Role of Protein Kinase Czeta in Ras-mediated Transcriptional Activation of Vascular Permeability Factor/Vascular Endothelial Growth Factor Expression*

Soumitro Pal, Kaustubh Datta, Roya Khosravi-FarDagger, and Debabrata Mukhopadhyay§

From the Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

Received for publication, August 28, 2000, and in revised form, October 13, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a multifunctional cytokine, is regulated by different factors including degree of cell differentiation, hypoxia, and certain oncogenes namely, ras and src. The up-regulation of VPF/VEGF expression by Ras has been found to be through both transcription and mRNA stability. The present study investigates a novel pathway whereby Ras promotes the transcription of VPF/VEGF by activating protein kinase Czeta (PKCzeta ). The Ras-mediated overexpression of VPF/VEGF was also found to be inhibited by using the antisense or the dominant-negative mutant of PKCzeta . In co-transfection assays, by overexpressing oncogenic Ha-Ras (12 V) and PKCzeta , there was an additive effect up to 4-fold in activation of Sp1-mediated VPF/VEGF transcription. It has been shown through electrophoretic mobility shift assay that Ras promoted the PKCzeta -induced binding of Sp1 to the VPF/VEGF promoter. In the presence of PDK-1, a major activating kinase for PKC, the Ras-mediated activation of VPF/VEGF promoter through PKCzeta was further increased, suggesting that PKCzeta can serve as an effector for both Ras and PDK-1. In other experiments, with the use of a dominant-negative mutant of phosphatidylinositol 3-kinase, the activation of VPF/VEGF promoter through Ras, PDK-1, and PKCzeta was completely repressed, indicating phosphatidylinositol 3-kinase as an important component of this pathway. Taken together, these data elucidate the signaling mechanism of Ras-mediated VPF/VEGF transcriptional activation through PKCzeta and also provide insight into PKCzeta and Sp1-dependent transcriptional regulation of VPF/VEGF.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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The growth and metastasis of tumors depend on the development of an adequate blood supply via angiogenesis which is attributed in large part to the production of angiogenesis promoting growth factors by tumor (1, 2). Although many angiogenic factors have been described, one in particular stands out for its potency and specificity, namely vascular permeability factor/vascular endothelial growth factor (VPF1/VEGF) (3, 4). VPF/VEGF is overexpressed by a wide variety of human tumors and plays a critical role in tumorigenesis. Although constitutively expressed by many tumor cells, VPF/VEGF expression is substantially up-regulated by hypoxia, cytokines, hormones, and certain oncogenes including activated forms of src and ras (5-7). We have recently shown that protein kinase signaling pathways also play an important role in tumor angiogenesis (8, 9). Among the protein kinase C (PKC) family, the isoform PKCzeta plays a critical role in regulating VPF/VEGF overexpression.

PKCzeta represents an atypical PKC isoform in that: 1) it lacks the C2 domain making its kinase activity Ca2+ independent, and 2) it possesses only one zinc finger region in its regulatory domain (10). Consequently, PKCzeta does not bind Ca2+ and cannot be activated by diacylglycerol or phorbol esters (11). In addition, prolonged treatment with phorbol esters does not down-regulate PKCzeta (10), and most PKC inhibitors do not decrease PKCzeta activity (11). PKCzeta is found to be involved in a wide range of physiological processes including mitogenesis, protein synthesis, cell survival, and transcriptional regulation (12, 13). Like many other protein kinases, PKCzeta requires phosphorylation within its activation loops to express full catalytic potential (13, 14).

For the past few years, research has focused on the role of the oncogene in the signaling pathways controlling cell growth and differentiation (15, 16). Ras has also been found to be involved both in transcriptional and post-transcriptional up-regulation of VPF/VEGF expression, and thus angiogenesis (17). The persistent activation of signaling pathways induced by Ras accounts for overexpression of VPF/VEGF in a significant fraction of human tumors (7, 17). PKCzeta could have an important cross-talk with Ras (18). Several reports have shown that cell stimulation activates Ras which triggers a number of important serine/threonine kinases that have MAP kinase kinase (MEK) as substrate, such as MEK kinase, c-Raf-1, and B-Raf, culminating in the activation of MAP kinase (MAPK) (19-22). The kinase-deficient mutants of Raf-1, MEKs, and MAPKs have been shown to block Ras-mediated signaling events and transformation (23, 24). It has been suggested that Ras may function primarily to promote the translocation of Raf-1 from the cytosol to the plasma membrane, where subsequent Ras-independent events trigger Raf-1 kinase activation (25). In some studies, it has clearly been shown that PKCzeta may also serve as a downstream target of Ras (26). For example, both proteins have been found to be critically involved in the activation of NF-kappa B (27, 28). It appears that the mechanism whereby PKCzeta controls cell signaling could, at least in part, implicate the channeling of Ras signals in the activation of MAPK (26). However, despite the evidence that Raf-1 is a critical downstream effector of Ras function, there is increasing evidence that Ras may mediate its action through the activation of multiple downstream effector-mediated pathways (29). For example, the existence of Raf-independent Ras signaling pathways is suggested by the expanding roster of candidate Ras effectors that have been identified (30-33). Like Raf-1, these functionally diverse proteins, including Rho family members, show preferential binding to the active GTP-bound form of Ras and this interaction requires an active Ras effector domain (amino acids 32 to 40).

Some recent findings have shown that the enzyme phosphoinositide-dependent protein kinase-1 (PDK-1) is at the hub of many signaling pathways, activating PKB and PKC isoenzymes as well as p70-S6 kinase and perhaps PKA (13, 34-36). It has been shown that PDK-1 induces PKCzeta phosphorylation in vivo, leading to its activation (13). Furthermore, PDK-1 directly phosphorylates PKCzeta at the activation-loop Thr410 residue in vitro (13, 14, 37). As PKCzeta phosphorylation/activation is almost completely blocked by coexpression of dominant-negative PDK-1 or by mutation of Thr410, it is likely that a pre-requisite for PKCzeta activation is phosphorylation of Thr410 (13, 38). PKCzeta is found to be associated with PDK-1 in the same complex, and also identified as its in vivo substrate along with Akt/PKB and p70 S6 kinase (39). It has also been shown that PDK-1 is recruited in the signaling pathways through PI 3-kinase and serves as a multifunctional downstream effector (13, 37, 39).

There are several reports that PKCzeta is activated by important lipid intracellular mediators like phosphatidic acid (40), phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3) (11), and ceramide (41). It has been shown that insulin and insulin-like growth factor-1 leads to the activation of PKCzeta which can be inhibited by chemical inhibitors of PI 3-kinase (42). Similarly, activation of PI 3-kinase by lipopolysaccharide leads to the activation of PKCzeta which is sensitive to PI 3-kinase inhibitors and a dominant-negative PI 3-kinase mutation (43). Taken together, these observations pinpoint PKCzeta as a target of important lipid second messengers and support its role in cell signaling.

In previous studies, we have shown that PKCzeta plays a significant role in promotion of tumor angiogenesis by stimulating the expression of VPF/VEGF (8, 9). In addition to rendering microvessels hyperpermeable, VPF/VEGF stimulates endothelial cells to migrate and divide and profoundly alters their pattern of gene expression (3, 4, 44-47). We have shown that PKCzeta interacts with and phosphorylates the transcription factor Sp1 and increases the VPF/VEGF promoter activity in human fibrosarcoma (HT1080) and renal cell carcinoma (786-0) cell lines, where basal level of VPF/VEGF is also very high (9). In the present study, we dissect the upstream signaling pathways, with special emphasis on Ras, that is required for activation of PKCzeta to promote Sp1-mediated VPF/VEGF transcription. Our observations clearly indicate that coordinated signaling through Ras, PDK-1, and PI 3-kinase may be required to mediate PKCzeta -induced activation of VPF/VEGF promoter.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Cell Culture-- Human fibrosarcoma (HT1080) and renal cell carcinoma (786-0) cell lines were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Hyclone Laboratories).

