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Vol. 16, Issue 9, 4153-4162, September 2005

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Involvement of PI3K and MAPK Signaling in bcl-2-induced Vascular Endothelial Growth Factor Expression in Melanoma Cells

Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, 00158 Rome, Italy

Submitted December 17, 2004; Accepted June 16, 2005
Monitoring Editor: Gerard Evan

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We have previously demonstrated that bcl-2 overexpression in tumor cells exposed to hypoxia increases the expression of vascular endothelial growth factor (VEGF) gene through the hypoxia-inducible factor-1 (HIF-1). In this article, we demonstrate that exposure of bcl-2 overexpressing melanoma cells to hypoxia induced phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2 proteins. On the contrary, no modulation of these pathways by bcl-2 was observed under normoxic conditions. When HIF-1 expression was reduced by RNA interference, AKT and ERK1/2 phosphorylation were still induced by bcl-2. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways reduced the induction of VEGF and HIF-1 in response to bcl-2 overexpression in hypoxia. No differences were observed between control and bcl-2-overexpressing cells in normoxia, in terms of VEGF protein secretion and in response to PI3K and MAPK inhibitors. We also demonstrated that RNA interference-mediated down-regulation of bcl-2 expression resulted in a decrease in the ERK1/2 phosphorylation and VEGF secretion only in bcl-2-overexpressing cell exposed to hypoxia but not in control cells. In conclusion, our results indicate, for the first time, that bcl-2 synergizes with hypoxia to promote expression of angiogenesis factors in melanoma cells through both PI3K- and MAPK-dependent pathways.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Angiogenesis is a complex and multilevel process of new capillary formation on the basis of already existing blood vessels. Physiologically, it is a very strictly regulated process, which results in a balance between stimulatory (angiogenic) and inhibitory (angiostatic) factors to control the correct development of blood vessels. Angiogenesis is a rate-limiting step in tumor growth and progression, and many tumors develop a severely hypoxic microenvironment and secrete angiogenesis-stimulating factors (Folkman et al., 1971; Talks et al., 2000). Vascular endothelial growth factor (VEGF), a potent and specific mitogen for vascular endothelial cells, is a critical mediator of angiogenesis. Its expression is induced in cancer cells as a result of hypoxia and multiple genetic alterations, including p53 and PTEN loss-of-function, RAS and SRC gain-of-function, and autocrine tyrosine kinase signaling pathways (Mukhopadhyay et al., 1995; Arbiser et al., 1997; Petit et al., 1997; Akagi et al., 1998; Ellis et al., 1998; Yen et al., 2000). Based on the analysis of melanoma and breast cancer cells recently reported by our group (Biroccio et al., 2000; Iervolino et al., 2002; Del Bufalo et al., 2003; Trisciuoglio et al., 2004), this list can now be extended to include the increased VEGF expression resulting from bcl-2 overexpression. Hypoxia- and growth factor-induced VEGF expression in tumor cells is regulated by the hypoxia inducible factor 1 (HIF-1) (Jiang et al., 1997; Mazure et al., 1997; Ravi et al., 2000; Zhong et al., 2000; Zundel et al., 2000; Laughner et al., 2001; Fukuda et al., 2002), a transcription factor that is composed of an hypoxia-inducible-HIF-1 subunit and a constitutive expressed HIF-1 subunit (Wang and Semenza, 1995; Semenza, 2002a). The unique feature of HIF-1 is the regulation of HIF-1 expression and activity based upon the cellular O₂ concentration. HIF-1 expression and activity also are regulated by major signal transduction pathways, including those involving phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) (Berra et al., 2000; Semenza, 2002b). Stimulation of cells with a variety of growth factors and cytokines induces HIF-1 target gene expression under nonhypoxic conditions through activation of a variety of signal transduction pathways, including PI3K and the downstream serine-threonine protein kinase AKT (protein kinase B) and FKBP/rapamycin-associated protein (mTOR). Receptor tyrosine kinase activity also leads to signaling via the ERK and p38 MAP kinase pathways. PI3K and AKT also have been implicated in the induction of HIF-1 expression by hypoxia (Berra et al., 2000; Semenza, 2002b).

