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[Cancer Research 59, 4804-4807, October 1, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Extracellular Signal-regulated Kinase Activation Is Required for Up-Regulation of Vascular Endothelial Growth Factor by Serum Starvation in Human Colon Carcinoma Cells1

Young D. Jung, Kayo Nakano, Wenbiao Liu, Gary E. Gallick and Lee M. Ellis2

Departments of Cancer Biology [Y. D. J., K. N., W. L., G. E. G., L. M. E.] and Surgical Oncology [L. M. E.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Vascular endothelial growth factor (VEGF) is a potent angiogenic factor important for colon cancer neovascularization. In previous studies, serum starvation led to induction of VEGF in human colon carcinoma cells. We investigated the possible participation of mitogen-activated protein kinases in serum starvation induction of VEGF in the HT29 human colon carcinoma cell line. The extracellular signal-regulated kinases (Erks) 1 and 2 were activated after 3–6 h of serum starvation. Using transient transfection of VEGF promoter-reporter constructs, serum starvation led to an increase in VEGF promoter activity. An inhibitor of phosphorylation of Erk-1/2 blocked the increase of VEGF expression and promoter activity induced by serum starvation. Serum starvation activates several mitogen-activated protein kinases, but activation of Erk-1/2 is critical for the up-regulation of VEGF mRNA in colon carcinoma cells.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Angiogenesis is an active process that is dependent upon the balance of positive and negative effector molecules. VEGF3 is an endothelial cell-specific mitogen that is the one most closely associated with inducing and maintaining the neovasculature in human colon cancer (1) . VEGF expression has been reported to increase markedly under stress conditions. Hypoxia causes a rapid increase in VEGF expression in numerous cell lines in vitro. In human tumors, tumor cells adjacent to necrotic areas representing hypoxic regions also demonstrate an increase in VEGF expression (2) . The signal transduction pathway responsible for hypoxia-induced VEGF expression has been well characterized and leads to activation of Akt and ultimately downstream activation of hypoxia-inducible factor-1 (HIF)-1 (3) . However, various other stresses and stimuli lead to VEGF induction, including cell density (4) and serum starvation (5) . The signal transduction pathway(s) responsible for induction of VEGF due to these stress conditions is less well defined. Although hypoxia is one consequence of a poorly vascularized tumor, another consequence of poor tumor perfusion is growth factor deprivation, an in vitro corollary to serum starvation. To begin to elucidate the signal transduction pathway(s) responsible for VEGF induction by serum starvation, we examined the roles of several MAPK family members as intermediates of growth and stress pathways. We further examined Akt, an intermediate of HIF-1 induction of VEGF mRNA (3) . Our data demonstrate that serum starvation results in the induction of VEGF mRNA in HT29 human colon carcinoma cells by activation of the mitogenic pathway mediated by Erks 1 and 2. These data suggest that common intracellular signal transduction pathways mediate multiple processes essential for tumor growth and survival. Furthermore, multiple signal transduction pathways lead to induction of VEGF, which may represent a survival mechanism for cancer cells.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Lines and Culture Conditions.
The human colon cancer cell line HT29 was obtained from the American Type Culture Collection (Manassas, VA). These cells were cultured and maintained in MEM supplemented with 10% fetal bovine serum, 2 units/ml penicillin/streptomycin, vitamins, 1 mM sodium pyruvate, 2 mM L-glutamine, and nonessential amino acids at 37°C in 5% CO2 and air. For induction of serum starvation, cells were grown to 100% confluence in MEM and 10% fetal bovine serum, and the medium was then changed to serum-free medium. Cells were grown to 100% confluence to avoid any variations in VEGF expression that might occur when cells are grown to a lesser confluence, as demonstrated previously (4) . To determine the effects of serum starvation on intracellular protein kinase levels and phosphorylation, cells grown under these serum-free conditions were harvested after various time periods, and cell lysates were obtained. Total and phosphorylated protein levels were determined by Western blot analyses as described below.