Plasmids-- All the VPF/VEGF reporter constructs used in transient transfection assays contain sequences derived from the human VPF/VEGF promoter driving expression of firefly luciferase. The 0.35- and 0.07-kb deletion mutant constructs were made by polymerase chain reaction from the 2.6-kb VPF/VEGF promoter fragment and subcloned into pGL-2 Basic vector (Promega) as described earlier (48). The overexpressed PKCzeta and a kinase inactive PKCzeta cDNA (PKCzeta KW; LYS-275 to tryptophan substitution), both subcloned into pCMV2FLAG vector were generous gifts from Alex Toker (13). All Ras expression constructs encode mutant versions of the transforming human Ha-Ras(G12V). The pDCR-ras(G12V), pDCR-ras(G12V,T35S), and pDCR-ras(G12V,E37G) mammalian constructs encode effector domain mutants of Ha-Ras(G12V) in which expression is under the control of the cytomegalovirus promoter (31). The Ras(Q61L,C186S) is the dominant inhibitory mutant of Ras (31). The Myc-PDK-1, Myc-PDK-1.K/N, and GST-Delta p85 were generous gifts from Alex Toker (18). The kinase-inactive variant Myc-PDK-1.K/N was made by mutating the conserved critical Lys110 residue to Asn.

Antiserum and Oligonucleotides-- A polyclonal anti-rabbit antibody directed against the phosphorylated activation loop Thr410 of PKCzeta was received from Alex Toker as a generous gift (13). All PKC oligonucleotides were synthesized as phosphorothioate derivatives from Genemed Synthesis (San Francisco, CA) (49).

Northern Blot Analysis-- RNA, isolated by the single-step acid-phenol extraction method (8, 49), was separated on a formaldehyde-agarose gel, transferred to a GeneScreen membrane by using 10 × SSC, and probed with random primer-labeled cDNAs in a solution containing 0.5 M sodium phosphate (pH 7.2), 7% SDS, 1% bovine serum albumin, 1 mM EDTA, and sonicated herring sperm DNA (50 µg/ml) at 68 °C. Blots were washed three times with a solution containing 40 mM sodium phosphate (pH 7.2), 0.5% SDS, 0.5% bovine serum albumin, and 1 mM EDTA at 68 °C and quantitated by laser densitometry.

Immunoprecipitations-- As described earlier, cells were washed twice with cold phosphate-buffered saline, lysed with ice-cold lysis buffer (50 mM Tris, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM Na3VO4, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 0.5% aprotinin, and 2 mM pepstatin A), incubated for 10 min on ice, and centrifuged for 10 min at 4 °C (8, 48). Immunoprecipitations were carried at antibody excess, using 0.5 mg of total protein either with a mouse monoclonal antibody (1 µg) directed against Ras (Transduction Laboratories) or a rabbit polyclonal antibody (1 µg) directed against PKCzeta (Chemicon International Inc). Immunocomplexes were captured with protein A-agarose beads (Amersham Pharmacia Biotech). After three washes with cell lysis buffer, bead-bound proteins were subjected to Western blot analysis.

Western Blot Analysis-- Protein samples were mixed with 2 × sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 10% beta -mercaptoethanol, 4% sodium dodecyl sulfate (SDS), and 0.0025% bromphenol blue), boiled, and run on 7.5-10% polyacrylamide gels with Tris glycine-SDS running buffer (Bio-Rad). Agarose beads with bound proteins were handled in the same manner and directly loaded on the gel. Size-separated proteins were transferred to a polyvinylidene difluoride membrane (PerkinElmer Life Sciences) at 70 volts. For immunodetection, the membranes were blocked with 5% milk or 2% bovine serum albumin in phosphate-buffered saline-Tween 20 (PBST) and then coated with primary antibody. After washings, the membranes were incubated with peroxidase-linked secondary antibody and the reactive bands were detected by chemiluminescent substrate.

Transfection Assays-- Cells were plated at 2-3 × 105 cells/60-mm dish 1 day before transfection with VPF/VEGF promoter-luciferase construct and expression plasmids using the calcium-phosphate precipitation method (50). The expression was normalized with a control empty expression vector. Cells were harvested for luciferase assay 40 h after transfection. Luciferase activity was measured using the luciferase assay kit (Promega). In all co-transfection experiments, transfection efficiency was normalized by assaying beta -galactosidase activity using the beta -galactosidase gene under control of cytomegalovirus immediate early promoter as internal control. 786-0 cells were transfected using Effectene transfection reagent (Qiagen), following manufacturer's protocol. 1:25 ratio of DNA to Effectene was used for all the experiments. For all the transfection assays, average results from three independent experiments were plotted.

Nuclear Extract Preparation and Electrophoretic Mobility Shift Assays (EMSAs)-- Nuclear extracts were prepared from HT1080 cells following a standard protocol (48), with modifications. Cells were washed in cold phosphate-buffered saline, suspended in buffer A (10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 10 µg/ml aprotinin, 3 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride) and incubated for 15 min on ice. Cells were then lysed with 0.5% Nonidet P-40 and the pellets were resuspended in buffer C (50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol, 3 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride). Following incubation on a rotating rack for 25 min, samples were centrifuged at 14,000 rpm for 10 min. Clear supernatants, containing the nuclear proteins were collected and stored at -70 °C.

EMSAs were performed as described previously (48). Briefly, EMSA binding reaction mixtures (25 µl) contained 20 mM HEPES (pH 8.4), 100 mM KCl, 20% glycerol, 0.1 mM EDTA, 0.2 mM ZnSO4, 0.05% Nonidet P-40, and 1 µg of bovine serum albumin. Extract protein and 200 ng of poly(dA-dT)·poly(dA-dT) were added at room temperature 10 min prior to addition of ~0.1 ng of radiolabeled oligonucleotide probe. After 20 min incubation at 4 °C, samples were run on 7% acrylamide gel in 1 × TAE (40 mM Tris acetate, 1 mM EDTA) buffer.

The radiolabeled oligonucleotide used in EMSA studies was a 188-base pair polymerase chain reaction-generated fragment (base pair -195 to -7, relative to the transcription start site) of the VPF/VEGF promoter containing the four putative Sp1-binding sites (48).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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PKCzeta , an Intermediary Molecule for Ras-mediated Overexpression of VPF/VEGF-- In the present study we demonstrate that oncogene Ha-Ras(G12V) promoted the Sp1-mediated VPF/VEGF transcriptional activation in human fibrosarcoma (HT1080) and renal cell carcinoma (786-0) cell lines (Fig. 1). 786-0 and HT1080 cells were co-transfected with a 2.6-kb VPF/VEGF promoter-luciferase construct and plasmid containing Ha-Ras(G12V). VPF/VEGF reporter activity was increased up to 2-fold in comparison with cells transfected with expression vector alone. To define the region of the VPF/VEGF promoter that is responsive to Ras, we utilized two different 5' deletions of the 2.6-kb promoter-reporter vector and co-transfected these deletions with a plasmid containing Ha-Ras(G12V). Ras increased the reporter activity by 2-fold in the 0.35-kb segment of the VPF/VEGF promoter that contains the Sp1-binding site, although there was no change of reporter activity in the 0.07-kb VPF/VEGF promoter having the deleted Sp1-binding site (Fig. 1) (48). These results suggest that transforming human Ras itself activates VPF/VEGF transcription in a Sp1-dependent manner.