We previously demonstrated that bcl-2 overexpression in human melanoma cells exposed to hypoxic conditions increases VEGF promoter activity and mRNA stability and enhances HIF-1 DNA binding activity (Iervolino et al., 2002). However, the steps that participate in VEGF expression mediated by HIF-1 activity in response to bcl-2 overexpression in human melanoma cells exposed to hypoxia are still unknown. We have demonstrated that the MAPK pathway is implicated in signal transduction by bcl-2 (Trisciuoglio et al., 2004). In particular, our studies showed that ERK1/2 is activated in bcl-2-overexpressing cancer cells under hypoxic conditions (Trisciuoglio et al., 2004). However, to which extent this route may participate in VEGF accumulation by bcl-2 in human melanoma cells is still unsolved. Because VEGF up-regulation via both PI3K and MAPK pathways has been demonstrated (Berra et al., 2000; Semenza, 2002b) and the ability of MAPK to regulate HIF-1 may be stimulus- and/or cell type specific (Rak et al., 2000), we investigated the role of PI3K and MAPK pathways on bcl-2 induced VEGF/HIF-1 up-regulation in melanoma cells exposed to hypoxia. In this article, we provide evidence that the activity of both signal transduction pathways is required for the HIF-1-mediated induction of VEGF expression by bcl-2.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cell Cultures, Hypoxic Exposure, and Inhibitor Treatments
M14 human melanoma parental cells, MN8 control and bcl-2-overexpressing (MB5 and MB6) clones, previously obtained after transfection (Iervolino et al., 2002), were used for all the experiments. The cells were cultured in a humidified atmosphere with 95% air/5% CO₂ (normoxia) or in specially designed aluminium chambers flushed with a gas mixture containing 5% CO₂ and 95% N₂ (hypoxia). Under hypoxic conditions (24 h), oxygen concentration remains at 1% throughout the incubation period. In some experiments, UO126 (5 and 10 µM; Promega, Madison, WI) and PD98059 (25 and 50 µM; Promega), which are selective pharmacological inhibitors of ERK, and wortmannin (Wort) (35 and 50 nM; Calbiochem, San Diego, CA) and LY29402 (20 and 40 µM; Calbiochem), which are selective pharmacological inhibitors of PI3K, were added before exposure to normoxia or hypoxia. At the end of treatment, the cells were harvested, counted, and used for protein preparation and/or total RNA extraction.

RNA Interference (RNAi) Experiments
For silencing of bcl-2, cells were transfected with different amount (2–6 µg) of the RNAi expression vector against bcl-2 (IMG-819; Imgenex, San Diego, CA) or with negative control vectors (IMG-700; Imgenex) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Twenty-four hours after transfection, the cells were exposed in serum-free medium under hypoxic conditions for 24 h.

For silencing of HIF-1, two oligonucleotides consisting of ribonucleosides except for the presence of 2'-deoxyribonucleosides (dTdT) at the 3' end, 5'-AGAGGUGGAUAUGUGUGGGdTdT-3' and 5'-CCCACACAUAUCCACCUCUdTdT-3', were synthesized and annealed (Dharmacon Research, Lafayette, CO). The cells were exposed to 100 nM small interference RNA (siRNA) in the presence of Lipofectamine 2000 (Invitrogen) for 48 h and then exposed in serum-free medium under hypoxic conditions for 24 h. Control experiments were performed using siRNA directed against unrelated mRNA.

Analysis of Apoptosis
To measure the percentage of apoptotic cells, 1 x 10⁶ adherent cells were stained with fluorescein isothiocyanate (FITC)-conjugated annexin V using the Vybrant apoptosis kit (Molecular Probes, Eugene, OR) according to manufacturer's instructions and analyzed by flow cytometry, while simultaneously assessing membrane integrity by propidium iodide exclusion.

VEGF and Interleukin (IL)-8 Analysis
Enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) was used to determine the amount of VEGF and IL-8 proteins in the conditioned medium. Northern blot analysis was performed using a 600-base pairs fragment of the plasmid specific for VEGF and the probe for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (Biroccio et al., 2000). Densitometric analysis was performed after Northern blot analysis.

Protein Extracts
Total protein extracts of cell cultures were prepared as described previously (Iervolino et al., 2002). Cytosolic extracts were harvested by lysing cells with buffer containing 10 mM HEPES, pH 7.5, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.7% Nonidet P-40, and inhibitors proteases and phosphatases for 10 min at 4°C and centrifuged at 10,000 rpm for 15 min, and then supernatants (cytosolic extract) were harvested. Pellets were further lysed with buffer containing 20 mM HEPES, pH 7.5, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, and supernatants (nuclear extract) were harvested after centrifugation at 12,000 rpm at 4°C for 10 min.

