in situ hybridization and immunocytochemistry techniques showed that expression of ßPDGFR was not detectable in the endothelial cells of normal human brain but was induced in the vasculature of low- and high-grade gliomas, particularly in the hyperplastic endothelium of glioblastoma multiforme (30 , 31 , 46) . Up-regulation of ßPDGFR has also been reported for the capillary endothelial cells in human carcinoid tumors, in wounds, and in inflammatory tissues (32 , 47 , 48) . These observations suggest that PDGF may have a direct effect on endothelial cells undergoing angiogenesis. In this regard, PDGF may act as a mitogen and directly stimulate endothelial cell proliferation (49) , or it may induce VEGF expression in endothelial cells, which in turn causes an autocrine stimulation through VEGF receptors. Here, we demonstrated that expression of ßPDGFR in normal endothelial cells created an autocrine PDGF induction of VEGF expression, which was markedly augmented by exposure to exogenous PDGF. The VEGF proteins that were secreted were functional and capable of stimulating endothelial cell mitogenesis and migration. These results may be important to understanding the mechanisms of both autocrine and paracrine PDGF action in up-regulating VEGF expression in many pathological conditions where ßPDGFR is induced in neovascular endothelial cells. For example, this may explain the in vivo effect of PDGF on the development of a prominent blood vessel network in xenotransplanted human melanoma tumors (50) . Studies of transgenic mouse models of tumorigenesis and human breast and cervical cancers have revealed that the angiogenic switch occurs in the early stages of tumor development, even preceding the appearance of solid tumors (51) . In the case of glioma tumorigenesis, PDGF-induced VEGF expression may contribute not only to the expansion of an established tumor but also to the regulation of the angiogenic switch for initial tumor development, because induction of ßPDGFR on endothelial cells can occur in early stages of glioma formation (30 , 31 , 46) . These data emphasize the importance of epigenetic changes such as up-regulation of PDGF and its receptors in regulating angiogenic switch in the early stages of tumor development, as opposed to hypoxia, which may be a major stimulus for neovascularization in an established tumor where the hypoxic condition becomes evident.
These findings extend the actions of VEGF from its usual paracrine mechanism whereby tumor cells secrete it to stimulate endothelial cell migration and proliferation. There is substantial evidence to indicate that endothelial cells themselves are capable of expressing VEGF as well. VEGF transcripts have been identified in several cultured endothelial cells such as rat brain capillary endothelial cells, bovine glomerular and retinal endothelial cells (52, 53, 54) , and in diabetic neovascular membranes (55) . Although many types of human endothelial cells express no VEGF under quiescent conditions, they can express VEGF in response to hypoxia (56 , 57) and, as we showed here, in response to external sources of PDGF.
We have also established PI3K as a mediator essential in ßPDGFR-mediated signaling leading to VEGF induction in endothelial cells by demonstrating that PDGF-induced VEGF expression was blocked by the PI3K inhibitors, wortmannin and LY24009, and by the dominant interfering p85 subunit of PI3K. This was further supported by the observations that whereas a mutant receptor that does not activate PI3K as well as three other receptor binding proteins expressed a minimal level of VEGF, restoring the PI3K binding sites to the receptor rescued nearly complete VEGF induction. We also showed that this induction was independent of PKC, in contrast to a previous report that NIH3T3 mouse fibroblasts cells require PKC for PDGF-dependent VEGF expression (19) , possibly due to cell type specificity. It is also noteworthy that the induction of VEGF transcription by hypoxia in Ras-transformed NIH3T3 mouse fibroblasts appears to require PI3K as well (58) .
