Focal Adhesion Kinase, Rap1, and Transcriptional Induction of Vascular Endothelial Growth Factor

Essam A. Sheta, Michael A. Harding, Mark R. Conaway, Dan Theodorescu

Affiliations of authors: E. A. Sheta, M. A. Harding, D. Theodorescu (Department of Molecular Physiology and Biological Physics), M. R. Conaway (Department of Health Evaluation Sciences), University of Virginia Health Sciences Center, Charlottesville.

Correspondence to: Dan Theodorescu, M.D., Ph.D., Box 422, University of Virginia Health Sciences Center, Charlottesville, VA 22908 (e-mail: Theodorescu{at}virginia.edu).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Signals from a cell's environment are sensed by receptors, which activate pathways that, in turn, transmit the signals to the nucleus, informing decisions on growth, angiogenesis, and other cell functions. Transcription of vascular endothelial growth factor (VEGF), a potent angiogenic factor, can be induced by cell–cell contact. In the current work, we sought to determine if this induction is dependent on transformation of cells to a malignant phenotype and subsequently to determine which signaling molecules mediate activation of VEGF transcription. Methods: Normal and transformed prostate epithelial cell lines were examined at various cell densities to simulate the effect of increased cell contact on expression of VEGF messenger RNA. Transformed cells were also cotransfected with a VEGF promoter-reporter construct and with constructs that express dominant negative or activated versions of signal transduction proteins hypothesized to be involved in the cell–cell contact process, and reporter activity was assessed at various cell densities. All P values are two-sided. Results: Direct cell–cell contact, but not extracellular matrix components, resulted in transcriptional activation of a VEGF promoter-reporter construct in malignant (P<.0001) but not in nonmalignant (P = .37) prostate cells. This process was mediated via a mitogen-activated protein kinase (MAPK); it required the activity of focal adhesion kinase (FAK), Rap1, and Raf and was Ras independent. In addition, transcriptional activation of a Ras-sensitive Elk-1 chimeric reporter by cell–cell contact suggests that Rap1 is a key factor in regulating the specificity of convergent MAPK-signaling pathways arising from different upstream extracellular stimuli. Conclusions: Cell contact induction of VEGF transcription via FAK and Rap1 provides a novel Ras-independent, but transformation-dependent, mechanism for stimulus-specific regulation of tumor VEGF expression via MAPK.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cells need to sense their environment to make decisions on growth, angiogenesis, differentiation, motility, or other functions. Environmental signals are detected by receptors that bind ligands, such as growth factors, extracellular matrix (ECM), or cell-surface molecules on adjacent cells. These receptors, in turn, activate signaling pathways that communicate the state of the environment to the nucleus. Upon malignant transformation, signaling processes can be subverted, leading to a dysregulation of normal cellular functions. Consistent with this hypothesis, several investigators have demonstrated how the induction of vascular endothelial growth factor (VEGF), a powerful stimulator of angiogenesis (1), can be regulated by mutant forms of genes such as Ras (2), Raf (3), and Src (4) that contribute to the tumorigenic phenotype.

These observations also suggest that nonmutated forms of the products of these same genes may be candidate regulators of VEGF expression in response to extrinsic microenvironmental signals mediated by receptors for growth factors or ECM components (5). Increased transcription of VEGF messenger RNA (mRNA) in response to cell density was demonstrated in human colon (6) and renal (7) cancer cell lines and was dependent on soluble factors in the former but not in the latter cell lines. It is interesting that, in both cases, the mRNA levels were related to Src activation (79), suggesting that both soluble factors and cell contact can regulate VEGF expression via Src. However, these experiments did not determine whether VEGF induction was dependent on the tumorigenic phenotype and if the increase in VEGF mRNA was transcriptional or post-transcriptional, and they did not define mechanistically the signaling pathways involved.

Several known pathways exist that can transmit cell contact information to intracellular signaling molecules. The cadherins are a family of transmembrane glycoproteins responsible for calcium-dependent, cell–cell adhesion mediated by a group of cytoplasmic proteins, the catenins, that act inside the cell to couple the cadherin molecule to the microfilament cytoskeleton (10). Dysfunction of cadherin/catenin-dependent cell–cell adhesion has been demonstrated in prostate cancer cell lines (11) and in prostate tumors (12) and is believed to contribute to the acquisition of an invasive phenotype. Focal adhesion kinase (FAK) is directly activated by integrin clustering-mediated cell adhesion (13) in response to fibronectin, an ECM component. Upon cell adhesion, activation of FAK by autophosphorylation creates phosphotyrosine-binding sites for SH2 domain proteins, including Grb2 (14), phosphatidylinositol 3-kinase (PI3K) (15), and Src family kinases (16). These molecules have, in turn, been implicated in many aspects of the Ras-signaling cascade, suggesting possible links between extrinsic and intrinsic factors regulating VEGF. It is now also evident that the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK), or MEK, is a downstream target of FAK in integrin-initiated signaling pathways (1719). Tracing the pathway of integrin-mediated MAPK activation backward from MAPK reveals that MEK is responsible for direct activation of MAPK (20). Although the exact role of MAPK activity in cancer progression is not clearly defined, Gioeli et al. (21) have recently reported that, in prostate tumors, the level of activated MAPK increases with tumor stage and tumor grade. While integrin-mediated activation of MAPK stimulated by cell adhesion is mainly dependent on Ras (17,22), substantial evidence suggests the existence of Ras-independent mechanisms of such activation (23,24). A pathway leading to MAPK stimulation that may be independent of Ras is one mediated by Rap1 (25), which has been found to be functionally active in prostate cancer cells (26). Despite a possible link of FAK to Rap1 via Crk (27), Crk has so far not been linked to FAK or Src in a functionally important signaling pathway, such as one mediating the expression of target genes involved in angiogenesis.

