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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2002 as doi:10.1096/fj.02-0122fje. |
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Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, Rome, Italy; and
* Human Anatomy and Histology Department, University of Bari Medical School, Italy
3Correspondence: Experimental Chemotherapy Laboratory, Regina Elena Cancer Institute, Via delle Messi d’Oro 156, 00158 Rome, Italy. E-mail: delbufalo{at}ifo.it
SPECIFIC AIM
Bcl-2 overexpression in breast and prostate carcinoma increases hypoxia-induced VEGF expression and angiogenesis. In this study we evaluated the role of bcl-2 in melanoma angiogenesis and the mechanisms involved in bcl-2-induced VEGF expression. Thus, the M14 human melanoma cell line has been transfected with a bcl-2 expression vector and the effect of bcl-2 overexpression on in vitro and in vivo angiogenesis has been investigated.
PRINCIPAL FINDINGS
1. Bcl-2 overexpression increases hypoxia-induced VEGF production and angiogenesis
Bcl-2 transfectants (MB5 and MB6) grown in hypoxia for 16 or 24 h showed an increase in VEGF protein and mRNA expression of 
threefold compared with the parental cell line (M14) and the control clone (MN8). No difference in VEGF expression at mRNA and protein level was observed when bcl-2 transfectants cultured in normoxic conditions were compared with the control lines.
To evaluate the functional activity of secreted VEGF, we examined EA.hy926 vessel morphogenesis on Matrigel, proliferation, and chick chorioallantoic membrane (CAM) vascularization in the presence of conditioned media (CM) obtained from M14, control clone, and bcl-2 transfectants grown for 24 h under hypoxic conditions. When EA.hy926 cells were exposed to CM of two bcl-2 transfectants, the cells became elongated, forming thin cords of interconnecting cells whereas endothelial cells showed only a reduced spread when exposed to CM of the control cells. Similarly, CM from two bcl-2 transfectants increased cell proliferation by fourfold compared with CM from the control cells. Macroscopic and microscopic observation of CAM showed a greater number of blood vessels and intersection points and a higher microvessel density after exposure to bcl-2 transfectants CM compared with M14 CM or vehicle alone. The use of VEGF specific antibodies validated the role of VEGF on bcl-2-induced in vitro and in vivo angiogenesis.
2. Bcl-2 overexpression increases VEGF mRNA stabilization and promoter activity in hypoxic conditions
Because VEGF activity can be modulated by mRNA stabilization and transcriptional regulation, to gain information about the mechanism by which bcl-2 synergizes with hypoxia to induce VEGF expression, we checked both these control points (Fig. 1
). Analysis of the Northern blot shows that when the M14 cells were grown under hypoxia, VEGF mRNA had a half-life of 180 min, whereas MB6 bcl-2 transfectant, grown at the same experimental conditions showed an extended half-life of 330 min.
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To determine whether trans-activation of the VEGF promoter occurred differently in control cells and bcl-2 transfectants, a series of promoters coupled to the luciferase reporter gene was used. The 1511 bp human VEGF promoter-reporter gene that includes 1175 bp from the start site of transcriptional and 336 bp of the 5' untranslated region of VEGF promoter was used. VEGF promoter activity was similar in all cells tested in normoxic conditions regardless the level of bcl-2 protein expression. Exposure to 24 h hypoxia induced two- to threefold increase in promoter activity in control cells whereas sevenfold enhancement was observed in bcl-2 overexpressing cells (Fig. 1C
). The 385 bp fragment containing one HIF-1 element exhibited a 5-fold increase after hypoxic conditions in control cells and displayed 12-fold enhancement in bcl-2 transfectants. To further investigate VEGF promoter activity under hypoxic conditions, we used the wild-type (wt) HIF-1 binding-site polymer that contains four copies of HIF-1 element. This binding-site polymer exhibited an 8-fold increase in the activity when exposed to hypoxia in control cells, whereas the same promoter region displayed 20-fold increase in the activity of bcl-2 transfectants. The same promoter region containing five copies of a mutation in the putative HIF-1 binding site (mut HIF-1 binding-site polymer) displayed no increase in activity for any line exposed to low oxygen conditions.