Western Blot Analysis
Nuclear, cytosolic, and total extracts (60 µg) were fractionated by SDS-PAGE, transferred to a nitrocellulose filter, and subjected to immunoblot assays. For analysis of HIF-1 and HIF-1, nuclear proteins were analyzed using monoclonal antibodies against HIF-1 (H167; Novus Biologicals, Littleton, CO) or HIF-1 (Transduction Laboratories, San Diego, CA) at 1:500 dilution. Antibodies (Cell Signaling Technology, Beverly, MA) specific for phosphorylated (Thr202/Tyr204) or total ERK1/2, phosphorylated (Ser473) or total AKT were used at 1:1000 dilution. Immunoreactive bands were visualized using horseradish peroxidase-coupled goat anti-rabbit immunoglobulin and the ECL detection system (Amersham Biosciences, Piscataway, NJ). To check the amount of proteins transferred to the nitrocellulose membranes, -actin was used as control and detected by anti--actin polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution. Densitometric analysis was performed after Western blot analysis.

Promoter Activity
Promoter activity was evaluated using the VEGF 1511 fragment of the VEGF promoter construct, and the HIF-1-like wild-type (wt; 808-base pair) binding site polymers as reported previously (Iervolino et al., 2002). Twenty-four hours after transfection with plasmids legated to luciferase, half of the cells were subjected to normoxia and the other half was kept under hypoxia in the presence or absence of UO126, PD98059, Wort, or LY29402. Samples were collected 24 h after the induction of hypoxia and analyzed for luciferase and -galactosidase activity. Relative luciferase expression was determined as ratio of -galactosidase activity. The mean of five independent experiments was calculated for each condition. To normalize for transfection efficiency, PEQ-176 plasmid (0.2 µg) was included in the transfections.

Electrophoretic Mobility Shift Assay (EMSA)
EMSA was performed, as described previously (Iervolino et al., 2002), after exposure to hypoxia for 24 h in the presence or absence of UO126, PD98059, Wort, or LY29402. The following double-strand oligomer as radiolabeled probes or cold competitor: HIF-1 (-985 to –951 of human VEGF 5' gene promoter), 5'TCGACCACAGTGCATACGTGGGCTCCAACAGGTCCTCTTC-3'.

Statistical Analysis
The statistical significance of the differences between M14 cells, MN8 control clone, and bcl-2 transfectants was determined by the Student's t test for unpaired data (two sided).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Effect of bcl-2 Overexpression on AKT and ERK1/2 Phosphorylation and Cellular Distribution
We previously demonstrated that ERK kinase phosphorylation is induced in bcl-2-overexpressing melanoma clones exposed to hypoxic conditions for 24 h (Trisciuoglio et al., 2004). Because phosphorylation of cytosolic ERK1/2 leads to their activation and translocation within the nucleus (Brunet et al., 1999; Volmat et al., 2001), we first confirmed ERK activation by Western blot. Using an anti-phospho-ERK antibody that recognizes the active forms of ERK1/2, an increase in both ERK1 and ERK2 phosphorylation, of about two- to threefold, was observed in total extracts from MB5- and MB6 bcl-2-overexpressing cells exposed to hypoxia for 24 h compared with the M14 parental line and MN8 control clone exposed at the same experimental conditions (Figure 1A). As evidenced in Figure 1B, bcl-2 transfectants showed an increased amount (about fivefold) of nuclear phosphorylated ERK1/2 compared with control cells: whereas ERK1/2 were slightly detectable in nuclei of control cells, they were very evident in nuclei of bcl-2-overexpressing clones. The difference between control and bcl-2-overexpressing cells, even though to a lesser extent (about a threefold increase), was also evident when comparing the amount of cytoplasmatic phosphorylated ERK1/2. These results were confirmed by immunofluorescence experiments that showed an elevated phospho-ERK nuclear translocation in bcl-2 transfectants compared with the control cells (our unpublished data).

View larger version (31K): [in this window] [in a new window]   Figure 1. Effect of bcl-2 overexpression on AKT and ERK1/2 phosphorylation and cellular distribution under normoxic and hypoxic conditions. Western Blot analysis of bcl-2 and ERK1/2 expression and phosphorylation in total extracts (A), ERK1/2 expression and phosphorylation in nuclear and cytoplasmatic protein extracts (B), and AKT expression and phosphorylation in total extracts (C). Western Blot analyses were performed 24 h after exposure to hypoxia. (D) ERK1/2 and AKT expression and phosphorylation in total extracts of cells exposed to normoxia for 24 h. -actin expression served as controls for uniformity of protein gel loading and blotting. Representative experiment out of two is shown.