In this article, we demonstrate that cell contact can induce VEGF gene transcription in malignant but not in benign prostatic epithelial cells and that such induction occurs via an FAK-dependent MAPK pathway. In addition, we show that such transcriptional induction is mediated via Rap1 and Raf in a Rasindependent manner. These results outline a novel transformationrelated signaling pathway regulating VEGF gene transcription and highlight the role of Rap1 as a stimulus-specific downstream effector of FAK function.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell Culture and Northern Blot Analysis

LnCaP and PC3 prostate cancer cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in T-medium (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD) containing 5% fetal bovine serum. The PrEC primary prostate epithelial cells (catalog No. CC-2555), purchased from Clonetics (Walkersville, MD), are isolated from normal prostate tissue and have a finite life span in culture (28). PrEC cells were grown in medium provided by the manufacturer. For northern blot analysis, cells were plated at various densities or for varying times, as indicated in the figure legends. Cell numbers, also indicated in the figure legends, reflect the relative number of cells that were present per unit of surface area, corresponding to the surface area of a single well of a six-well tissue culture plate. In this way, the levels of induction of VEGF mRNA could be compared on a per cell basis with those observed during the experiments using the VEGF promoter-reporter constructs described below. Northern blot analyses were carried out as described previously (29). Equal loading of the RNA samples was confirmed by visualization of the 18S ribosomal RNA band (30). The relative intensity of the VEGF bands was assessed by densitometry and analysis with ImageQuant V3.3 software (Molecular Dynamics, Sunnyvale, CA).

Plasmid DNA Transfection, Pharmacologic Inhibitors, and Growth Factors

DNA transfections were performed by electroporation using a Bio-Rad Gene Pulser II RF Module (Bio-Rad Laboratories, Richmond, CA). Cells were treated with pharmacologic inhibitors 6 hours before they were assayed for VEGF promoter-reporter activity. At that time point, the medium was removed and replaced with that containing the drugs at the concentrations indicated in the figure legends. The PI3K inhibitors, wortmannin (31) and LY294002 (32), were purchased from Sigma Chemical Co. (St. Louis, MO). PD098059 (33), a specific inhibitor of MAPK/ERK, was purchased from Promega Corp. (Madison, WI), and SB 203580 (34), a specific inhibitor of p38 MAPK, was purchased from A. G. Scientific (San Diego, CA). Murine epidermal growth factor (EGF) was purchased from Sigma Chemical Co.

Plasmid Constructs

A clone containing the part of the coding sequence of VEGF (35) was used for probe construction. The entire VEGF gene promoter region in pGL2 (36) was subcloned into the pGL3-basic vector (Promega Corp.). The resulting VEGF reporter construct, VEGF-Fluc, consists of 2.65 kilobase pairs of the VEGF gene extending into exon 1 but lacking a translation initiation site or extraneous eukaryotic promoter or enhancer elements, coupled to the firefly luciferase (F-luc) coding region and a simian virus 40 (SV40) intron and polyadenylation signals. The control reporter pRL-thymidine kinase (pRL-TK) construct that contains the renilla luciferase (R-luc) gene and is driven by the herpes simplex virus thymidine kinase (HSV-TK) promoter was purchased from Promega Corp. The pBluescript (Stratagene, San Diego, CA) was used as a carrier to equalize the total amount of DNA used in transfections.

SrcA430 dominant negative construct in pcDNA3 (37), RasV12 activated, RasN17, RapN17 dominant negatives, and kinase dead dominant negative Raf constructs in pEXV (38), FRNK expression vector in pRK5 (13), GAL4-Elk-1 construct encoding the DNA-binding domain of GAL4 (residues 1–147) linked to the carboxyl-terminal transcription activation domain of Elk-1 (residues 307–428), and the GAL4-E1b-luciferase reporter gene have been described previously (39). The activated MEK and pc22W-F-Raf, as well as the dominant negative MEK expression constructs (40), were used.

Transient Expression, Conditioned Medium, and ECM Assays

Plasmid DNA was prepared by the EndoFree Plasmid Maxi Kit obtained from Qiagen (Valencia, CA) with the use of the manufacturer's procedure. After transfection, the cells were allowed to recover for 24 hours before all assays. For coculture assays, transfected cells were then trypsinized and plated either at different cell numbers or at a fixed number on top of variable numbers of nontransfected cells (referred to as the "cell contact layer") in six-well plates. This latter experimental design was used subsequently to measure the effect of different DNA constructs on VEGF promoter activity and will be referred to as the "cell contact assay." This latter analysis was modified in two ways to exclude the possibility of a diffusible factor as the mediator of the cell density VEGF induction in target cells. First, we separated the transfected cells from the nontransfected ones by a permeable Transwell membrane (Corning Costar, Cambridge, MA). The mesh size of this membrane (0.4 µm) allows the diffusion of all growth factors but not cell passage. Second, after the cells were incubated for 48 hours, we harvested medium from pBluescript-transfected cells grown densely (1.0 x 106 cells per well). This conditioned medium was applied to sparsely grown cells (0.25 x 106 cells per well) cotransfected with VEGF-Fluc and pRL-TK. After a further 48-hour incubation, analysis was carried out as described below.

BD Biocoat® Variety Pack 2 (catalog No. 354431; Becton Dickinson, Bedford, MA) ECM-precoated plates were used to test the hypothesis that an ECM component is responsible for the cell density transcriptional induction of VEGF. Six-well plates precoated with collagen types I and IV, fibronectin, laminin, and poly-D-lysine (control) were used to plate 1.0 x 106 per well (dense) and 0.25 x 106 per well (sparse) of LnCaP cells cotransfected with VEGF-Fluc and pRL-TK. After a further 48-hour incubation, analysis was carried out as described below.

To normalize for transfection efficiency, plating efficiency, and number of cells per plate, we used a similar ratio of VEGF-Fluc/pRL-TK DNA in all transient transfection experiments. For the determination of both VEGF-Fluc (i.e., F-luc) and control pRL-TK promoter (i.e., R-luc) activities, cells were lysed in Passive Lysis Buffer (Promega Corp.) and assayed simultaneously for F-luc and R-luc according to the instructions on the Promega Dual Luciferase Assay Kit in a Turner Designs (Sunnyvale, CA) TD-20/20 luminometer. The relative activity of each luciferase was recorded as relative light units (RLU). To normalize for variations in both transfection and plating efficiencies as well as to calculate the effect of cell density on the promoter on a per cell basis, we used the ratio of F-luc RLU/R-luc RLU. We further calculated the fold induction of luciferase activity by dividing the above-mentioned ratio for each cell number by the ratio observed at the lowest cell number in each experiment. All assays were performed from multiple samples and were done at least twice. For the analysis, data were averaged across all samples.