3. HIF-1
protein expression and nuclear DNA binding activity is increased in bcl-2 transfectants exposed to hypoxia
Because VEGF promoter contains hypoxia-responsive elements that specifically bind HIF-1 transcription factor, we evaluated HIF-1 expression at the mRNA and protein levels and HIF-1 DNA binding activity (Fig. 2
). As reported in Fig. 2A
, similar levels of HIF-1 mRNA were found in control and bcl-2 overexpressing cells under normoxic or hypoxic conditions. At the protein level, no differences between control and bcl-2 overexpressing cells were observed under normoxic conditions. After 24 h of hypoxic conditions, an increase in the lower and the higher molecular weight forms of HIF-1 protein was observed in control cells and bcl-2 transfectants. When the levels of HIF-1 protein found under hypoxia were compared with normoxia, an increase of twofold was found for M14 cells and fourfold in bcl-2 transfectants. DNA binding activity of HIF-1 was analyzed by EMSA. As evident in Fig. 2B
, the two bcl-2 overexpressing clones show a significant increase of the HIF-1 DNA binding activity compared with the parental M14 cells. The difference was already detectable 16 h after hypoxia and was more evident when the exposure to hypoxia was prolonged to 24 h. Cold competition and supershift analysis confirmed the specificity of DNA binding complexes. Results obtained with the MN8 control clone were comparable to those of M14 cells.
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CONCLUSIONS AND SIGNIFICANCE
We first demonstrated the relevance of bcl-2 on melanoma angiogenesis evident from experiments on endothelial cell proliferation, morphogenesis, and CAM vascularization. The bcl-2-mediated angiogenesis correlated with increased VEGF protein and mRNA expression, and the specific role of VEGF was proved using a specific VEGF165 neutralizing antibody. We also found that the increase in VEGF protein and mRNA expression and angiogenesis observed in bcl-2 transfectants is not due to enhanced cell survival.
As reported in Fig. 3
, we have evidence that VEGF mRNA stabilization and HIF-1-mediated VEGF transcriptional activity are important control points in bcl-2/hypoxia-induced VEGF expression and angiogenesis. An increased half-life of the VEGF message of twofold was observed in bcl-2 transfectants compared with control cells. Transfection experiments with a reporter gene under the control of the VEGF promoter clearly demonstrated that bcl-2 overexpression also enhanced hypoxia-induced VEGF transcriptional activity. The increase in transcriptional activity was further enhanced when cells were transfected with reporter constructs carrying one or four copies of HIF-1 element. The demonstration that the same promoter region, containing five copies of a mutation in the putative HIF-1 binding site, displayed no increase in activity for all the lines exposed to low oxygen conditions confirmed the importance of HIF-1 sequence in the transcriptional activation of VEGF.
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In agreement with data demonstrating no significant induction of HIF-1 mRNA by hypoxia, we found that HIF-1 mRNA levels were not induced by hypoxia and were similar among control cells and bcl-2 transfectants. On the contrary, we demonstrated an enhancement in the protein expression in bcl-2 transfectants compared with control cells. This increase was paralleled by an enhancement in HIF-1 DNA binding activity. It is possible that the increase in HIF-1 protein is responsible for the enhancement in DNA binding activity of HIF-1. Other mechanisms can also be responsible in forming active heterodimers or interacting with comodulators of its activity.
To our knowledge, this is the first demonstration that VEGF mRNA stabilization and HIF-1-mediated VEGF transcriptional activity are important control points in bcl-2/hypoxia-induced VEGF expression, indicating that distinct molecular mechanisms are involved in this phenomenon (Fig. 3)
. Supporting our data, other oncogenes have demonstrated to induce up-regulation of VEGF through enhancement of mRNA stabilization and transcriptional activity.
Several hypothesis can be formulated regarding the possible mechanisms by which bcl-2 induces VEGF mRNA stabilization and HIF-1-mediated VEGF transcriptional activity. Because we previously demonstrated that bcl-2 overexpression inhibits mitochondrial metabolism and HIF-1 hydroxylase, which requires dioxygen, is inactive in hypoxia, it is possible that bcl-2 protein can directly or indirectly modify posttranslational hydroxylation of HIF-1. It is also possible that bcl-2 acts on the expression of sequence-specific RNA binding proteins responsible of VEGF mRNA stabilization. Bcl-2 should increase VEGF expression through the enhancement of the activity of transcriptional factors other than HIF-1. We earlier demonstrated that bcl-2 increase NF-
B transcriptional activity in a human breast carcinoma line. Since NF-B signaling blockade has been demonstrated to inhibit in vitro and in vivo expression of VEGF, it is possible that bcl-2 affects VEGF expression in hypoxic conditions through modulation of the activity of NF-B or other transcription factors.
In this study we provide a molecular characterization of the mechanism for the synergistic effect between bcl-2 and hypoxia on VEGF expression. The fact that VEGF levels correlate with disease progression in melanoma supports the idea that direct or indirect inhibition of this molecule may be useful for this neoplasia. Clarification of the molecular mechanisms through which bcl-2 regulates VEGF expression and consequently angiogenesis, is of great benefit because it might lead to the establishment of new targets for tumor therapy based.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0122fje; to cite this article, use FASEB J. (July 1, 2002) 10.1096/fj.02-0122fje 
2 These authors contributed equally to this work.
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