Our data on PI3K-dependent VEGF induction in endothelial cells suggest a novel function for PI3K in angiogenesis. This complements and extends previous data that have implicated PI3K in tumor-promoting functions such as transformation, cell survival, anchorage-independent growth, and cell motility and invasion (59, 60, 61, 62, 63)
. It will be intriguing to investigate which of the downstream effectors of PI3K are involved in PDGF-dependent VEGF induction in endothelial cells. The known downstream effectors of PI3K include several PKC isoforms (PKC
, PKC
, and PKC
), ribosomal p70S6K, the serine/threonine kinase Akt (also known as PKB- and RAC-), and the small G protein Rac, each of which mediates distinct biological responses (64)
. Recent reports also suggest that PI3K may intersect with the Raf/mitogen-activated protein kinase pathway leading to gene regulation (65)
. In any case, the present studies suggest that under certain pathological conditions, VEGF can be activated through the activation of ßPDGFR expressed on endothelial cells, thus modulating endothelial cell functions through an autocrine pathway that may enhance the paracrine effects of VEGF in stimulating angiogenesis. This induction is largely mediated by the PI3K signaling pathway, suggesting that PI3K plays a role in angiogenesis and is therefore a potential target for therapeutic inhibition of angiogenesis and tumorigenesis.
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ACKNOWLEDGMENTS |
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SH2-N in pSG5, and members of the laboratory for critical reading of the manuscript. |
FOOTNOTES |
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1 D. W. was supported in part by the Rebert Steel Foundation for Pediatric Cancer Research. 
2 To whom requests for reprints should be addressed, at Ludwig Institute for Cancer Research, San Diego Branch, 9500 Gilman Drive, La Jolla, CA 92093-0660. Fax: (619) 534-7816; E-mail: dbwang{at}ucsd.edu
3 The abbreviations used are: VEGF, vascular endothelial growth factor; PKC, protein kinase C; PLC, phospholipase C-1; GAP, GTPase-activating protein of Ras; SFM, serum-free medium; CM, conditioned medium; PAE, porcine aortic endothelial; TPA, phorbol 12-tetradecanoate 13-acetate; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BrdUrd, bromodeoxyuridine.
Received 10/16/98. Accepted 1/29/99.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSImmunoprecipitation and Western...In Vitro PI3K Activity...RESULTSDISCUSSIONREFERENCES |
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1, and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor’s mitogenic signal. Cell, 73: 321-334, 1993.[Medline]
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 G. Boguslawski, P. W. McGlynn, K. A. Harvey, and A. T. Kovala SU1498, an Inhibitor of Vascular Endothelial Growth Factor Receptor 2, Causes Accumulation of Phosphorylated ERK Kinases and Inhibits Their Activity in Vivo and in Vitro J. Biol. Chem., February 13, 2004; 279(7): 5716 - 5724. [Abstract] [Full Text] [PDF]
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 D. J. Brat and T. B. Mapstone Malignant Glioma Physiology: Cellular Response to Hypoxia and Its Role in Tumor Progression Ann Intern Med, April 15, 2003; 138(8): 659 - 668. [Full Text] [PDF]
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P. Guo, B. Hu, W. Gu, L. Xu, D. Wang, H.-J. S. Huang, W. K. Cavenee, and S.-Y. Cheng Platelet-Derived Growth Factor-B Enhances Glioma Angiogenesis by Stimulating Vascular Endothelial Growth Factor Expression in Tumor Endothelia and by Promoting Pericyte Recruitment Am. J. Pathol., April 1, 2003; 162(4): 1083 - 1093. [Abstract] [Full Text] [PDF]
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 S. Kiriakidis, E. Andreakos, C. Monaco, B. Foxwell, M. Feldmann, and E. Paleolog VEGF expression in human macrophages is NF-{kappa}B-dependent: studies using adenoviruses expressing the endogenous NF-{kappa}B inhibitor I{kappa}B{alpha} and a kinase-defective form of the I{kappa}B kinase 2 J. Cell Sci., February 15, 2003; 116(4): 665 - 674. [Abstract] [Full Text] [PDF]