Because PI3K and AKT have also been implicated in the induction of angiogenesis in response to hypoxia (Richard et al., 1999; Berra et al., 2000, Semenza, 2002b; Arsham et al., 2004), we evaluated AKT phosphorylation in parental cells and bcl-2 transfectants exposed to hypoxia for 24 h. Using an anti-phospho-AKT (Ser-473) antibody that recognizes the active form of AKT (Figure 1C), an increase in AKT phosphorylation of about five- to sixfold was observed in bcl-2 transfectants compared with the parental line or control clone. Using a polyclonal antibody raised against total AKT, no modification in the level of AKT kinase expression was detected, indicating that the increase in AKT phosphorylation was not due to an increase in total AKT protein. ERK1/2 and AKT phosphorylation under hypoxic conditions also were observed after transient transfection of M14 cells with a bcl-2 expression vector (our unpublished data). Thus, indicating that the effects seen were a direct consequence of bcl-2 overexpression and did not represent a chronic adaptation to higher levels of bcl-2.

Experiments also were performed to evaluate the effect of bcl-2 overexpression on ERK1/2 and AKT phosphorylation under normoxic conditions. As reported in Figure 1D, a similar ERK1/2 and AKT phosphorylation was observed between the parental, control, and bcl-2-overexpressing cells.

Effect of PI3K and MAPK Inhibitors on the VEGF Protein, mRNA, and Promoter Activity
To determine the signal transduction pathways mediating the effects of bcl-2 on VEGF protein expression, M14 parental cells, MN8 control clone and two bcl-2 transfectants (MB5 and MB6) were pretreated with PI3K (Wort and LY29402) and ERK (UO126 and PD98059) inhibitors, and the level of VEGF protein secretion was determined by ELISA assay after exposure to normoxic or hypoxic conditions for 24 h.

As demonstrated previously (Iervolino et al., 2002) and reported in Figure 2A, no differences where observed between control cells and bcl-2 transfectants, exposed to normoxic conditions, in terms of VEGF protein secretion: the VEGF protein ranged from 1656 ± 284 to 1880 ± 335 pg/10⁶ cells/24 h for the different lines (p > 0.05). VEGF secretion was decreased by PI3K (50%) and ERK1/2 (70%) inhibitors to the same extent in both parental and bcl-2-overexpressing cells when exposed to normoxia.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of PI3K and ERK inhibitors on the VEGF protein expression. Expression of VEGF was evaluated by ELISA in conditioned medium of M14 parental line (white columns), MN8 control clone (black columns), and bcl-2 transfectants (dark and light gray columns) grown under normoxic (A) or hypoxic (B) conditions for 24 h in absence or in presence of wortmannin (50 nM) or UO126 (10 µM). Two representative experiments out of six with standard deviations are reported. * Indicates that values are significantly different from the corresponding value of control (*p < 0.05, **p < 0.001).

We further investigated the effect of PI3K and MAPK inhibitors on VEGF mRNA-induction by bcl-2/hypoxia in MB5 and MB6 bcl-2-overexpressing clones (Figure 3A). As expected (Iervolino et al., 2002), 24-h hypoxia induced about a three- to fourfold increase in mRNA expression in MB5 and MB6 clones compared with normoxic conditions. The two inhibitors reduced the induction of VEGF mRNA expression by bcl-2 in both bcl-2 transfectants. In particular, under these conditions the accumulation of VEGF mRNA, after GAPDH normalization, was reduced by 30–40% in Wort-treated cells and by 50–70% in UO126-treated cells (Figure 3A).

View larger version (32K): [in this window] [in a new window]   Figure 3. Effect of PI3K and ERK inhibitors on VEGF mRNA expression and promoter activity. VEGF mRNA expression by Northern blot analysis (A) and VEGF transcriptional activity (B) were performed in M14 parental line (white columns), MN8 control clone (black columns), and two bcl-2 transfectants (dark and light gray columns) grown under normoxic or hypoxic conditions for 24 h in the absence or presence of Wort or UO126. (A) Fold increase relative to normoxia was calculated by densitometric analysis. A representative experiment out of two is shown. (B) Cells were transfected with a reporter plasmid (VEGF 1511 base pairs fragment of the VEGF promoter) and exposed to hypoxia before assaying for luciferase activity. Luciferase values were normalized for transfection efficiency (luciferase/galactosidase ratios). A representative experiment out of six with standard deviations is reported. * Indicates that values are significantly different from the corresponding value of control (*p < 0.05, **p < 0.001).