Statistical Analysis

The experiments are factorial designs in two complete randomized blocks, and experimental replications serve as the blocking factor. One exception is the experiment displayed in Fig. 2Go, B, where a complete randomized block without factorial structure on the treatments was used. In the factorial experiments, the treatments are combinations of cell number and either cell line (i.e., PrEC, LnCaP, or PC3) or construct (FRNK, Ras, Rap1, etc.). Analysis-of-variance (ANOVA) methods appropriate for these experimental designs were used to estimate the treatment effects and the error mean square. Residual plots and plots of within-cell standard deviation versus within-cell mean indicated that the use of the F-luc/R-luc ratio as a dependent variable would violate the constant variance assumption of the ANOVA methods. Consequently, analyses were conducted with the use of the natural log of the F-luc/R-luc ratio. Diagnostic plots using the transformed variable did not indicate violations of the model assumptions.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2. Cells were transiently transfected with 5 µg of the vascular endothelial growth factor (VEGF)-Fluc (F-luc, i.e., firefly luciferase) reporter construct and 0.5 µg of pRL-thymidine kinase (R-luc, i.e., renilla luciferase) plasmid as a control reporter. After electroporation, cells were allowed to recover for 24 hours and then trypsinized and replated in six-well plates. Cells were allowed to grow for a further 48 hours and then lysed and assayed for F-luc and R-luc. We normalized the VEGF-Fluc activity on a per cell basis by computing the F-luc/R-luc ratio. A) VEGF promoter activity as a function of cell density: Transfected cells were replated at either sparse (0.1 x 106 cells per well) or confluent (1.0 x 106 cells per well) densities. Fold induction is in relation to the lowest cell number. Estimates and 95% confidence intervals for the fold induction between confluent and sparse densities for PrEC, LnCaP, and PC3 cells are shown. The confidence intervals are adjusted for multiple comparisons with the use of the Bonferroni procedure, and all P values are two-sided. Statistically significant differences in fold induction between confluent and sparse densities are found for the LnCaP and PC3 cell lines (P<.0001) but not for the PrEC cell line (P = .37). The cell lines are significantly different from each other (P<.0001) with respect to the fold induction between confluent and sparse densities. B) VEGF promoter activity as a function of cell number in contact layer: Transfected LnCaP cells (0.25 x 106) were directly plated on top of variable numbers of nontransfected cells (contact layer), which were placed in six-well plates 24 hours earlier. The fold induction is in relation to transfected cells plated with no contact layer. Estimates and 95% confidence intervals for the fold induction between cell number in contact layer relative to zero cells in contact layer are shown. The confidence intervals are adjusted for multiple comparisons with the use of the Bonferroni procedure. Statistically significant differences (P<.0001) in fold induction between cell number and control are found for 0.25 x 106, 0.5 x 106, and 1.0 x 106 cells in the contact layer.

 
Contrasts were used to estimate the effects of interest. For example, contrasts were used to estimate the increase in induction in the transient transfection assays between confluent and sparse densities within each cell line (i.e., PrEC, LnCaP, or PC3). Confidence intervals for the effects of interest were computed with the use of the transformed dependent variable. The antilog of the end points of these intervals was used to obtain confidence intervals in the original scale. Within each analysis, the Bonferroni adjustment was used to control the overall type I error rate at 0.05 and the overall confidence level at 95%. All of the statistical tests are two-sided; P values less than .05 are considered to be statistically significant. Data shown were plotted with the use of the STATISTICA for Windows computer program (StatSoft, Inc., Tulsa, OK).


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
VEGF mRNA in Densely Plated Benign and Malignant Prostate Epithelial Cells

To gain initial insights into the regulation of VEGF in response to cell density, we plated LnCaP cells at a fixed sparse density (6.25 x 104 cells per six-well plate unit) and allowed them to grow for 2–8 days before they were harvested. The VEGF mRNA level increased as a function of cell time and thus cell density, compared on a per cell basis (equal amounts of RNA loaded) (Fig. 1Go, A). To determine the dependency of this response on transformation status, we plated LnCaP and PrEC cells at different densities and allowed them to grow for 48 hours before they were harvested. The VEGF mRNA level was threefold to fourfold higher when the highest and lowest densities were compared on a per cell basis (equal amounts of RNA loaded) in LnCaP cells but showed no increase in PrEC cells (Fig. 1Go, B). The cell numbers indicated were selected because they represent conditions where essentially no cell–cell contact is present (in a range between 0.1 x 106 to 0.25 x 106 cells per well) or cells are confluent (in a range between 0.7 x 106 to 1.0 x 106 cells per well). The time course and cell densities used in this experiment are the same as those used in subsequent experiments employing the VEGF-Fluc construct. To determine whether VEGF is transcriptionally regulated as a function of cell density, we transiently cotransfected VEGF-Fluc and pRL-TK into PrEC, LnCaP, and PC3 cells. Various numbers of transiently transfected cells were then plated in six-well plates. After a 48-hour incubation, induction of luciferase activity was assayed and was found to be twofold to fivefold higher on a per cell basis in dense cells than in sparse cells (Fig. 2Go, A) but only in malignant cells. To further verify that this effect was not due to a larger number of dead cells at one density compared with another, we assessed the magnitude of this induction by normalizing the LnCaP data for cell counts assessed with the use of a hemocytometer and vital dye. These results revealed similar levels of induction whether the data were normalized on a per cell basis either to cell counts at the end of incubation or to the cotransfected pRL-TK (R-luc), the latter as described in the "Materials and Methods" section (data not shown).



View larger version (56K):
[in this window]
[in a new window]
 
Fig. 1. Vascular endothelial growth factor (VEGF) messenger RNA (mRNA) expression as a function of cell density: Northern blot analysis of 7 µg of total cellular RNA was carried out. After the mRNA was transferred to nylon membranes, methylene blue staining of 18S ribosomal RNA served as a loading control. The membranes were hybridized with a 32P-labeled VEGF complementary DNA probe and autoradiographed. The relative steady-state level of VEGF mRNA was quantitated by densitometry. Fold induction indicates the level of VEGF mRNA relative to the first sample. A) LnCaP cells were plated at a fixed sparse density (6.25 x 104 cells per six-well plate unit) and allowed to grow for the indicated time intervals before isolation of RNA. B) LnCaP or PrEC cells were plated at various densities, and RNA was isolated 48 hours later. Results shown are representative of data obtained from independent experiments.