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Fig. 1.   Effect of oncogenic Ras on VPF/VEGF promoter activity. Human renal cell carcinoma (786-0) and fibrosarcoma (HT1080) cell lines were co-transfected with 2.6-, 0.35-, or 0.07-kb VPF/VEGF promoter-luciferase constructs (1.0 µg) and Ha-Ras(G12V) (1.2 µg) expression vectors. Cells were harvested for luciferase assays 40 h after transfection, and fold activation was calculated as relative to the activity of same reporter construct co-transfected with an empty expression vector (pDCR). The raw values of luciferase activities of 2.6-, 0.35-, or 0.07-kb VPF/VEGF promoters are 32,194 ± 180, 31,534 ± 177, and 19,532 ± 140, respectively, in 786-0 cells. In case of HT1080 cells, these values are 8,606 ± 92, 8,230 ± 90, and 4,115 ± 64, respectively. Open bars represent the empty expression vector, while the black bars represents Ha-Ras(G12V) expression vector.

To elucidate the role of PKCzeta in Ras-mediated VPF/VEGF overexpression, we studied the effect of the dominant-negative mutant (kw) and the antisense (AS) oligonucleotide of PKCzeta on the expression of VPF/VEGF mRNA in HT1080 and 786-0 cells. In these two cell lines, the oncogenic Ras is already activated and as a result the basal level of VPF/VEGF is very high. Through Northern blot analysis, we demonstrate that the PKCzeta (kw) and PKCzeta (AS) clearly reduced the VPF/VEGF mRNA expression level by 50 and 70%, respectively (at their highest doses) in HT1080 cells (Fig. 2). We also found the same pattern of inhibition of VPF/VEGF expression in 786-0 cells (data not shown). Previously, we have shown that this oligonucleotide could effectively reduce the PKCzeta protein level, whereas the other PKC isoforms remained unchanged (49). There was no change in VPF/VEGF expression with the antisense oligonucleotide of PKCbeta (data not shown). This result suggests that PKCzeta plays a critical role as an intermediary molecule in Ras-mediated overexpression of VPF/VEGF. We recently showed that PKCzeta can also promote the Sp1-dependent transcription of VPF/VEGF in HT1080 and 786-0 cell lines (9). We demonstrated that co-transfection of HT1080 and 786-0 cells with a plasmid overexpressing PKCzeta (at a concentration of 0.6 µg) and different deletion mutants of VPF/VEGF promoter luciferase constructs results in activation of Sp1-mediated transcription, whereas expression of a dominant-negative mutant of PKCzeta represses this activation (9). Interestingly, with the increase in the dose of PKCzeta overexpressing plasmid beyond 1.2 µg concentration, the VPF/VEGF transcription was decreased and gradually came down to the basal level (Fig. 3). These results indicate that PKCzeta -mediated VPF/VEGF transcription might be a rate-limiting step and required activation through upstream signaling pathways.



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Fig. 2.   Effect of dominant-negative mutant and antisense oligonucleotide of PKCzeta on VPF/VEGF mRNA expression. Total RNA (5 µg) was extracted from HT1080 cells that had been transfected with different concentrations of the dominant-negative mutant and the antisense oligonucleotide of PKCzeta and subjected to Northern blot analysis. The blot was probed with 32P-labeled VPF/VEGF cDNA. Fold expression was calculated by densitometry using 36B4 ribosome-associated mRNA expression as a normalization control. The lower panel shows ethidium staining of the RNA samples prior to transfer.



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Fig. 3.   Dose-dependent effect of overexpressed PKCzeta on VPF/VEGF transcription. HT1080 cells were co-transfected with 0.35-kb VPF/VEGF promoter-luciferase construct (1.0 µg) and increasing concentrations (0.2-2.0 µg) of wt-PKCzeta cDNA. Cells were harvested for luciferase assay 40 h after transfection, and fold activation was calculated as relative to the activity of the same reporter construct co-transfected with an empty expression vector (pCMV-FLAG). In all the doses of wt-PKCzeta , the amount of total DNA was balanced by the empty expression vector.

Ras and PKCzeta Are Present in the Same Complex-- Since several reports clearly indicated PKCzeta as a critical step downstream of Ras (12, 18, 26, 51) and as Ras was also found to be involved in VPF/VEGF transcriptional regulation (17), here we set out to dissect whether there was any association between PKCzeta and Ras. To this end, we prepared the lysates of HT1080 and 786-0 cells and immunoprecipitated with anti-Ha-Ras antibody followed by immunoblotting with antibody directed against PKCzeta . We found a strong band corresponding to PKCzeta in the immunoprecipitates prepared from both the cell lines (Fig. 4A). This experiment demonstrated that PKCzeta and Ras were present in the same complex, but did not elucidate whether these proteins might interact directly with each other. To test for this possibility, we performed in vitro association experiment using GST-Ras fusion protein and recombinant PKCzeta isoform. Bacterially expressed GST protein alone or GST protein fused to activated form (GTP-bound) of Ras was bound to glutathione-Sepharose beads, and these were mixed with purified recombinant PKCzeta in a buffer designed to approximate intracellular ionic concentrations. After suitable incubation and extensive washing with the same buffer, the bound proteins were separated by SDS-PAGE and subjected to Western blotting with antibodies to PKCzeta . But we did not observe any significant association of PKCzeta with immobilized activated Ras (data not shown), which reflected that, although PKCzeta and Ras were present in the same complex, they may not interact directly. We next sought to determine whether association of Ras could activate PKCzeta . As it has been shown previously that phosphorylation at the activation loop Thr410 residue is an important step for activation of PKCzeta (13), we made use of a phospho-specific antibody raised against the phosphorylated activation loop sequence. This antibody can specifically recognize PKCzeta when phosphorylated at Thr410. We transfected the human HT1080 cells either with an expression plasmid for PKCzeta alone or in combination with a plasmid containing human Ha-Ras(G12V). Lysates were prepared from the transfected cells, immunoprecipitated with an antibody specific to PKCzeta and then subjected to Western blot analysis with phospho-specific PKCzeta antibody. A band of phosphorylated PKCzeta was detected in case of cells transfected with the combination of PKCzeta and Ha-Ras(G12V) (Fig. 4B). This result reveals that Ras plays a significant role in regulating phosphorylation of the activation loop Thr410 of PKCzeta .



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Fig. 4.   Ras associates with and also phosphorylates PKCzeta . A, lanes 2 and 3, extracts were prepared from both 786-0 and HT1080 cells and immunoprecipitated with a monoclonal antibody directed against Ha-Ras. In lanes 1 and 4, 786-0 cell extracts were immunoprecipitated with antibody against PKCzeta and c-Src, respectively. B, HT1080 cells were transfected with different combinations of wt-PKCzeta (0.6 µg), Ha-Ras(G12V) (1.2 µg), PDK-1 (2.0 µg), and PI 3-kinase dominant-negative mutant (p85DN) (2.0 µg). Cells were lysed 40 h after transfection and the cellular extracts were immunoprecipitated using PKCzeta polyclonal antibody. All the immunoprecipitates (IP) were then captured by protein A-Sepharose beads. After thorough washings, the Sepharose beads were boiled in SDS buffer and separated by SDS-PAGE. Western blottings (Blot) were performed by using (A) PKCzeta polyclonal antibody and (B) a phospho-specific antibody, raised against the phosphorylated (T410) activation loop sequence of PKCzeta . In the lower panel of B, the protein extracts from the cells transfected with different combinations of PKCzeta , Ha-Ras(G12V), PDK-1 and p85DN were separated by SDS-PAGE and blotted with PKCzeta polyclonal antibody.