To examine whether inhibition of Erk-1/2 activity would inhibit VEGF mRNA expression, HT29 cells were grown in serum-free medium and treated with or without 50 µM PD98059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one], a specific inhibitor of MAPKK (MEK1) (New England Biolabs Inc., Beverly, MA), and an upstream effector of Erk-1/2 (6) . Levels of VEGF mRNA were then measured by Northern blotting as described below. To confirm that PD98059 could inhibit the phosphorylation of Erk-1/2 while simultaneously blocking VEGF induction, cells were treated with PD98059 1 h prior to being exposed to serum-free medium, and total and phosphorylated protein levels were determined.

Western Blot Hybridization.
Prior to lysis, cells were rinsed twice with ice-cold PBS and lysed with protein lysis buffer [20 mM sodium phosphate (pH 7.4), 150 mM sodium chloride, 1% Triton X-100, 5 mM EDTA, 5 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 1 µg/ml leupeptin, and 500 µM Na3VO4]. Protein was quantitated spectrophotometrically. Aliquots (100 µg) of the protein were subjected to electrophoresis on 8% polyacrylamide gels. The protein was then transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) by electrotransfer. Following blocking with 5% milk in 0.5% Tween 20 in PBS, the membrane was probed with the primary antibody [1:1000 dilution of rabbit polyclonal antiphosphospecific p44/42 MAPK (Erk-1/2) antibody, antiphosphospecific P38 MAPK antibody, antiphosphospecific Akt antibody, or antiphosphospecific JNK antibody (New England Biolabs Inc.)]. The membranes were then washed and treated with the secondary antibody labeled with horseradish peroxidase (antirabbit immunoglobulin from donkey at a 1:3000 dilution; Amersham Pharmacia Biotech, Arlington Heights, IL). Using a commercially available chemiluminescence kit (Amersham Pharmacia Biotech), protein bands were visualized. For assaying total protein levels, the membrane was washed with stripping solution [100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7)] for 30 min at 50°C and reprobed with rabbit polyclonal anti-p44/42, anti-p38, anti-Akt, or anti-JNK antibody (all at a 1:1000 dilution).

mRNA Extraction and Northern Blot Analysis.
Total RNA was extracted from cells using TRI-Reagent (Molecular Research Center, Inc., Cincinnati, OH). Northern blot hybridization was performed as previously described (4) . A human VEGF specific 204-bp cDNA probe was a gift of Dr. Brygida Berse (Harvard Medical School, Boston, MA), and a glyceraldehyde-3-phosphate dehydrogenase probe was purchased from the American Type Culture Collection. The VEGF probe identifies all alternatively spliced forms of VEGF mRNA transcripts. Probes were purified by agarose gel electrophoresis using a QIAEX gel extraction kit (Qiagen, Inc., Chatworth, CA). Each cDNA probe was radiolabeled with [{alpha}-32P]deoxyribonucleotide triphosphate by the random-priming technique using the Rediprime labeling system (Amersham Pharmacia Biotech). Aliquots (25 µg) of total RNA were subjected to electrophoresis in 1% denaturing formaldehyde-agarose gels. The RNA was transferred to a Hybond-N+ positively charged nylon membrane (Amersham Pharmacia Biotech) overnight by capillary elution and UV cross-linked at 120,000 µJ/cm2 by using an UV Stratalinker 1800 (Stratagene, La Jolla, CA). After prehybridization of blots for 3–4 h at 65°C in Rapid-hybridization buffer (Amersham Pharmacia Biotech), the membranes were hybridized overnight at 65°C with the cDNA probe for VEGF or glyceraldehyde-3-phosphate dehydrogenase. The probed nylon membranes were washed and exposed to radiographic film (Life Technologies, Inc., Grand Island, NY).