Effect of bcl-2 RNAi on bcl-2 Expression, Apoptosis, and ERK1/2 Phosphorylation
To confirm the results obtained through up-regulation of bcl-2, we specifically silenced the bcl-2 gene. For this purpose, the MN8 control clone and bcl-2-overexpressing cells were transfected with RNAi-targeting bcl-2 mRNA (bcl-2 RNAi) and then exposed to hypoxia for 24 h. As a control for specificity of RNAi, cells were transfected with a scrambled RNAi vector. Western blot analysis demonstrated that bcl-2 RNAi severely suppressed expression of bcl-2 in a dose-dependent manner compared with untransfected or control transfected cells (Figure 4A). A decrease in bcl-2 expression had already been observed after transfection with the lowest concentration (2 µg) of bcl-2 RNAi both in parental (about a twofold decrease) and bcl-2-overexpressing (about a sixfold decrease) cells, whereas the bcl-2 protein was not detectable after transfection with the highest concentration (6 µg). No effects of RNAi were observed on the expression of -actin, which was used as an internal control for specificity and loading. Cytofluorimetric analysis for detection of apoptosis revealed the presence of apoptotic cells in the control cells (30%) and bcl-2 overexpressing clones (25%) only when transfected with the highest concentration (6 µg) of bcl-2 RNAi (Figure 4B). Then, we evaluated the impact of reduced bcl-2 expression on ERK1/2 phosphorylation (Figure 4C). Bcl-2 RNAi induced a dose-dependent decrease in ERK1/2 phosphorylation in bcl-2 overexpressing cells compared with untreated cells: about a 10-fold decrease in ERK1/2 phosphorylation was already evident after transfection with the lowest concentration (2 µg) of bcl-2 RNAi. On the contrary, no modulation of ERK1/2 phosphorylation was evidenced in the MN8 control clone after down-regulation of the bcl-2 protein (Figure 4C).

View larger version (46K): [in this window] [in a new window]   Figure 4. Effect of bcl-2 RNAi on the bcl-2 expression, induction of apoptosis, ERK1/2 phosphorylation, and VEGF protein levels. (A) Western blot analysis of bcl-2 protein in MN8 control clone and MB5 bcl-2-overexpressing clone transfected with different amount (2–6 µg) of the RNAi expression vector against bcl-2 (bcl-2 RNAi) or with negative control vector (6 µg) and then exposed for 24 h to hypoxia. -actin was used as a control for equal protein loading. A representative experiment out of two is shown. (B) Detection of apoptosis by annexin V/propidium iodide (PI) staining in MN8 control clone and bcl-2-overexpressing clones (MB5 and MB6) transfected with bcl-2 RNAi or with negative control vector and then exposed for 24 h to hypoxia. The percentage of apoptotic cells (annexin V+/PI-) is reported. (C) Western Blot analysis of ERK1/2 expression and phosphorylation in total extracts of MN8 control cells and MB5 bcl-2-overexpressing clone transfected with the bcl-2 RNAi expression vector or with negative control vector and then exposed for 24 h to hypoxia. (D) Expression of VEGF protein evaluated by ELISA in conditioned medium of M14 parental line (white columns), MN8 control clone (black columns), and bcl-2-overexpressing cells (dark and light gray columns) transfected with the bcl-2 RNAi or with negative control vector and then grown under hypoxic conditions for 24 h. * Indicates that values are significantly different from the corresponding value of control (*p < 0.01).

Effect of PI3K and MAPK Inhibitors on HIF-1 Protein Expression and DNA Binding Activity
We previously demonstrated that Bcl-2 overexpression in human melanoma cells increases HIF-1 expression and DNA binding activity (Iervolino et al., 2002). To examine the contribution of the MAPK and PI3K signaling pathways to bcl-2/hypoxia-induced HIF-1 protein expression, we exposed the cells to 24 h hypoxia after treatment with UO126 and Wort. As shown in Figure 5A, the levels of HIF-1 protein was increased by about threefold in bcl-2-overexpressing clones exposed to hypoxia compared with parental cells at the same experimental conditions. The expression of HIF-1 protein was not detected in bcl-2 transfectants pretreated with UO126 and was almost absent in cells pretreated with Wort (Figure 5A). No changes in HIF-1 were observed after bcl-2 overexpression and treatment with inhibitors.