Ras Activates PKCzeta for Sp1-mediated VPF/VEGF Transcription-- We attempted to explore the involvement of Ras in PKCzeta -mediated activation of VPF/VEGF transcription. To this end, HT1080 cells were co-transfected with a 0.35-kb VPF/VEGF promoter-luciferase construct and the plasmid containing either overexpressed PKCzeta or transforming human Ha-Ras(G12V). Both PKCzeta and Ha-Ras(G12V) increased the VPF/VEGF reporter activity by 2- and 2.5-fold, respectively, in comparison to the cells transfected with expression vector alone (Fig. 5A). Interestingly, when we transfected the cells with a combination of PKCzeta and Ha-Ras(G12V), the VPF/VEGF reporter activity increased up to 4-fold. A dominant negative mutant of transforming human Ras (Ras(Q61L,C186S), which prevents downstream signaling) decreased the PKCzeta -mediated reporter activation almost to the control level (Fig. 5A). In 786-0 cells, we also observed a similar type of activation of VPF/VEGF promoter activity through PKCzeta and Ha-Ras(G12V) (Fig. 5B). Expression of both PKCzeta and Ras in the transfected cells were confirmed through Western blot analysis (Fig. 5C). Together these results indicate that PKCzeta is a key activator of Sp1-mediated VPF/VEGF transcription and oncogenic ras plays a significant role in activation of PKCzeta for such transcriptional regulation. Interestingly, a dominant-negative mutant of PKCzeta completely blocked the Ras-mediated transcriptional activation of VPF/VEGF (Fig. 5A). Moreover, the combination of PKCzeta kw and Ras(Q61L,C186S) reduced the VPF/VEGF transcriptional activation to the same extent when these two mutants were used individually which suggests that PKCzeta and Ras are in the same signaling pathway (Fig. 5A).



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Fig. 5.   Ras promotes transcription of VPF/VEGF through PKCzeta . A, HT1080; and B, 786-0 cells were co-transfected with 0.35-kb VPF/VEGF promoter-luciferase construct (1.0 µg) and different combinations of wt-PKCzeta (0.6 µg), Ha-Ras(G12V) (1.2 µg), Ras(Q61L,C186S) (2.0 µg) or PKCzeta (KW) (2.0 µg) cDNAs. In all the transfection experiments, cells were harvested for luciferase assay 40 h after transfection, and expression in each experiment was normalized to respective empty expression vector. Fold activation was calculated as relative to the activity of the same reporter construct, co-transfected with the control vector. C, expression of PKCzeta and Ras in the transfected samples of HT1080 cells were confirmed by Western blot analysis, using PKCzeta (polyclonal) and Ras (monoclonal) antibodies.

We also observed by EMSA, that in presence of Ras, the Sp1-mediated transcription of VPF/VEGF through PKCzeta was further increased. We made use of a 188-base pair VPF/VEGF promoter fragment that contained all the four Sp1-binding sites and performed EMSA with the nuclear extracts of HT1080 cells, transfected with overexpressed PKCzeta in the presence or absence of Ha-Ras(G12V). As shown in Fig. 6A, overexpression of PKCzeta promoted the binding of Sp1 to the VPF/VEGF promoter and in presence of Ras, this binding was further increased. This specific protein-DNA complex formation was competed away with 10-fold molar excess of Sp1 consensus oligonucleotide. Interestingly, the dominant-negative mutant of PKCzeta (PKCzeta kw) significantly reduced the Ras-induced binding of Sp1 to the VPF/VEGF promoter (Fig. 6B). These results again clearly indicate that PKCzeta induces VPF/VEGF transcription through Sp1, and for which it needs activation through Ras.



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Fig. 6.   Ras promotes the PKCzeta -induced binding of Sp1 to the VPF/VEGF promoter. A-C, by using a 188-base pair VPF/VEGF promoter fragment (having all the four putative Sp1-binding sites) as the probe, EMSA were performed with partially purified nuclear extracts of HT1080 cells transfected with different combinations of PKCzeta (0.6 µg), Ha-Ras(G12V) (1.2 µg), PDK-1 (2.0 µg), and the dominant-negative (DN) mutants of PKCzeta and PI 3-kinase (2.0 µg). Nuclear extracts were prepared from the transfected cells 40 h after transfection. A, unradiolabeled Sp1 consensus oligonucleotide (oligo) (10-fold molar excess) was added to the binding reaction mixture of the control sample (without any transfection) run in lane 6, to show that it can compete away the specific protein-DNA complexes.

Observations from different laboratories clearly indicate that oncogenic Ras-mediated transformation needs different downstream targets, one of which involves Raf-1/MEK/MAPK pathways (23, 24, 52). From our observations, it appears that PKCzeta could be an intermediary member of this Raf/MEK/MAPK signaling cascade, which is downstream of Ras. Although Raf-1 is a critical downstream of Ras, it has also been demonstrated that oncogenic Ras-mediated transformation occurs through both Raf-dependent and Raf-independent pathways (29, 30, 32, 33, 53, 54). Here we set out to determine which particular pathway of Ras signaling is involved in VPF/VEGF transcriptional activation.

Both Raf-dependent and Raf-independent Pathways Are Involved in Channeling Ras Signals for PKCzeta -mediated Activation of VPF/VEGF Transcription-- To define the role of Raf-dependent and Raf-independent pathways in promoting Ras activity, we selected two different effector loop mutants of Ha-Ras. The mutant Ha-Ras(G12V,T35S) retained full-length Raf-1 binding activity while the other mutant, Ha-Ras(G12V,E37G) failed to bind and activate Raf-1 but could effectively activate some of the other Ras effector-mediated pathways, like the Rho pathway (31, 55).

Next, we co-transfected the HT1080 cells with 0.35-kb VPF/VEGF promoter-luciferase construct and either the Ras mutants alone or in combination with PKCzeta . In the presence of Ha-Ras(G12V,T35S) or Ha-Ras(G12V,E37G) alone, there was an almost 1.7-fold activation of VPF/VEGF reporter activity (Fig. 7). When the cells were transfected with these two Ras effector mutants in presence of PKCzeta , the VPF/VEGF reporter activity was increased up to 3-fold (Fig. 7). These results are consistent with the hypothesis that Ras plays a critical role in PKCzeta -mediated activation of VPF/VEGF transcription. The above experiments also clearly suggest the involvement of both Raf-dependent and Raf-independent pathways in channeling of Ras signals in the activation of PKCzeta . Earlier studies have also shown that the Ha-Ras(G12V,E37G) mutant could complement the transforming activity of the Ha-Ras(G12V,T35S) mutant, indicating that Raf-independent pathways activated by Ras can contribute to transformation (31, 55). The effector loop mutant Ha-Ras(G12V,E37G) mainly involves the RhoA, Rac1, or CDC42 among the Rho family member of proteins.



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Fig. 7.   Activation of PKCzeta -mediated VPF/VEGF transcription through different effector loop mutants of Ha-Ras(G12V). HT1080 cells were co-transfected with 0.35-kb VPF/VEGF promoter-luciferase construct (1.0 µg) and different combinations of wt-PKCzeta (0.6 µg) and two effector loop mutants of Ha-Ras(G12V), Ha-Ras(G12V,T35S) and Ha-Ras(G12V,E37G) (each 1.2 µg) expression vectors. The cells were harvested for luciferase assay 40 h after transfection and expression in each experiment was normalized with respective empty expression vector. Fold activation was calculated as relative to the activity of the same reporter construct, co-transfected with the control vector.

PDK-1 Plays a Crucial Role in Activation of PKCzeta for VPF/VEGF Transcription-- Over the last few years, PDK-1 was identified as the first known upstream activating kinase for PKC (36, 37, 46). It has been shown that PDK-1 could phosphorylate and activate PKCzeta in vivo, and this activation was due to phosphorylation of threonine 410 in PKCzeta activation loop (13). Based on this observation, we explored the role of PDK-1 in PKCzeta -mediated transcriptional activation of VPF/VEGF promoter. The HT1080 cells were co-transfected with 0.35-kb VPF/VEGF promoter and different combinations of PKCzeta and PDK-1. As shown in Fig. 8A, overexpression of PDK-1 increased the PKCzeta -mediated activation of VPF/VEGF promoter about 3.3-fold, suggesting PDK-1 was capable of activating PKCzeta . When we used a dominant-negative mutant of either PKCzeta or PDK-1, the activation of VPF/VEGF promoter was reduced almost to control level (Fig. 8A). From the above results, it is likely that PKCzeta needs activation through PDK-1, as the kinase-inactive variant of PDK-1 in which the critical Lys110 residue was mutated to an Asn (PDK-1 Lys/Asn), could effectively block the activation of VPF/VEGF transcription through PKCzeta .



