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).
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ABSTRACT |
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INTRODUCTION |
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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, cellcell 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 cellcell 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.
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MATERIALS AND METHODS |
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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 1147) linked to the carboxyl-terminal transcription activation domain of Elk-1 (residues 307428), 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. 2, 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.
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RESULTS |
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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 28 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. 1, 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. 1
, B). The cell numbers indicated were selected because they represent conditions where essentially no cellcell 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. 2
, 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).
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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. 1. 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. 2
, 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. 3, 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 (3060 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.
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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. 4, 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).
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DISCUSSION |
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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 cellcell 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 cellcell 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. 5). In this way, the effect of a variety of factors, such as cellcell 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 cellcell 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.
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NOTES |
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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.
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Manuscript received October 26, 1999; revised April 24, 2000; accepted May 2, 2000.
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