View larger version (51K): [in this window] [in a new window]   Figure 5. Effect of bcl-2 overexpression on HIF-1 protein expression, DNA binding activity and transcriptional activity under hypoxic conditions. Western blot analysis of HIF-1 and HIF-1 protein expressions (A), EMSA (B) and HRE transcriptional activity (C) were performed in the nuclear extracts of M14 parental line and two bcl-2 transfectants (MB5, MB6) grown under hypoxic conditions for 24 h in the presence or absence of UO126 (5 µM) or Wort (35 nM). (B) EMSA was carried out using the double-strand oligomer as radiolabeled probes or cold competitor (cold). Supershift assay (SS) of HIF-1 DNA-binding complexes in MB6 cells was performed by addition of anti-HIF-1 antibody (antibody) to the reaction mixture. Representative experiment out of two is shown. (C) M14 parental line (white columns), MN8 control clone (black column), and two bcl-2 transfectants (dark and light gray columns) were transfected with a pHRE-Luc reporter plasmid. After 24 h, the cells were exposed to normoxia, hypoxia, or hypoxia in presence of UO126 or wortmannin. Luciferase values were normalized for transfection efficiency (luciferase/galactosidase ratios). Results are means of five independent experiments. * Indicates that values are significantly different from the corresponding value of control (*p < 0.05, **p < 0.01). (D) Analysis of HIF-1, HIF-1 protein expression and ERK1/2 and AKT expression and phosphorylation in total extracts obtained from MN8 control line and bcl-2 trasfectants (MB5, MB6), after transfection with small interference RNA directed against HIF-1 (siRNA) or with small interference RNA directed against unrelated control mRNA. -actin expression served as controls for uniformity of protein gel loading and blotting. Representative experiments out of two is shown.

Because we previously found the ability of bcl-2 to increase hypoxic responsive element (HRE) promoter activity in hypoxia (Iervolino et al., 2002), we evaluated the role of the PI3K and MAPK signaling pathways in the transcriptional activation of HIF-1 by bcl-2/hypoxia. Parental and bcl-2-overexpressing cells were transiently transfected with a pHRE-Luc reporter plasmid, which contains four concatamerized HIF-1 binding sites, and then exposed to normoxia or hypoxia for 24 h in the presence or absence of PI3K and MAPK inhibitors. As shown in Figure 5C, HRE promoter activity in bcl-2 transfectants under hypoxic conditions was about twofold higher than in M14 parental cells exposed to the same experimental conditions. Although a decrease of 25% in HRE promoter activity was observed in parental cells treated with UO126, no inhibition was revealed after treatment with Wort. On the contrary, the increase in HIF-1-dependent reporter gene transcription observed in bcl-2-overexpressing clones exposed to hypoxia was significantly reduced by cellular pretreatment with either Wort (30% reduction) or UO126 (70% reduction). Results obtained with PD98059 or LY29402 were similar to those obtained when using UO126 and Wort, respectively (our unpublished data). Results obtained with the MN8 control clone were superimposable to those obtained when using M14 (our unpublished data).

To evaluate whether MAPK and PI3K pathways were still induced in hypoxia by bcl-2 in the absence of HIF-1 activity, siRNA was used to reduce the expression of HIF-1 transcription factor. MN8 control and bcl-2-overexpressing cells were transfected with siRNA-targeting HIF-1 mRNA (HIF-1 siRNA) and then exposed to hypoxia for 24 h. As a control for specificity of siRNA, cells were transfected with siRNA directed against unrelated control mRNA. Western blot analysis demonstrated that HIF-1 siRNA severely suppressed expression of HIF-1 in both the MN8 control cells and bcl-2 transfectants compared with control transfected cells (Figure 5D). As expected, no effects of HIF-1 siRNA were observed on the expression of HIF-1, and VEGF expression was reduced both in parental and bcl-2 overexpressing cells (our unpublished data). Thus, we also evaluated the impact of reduced HIF-1 expression on ERK1/2 and AKT phosphorylation. No modulation of ERK1/2 and AKT phosphorylation was observed in parental or bcl-2-overexpressing cells after HIF-1 silencing.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Several studies have demonstrated that in addition to mediating antiapoptotic (Cory et al., 2003) and proliferative signals (Quinn and Richardson, 2004), bcl-2 also promotes tumor angiogenesis (Iervolino et al., 2002; Trisciuoglio et al., 2004). Moreover, Bcl-2 regulates gene expression and modulates the transactivity of several transcription factors (Feng et al., 1999; Ricca et al., 2000; Schwarz et al., 2002; Cory et al., 2003; Quinn and Richardson, 2004).