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Fig. 8.   Role of PDK-1 in PKCzeta and Ras-induced activation of VPF/VEGF transcription. A, HT1080; and B, 786-0 cells were co-transfected with 0.35-kb VPF/VEGF promoter-luciferase construct (1.0 µg) and different combinations of wt-PKCzeta (0.6 µg), Ha-Ras(G12V) (1.2 µg), wt-PDK-1 (Myc-tagged) (2.0 µg), PDK-1(KN) (Myc-tagged) (2.0 µg), and PKCzeta (KW) (2.0 µg) expression vectors. The cells were harvested for luciferase assay 40 h after transfection, and expression in each experiment was normalized with a respective empty expression vector. Fold activation was calculated as relative to the activity of the same reporter construct, co-transfected with control vector. C, expression of PKCzeta , Ras, and PDK-1 in the transfected samples of HT1080 cells were confirmed by Western blot analysis, using PKCzeta (polyclonal), Ras (monoclonal), and Myc (monoclonal) antibodies.

We next sought to determine whether the combination of both PDK-1 and Ras can activate PKCzeta . Fig. 8A indeed shows that in presence of both PDK-1 and transforming Ha-Ras(G12V) the PKCzeta -mediated activation of VPF/VEGF promoter activity was increased ~4.5-fold. In 786-0 cells, we also observed a similar type of activation of VPF/VEGF promoter through PDK-1, Ras, and PKCzeta (Fig. 8B). In a separate experiment, it has been shown that the Ras-mediated phosphorylation of PKCzeta was further increased in presence of PDK-1 (Fig. 4B). We also found through EMSA that in presence of PDK-1, the binding of Sp1 to the VPF/VEGF promoter through Ras and PKCzeta was further increased (Fig. 6A). Together, these results clearly indicate that PKCzeta needs upstream signaling through both PDK-1 and Ras for VPF/VEGF transcription. Interestingly, when we used the dominant-negative mutant of PDK-1, the activation of VPF/VEGF promoter mediated by the combination of Ras and PKCzeta was not lowered significantly (Fig. 8A). This suggests that although both PDK-1 and Ras activate PKCzeta , they mediate their action through two different signaling pathways.

PI 3-Kinase Acts as Major Upstream Effector to Activate PKCzeta -- PI 3-kinases and their lipid products play a crucial role in various aspects of cell function (37, 43, 53). Interestingly, PI 3-kinase may regulate PKCzeta by generation of activating molecules (e.g. phosphatidylinositol 1,4,5-trisphosphate) and/or by acting as a "linker" protein to bring PKCzeta in contact with other activating molecules (11, 40, 56). PI 3-kinase consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (57). It has been shown previously that PI 3-kinase interacts directly with Ras through its catalytic subunit and the effector site of Ras in a GTP-dependent manner (33). Recent reports have shown that PDK-1 also binds with high affinity to the PI 3-kinase lipid product phosphatidylinositol 3,4,5-triphosphate (PtdIns-3,4,5-P3) (18). As PDK-1 also interacts with PKCzeta , their association reveals extensive cross-talk between the enzymes in the PI 3-kinase signaling pathway.

To elucidate the involvement of PI 3-kinase in Ras, PDK-1, and PKCzeta -mediated up-regulation of VPF/VEGF transcription, HT1080 cells were co-transfected with 0.35-kb VPF/VEGF promoter and different combinations of Ha-Ras(G12V), PDK-1, and PKCzeta in the presence or absence of a dominant-negative mutant (Delta p85) of PI 3-kinase. As shown in Fig. 9, Delta p85 reduced the PDK-1 and PKCzeta -mediated activation of VPF/VEGF promoter to the basal level. In the presence of Delta p85 alone, the VPF/VEGF promoter activation was almost completely shut down. This observation clearly demonstrates that PKCzeta and PDK-1 need signaling through PI 3-kinase, as shown by another group (13). Through EMSA, it has also been shown that Delta p85 significantly inhibited the PKCzeta -induced binding of Sp1 to the VPF/VEGF promoter sequence (Fig. 6C). Interestingly, Delta p85 could not significantly decrease the individual effect of Ras or the additive effect of Ras and PKCzeta in mediating activation of VPF/VEGF promoter (Fig. 9). But in the presence of Delta p85, the Ras-mediated phosphorylation of PKCzeta was significantly reduced (Fig. 4B). These data reflect that, although Ras-mediated activation of PKCzeta is dependent on PI 3-kinase, the Ras-induced transcription of VPF/VEGF through PKCzeta is partially dependent upon the PI 3-kinase pathways. It appears that for VPF/VEGF transcription, PI 3-kinase activates PKCzeta mainly through PDK-1.



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Fig. 9.   Role of PI 3-kinase in PKCzeta , Ras, and PDK-1-induced transcriptional activation of VPF/VEGF. HT1080 cells were co-transfected with 0.35-kb VPF/VEGF promoter-luciferase construct (1.0 µg) and different combinations of wt-PKCzeta (0.6 µg), Ha-Ras(G12V) (1.2 µg), wt-PDK-1 (Myc-tagged) (2.0 µg), and PI 3-kinase dominant negative mutant (p85DN) (2.0 µg) expression vectors. The cells were harvested for luciferase assay 40 h after transfection, and expression in each experiment was normalized with respective empty expression vector. Fold activation was calculated as relative to the activity of the same reporter construct, co-transfected with a control vector.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VPF/VEGF, a multifunctional cytokine, was originally discovered because of its ability to increase the permeability of microvessels, primarily post-capillary venules and small veins, to circulating macromolecules (4, 44-47). It plays an important role in both pathological and physiological angiogenesis (3, 4). Although constitutively expressed by many tumor cells and transformed cell lines, VPF/VEGF expression is also subject to regulation by both PKC and cAMP-dependent kinase pathways (58) and by mechanisms involving alterations in both mRNA transcription and stability (59, 60). Other factors that can regulate VPF/VEGF expression include the degree of cell differentiation; local concentrations of oxygen, glucose, and serum; cytokines; hormones; prostaglandins; modulators of PKC; calcium influx; the electron transport chain; depolarizing agents; angiotensin II; stimulators of adenylate cyclase; nitric oxide; and expression levels of certain oncogenes, like src or ras (5-7, 17, 61, 62).

The data presented here investigate a novel mechanism by which transforming human Ras regulated the transcription of VPF/VEGF through PKCzeta , finally resulting in tumor angiogenesis. By blocking PKCzeta with the use of its dominant-negative mutant or antisense oligonucleotide, we demonstrate that PKCzeta acts as an important intermediary molecule in Ras-mediated overexpression of VPF/VEGF (Fig. 2). In accord with earlier work, we also found that PKCzeta promoted the Sp1-mediated transcription of VPF/VEGF in human HT1080 and 786-0 cells where the oncogene ras was already activated. With the increase in the dose of PKCzeta , there was gradual activation of Sp1-mediated VPF/VEGF transcription, but from a certain dose (1.2 µg), this activation was lowered and then gradually came down to the basal level (Fig. 3). These findings clearly suggest that PKCzeta -mediated activation of VPF/VEGF transcription is a rate-limiting step and may need activation through its upstream signaling pathways. Thus, if we overexpress PKCzeta beyond a certain concentration, it acts like a dominant-negative factor since it is no longer activated. Here we attempt to dissect the role of Ras, along with PDK-1 and PI-3 kinase in activating PKCzeta and show that they play a significant role in PKCzeta -mediated activation of VPF/VEGF transcription.