 
Effects of Cell–Cell Contact, Soluble Factors, and Known ECM Components on VEGF Transcription

To determine whether the observed transcriptional regulation of VEGF is secondary to secretion of a diffusible factor or is due to cell contact, we repeated the experiment described above with an equal number of VEGF-Fluc/pRL-TK-transfected LnCaP cells separated from increasing numbers of nontransfected LnCaP cells by a permeable Transwell polycarbonate membrane and incubated for 48 hours. To ensure that the transfected cells in this experiment retained the capacity of VEGF promoter induction, we repeated the above experiment with the simultaneous plating and assessment of transfected cells under sparse and dense conditions, as described in Fig. 1Go. No induction was seen with the cells separated by the filter (data not shown), and a twofold to 2.5-fold induction was observed when cells were plated at various densities (data not shown). To further confirm the absence of a diffusible factor effect, we harvested the conditioned medium from confluent cells as described and applied it to cells that had been transfected with the VEGF-Fluc/pRL-TK and subsequently plated sparsely (0.25 x 106 cells per well). No induction of the VEGF reporter was observed with this conditioned medium. To further confirm the requirement for cell contact, we plated a fixed number (0.25 x 106 cells per well) of VEGF-Fluc/pRL-TK-transfected cells directly on top of various densities of nontransfected cells. This experiment revealed a twofold to threefold induction of VEGF promoter activity for 0.25 x 106 per well transfected LnCaP cells plated on top of this cell contact layer of nontransfected confluent LnCaP cells (1 x 106) versus transfected cells plated without the cell contact layer (Fig. 2Go, B). In addition, no increase in VEGF-Fluc induction over control (poly-D-lysine) was noted in sparse cells when they were plated on several purified ECM components (data not shown). Taken together, these results indicate that induction of VEGF gene transcription by increasing the cell density requires direct cell contact and is unlikely to be mediated by a diffusible factor or by known ECM components, such as collagen types I and IV, fibronectin, or laminin. Because we subsequently used this assay to test the effect of different signaling molecules on VEGF promoter activity in response to cell contact, we will employ the term "cell contact assay" to refer to this experimental design.

FAK and MAPK and Cell Contact-Mediated Transcriptional Induction of VEGF

Cell-surface interactions in epithelial cells are known to be mediated by FAK via tyrosine phosphorylation (14,4143), which subsequently initiates multiple downstream signaling cascades. We have explored the possible involvement of FAK and MAPK in the transcriptional induction of VEGF in response to cell contact by cotransfecting LnCaP cells with the VEGF reporter and FRNK. FRNK, the carboxy-terminal domain of FAK, is known to act as a dominant negative modulator of FAK function (13). Cotransfection with FRNK suppressed VEGF promoter activity (Fig. 3Go, A). Since it has been reported recently that, upon cell adhesion, PI3K binds to FAK (15,44), we tested the involvement of PI3K by applying two PI3K-specific inhibitors, wortmannin and LY294002. In our standard cell contact assay, wortmannin (1 µM) and LY294002 (30–60 µM) suppressed the VEGF promoter activity, suggesting that the cascade for VEGF expression may be mediated through effectors downstream of PI3K (data not shown). While we realize that FRNK may have additional as yet undiscovered functions separate from its negative regulatory effect on FAK, taken together, these results suggest that FAK and PI3K are involved in the VEGF induction by cell contact.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3. A) Effect of FRNK on cell contact-modulated vascular endothelial growth factor (VEGF) promoter activity: LnCaP cells were transiently transfected with 5 µg of the VEGF-Fluc (F-luc, i.e., firefly luciferase) construct, 0.5 µg of pRL-thymidine kinase (R-luc, i.e., renilla luciferase) as a reporter of transfection efficiency, and either 5 µg of pRK5 MYC-FRNK or pBluescript (PBS) and then processed; results were calculated as for Fig. 2Go, B. Estimates and Bonferroni-adjusted 95% confidence intervals for fold induction between 0.25 x 106 cells relative to plastic for pBluescript and FRNK are shown. All P values are two-sided. There is a statistically significant difference in fold induction for pBluescript (P<.0001) but not for FRNK (P = .11). The pBluescript and FRNK constructs are significantly different from each other in the fold induction relative to plastic (P<.002). B) Modulation of VEGF promoter activity by mitogen-activated protein kinase/extracellular-regulated kinase (MEK): LnCaP cells were transiently transfected with 3 µg of the VEGF-Fluc (F-luc) construct, 0.5 µg of pRL-thymidine kinase (R-luc) as a reporter of transfection efficiency, and 3 µg of a construct expressing either dominant negative MEK (dnMEK), activated MEK (actMEK), or pBluescript (PBS). Estimates and Bonferroni-adjusted 95% confidence intervals for fold induction between 0.25 x 106 cells relative to plastic for pBluescript, actMEK, and dnMEK. There is a statistically significantly greater induction for pBluescript (P<.0001), whereas there is a significantly lower induction with actMEK (P = .03) and dnMEK (P = .03).

 
Since MAPKs are known downstream mediators of FAK, we tested the possible involvement of two distinct MAPK-signaling pathways (4547), namely, those leading to activation of either the extracellular-regulated kinases (MEK/ERK) or p38 MAPK in cell contact-mediated induction of VEGF promoter activity. During an LnCaP cell contact experiment, PD098059 (50–100 µM), an inhibitor of MEK1 and MEK2 activation by Raf (33), had an inhibitory effect on the VEGF reporter (data not shown). These results were further confirmed with the use of dominant negative and activated MEK. The dominant negative MEK completely inhibited the cell contact-mediated activity of the VEGF promoter (Fig. 3Go, B). On the other hand, activated MEK enhanced VEGF promoter activity by approximately 10-fold (data not shown) and did not have an additive effect with cell contact (Fig. 3Go, B). In addition, the application of SB 203580 (50–100 µM), which specifically inhibits p38 MAPK activity (34), had no appreciable effect on VEGF promoter activity (data not shown), suggesting that p38 MAPK activity is not required for cell contact-mediated induction of VEGF-Fluc. Taken together, these experiments suggest that the FAK/PI3K act via the MEK-signaling cascade to regulate the VEGF promoter in response to cell contact.