VEGF Promoter-Reporter Activity in Response to Serum Starvation.
The role of transcriptional regulation of VEGF by serum starvation was examined using transient transfection with a VEGF promoter (luciferase)-reporter construct (full-length VEGF promoter cDNA was kindly provided by J. Abraham, Scios Nova Inc., Mountain View, CA, and was subcloned into pGL3 using standard techniques; Ref. 5 ). The following plasmids were used: pGL3-VEGF (containing the human VEGF promoter linked to the firefly luciferase reporter gene; Promega, Madison, WI), pRLTK (an internal control plasmid containing the herpes simplex thymidine kinase promoter linked to a constitutively active Renilla luciferase reporter gene), and pGL3 (plasmid vector alone as a negative control). HT29 cells (0.5–1.0 x 106) were seeded in six-well plates, and the pRLTK and pGL3-VEGF constructs were cotransfected into cells using the Lipofectin method (Life Technologies, Inc.) or FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) as outlined by the manufacturers. pRLTK and pGL3 were cotransfected as an negative control. After cells were incubated in the transfection medium for 20 h, the medium was changed to standard medium, and cells were incubated for 24 h. Cells were then incubated under serum-free condition for various time periods. To determine whether MEK1 inhibition by PD98059 could inhibit the increase in VEGF promoter activity, cells were treated with PD98059 1 h prior to being exposed to serum-free medium as described above. Cells were harvested with passive lysis buffer (Dual-Luciferase reporter assay system; Promega), and luciferase activity was determined using a single sample luminometer, as outlined in the manufacturer’s protocol.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Expression of VEGF mRNA by Serum Starvation.
To determine the effects of serum starvation on VEGF expression, HT29 cells were grown to 100% confluence in MEM supplemented with 10% fetal bovine serum. The medium was then changed to serum-free medium, and the cells were harvested at intervals thereafter. To determine the effects of these conditions on VEGF expression, Northern blot analysis for VEGF mRNA expression was performed. As shown in Fig. 1Citation , no significant change in VEGF mRNA expression was observed during the first 24 h of serum-free conditions. However, VEGF mRNA increased abruptly more than 4-fold between 24 and 48 h of growth in serum-free medium. These results confirm that serum starvation leads to induction of VEGF.



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  		Fig. 1. Effect of serum starvation on VEGF mRNA expression in colon carcinoma cells. Cells were grown to 100% confluence for various time periods in serum-free medium. Northern blots were done for VEGF expression. VEGF expression increased more than 4-fold between 24 and 48 h of growth in serum-free medium. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
Effect of Serum Starvation on Expression Level and Phosphorylation of MAPKs.
To determine the signaling intermediates activated by serum starvation, cells grown under conditions identical to those for VEGF induction were exposed to serum-free conditions for various times. Cells were lysed for analyses of signaling intermediates. Phosphorylated and total Erk-1/2 (p44/42), P38, and JNK were examined. JNK was expressed at very low levels in HT29 cells, and JNK activity was not induced by serum starvation (data not shown). Results for other MAPKs are shown in Fig. 2ACitation . Relative expression levels of Erk-1/2 and P38 were unchanged after serum starvation. However, Erk-1/2 phosphorylation and P38 phosphorylation increased 9- and 2.5-fold, respectively, with serum starvation, indicative of activation of each of these MAPK family members. These results suggest that several stress pathways are activated by serum starvation.



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  		Fig. 2. Effect of serum starvation with or without the addition of PD98059 on Erk-1/2, P38, and Akt levels and phosphorylation in colon carcinoma cells. A, HT29 cells were grown to 100% confluence, and the medium was changed to serum-free medium. Cells were then harvested at the indicated time points, and Western blots were done for total and phosphorylated Erk-1/2, P38, and Akt levels in colon carcinoma cells. B, to determine whether PD98059 blocks phosphorylation of Erk-1/2, P38, and Akt, cells were treated under identical conditions except that PD98059 was added 1 h prior to serum starvation.

 
Effect of Serum Starvation on Expression Level and Phosphorylation of Akt.
Because hypoxia is known to increase expression of VEGF mRNA through activation of Akt, we examined the effect of serum starvation on this kinase. Relative expression levels of Akt (Fig. 2A)Citation were increased after 1 h of serum starvation, with return to baseline levels at times thereafter. Phosphorylated Akt demonstrated a 2–3-fold increase at 1 and 3 h of serum starvation.