Ras is a point of convergence for many signaling pathways and plays a significant role in expression of VPF/VEGF (17). Here, we demonstrate that Ras promotes VPF/VEGF transcription in a Sp1-dependent manner (Fig. 1). It has been reported that Ras triggers activation of series of kinases known as the MAPK cascade (Ras > MEK > MAPK) (16, 20-22). Raf-1 serine/threonine kinase has been reported to be one of the downstream effectors of Ras (23, 24, 52). Raf-1 in turn activates MAPK kinases (MEK1 and MEK2), which in turn activate p42 and p44 MAPKs/extracellular signal-regulated kinases (19, 63, 64). Activated MAPKs then translocate into the nucleus, where they phosphorylate and activate nuclear transcription factors (65, 66), resulting in immediate early gene induction. As from the previous reports, PKCzeta appears to be located downstream of Ras (12, 18, 28, 51), it seems conceivable that PKCzeta could be critically involved in channeling Ras signals for activation of MAPK resulting in up-regulation of VPF/VEGF transcription. In the current study, we observe that PKCzeta or Ras alone increased the VPF/VEGF transcription in human HT1080 or 786-0 cells up to 2- and 2.5-fold, respectively (Fig. 5, A and B). But when the cells were transfected in combination with PKCzeta and Ha-Ras(G12V), the transcriptional activation was increased up to 4-fold (Fig. 5, A and B). A dominant-negative mutant of Ha-Ras blocked the PKCzeta -mediated activation of VPF/VEGF transcription (Fig. 5A). The dominant-negative mutant of PKCzeta reduced the VPF/VEGF transcription below the control level (Fig. 5A). These results clearly indicate that PKCzeta needs signaling through Ras for activation. It may also play an important role in channeling a part of Ras signal to its downstream targets to promote tumor angiogenesis. Observations from others have prompted suggestions that PKCzeta is a required step of Ras-mediated mitogenic signaling and that Ras directly interacts in vitro with the regulatory domain of PKCzeta as well as that the association of PKCzeta with Ras in vivo is triggered by platelet-derived growth factor (51). In our studies, although we were unable to detect any direct interaction between PKCzeta and Ras, these two proteins were found to be present in the same immunocomplex (Fig.-4A). Therefore, it is quite clear that Ras-mediated signaling is an important step for PKCzeta activation and thus increasing VPF/VEGF promoter activity. We have also confirmed this through EMSA, where Ras promoted the PKCzeta -induced binding of Sp1 to the VPF/VEGF promoter (Fig. 6A). Moreover, it is also evident that even the activated form of Ras cannot bypass PKCzeta in regulating VPF/VEGF transcription, as the dominant-negative mutant of PKCzeta clearly inhibited the Ras-induced binding of Sp1 to the VPF/VEGF promoter (Fig. 6B).

We next set out to dissect the role of Raf-independent downstream effectors that are involved in mediating Ras signals. Mutations in the Ras effector domain (residues 32-40) can impair Ras transforming activity and interaction with effector proteins without causing alterations in intrinsic GDP and GTP regulation (31, 67). We made use of two different effector loop mutants of Ha-Ras(G12V). Ha-Ras(G12V,T35S) retained the full-length Raf-1 binding activity, while Ha-Ras(G12V,E37G) was impaired in its ability to bind to Raf-1. Consistent with its impaired ability to up-regulate Raf-1 kinase activity, it has been demonstrated that cells expressing the later mutant show no significant increase in p42 and p44 MAPK activities (31, 55). In contrast, both Ha-Ras(G12V)- and Ha-Ras(G12V,T35S)-expressing cells show elevated MAPK activities. Recent studies have demonstrated that, like Rho family proteins, Ha-Ras(G12V,E37G) can cause activation of the stress-activated protein kinase (SAPK/JNK) (16, 24, 68). It has also been shown to mediate its functions through Rho family of proteins, mainly, RhoA, Rac1, and CDC42 (24, 31). In this study, we observed that both Ha-Ras(G12V,T35S) and Ha-Ras(G12V,E37G) caused significant activation of VPF/VEGF transcription through PKCzeta (Fig. 7). This result clearly suggests that Ras-induced activation of PKCzeta for VPF/VEGF transcription is mediated through both Raf-dependent and Raf-independent pathways.

Recent works have identified PDK-1, constitutively active in mammalian cells, as a major upstream activating kinase for PKCzeta (13, 34-36). Like Akt/PKB and p70 S6K, PKCzeta has also been shown to be one of the in vivo substrates of PDK-1 (13, 39). Phosphorylation of PKCzeta Thr410 by PDK-1 leads to activation of the enzyme, both in vivo and in vitro (38). In the present study, we observe that PDK-1 plays a significant role in promoting VPF/VEGF transcription induced by PKCzeta . PDK-1 increased PKCzeta -mediated activation of VPF/VEGF transcription up to 3.3-fold in human HT1080 and 786-0 cells (Fig. 8, A and B). A dominant-negative mutant of either PDK-1 or PKCzeta completely blocked the transcription of VPF/VEGF (Fig. 8A). The mutant PKCzeta reduced the VPF/VEGF transcription well below the control level (Fig. 8A). All of these observations clearly present PDK-1 as an activator of PKCzeta . Interestingly, when we transfected the HT1080 or 786-0 cells with combinations of both Ras and PDK-1, there was an additive effect up to 4.5-fold in PKCzeta -mediated activation of VPF/VEGF transcription (Fig. 8, A and B). This suggests that upstream signaling from both Ras and PDK-1 is essential for the activation of PKCzeta . In EMSA, we have shown that PDK-1 promoted the Ras and PKCzeta -induced binding of Sp1 to the VPF/VEGF promoter (Fig. 6A). We have also demonstrated that the signaling through Ras and PDK-1 are mediated by two distinctly different pathways, as the dominant-negative mutant of PDK-1 could not effectively block PKCzeta and Ras-induced transcriptional activation of VPF/VEGF promoter (Fig. 8A). Through the use of a phospho-specific antibody, it has been shown that both Ras and PDK-1 stimulate phosphorylation of the PKCzeta activation loop Thr410 (Fig. 4B). Observations from other laboratories (13, 38) have demonstrated that PKCzeta phosphorylation/activation is almost completely blocked by coexpression of dominant-negative PDK-1 or by mutation of Thr410. Thus, it is likely that a prerequisite for PKCzeta activation is phosphorylation of Thr410. Taken together, these observations indicate that Ras and PDK-1 constitute two distinct pathways, both of which are required for PKCzeta -mediated VPF/VEGF transcriptional activation.

PKCzeta has received considerable attention in recent years as it has been implicated as a downstream target of PI 3-kinase (11, 40, 42, 43). PDK-1 also serves as an important member of PI 3-kinase pathways (13, 37, 39). The observation that PKCzeta associates with PDK-1 in vivo suggests considerable cross-talk between effector molecules in the PI 3-kinase signaling pathway (13, 37). On the other hand, PI 3-kinase also interacts with Ras·GTP but not with Ras·GDP and is activated both in vitro and in vivo as a result of this interaction (53, 69). We have found that a dominant-negative mutant of PI 3-kinase significantly reduces the activation of PKCzeta through Ras (Fig. 4B). In the present study, we have shown that PI 3-kinase acts as a major activator for PDK-1 and PKCzeta -mediated pathway, regulating VPF/VEGF transcription. The dominant-negative mutant of PI 3-kinase significantly reduced the PKCzeta -induced binding of Sp1 to the VPF/VEGF promoter sequence as well as inhibited the PDK-1 and PKCzeta -induced activation of VPF/VEGF promoter almost to the basal level (Figs. 6C and 9). These observations suggest that both PDK-1 and PKCzeta need activation through PI 3-kinase. In contrast, the dominant-negative mutant of PI 3-kinase could not lower the Ras and PKCzeta -mediated VPF/VEGF transcription below the control level (Fig. 9). Moreover, the mutant PI 3-kinase failed to reduce the VPF/VEGF transcriptional activation mediated by Ras alone (Fig. 9). Thus, although Ras interacts with PI 3-kinase, it may not need activation through PI 3-kinase to promote VPF/VEGF transcription.