Our previous results, demonstrating that forced overexpression of bcl-2 in melanoma cells exposed to hypoxia enhances HIF-1-mediated-VEGF expression (Iervolino et al., 2002) and induces ERK1/2 activation (Trisciuoglio et al., 2004), persuaded our group to investigate the participation of MAPK and PI3K signaling pathways in bcl-2-mediated VEGF/HIF-1 expression and activity. Even though the role of MAPK and PI3K pathways in the process of HIF-1/VEGF activation is still controversial (Alvarez-Tejado et al., 2002; Stiehl et al., 2002), several studies evidenced that these two specific intracellular signaling pathways are actively involved in angiogenesis mediated by VEGF (Berra et al., 2000; Zhong et al., 2000; Semenza, 2002; Sang et al., 2003). Recognizing which signal transduction pathway is active in a tumor-overexpressing bcl-2 may have prognostic information and also may identify a potential target for antiangiogenic therapy.

Our results demonstrated that the effect of bcl-2 overexpression on VEGF expression, ERK and AKT phosphorylation, and on the response to PI3K and MAPK inhibitors was strictly dependent on the oxygen concentration. In fact, all of these parameters were similar in the control and bcl-2-overexpressing cells exposed to normoxic conditions, and as reported for other tumor histotypes (Chelouche-Lev et al., 2004), both PI3K and MAPK pathways were essential for the constitutive expression of VEGF in normoxia. On the contrary, under hypoxic conditions increased VEGF secretion, AKT and ERK phosphorylation were evidenced in both transiently and stably bcl-2-overexpressing cells. A strong involvement of PI3K and MAPK in the induction of HIF-1-mediated VEGF expression by bcl-2 also was evidenced. Moreover, when HIF-1 expression was reduced by siRNA, AKT and ERK1/2 phosphorylation were still induced by bcl-2, indicating that the role of bcl-2 as an enhancer in PI3K and MAPK pathways under hypoxic conditions is not related to HIF-1 induction.

The cooperation of bcl-2 overexpression with hypoxia on VEGF and HIF-1 expression confirmed in the present study, corroborates the importance of two parallel pathways for induction of HIF-1 in human cancer, one based on physiological stimulation and the other on genetic alterations.

Confirming the involvement of the MAPK pathway in bcl-2 overexpression in hypoxia (Trisciuoglio et al., 2004), the present study shows that bcl-2 and hypoxia cooperate to promote ERK phosphorylation and translocation to the nucleus. The evidence that the phosphorylation of ERK1/2 regulatory sites can drive their translocation to the nucleus where ERK exerts part of its biological activity (Brunet et al., 1999) and that ERK1/2 phosphorylate HIF-1 and enhance HIF-1 transcriptional activity (Feng et al., 2004) indicates a possible mechanism by which bcl-2 increases HIF-1-mediated VEGF induction in our experimental models. Furthermore, the previously demonstrated effect of MAPK signaling on HIF activation through p300/CBP can be postulated as a possible mechanism by which bcl-2/hypoxia induces VEGF expression (Sang et al., 2003). Moreover, the fact that RNAi-mediated down-regulation of bcl-2 expression resulted in a decrease in the ERK1/2 phosphorylation and VEGF secretion in bcl-2 transfectants exposed to hypoxia, but not in the control cells under the same experimental conditions, confirms the importance of bcl-2 on angiogenesis in conditions of low oxygen concentration. From a clinical perspective, these findings are relevant only for melanoma expressing high bcl-2 protein levels.