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N. Pore, S. Liu, D. A. Haas-Kogan, D. M. O'Rourke, and A. Maity PTEN Mutation and Epidermal Growth Factor Receptor Activation Regulate Vascular Endothelial Growth Factor (VEGF) mRNA Expression in Human Glioblastoma Cells by Transactivating the Proximal VEGF Promoter Cancer Res., January 1, 2003; 63(1): 236 - 241. [Abstract] [Full Text] [PDF]
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S. A. Woods, C. J. McGlade, and A. Guha Phosphatidylinositol 3'-kinase and MAPK/ERK kinase 1/2 differentially regulate expression of vascular endothelial growth factor in human malignant astrocytoma cells Neuro-oncol, October 1, 2002; 4(4): 242 - 252. [Abstract] [PDF]
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C. Treins, S. Giorgetti-Peraldi, J. Murdaca, G. L. Semenza, and E. Van Obberghen Insulin Stimulates Hypoxia-inducible Factor 1 through a Phosphatidylinositol 3-Kinase/Target of Rapamycin-dependent Signaling Pathway J. Biol. Chem., July 26, 2002; 277(31): 27975 - 27981. [Abstract] [Full Text] [PDF]
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C. B. Knobbe, A. Merlo, and G. Reifenberger Pten signaling in gliomas Neuro-oncol, July 1, 2002; 4(3): 196 - 211. [Abstract] [PDF]
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 L. J. Metheny-Barlow, B. Flynn, H. E. van Gijssel, A. Marrogi, and B. I. Gerwin Paradoxical Effects of Platelet-Derived Growth Factor-A Overexpression in Malignant Mesothelioma . Antiproliferative Effects In Vitro and Tumorigenic Stimulation In Vivo Am. J. Respir. Cell Mol. Biol., June 1, 2001; 24(6): 694 - 702. [Abstract] [Full Text] [PDF]
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E. Y. Chen, N. M. Mazure, J. A. Cooper, and A. J. Giaccia Hypoxia Activates a Platelet-derived Growth Factor Receptor/Phosphatidylinositol 3-Kinase/Akt Pathway That Results in Glycogen Synthase Kinase-3 Inactivation Cancer Res., March 1, 2001; 61(6): 2429 - 2433. [Abstract] [Full Text]
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 B. D. Smolich, H. A. Yuen, K. A. West, F. J. Giles, M. Albitar, and J. M. Cherrington The antiangiogenic protein kinase inhibitors SU5416 and SU6668 inhibit the SCF receptor (c-kit) in a human myeloid leukemia cell line and in acute myeloid leukemia blasts Blood, March 1, 2001; 97(5): 1413 - 1421. [Abstract] [Full Text] [PDF]
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S. Xiong, R. Grijalva, L. Zhang, N. T. Nguyen, P. W. Pisters, R. E. Pollock, and D. Yu Up-Regulation of Vascular Endothelial Growth Factor in Breast Cancer Cells by the Heregulin-{beta}1-activated p38 Signaling Pathway Enhances Endothelial Cell Migration Cancer Res., February 1, 2001; 61(4): 1727 - 1732. [Abstract] [Full Text]
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A. Maity, N. Pore, J. Lee, D. Solomon, and D. M. O’Rourke Epidermal Growth Factor Receptor Transcriptionally Up-Regulates Vascular Endothelial Growth Factor Expression in Human Glioblastoma Cells via a Pathway Involving Phosphatidylinositol 3'-Kinase and Distinct from That Induced by Hypoxia Cancer Res., October 1, 2000; 60(20): 5879 - 5886. [Abstract] [Full Text]
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 W. Zundel, C. Schindler, D. Haas-Kogan, A. Koong, F. Kaper, E. Chen, A. R. Gottschalk, H. E. Ryan, R. S. Johnson, A. B. Jefferson, et al. Loss of PTEN facilitates HIF-1-mediated gene expression Genes & Dev., February 15, 2000; 14(4): 391 - 396. [Abstract] [Full Text]
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R. Bagheri-Yarmand, R. K. Vadlamudi, R.-A. Wang, J. Mendelsohn, and R. Kumar Vascular Endothelial Growth Factor Up-regulation via p21-activated Kinase-1 Signaling Regulates Heregulin-beta 1-mediated Angiogenesis J. Biol. Chem., December 8, 2000; 275(50): 39451 - 39457. [Abstract] [Full Text] [PDF]
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B.-H. Jiang, J. Z. Zheng, M. Aoki, and P. K. Vogt Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells PNAS, February 15, 2000; 97(4): 1749 - 1753. [Abstract] [Full Text] [PDF]
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). The larger minor may be VEGF189 (
). WT, wild type. B, for better quantitation, amount of VEGF proteins presented in CM was analyzed by VEGF ELISA using non-purified CM; bars, SD.
, + PDGF.