Roles of Rap1, Raf, and Ras in the Transcriptional Regulation of VEGF

Since mutationally activated forms of both Ras (48) and Src (49) genes have been shown to induce VEGF, we asked whether these genes could be the mediators of VEGF regulation by cell contact. Thus, we repeated the cell contact assay by cotransfection of either dominant negative Src or Ras constructs into LnCaP cells. It is interesting that the pEXV-RasN17 construct encoding a dominant negative form of Ras did not have any impact on the VEGF promoter activity, whereas SrcA430, the dominant negative form of Src, resulted in inhibition (Fig. 4Go, A). Since the lack of an effect of the dominant negative RasN17 construct could be due to its inactivity in our system, we tested the ability of this construct to inhibit a well-characterized Ras-signaling pathway, namely, the activation of the Elk-1 transcription factor by EGF treatment. Thus, a construct consisting of GAL4 cis-acting elements linked to luciferase (GAL4-luc) was cotransfected with a construct expressing a chimeric protein consisting of the GAL4 DNA-binding domain coupled to the Elk-1 activation domain. These plasmids were transfected in combination with either RasN17 or pBluescript. After 16 hours of serum starvation, the cells were treated for 2 hours with 10 ng/mL EGF. In the transfections with pBluescript, there was a 14-fold induction of Elk-1 activity, whereas the activity was inhibited in cells transfected with RasN17 (data not shown), indicating the functional competence of the RasN17 construct. To demonstrate the competence of Ras to induce VEGF in LnCaP cells, we cotransfected RasV12, a mutationally activated form of Ras, into these cells and observed a threefold to fourfold induction of the VEGF reporter (data not shown).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4. A) Effect of Src and Ras on cell contact-mediated induction of vascular endothelial growth factor (VEGF) promoter activity: The experimental promoter activity (VEGF) was normalized for transfection efficiency on a "per cell basis" by computing the F-luc (i.e., firefly luciferase)/R-luc (i.e., renilla luciferase) ratio. Results are the average of multiple samples, and the values represent the average of two independent experiments. LnCaP cells were transiently transfected with 5 µg of the VEGF-Fluc (F-luc) reporter construct, 0.5 µg of pRL-thymidine kinase (R-luc) plasmid as a control reporter, and 5 µg of either pcDNA3-SrcA430 (SRC), pEXV-RasN17 (Ras), or pBluescript (pBS) and then processed; results were calculated as for Fig. 2Go, B. Estimates and Bonferroni-adjusted 95% confidence intervals for fold induction between 0.5 x 106 cells and 1.0 x 106 cells relative to plastic for pBluescript, Ras, and SRC are shown. All P values are two-sided. At both cell numbers, significantly greater induction (P<.0001) was observed for pBluescript and Ras. At 0.5 x 106 cells in the contact layer, there was a small increase in induction with SRC (P = .06) as well as a slightly greater amount of induction with SRC at 1.0 x 106 cells (P<.002). At both 0.5 x 106 and 1.0 x 106 cells, fold induction relative to plastic was significantly greater with pBluescript and Ras than with SRC (P<.001). B) Modulation of VEGF promoter activity by dominant negative and activated Raf: LnCaP cells were transiently transfected with 3 µg of the VEGF-Fluc (F-luc) construct, 0.5 µg of pRL-thymidine kinase as a reporter of transfection efficiency, and 3 µg of either a dominant negative pEXV-Raf (dnRaf) or pBluescript (PBS) and then processed; results were calculated as for Fig. 2Go, B. Estimates and Bonferroni-adjusted 95% confidence intervals for fold induction between 0.25 x 106 cells relative to plastic for pBluescript and dnRaf are shown. There is a statistically significant difference in fold induction for pBluescript (P<.0001) but not for dnRaf (P = .11). The pBluescript and dnRaf constructs are significantly different from each other in the fold induction relative to plastic (P = .001). C) Modulation of VEGF promoter activity by dominant negative Rap1: LnCaP cells were transiently transfected with 3 µg of the VEGF-Fluc (F-luc) construct, 0.5 µg of pRL-thymidine kinase as a reporter of transfection efficiency, and either 3 µg of dominant negative Rap1N17 (dnrap) or pBluescript (PBS) and then processed; results were calculated as for Fig. 2Go, B. Estimates and Bonferroni-adjusted 95% confidence intervals for fold induction between 0.25 x 106 cells relative to plastic for pBluescript and Rap1N17 are shown. There is a statistically significant difference in fold induction for pBluescript (P<.0001) as well as for Rap1N17 (P = .002). The pBluescript and Rap1N17 constructs are significantly different from each other in the fold induction relative to plastic (P<.0001).

 
Because some of the Ras-independent downstream signaling effects of Src are known to proceed via Raf, we repeated the cell contact assay with a dominant negative Raf construct that inhibited VEGF promoter activity in the cell contact assay (Fig. 4Go, B). LnCaP cells cotransfected with activated Raf have an approximately threefold induction of VEGF promoter activity, which is not further induced by cell contact. To further characterize the specificity of the cell contact signaling pathways leading to VEGF induction, we carried out a cell contact assay as described in Fig. 2Go, B, using LnCaP cells cotransfected with GAL4-luc and GAL4 DNA-binding domain/Elk-1 activation domain chimera. An induction of Elk-1 was found with increasing cell contact, indicating that Elk-1 is activated by the pathway that results in increased VEGF promoter activity in response to cell contact, which supports the data implicating MAPK and Raf in this process. Since our results suggested that a Ras-independent, FAK-signaling pathway was mediating VEGF transcription in response to cell contact, we sought to evaluate the role of Rap1, a Ras-like guanosine triphosphatase (GTPase) known to be involved in Ras-independent MAPK activation (50). It is interesting that Rap1 has an effector domain virtually identical to that of Ras, indicating that both GTPases may interact with similar effectors. Since various isoforms of Raf have been shown to interact differently with Ras and Rap1 (51), the involvement of Rap1 in the density regulation of VEGF transcription seemed to offer a solution to explain how cell contact can be mediated by Raf and MAPK without the involvement of Ras activation. Hence, we repeated the cell contact assay with a dominant negative Rap1 construct (52), which indicated that this construct reduced the VEGF promoter induction in the cell contact assay (Fig. 4Go, C).