In summary, our results indicate that Ras-mediated VPF/VEGF transcription occurs mainly through PKCzeta , in a Sp1-dependent manner. PDK-1 and PI-3 kinase also play an important Ras-independent role in exerting an additive effect in this signaling cascade. All of these mechanisms which up-regulate VPF/VEGF may have important connections with tumor angiogenesis where mutant Ras alleles contribute to solid tumor development and metastasis. It appears that in addition to hypoxia, different growth factors or hormones, the activation of oncogenes such as Ras, also play a significant role in tumor angiogenesis in a stable manner by stimulating constitutive expression of VPF/VEGF. Moreover, possibilities of common pathways for different stimuli to promote VPF/VEGF expression cannot be ignored. Therefore, understanding the signaling pathways for the activation of VPF/VEGF expression and designing inhibitory molecule(s) of these signaling cascades might have comprehensive effects in tumor angiogenesis, progression, and metastasis.


    ACKNOWLEDGEMENTS

We thank Dr. Alex Toker for PDK-1, PDK-1 DN, p85 DN expression vectors and also for anti-phospho-PKCzeta antibodies; Dr. Jack Lawler for helpful comments on the manuscript; Rinku Pal and Alexis Bywater for technical assistance.


    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant CA78383, the Massachusetts Public Health, and under terms of a contract from the National Foundation for Cancer Research (to D. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Howard Temin awardee supported by National Institutes of Health Grant CA 78396).

§ Eugene P. Schonfeld Medical Research awardee from the National Kidney Cancer Association. To whom correspondence should be addressed: Dept. of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Ave., RN 270H, Boston, MA 02215. Tel.: 617-667-7853; Fax: 617-667-3591; E-mail; dmukhopa@caregroup.harvard.edu.

Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M007818200


    ABBREVIATIONS

The abbreviations used are: VPF, vascular permeability factor; VEGF, vascular denothelial growth factor; PKCzeta , protein kinase Czeta ; MAP, mitogen-activated protein; MAPK, mitogen activated protein kinase; PI 3-kinase, phosphatidylinositol 3-kinase; PI(3, 4,5)P3, phosphatidylinositol 3,4,5-triphosphate; kb, kilobase(s); EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