As an additional parameter to confirm the involvement of PI3K and MAPK on VEGF/HIF-1 expression, we have shown that the inhibition of MAPK and PI3K pathways successfully reduced bcl-2 induction of these molecules under hypoxic conditions. In particular, using selective pharmacologic inhibitors of PI3K (Wort or LY29402) and MAPK (UO126 or PD98059), we have found that the induction of VEGF and HIF-1 in bcl-2-overexpressing cells is dependent upon activity of both the MAPK and PI3K pathways. In particular, VEGF protein and mRNA expression and VEGF promoter activity induced after bcl-2 transfection in hypoxia were reduced in a dose-dependent manner by inhibition of either MAPK or PI3K pathways. The induction of the HIF-1 protein, DNA binding, and transcriptional activity also were partially or completely blocked by PI3K and MAPK pathways. These data are in agreement with results demonstrating PI3K and MAPK signaling-mediated HIF-1 and VEGF induction in several experimental models and in response to different stimuli (Fukuda et al., 2002; Semenza, 2002b).

Together, our results point to the important role of the MAPK and PI3K signaling pathways in the modulation of the bcl-2 effect on VEGF/HIF-1 expression and indicate that at least two different signal transduction pathways are activated by bcl-2 overexpression in melanoma cells exposed to hypoxic conditions. The inhibitory effect of UO126 was more evident than the inhibitory effect of Wort for all the experiments performed and at the concentrations used. Although the inhibition of MAPK pathway seems to affect VEGF/HIF-1 expression more than the inhibition of PI3K, it should be considered that the effect of PI3k inhibition is more specific for the bcl-2-overexpressing clones than for parental cells. Even though it is difficult to ascertain which pathway plays a more important role in our model system, the involvement of PI3K and MAPK in the induction of HIF-1-mediated VEGF expression by bcl-2 in hypoxia, represent two important signaling pathways. These data are in agreement with those demonstrating the importance of both the PI3K and MAPK pathways on HIF-1 and VEGF expression (Jiang et al., 2000; Sang et al., 2003).

The signal transduction pathway involving PI3K, AKT, and FRAP has been shown to regulate the translation of several proteins, including VEGF (Graff and Zimmer, 2003), via phosphorylation of eukaryotic initiation factor 4E-binding protein 1 and p70 ribosomal protein S6 kinase (Gingras et al., 1999; Peterson et al., 1999; Fukuda et al., 2002), and ERK also has been shown to phosphorylate the eukaryotic initiation factor 4E-binding protein 1 (Haystead et al., 1994; Fukuda et al., 2002). Thus, it is possible that PI3K and MAPK pathways are responsible for the increased VEGF and HIF-1 protein expression induced by bcl-2/hypoxia in our experimental model through this mechanism.

Regarding the possible mechanism by which bcl-2 modulates transcription factors and gene expression in our experimental model, it is possible that bcl-2 activates ERK signaling through the RAS/RAF/MEK pathway. Recently, upstream activators of ERK1/2, such as RAS and RAF, have been demonstrated to play a role in bcl-2-induced gene expression in nontumoral cells such as rabbit lens epithelial cells (Feng et al., 2004), and bcl-2 overexpression in PC12 pheochromocytoma cells has been found to affect cellular signaling pathway via an up-regulation of Ras (Schwarz et al., 2002).

Because we previously demonstrated the ability of bcl-2 to modulate Sp1 DNA binding activity through the ERK signaling pathway, we do not exclude a possible involvement of Sp1 in VEGF modulation by bcl-2. In fact, data from other groups evidenced that Sp1 is required for PI3K-mediated induction of VEGF (Pore et al., 2004) and that phosphorylation of Sp1 by ERK1/2 MAPK is a crucial event for the regulation of VEGF (Berra et al., 2000, Milanini-Mongiat et al., 2002). We are currently investigating the involvement of Sp1 and the RAS/RAF pathway on bcl-2-induced HIF-1/VEGF expression.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
D. T. and A. I. are the recipient of a fellowship from the Italian Foundation for Cancer Research. We are grateful to Dr. Adele Petricca for secretarial assistance in preparation of the manuscript, to Paula Franke for revising the English language, and to Drs. Maurizio Fanciulli and Oreste Segatto for critical reading of the manuscript. This study was supported by Ministero della Sanità (D.D.B.) and Italian Association for Cancer Research (D.D.B.).

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E04-12-1087) on June 29, 2005.

Abbreviations used: ERK1/2, extracellular signal-regulated kinase 1/2; HIF-1, hypoxia-inducible factor-1; HRE, hypoxic responsive element; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; VEGF, vascular endothelial growth factor; Wort, wortmannin.

^* These authors contributed equally to this work.

Address correspondence to: Donatella Del Bufalo (delbufalo{at}ifo.it).

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