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
By altering in vitro cell density to simulate changes in cell–cell interactions in vivo, our results (Fig. 5Go) suggest the existence of a tumor-specific pathway that may transduce microenvironmental signals regulating the transcriptional regulation of VEGF in prostate cancer cells. Since normal prostate epithelial cells do not induce VEGF transcription in response to cell contact, one could speculate that, at some point in transformation, prostate cells corrupt the regulation of this pathway, allowing them to utilize it to facilitate their continued growth. This alteration differs substantially from the induction of VEGF mRNA observed in response to mutated Raf or MEK transfection, since, in the former case, the cells remain responsive to microenvironmental stimuli, such as cell–cell contact.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5. Summary and hypothetical working model of the microenvironmental signaling network regulating vascular endothelial growth factor (VEGF) transcription: Focal adhesion kinase (FAK), Src, phosphatidylinositol 3-kinase (PI3K), Rap1, Raf, and mitogen-activated protein kinase/extracellular-regulated kinase (MEK) are key effectors in a Ras-independent signaling pathway mediating the cell contact-dependent transcriptional regulation of VEGF. Arrows do not necessarily indicate direct physical relationship between molecules. Gray shading around FAK, Src, and PI3K represents their known association in signaling complexes. dnMEK = dominant negative MEK; dnRaf = dominant negative Raf; ECM = extracellular matrix; EGF = epidermal growth factor.

 
We believe that the current study is the first to demonstrate the involvement of FAK in the process of transcriptional regulation of VEGF. Since a primary role of FAK is in the cooperative regulation and integration of signals initiated by cell adhesion (53) in response to integrin activation, our results raise the interesting possibility that alterations in the integrin repertoires associated with cellular transformation or progression may play a role in the transcriptional regulation of VEGF via FAK. This latter hypothesis is made even more plausible by the observation that normal prostate epithelial cells express several integrins including alpha 2, 3, 4, 5, 6, v, beta 1, and beta 4 (54), while expression of alpha 2, alpha 4, alpha 5, alpha v, and beta 4 integrins is lost in prostate carcinoma cells (55). Thus, during tumor progression, prostate cells may lose the growth-suppressive and invasion-suppressive microenvironmental signals mediated by the cadherin/catenin (56) complexes, while simultaneously responding with increased angiogenesis via FAK-mediated signaling. Although we have not observed VEGF induction with known ECM components, such as collagen types I and IV, fibronectin, or laminin, yet undiscovered stimuli of the integrin-mediated VEGF signaling may still account for this effect.

While integrin-mediated cell adhesion has been shown to strongly activate MAPK via the Ras-signaling pathway (22), there is also substantial evidence for Ras-independent FAK/integrin-mediated activation of MAPK in other experimental systems. For example, transfection of dominant negative RasN17 did not affect activation of MEK by adhesion to fibronectin (23). In addition, high levels of expression of the N-terminal portion of Raf, which has a binding site for Ras and completely blocks the activation of MAPK by oncogenic Ras, also failed to inhibit the integrin-mediated activation of the MAPK pathway (57). In this article, we demonstrate a mechanism by which Ras-independent, FAK-initiated MAPK activation can occur via Rap1.

Recent data from several laboratories (8,9,58) have shown that Src can be activated by cell density and that this activation, in turn, leads to an increase in VEGF mRNA. It is interesting that, in some cases, this effect was dependent on a soluble factor (6,9), while, in other cases, it was dependent on cell–cell contact (7), suggesting that Src can serve as a ubiquitous messenger of a variety of extracellular stimuli. It is also interesting that, while in Src-transformed cells the adaptor protein Shc is tyrosine phosphorylated (59), presumably by Src itself, resulting in its association with Grb2 and with mSOS, our data do not seem to implicate Ras in mediating the cell contact transcriptional induction of VEGF. Instead, we identify Rap1 as the mediator of this effect, suggesting that Rap1 may serve to narrow the specificity of Src activation to activation by cell-surface interactions mediated by a putative FAK/PI3K/Src complex (16).

One of the most intriguing observations in our study is the apparent specificity of cell–cell stimulation of VEGF transcription via MAPK without dependence on the activity of Ras. This finding is especially interesting, since it does not appear to be due to an inability of Ras to induce VEGF transcription in this system. The functional importance of Rap1 in this process is a novel observation that represents the first connection of this molecule to a step in the regulation of tumor angiogenesis and to FAK. In addition, this finding leads us to speculate that different extracellular signals can use different repertoires of signaling molecules, while eventually converging on a common pathway (Fig. 5Go). In this way, the effect of a variety of factors, such as cell–cell contact and diffusible factors, found in the tumor microenvironment can be processed and integrated by the cellular signaling circuits. Whether VEGF stimuli using different but converging pathways act synergistically or additively remains to be determined. An alternative explanation of how the specificity of VEGF induction by cell–cell contact can be related to Rap1 rather than to Ras may be differential activation of Ras and Rap1 based on their respective cellular locations. This notion is supported by the observation that the predominant mechanism of Ras activation hinges on the association of guanine nucleotide exchange factors with membrane-bound receptors, while activation of Rap1 occurs through activation of highly motile second messengers, such as cyclic adenosine monophosphate. Taken together, these observations suggest that Ras mainly serves a function in the proximity of a surface receptor, whereas Rap1 functions more intracellularly, which is compatible with the subcellular localization of the two proteins.

Finally, cellular localization notwithstanding, since Ras and Rap1 have a different repertoire of binding partners, they likely trigger a different spectrum of downstream signaling events, even though, in some cases, their effects may converge, as is the case for transcriptional regulation of VEGF. For example, a candidate effector for mediating the Rap1 effect is B-Raf, a close relative of Raf1, that can bind to and is activated by Rap1 in vitro (51) and has recently been shown to be expressed in LnCaP cells (26). The Rap1/B-Raf complex may lead to transmission to a distinct repertoire of MEK family members than would a Ras/Raf-1 complex (60,61). Our results with the dominant negative forms of MEK and Raf do not exclude this hypothesis, since these constructs probably inhibit most isoforms of these molecules.

In conclusion, we have outlined a novel FAK- and Rap1-dependent, Ras-independent MAPK-signaling pathway mediating the cell contact-dependent transcriptional regulation of VEGF. This tumor-specific signaling cascade demonstrates how malignant cells utilize the extracellular environment to increase angiogenic signals that subsequently can lead to tumor progression. It also shows how the repertoire of intracellular signaling molecules can integrate different extracellular regulatory stimuli.


    NOTES
 
E. A. Sheta and M. A. Harding contributed equally to this work.

Supported in part by Public Health Service training grant 1T32DK0T166-01 from the National Institute of Diabetes and Digestive and Kidney Diseases (to M. A. Harding), National Institutes of Health, Department of Health and Human Services; and by a career development award by the American Cancer Society (to D. Theodorescu).