Effect of Erk-1/2 Inhibition on Serum Starvation Induction of VEGF.
To examine the specific role of Erk-1/2 on VEGF induction, HT29 cells were pretreated with PD98059 and then subjected to serum-free growth conditions. PD98059 at the doses used did not affect cell viability. PD98059 [an inhibitor of MEK1 (which is an upstream effector of Erk-1/2)] inhibited the phosphorylation of Erk-1/2 by more than 50% (Fig. 2)Citation . Importantly, P38 phosphorylation and Akt phosphorylation were unaffected by PD98059 (Fig. 2)Citation .

Growth under serum-free conditions in the presence or absence of PD98059 inhibited the induction of VEGF mRNA by serum starvation (Fig. 3)Citation . These results suggest that whereas serum starvation induces multiple MAPKs (Erk-1/2 and P38) and Akt, only activation of Erk-1/2 is required for serum starvation induction of VEGF. These results are consistent with PD98059s inhibiting MAPKK, as reported by others (7) . VEGF induction was examined after PD98059 addition and serum-free incubation; no increase in VEGF expression was observed under these conditions (data not shown).



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  		Fig. 3. Effect of serum starvation with or without the addition of PD98059 on VEGF mRNA levels in colon carcinoma cells. HT29 cells were grown to 100% confluence in 10% MEM, and the medium was changed to serum-free medium. Cells were exposed to PD98059 at the time of serum starvation. RNA was extracted, and Northern blots for VEGF were performed at 48 h. Ribosomal 18 S bands were used as internal control. PD98059 blocked induction of VEGF mRNA by serum starvation.

 
Effect of Serum Starvation on VEGF Promoter Activity.
To more completely examine the kinetics and mechanism for serum starvation induction of VEGF, transient transfections were performed with promoter-reporter constructs in HT29 cells using the full-length 5' promoter region of the VEGF promoter. Cells transfected with pGL3-VEGF (promoter-reporter construct) and pRLTK (internal control) demonstrated a increase in VEGF promoter activity secondary to serum starvation. This increase in VEGF promoter activity began at approximately 3 h and increased over time. Cells undergoing transfection of the VEGF promoter-reporter construct and pretreated with PD98059 demonstrated no increase in VEGF promoter activity secondary to serum starvation (Fig. 4)Citation .



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  		Fig. 4. Effect of serum starvation with or without the addition of PD98059 on the VEGF promoter activity in colon carcinoma cells. HT29 cells were cotransfected with pGL3-VEGF (VEGF promoter-luciferase reporter construct) and pRLTK (control for transfection efficiency). Transfection with pGL3 and pRLTK was performed as a negative control. Twenty-four h after transient transfection, medium was changed to serum-free medium. For the MEK inhibitor study, PD98059 was added to the medium 1 h prior to serum starvation. Cells were harvested at the time points indicated, protein was extracted, and luciferase activity was determined. All studies were done a minimum of three times, and results are standardized to reporter activity at t = 0 (defined as 1.0). Results are expressed as mean fold increase ± SE. VEGF promoter activity was increased in a time-dependent manner by serum starvation. PD98059 completely blocked the increase in VEGF promoter activity by serum starvation.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
VEGF, one of the most potent angiogenic growth factors, plays major roles in development and homeostasis (8) . In animal models and human tumor cells, aberrant expression of VEGF is common, resulting directly from many genetic alterations, including activation of proto-oncogenes such as Ras and Src (9) and inactivation of tumor suppressors such as P53 (10) . Multiple environmental signals regulate the expression of VEGF. Polypeptide growth factors induce VEGF, and this is likely to be mediated by the Ras/Raf pathway (11) . Hypoxia induces VEGF through activation of a pathway involving phosphatidylinositol 3-kinase, Akt, and HIF-1 (3) . Because the VEGF promoter predicts the binding of multiple positive and negative regulators of transcription, several other signaling cascades may be important in the regulation of VEGF expression (12) . With the recent interest in targeting VEGF expression for antineoplastic therapies (13) , an understanding of the stimuli that lead to VEGF expression and the signaling pathways used by these stimuli is important in determining the efficacy of inhibitors of the pathways.