1. Folkman, J. (1971) N. Engl. J. Med. 285, 1182-1186[Medline] [Order article via Infotrieve]
2. Folkman, J. (1996) Sci. Am. 275, 150-154[Medline] [Order article via Infotrieve]
3. Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V., and Ferrara, N. (1989) Science 246, 1306-1309[Medline] [Order article via Infotrieve]
4. Senger, D. R., Galli, S. J., Dvorak, A. M., Perruzzi, C. A., Harvey, V. S., and Dvorak, H. F. (1983) Science 219, 983-985[Medline] [Order article via Infotrieve]
5. Mukhopadhyay, D., Tsiokas, L., and Sukhatme, V. P. (1995) Cancer Res. 55, 6161-6165[Abstract]
6. Mukhopadhyay, D., Knebelmann, B., Cohen, H. T., Ananth, S., and Sukhatme, V. P. (1997) Mol. Cell. Biol. 17, 5629-5639[Abstract]
7. Rak, J., Mitsuhashi, Y., Bayko, L., Filmus, J., Shirasawa, S., Sasazuki, T., and Kerbel, R. S. (1995) Cancer Res. 55, 4575-4580[Abstract]
8. Pal, S., Claffey, K. P., Dvorak, H. F., and Mukhopadhyay, D. (1997) J. Boil. Chem. 272, 27509-27512[Abstract/Free Full Text]
9. Pal, S., Claffey, K. P., Cohen, H. T., and Mukhopadhyay, D. (1998) J. Biol. Chem. 273, 26277-26280[Abstract/Free Full Text]
10. Ono, Y., Fuji, T., Ogita, K., Kikkawa, U., Igarashi, K., and Nishizuka, Y. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3099-3103[Abstract]
11. Nakanishi, H., and Exton, J. H. (1992) J. Biol. Chem. 267, 16347-16354[Abstract/Free Full Text]
12. Berra, E., Diaz-Meco, M. T., Dominguez, I., Municio, M. M., Sanz, L., Lozano, J., Chapkin, R. S., and Moscat, J. (1993) Cell 74, 555-563[Medline] [Order article via Infotrieve]
13. Chou, M. M., Hou, W., Johnson, J., Graham, L. K., Lee, M. H., Chen, C.-S., Newton, A. C., Schaffhausen, B. S., and Toker, A. (1998) Curr. Biol. 8, 1069-1077[Medline] [Order article via Infotrieve]
14. Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403[Medline] [Order article via Infotrieve]
15. Egan, S. E., and Weinberg, R. A. (1993) Nature 365, 781-783[CrossRef][Medline] [Order article via Infotrieve]
16. Khosravi-Far, R., and Der, C. J. (1994) Cancer Metastasis Rev. 13, 67-89[Medline] [Order article via Infotrieve]
17. White, M. A., Nicolette, C., Minden, A., Polverino, A., Van Aelst, L., Karin, M., and Wigler, M. H. (1995) Cell 80, 533-541[Medline] [Order article via Infotrieve]
18. Diaz-Meco, M. T., Lozano, J., Municio, M. M., Berra, E., Frutos, S., Sanz, L., and Moscat, J. (1994) J. Biol. Chem. 269, 31706-31710[Abstract/Free Full Text]
19. Kyriakis, J. M., App, H., Zhang, X.-F., Banerjee, P., Brautigan, D. L., Rapp, U. R., and Avruch, J. (1992) Nature 358, 417-421[CrossRef][Medline] [Order article via Infotrieve]
20. Quilliam, L. A., Khosravi-Far, R., Huff, S. Y., and Der, C. J. (1995) Bioessays 17, 395-404[Medline] [Order article via Infotrieve]
21. Roberts, T. M. (1992) Nature 360, 534-535[CrossRef][Medline] [Order article via Infotrieve]
22. Schlessinger, J. (1993) Trends Biochem. Sci. 18, 273-275[CrossRef][Medline] [Order article via Infotrieve]
23. Cowly, S., Paterson, H., Kemp, P., and Marshall, C. J. (1994) Cell 77, 841-852[Medline] [Order article via Infotrieve]
24. Khosravi-Far, R., Solski, P. A., Clark, G. J., Kinch, M. S., and Der, C. J. (1995) Mol. Cell. Biol. 15, 6443-6453[Abstract]
25. Leevers, S. J., Paterson, H. F., and Marshall, C. J. (1994) Nature 369, 411-414[CrossRef][Medline] [Order article via Infotrieve]
26. Berra, E., Diaz-Meco, M. T., Lozano, J., Frutos, S., Municio, M. M., Sanchez, P., Sanz, L., and Moscat, J. (1995) EMBO J. 14, 6157-6163[Abstract]
27. Devary, Y., Rosette, C., DiDonato, J. A., and Karin, M. (1993) Science 261, 1442-1445[Medline] [Order article via Infotrieve]
28. Dominguez, I., Diaz-Meco, M. T., Municio, M. M., Berra, E., Garcia de Herreros, A., Cornet, M. E., Sanz, L., and Moscat, J. (1992) Mol. Cell. Biol. 12, 3776-3783[Abstract]
29. Feig, L. A. (1993) Science 260, 767-768[Medline] [Order article via Infotrieve]
30. Howe, L. R., Leevers, S. J., Gomez, N., Nakielny, S., Cohen, P., and Marshall, C. J. (1992) Cell 71, 335-342[Medline] [Order article via Infotrieve]
31. Khosravi-Far, R., White, M. A., K., W. J., Solski, P. A., Chrzanowska-Wodnicka, M., Van aelst, L., Wigler, M. H., and Der, C. J. (1996) Mol. Cell. Biol. 16, 3923-3933[Abstract]
32. Kikuchi, A., Demo, S. D., Ye, Z.-H, Chen, Y.-W., and Williams, L. T. (1994) Mol. Cell. Biol. 14, 7483-7491[Abstract]
33. Rodriguez-Viciana, P., Warne, P. H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M. J., Waterfield, M. D., and Downward, J. (1994) Nature 370, 527-532[CrossRef][Medline] [Order article via Infotrieve]
34. Alessi, D. R., Deak, M., Casamayor, A., Caudwell, F. B., Morrice, N., and Norman, D. G. (1997) Curr. Biol. 7, 776-789[Medline] [Order article via Infotrieve]
35. Alessi, D. R., Kozlowski, M. T., Weng, Q. P., and Morrice, N. J. (1998) Curr. Biol. 8, 69-81[Medline] [Order article via Infotrieve]
36. Dutil, E. M., Toker, A., and Newton, A. C. (1998) Curr. Biol. 8, 1366-1375[Medline] [Order article via Infotrieve]
37. Le Good, J. A., Ziegler, W. H., Parekh, D. B., Alessi, D. R., Cohen, P., and Parker, P. J. (1998) Science 281, 2042-2045[Abstract/Free Full Text]
38. Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S. C., Hemmings, B. A., and Thomas, G. (1998) Science 279, 707-710[Abstract/Free Full Text]
39. Romanelli, A., Martin, K. A., Toker, A., and Blenis, J. (1999) Mol. Cell. Biol. 19, 2921-2928[Abstract/Free Full Text]
40. Nakanishi, H., Brewer, K. A., and Exton, J. H. (1993) J. Biol. Chem. 268, 13-16[Abstract/Free Full Text]
41. Lozano, J., Berra, E., Municio, M. M., Diaz-Meco, M. T., Dominguez, I., Sanz, L., and Moscat, J. (1994) J. Biol. Chem. 269, 19200-19202[Abstract/Free Full Text]
42. Mendez, R., Kollmorgen, G., White, M. F., and Rhoads, R. E. (1997) Mol. Cell. Biol. 17, 5184-5192[Abstract]
43. Herrera-Velit, P., Knutson, K. L., and Reiner, N. E. (1997) J. Biol. Chem. 272, 16445-16452[Abstract/Free Full Text]
44. Dvorak, H. F., Senger, D. R., and Dvorak, A. M. (1984) Dev. Oncol. 22, 96-114
45. Dvorak, H. F., Nagy, J. A., Dvorak, J. T., and Dvorak, A. M. (1988) Am. J. Pathol. 133, 95-109[Abstract]
46. Dvorak, H. F. (1990) Prog. Clin. Biol. Res. 354A, 317-30
47. Dvorak, H. F., Sioussat, T. M., Brown, L. F., Berse, B., Nagy, J. A., Sotrel, A., Manseau, E. J., Van de Water, L., and Senger, D. R. (1991) J. Exp. Med. 174, 1275-1278[Abstract]
48. Mukhopadhyay, D., Tsiokas, L., Zhou, X. M., Foster, D., Brugge, J. S., and Sukhatme, V. P. (1995) Nature 375, 577-581[CrossRef][Medline] [Order article via Infotrieve]
49. Shih, S. C., Mullen, A., Abrams, K., Mukhopadhyay, D., and Claffey, K. P. (1999) J. Biol. Chem. 274, 15407-15414[Abstract/Free Full Text]
50. Gorman, C. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051[Medline] [Order article via Infotrieve]
51. Diaz-Meco, M. T., Dominguez, I., Sanz, L., Dent, P., Lozano, J., Municio, M. M., Berra, E., Hay, R. T., Sturgill, T. W., and Moscat, J. (1994) EMBO J. 13, 2842-2848[Abstract]
52. Schaap, D., van der Wal, J., Howe, L. R., Marshall, C. J., and van Blitterswijk, W. J. (1993) J. Biol. Chem. 268, 20232-20236[Abstract/Free Full Text]
53. Rodriguez-Viciana, P., Warne, P. H., Khwaja, A., Marte, B. M., Pappin, D., Das, P., Waterfield, M. D., Ridley, A., and Downward, J. (1997) Cell 89, 457-467[Medline] [Order article via Infotrieve]
54. Russel, M., Lange-Carter, C. A., and Johnson, G. L. (1995) J. Biol. Chem. 270, 11757-11760[Abstract/Free Full Text]
55. White, F. C., Benehacene, A., Scheele, J. S., and Kamps, M. (1997) Growth Factors 14, 199-212[Medline] [Order article via Infotrieve]
56. Liao, D.-F., Monia, B., Dean, N., and Berk, B. C. (1997) J. Biol. Chem. 272, 6146-6150[Abstract/Free Full Text]
57. Hiles, I. D., Otsu, M., Volinia, S., Fry, M. J., Gout, I., Dhand, R., Panayotou, G., Ruiz-Larrea, F., Thompson, A., and Totty, N. F. (1992) Cell 70, 419-429[Medline] [Order article via Infotrieve]
58. Fischer, S., Sharma, H. S., Karliczek, G. F., and Schaper, W. (1995) Brain. Res. Mol. Brain Res. 28, 141-148[CrossRef][Medline] [Order article via Infotrieve]
59. Finkenzeller, G., Marme, D., Weich, H. A., and Hug, H. (1992) Cancer Res. 52, 4821-4823[Abstract]
60. Harada, S., Nagy, J. A., Sullivan, K. A., Thomas, K. A., Endo, N., Rodan, G. A., and Rodan, S. B. (1994) J. Clin. Invest. 93, 2490-2496[Medline] [Order article via Infotrieve]
61. Frank, S., Hubner, G., Breier, G., Longaker, M. T., Greenhalgh, D. G., and Werner, S. (1995) J. Biol. Chem. 270, 12607-12613[Abstract/Free Full Text]
62. Levy, A. P., Levy, N. S., Loscalzo, J., Calderone, A., Takahashi, N., Yeo, K. T., Koren, G., Colucci, W. S., and Goldberg, M. A. (1995) Circ. Res. 76, 758-766[Abstract/Free Full Text]
63. Dent, P., Haser, W., Haystead, T. A. J., Vincent, L. A., Roberts, T. M., and Sturgill, T. W. (1992) Science 257, 1404-1407[Medline] [Order article via Infotrieve]
64. Hofer, F., Fields, S., Schneider, C., and Martin, G. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11089-11093[Abstract/Free Full Text]
65. Hill, C. S., Marais, R., John, S., Wynne, J., Dalton, S., and Treisman, R. (1993) Cell 73, 385-406
66. Hill, C. S., and Treisman, R. (1995) Cell 80, 199-211[Medline] [Order article via Infotrieve]
67. Marshall, M. S. (1993) Trends Biochem. Sci. 18, 250-254[CrossRef][Medline] [Order article via Infotrieve]
68. Khosravi-Far, R., Chrzanowska-Wodnicka, M., Solski, P. A., Eva, A., Burridge, K., and Der, C. J. (1994) Mol. Cell. Biol. 14, 6848-6857[Abstract]
69. Kodaki, T., Woscholski, R., Hallberg, B., Rodriguez-Viciana, P., Downward, J., and Parker, P. J. (1994) Curr. Biol. 4, 798-806[Medline] [Order article via Infotrieve]


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