We thank Drs. M. E. Cox, S. J. Parsons, J. T. Parsons, and M. J. Weber at the University of Virginia for their many helpful suggestions.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

1 Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999;13:9–22.[Abstract/Free Full Text]

2 Rak J, Mitsuhashi Y, Bayko L, Filmus J, Shirasawa S, Sasazuki T, et al. Mutant ras oncogenes upregulate VEGF/VPF expression: implications for induction and inhibition of tumor angiogenesis. Cancer Res 1995;55:4575–80.[Abstract]

3 Grugel S, Finkenzeller G, Weindel K, Barleon B, Marme D. Both v-Ha-Ras and v-Raf stimulate expression of the vascular endothelial growth factor in NIH 3T3 cells. J Biol Chem 1995;270:25915–9.[Abstract/Free Full Text]

4 Mukhopadhyay D, Tsiokas L, Zhou XM, Foster D, Brugge JS, Sukhatme VP. Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature 1995;375:577–81.[Medline]

5 Clark EA, King WG, Brugge JS, Symons M, Hynes RO. Integrin-mediated signals regulated by members of the rho family of GTPases. J Cell Biol 1998;142:573–86.[Abstract/Free Full Text]

6 Koura AN, Liu W, Kitadai Y, Singh RK, Radinsky R, Ellis LM. Regulation of vascular endothelial growth factor expression in human colon carcinoma cells by cell density. Cancer Res 1996;56:3891–4.[Abstract]

7 Mukhopadhyay D, Tsiokas L, Sukhatme VP. High cell density induces vascular endothelial growth factor expression via protein tyrosine phosphorylation. Gene Exp 1998;7:53–60.

8 Kobayashi S, Okumura N, Nakamoto T, Okada M, Hirai H, Nagai K. Activation of pp60c-src depending on cell density in PC12h cells. J Biol Chem 1997;272:16262–7.[Abstract/Free Full Text]

9 Fleming RY, Ellis LM, Parikh NU, Liu W, Staley CA, Gallick GE. Regulation of vascular endothelial growth factor expression in human colon carcinoma cells by activity of src kinase. Surgery 1997;122:501–7.[Medline]

10 Aberle H, Schwartz H, Kemler R. Cadherin–catenin complex: protein interactions and their implications for cadherin function. J Cell Biochem 1996;61:514–23.[Medline]

11 Morton RA, Ewing CM, Nagafuchi A, Tsukita S, Isaacs WB. Reduction of E-cadherin levels and deletion of the alpha-catenin gene in human prostate cancer cells. Cancer Res 1993;53:3585–90.[Abstract]

12 Richmond PJ, Karayiannakis AJ, Nagafuchi A, Kaisary AV, Pignatelli M. Aberrant E-cadherin and alpha-catenin expression in prostate cancer: correlation with patient survival. Cancer Res 1997;57:3189–93.[Abstract]

13 Richardson A, Parsons T. A mechanism for regulation of the adhesion-associated proteintyrosine kinase pp125FAK. Nature 1996;380:538–40.[Medline]

14 Schlaepfer DD, Hunter T. Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases. Mol Cell Biol 1996;16:5623–33.[Abstract]

15 Chen HC, Appeddu PA, Isoda H, Guan JL. Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase. J Biol Chem 1996;271:26329–34.[Abstract/Free Full Text]

16 Schaller MD, Hildebrand JD, Shannon JD, Fox JW, Vines RR, Parsons JT. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol Cell Biol 1994;14:1680–8.[Abstract]

17 Schlaepfer DD, Hanks SK, Hunter T, van der Geer P. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature 1994;372:786–91.[Medline]

18 Miyamoto S, Teramoto H, Coso OA, Gutkind JS, Burbelo PD, Akiyama SK, et al. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J Cell Biol 1995;131:791–805.[Abstract]

19 Morino N, Mimura T, Hamasaki K, Tobe K, Ueki K, Kikuchi K, et al. Matrix/integrin interaction activates the mitogen-activated protein kinase, p44erk-1 and p42erk-2. J Biol Chem 1995;270:269–73.[Abstract/Free Full Text]

20 Ahn NG, Robbins DJ, Haycock JW, Seger R, Cobb MH, Krebs EG. Identification of an activator of the microtubule-associated protein 2 kinases ERK1 and ERK2 in PC12 cells stimulated with nerve growth factor or bradykinin. J Neurochem 1992;59:147–56.[Medline]

21 Gioeli D, Mandell JW, Petroni GR, Frierson HF Jr, Weber MJ. Activation of mitogen-activated protein kinase associated with prostate cancer progression. Cancer Res 1999;59:279–84.[Abstract/Free Full Text]

22 Clark EA, Hynes RO. Ras activation is necessary for integrin-mediated activation of extracellular signal-regulated kinase 2 and cytosolic phospholipase A2 but not for cytoskeletal organization. J Biol Chem 1996;271:14814–8.[Abstract/Free Full Text]

23 Chen Q, Lin TH, Der CJ, Juliano RL. Integrin-mediated activation of MEK and mitogen-activated protein kinase is independent of Ras. J Biol Chem 1996;271:18122–7.[Abstract/Free Full Text]

24 Brtva TR, Drugan JK, Ghosh S, Terrell RS, Campbell-Burk S, Bell RM, et al. Two distinct Raf domains mediate interaction with Ras. J Biol Chem 1995;270:9809–12.[Abstract/Free Full Text]

25 Bos JL, Franke B, M'Rabet L, Reedquist K, Zwartkruis F. In search of a function for the Ras-like GTPase Rap1. FEBS Lett 1997;410:59–62.[Medline]

26 Chen T, Cho RW, Stork PJ, Weber MJ. Elevation of cyclic adenosine 3`,5`-monophosphate potentiates activation of mitogen-activated protein kinase by growth factors in LNCaP prostate cancer cells. Cancer Res 1999;59:213–8.[Abstract/Free Full Text]

27 Matsuda M, Kurata T. Emerging components of the Crk oncogene product: the first identified adaptor protein. Cell Signal 1996;8:335–40.[Medline]

28 Campbell CL, Savarese DM, Quesenberry PJ, Savarese TM. Expression of multiple angiogenic cytokines in cultured normal human prostate epithelial cells: predominance of vascular endothelial growth factor. Int J Cancer 1999;80:868–74.[Medline]