In the present study, we examined the effects of serum starvation, one of the many consequences of vascular insufficiency, on VEGF expression. Previous work has demonstrated that serum starvation induces increased VEGF expression (5) . For this study, we used the well-characterized colon adenocarcinoma cell line HT29 to assess the signaling intermediates that might be responsible for increased VEGF mRNA expression. Specifically, we focused on MAPKs, as general mediators of a variety of signal transduction pathways, and on Akt, for its known role in mediating VEGF expression. Of the MAPKs, Erk-1/2 and P38 were activated by serum starvation. These results are not surprising, given the complex reaction of cells to this stress. Serum starvation has been shown variously to activate apoptosis pathways through NF-{kappa}B (14) , to up-regulate IFN regulatory factor in cultured Swiss 3T3 cells (15) , and to up-regulate expression of cyclins D and E in rat fibroblasts (16) . Our study also demonstrated increased phosphorylation of Akt after serum starvation, consistent with activation of stress pathways mediated by this kinase.

However, when cells were cultured in the presence of the specific MAPKK inhibitor PD98059, the induction of VEGF mRNA under serum-free growth conditions was blocked. Phosphorylation of Erk-1/2 was also blocked, but phosphorylation of P38 and phosphorylation of Akt were unaffected. These data suggest that Erk-1/2 activation, but not that of P38 or Akt, is required for induction of VEGF mRNA by serum starvation. Erk-1/2 became phosphorylated at relatively early time points, but VEGF mRNA expression did not increase until 24 h after serum starvation. We therefore examined the effects of serum starvation on VEGF promoter activity to better characterize the kinetics of these observations. VEGF promoter activity increased at time points temporally following Erk-1/2 activation. The MEK inhibitor, PD98059, completely blocked the increase of the VEGF promoter activity by serum starvation (Fig. 4)Citation . These data support the hypothesis that serum starvation up-regulates VEGF expression by induction of transcription of the VEGF gene via Erk-1/2 activation.

Although Erk-1/2 are generally considered mediators of the mitogenic response, Shimizu et al. (17) , in a rat model of myocardial infarction, have shown that Erk-1/2 activity is increased more than 4-fold in the myocardium after coronary artery ligation. This in turn leads to an increase in AP-1 binding activities. In other studies (18) , VEGF mRNA was also induced in the myocardium after infarction. The VEGF promoter has four AP-1 binding sites (19) . It is known that Erk-1/2 can activate the AP-1 pathway and thus may induce VEGF expression (20) . Studies in rat fibroblasts have demonstrated that Raf activation of Erk-1/2 leads to induction of VEGF, directly demonstrating that the mitogenic pathway is but one of several pathways that mediate VEGF induction (12) . Whether or not serum starvation uses this pathway remains to be determined.

Several of the binding motifs for transcription factors implicated as being stimulated by Erk-1/2 activation are also present in the 5' flanking region of the VEGF gene, including Ets, p300, SP-1, and Myc. Thus, the regulation of VEGF mRNA by environmental stimuli is likely to be very complex and involve the coordinate induction of several transcription factors.

In summary, multiple environmental stimuli important to tumor growth and metastasis may regulate the expression of VEGF mRNA. Some effects may be additive (12) , whereas others may be mediated by common pathways. A better understanding of the signaling pathways activated in response to environmental stimuli may be important to therapeutic strategies that target angiogenesis of tumor cells.


   ACKNOWLEDGMENTS
 
We thank Melissa Burkett for editorial assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by the Elvira Krause Fund for Cancer Research (to Y. D. J.), the Gillson Longenbaugh Foundation (to G. E. G. and L. M. E.), and NIH R-01 Grant CA65527 (to G. E. G.). Back

2 To whom requests for reprints should be addressed, at Department of Surgical Oncology, Box 106, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-6926; Fax: (713) 792-4689; E-mail: lellis{at}mdanderson.org Back

3 The abbreviations used are: VEGF, vascular endothelial growth factor; Erk, extracellular signal-regulated kinase; JNK, c-Jun N-terminal protein kinase; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MEK, mitogen-activated protein/Erk kinase; MEM, minimal essential medium. Back

Received 7/ 9/99. Accepted 8/17/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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