29 Theodorescu D, Cornil I, Fernandez BJ, Kerbel RS. Overexpression of normal and mutated forms of HRAS induces orthotopic bladder invasion in a human transitional cell carcinoma. Proc Natl Acad Sci U S A 1990;87:9047–51.[Abstract]

30 Zhong H, Simons JW. Direct comparison of GAPDH, beta-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia. Biochem Biophys Res Commun 1999;259:523–6.[Medline]

31 Wymann MP, Bulgarelli-Leva G, Zvelebil MJ, Pirola L, Vanhaesebroeck B, Waterfield MD, et al. Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol Cell Biol 1996;16:1722–33.[Abstract]

32 Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994;269:5241–8.[Abstract/Free Full Text]

33 Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A 1995;92:7686–9.[Abstract]

34 Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, et al. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 1995;364:229– 33.[Medline]

35 Cheng SY, Huang HJ, Nagane M, Ji XD, Wang D, Shih CC, et al. Suppression of glioblastoma angiogenicity and tumorigenicity by inhibition of endogenous expression of vascular endothelial growth factor. Proc Natl Acad Sci U S A 1996;93:8502–7.[Abstract/Free Full Text]

36 Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 1996;16:4604–13.[Abstract]

37 Chang JH, Gill S, Settleman J, Parsons SJ. c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation. J Cell Biol 1995;130:355–68.[Abstract]

38 Hartsough MT, Frey RS, Zipfel PA, Buard A, Cook SJ, McCormick F, et al. Altered transforming growth factor signaling in epithelial cells when ras activation is blocked. J Biol Chem 1996;271:22368–75.[Abstract/Free Full Text]

39 Roberson MS, Misra-Press A, Laurance ME, Stork PJ, Maurer RA. A role for mitogen-activated protein kinase in mediating activation of the glycoprotein hormone alpha-subunit promoter by gonadotropin-releasing hormone. Mol Cell Biol 1995;15:1531–9.

40 Catling AD, Schaeffer HJ, Reuter CW, Reddy GR, Weber MJ. A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function. Mol Cell Biol 1995;15:5214–25.[Abstract]

41 Calalb MB, Polte TR, Hanks SK. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol 1995;15:954–63.[Abstract]

42 Calalb MB, Zhang X, Polte TR, Hanks SK. Focal adhesion kinase tyrosine-861 is a major site of phosphorylation by Src. Biochem Biophys Res Commun 1996;228:662–8.[Medline]

43 Calautti E, Cabodi S, Stein PL, Hatzfeld M, Kedersha N, Paolo Dotto G. Tyrosine phosphorylation and src family kinases control keratinocyte cell–cell adhesion. J Cell Biol 1998;141:1449–65.[Abstract/Free Full Text]

44 King WG, Mattaliano MD, Chan TO, Tsichlis PN, Brugge JS. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation. Mol Cell Biol 1997;17:4406–18.[Abstract]

45 Lewis TS, Shapiro PS, Ahn NG. Signal transduction through MAP kinase cascades. Adv Cancer Res 1998;74:49–139.[Medline]

46 Ip YT, Davis RJ. Signal transduction by the c-Jun N-terminal kinase (JNK)—from inflammation to development. Curr Opin Cell Biol 1998;10:205–19.[Medline]

47 Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 1997;9:180–6.[Medline]

48 Rak J, Mitsuhashi Y, Bayko L, Filmus J, Shirasawa S, Sasazuki T, et al. Mutant ras oncogenes upregulate VEGF/VPF expression: implications for induction and inhibition of tumor angiogenesis. Cancer Res 1995;55:4575–80.[Abstract]

49 Mukhopadhyay D, Tsiokas L, Sukhatme VP. Wild-type p53 and v-Src exert opposing influences on human vascular endothelial growth factor gene expression. Cancer Res 1995;55:6161–5.[Abstract]

50 York RD, Yao H, Dillon T, Ellig CL, Eckert SP, McCleskey EW, et al. Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. Nature 1998;392:622–6.[Medline]

51 Ohtsuka T, Shimizu K, Yamamori B, Kuroda S, Takai Y. Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein. J Biol Chem 1996;271:1258–61.[Abstract/Free Full Text]

52 van den Berghe N, Cool RH, Horn G, Wittinghofer A. Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N). Oncogene 1997;15:845–50.[Medline]

53 Guan JL. Focal adhesion kinase in integrin signaling. Matrix Biol 1997;16:195–200.[Medline]

54 Cress AE, Rabinovitz I, Zhu W, Nagle RB. The alpha 6 beta 1 and alpha 6 beta 4 integrins in human prostate cancer progression. Cancer Metastasis Rev 1995;14:219–28.[Medline]

55 Allen MV, Smith GJ, Juliano R, Maygarden SJ, Mohler JL. Downregulation of the beta4 integrin subunit in prostatic carcinoma and prostatic intraepithelial neoplasia. Hum Pathol 1998;29:311–8.[Medline]

56 Murant SJ, Handley J, Stower M, Reid N, Cussenot O, Maitland NJ. Co-ordinated changes in expression of cell adhesion molecules in prostate cancer. Eur J Cancer 1997;33:263–71.[Medline]

57 Marte BM, Rodriguez-Viciana P, Wennstrom S, Warne PH, Downward J. R-Ras can activate the phosphoinositide 3-kinase but not the MAP kinase arm of the Ras effector pathways. Curr Biol 1997;7:63–70.[Medline]

58 Batt DB, Roberts TM. Cell density modulates protein-tyrosine phosphorylation. J Biol Chem 1998;273:3408–14.[Abstract/Free Full Text]

59 Schlaepfer DD, Broome MA, Hunter T. Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins. Mol Cell Biol 1997;17:1702–13.[Abstract]

60 Leevers SJ, Paterson HF, Marshall CJ. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 1994;369:411–4.[Medline]

61 Stokoe D, Macdonald SG, Cadwallader K, Symons M, Hancock JF. Activation of Raf as a result of recruitment to the plasma membrane. Science 1994;264:1463–7.[Medline]

Manuscript received October 26, 1999; revised April 24, 2000; accepted May 2, 2000.


This article has been cited by other articles in HighWire Press-hosted journals:


             
Copyright © 2000 Oxford University Press (unless otherwise stated)
Oxford University Press Privacy Policy and Legal Statement