Microenvironment Changes (in pH) Affect VEGF Alternative Splicing
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Vascular endothelial growth factor-A (VEGF-A) has several isoforms, which differ in their capacity to bind extracellular matrix proteins and also in their affinity for VEGF receptors. Although the relative contribution of the VEGF isoforms has been studied in tumor angiogenesis, little is known about the mechanisms that regulate the alternative splicing process. Here, we tested microenvironment cues that might regulate VEGF alternative splicing. To test this, we used endometrial cancer cells that produce all VEGF isoforms as a model, and exposed them to varying pH levels, hormones, glucose and CoCl2 (to mimic hypoxia). Low pH had the most consistent effects in inducing variations in VEGF splicing pattern (VEGF121 increased significantly, p < 0.001, when compared to VEGF145, 165 or 189). This was accompanied by activation of the p38 stress pathway and SR proteins (splicing factors) expression and phosphorylation. SF2/ASF, SRp20 and SRp40 down-regulation by siRNA impaired the effects of pH stimulation, blocking the shift in VEGF isoforms production. Taken together, we show for the first time that acidosis (low pH) regulates VEGF-A alternative splicing, may be through p38 activation and suggest the possible SR proteins involved in this process.
KeywordsAlternative splicing Endometrial carcinoma Microenvironment SR proteins VEGF
Dulbecco’s modified Eagle’s medium
enzyme-linked immunosorbent assay
extracellular signal-regulated kinase
fetal bovine serum
heterogeneous nuclear ribonucleoprotein
hypoxia-induced stability factor
mitogen-activated protein kinase
poly(A)-binding protein-interacting protein 2
real time RT-PCR
RNA recognition motif
- RS domain
domain rich in alternating serine and arginine residues
stress-activated protein kinase/Jun-amino-terminal kinase
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
small interfering RNA
vascular endothelial growth factor
Angiogenesis is important for the expansion of solid and hematologic cancers. In response to metabolic changes occurring within the tumor microenvironment, such as increased hypoxia and acidosis , a cascade of events takes place, resulting in the production of angiogenic “promoters” (stimulators) and a decrease in angiogenesis inhibitors [2, 3, 4]. One of the best known angiogenic stimulators, produced by most tumors, is the vascular endothelial growth factor (VEGF-A).
VEGF-A is produced by cells under stress, such as during hypoxia, resulting in tissue angiogenesis and oxygenation, although the molecular mechanisms regulating VEGF production in response to microenvironmental stimuli other than hypoxia, such as acidosis, are still poorly characterized .
Alternative splicing is a major mechanism for modulating the expression of cellular and viral genes and enables a single gene to increase its coding capacity. The VEGF isoforms mentioned above represent one family of proteins whose expression may be regulated by alternative splicing.
The family of SR (serine/arginine-rich) proteins has been implicated in splicing; they are characterized by an RNA recognition motif (RRM) and a C-terminal domain rich in alternating serine and arginine residues (the RS domain) . The RRMs determine RNA binding specificity, whereas the RS domain mediates protein-protein interactions that are thought to be essential for the recruitment of the splicing apparatus and for the splice site pairing.
In the present report, we studied the influence of microenvironment cues that could affect the VEGF-A gene splicing pattern, and determined the molecular mechanisms involved.
Microenvironment Changes Affect VEGF Alternative Splicing Pattern
VEGF Isoform Shift in Acidic pH is Accompanied by p38/MAPK Activation
Since acidic pH induced a shift in isoform production by RL95 cells, next we sought to define the signaling pathways that might be involved in this effect.
As shown in Fig. 4b, 8 h in acidic pH induced the activation of the stress signaling pathways p38 MAPK (p < 0.05) and SAPK/JNK, while ERK and Akt remained unchanged. In vitro blockade of the two signaling pathways using specific inhibitors demonstrated that cells cultured in the presence of the p38 pathway inhibitor (SB202190) did not respond to the acidic pH (Fig. 4c). Under these conditions the shift in VEGF isoform production was not observed (VEGF121 was not significantly different from all the other isoforms, p > 0.05), while the SAPK/JNK inhibitor SP600125 had little effect (VEGF121 vs VEGF165, p < 0.001, as observed in pH 5.5 condition). These data indicate that the p38 stress signaling pathway may be involved in the effects of acidic pH that result in modulation of the VEGF alternative splicing pattern.
SR Proteins Could be Involved in the Regulation of VEGF Isoforms
SR proteins have been described to be involved in the control of constitutive and alternative splicing of genes . To evaluate which SR proteins could be activated by the p38 stress signaling pathway and involved in VEGF isoform shift in acidic conditions, the modulation of SR proteins was investigated.
Since these proteins have RNA recognition motifs, we used bioinformatics software to search putative binding sites for these specific SR proteins, SF2/ASF, SRp20 and SRp40. This approach showed that SRp20 and SRp40 do not have putative binding sites at the exons of VEGF that are involved in alternative splicing, but SF2/ASF has putative binding sites at exons 5, 7 and 8 of VEGF. Therefore, we may speculate that the SF2/ASF protein may act on alternative splicing of VEGF by binding directly to the VEGF sequence and in contrary, the SRp20 and SRp40 could be exon-binding independent by recruiting splicing factors through its serine/arginine rich domain (SR proteins property already described by Wu and Maniatis in 1993) .
Since SR proteins are large families of proteins involved in the global mechanisms of alternative splicing, a more detailed study must be done in order to identify other SR or hnRNP (SR proteins antagonists, heterogeneous nuclear ribonucleoproteins) proteins that may regulate the VEGF isoforms pattern in acidic conditions.
Angiogenesis is an essential property of all tumors, allowing tumor expansion and contributing towards metastasis dissemination. Since it is a common feature of most malignancies, the importance of angiogenesis as a therapeutic target has been well documented . Regarding the molecular signals that control the tumor angiogenesis process, the increased production of pro-angiogenic factors by most tumors has received great attention, most notably the recognition of VEGF-A as a key angiogenic factor for the majority of tumors . VEGF-A has several isoforms, whose importance in the context of tumor angiogenesis has already been addressed. Although VEGF-A isoforms can bind differently to the ECM, they have also been attributed with different capacity to modulate the tumor vasculature [8, 16]. In detail, VEGF 121 has been described as more angiogenic and tumorigenic in breast  and prostate  cancer and also to specifically improve oxygenation in experimental breast tumors , while VEGF189 contributes to the establishment of distant metastasis of pulmonary adenocarcinoma . Given the lack of mechanistic information concerning the regulation of the VEGF alternative splicing process, in the present report we hypothesized that cues in the tumor microenvironment might selectively affect the VEGF splicing pattern, and studied the mechanisms involved in this effect.
Tumor microenvironment signals such as hypoxia and acidosis have been suggested to play a major role in the control of VEGF-A production, and consequently in modulation of angiogenesis . In fact, hypoxia and acidosis are common features of the majority of solid and hematologic malignancies, perhaps as a consequence of the tumor metabolic needs, or as a consequence of an altered (pro-malignant) microenvironment. Importantly, the extracellular pH has been recognized as an inducer of VEGF, and also to regulate the VEGF interactions with different cells and with components of the extracellular matrix .
In the present study, acidosis consistently affected the VEGF alternative splicing pattern produced by endometrial cancer cells (used as a model); this correlated to a shift in the usual VEGF isoform expression pattern, resulting in an significant increase in the VEGF121, that was not accompanied by the other VEGF isoforms.
The involvement of signaling pathways and splicing machinery had not been studied in the context of VEGF alternative splicing. In our report, we reveal the involvement of p38 and possible members of the SR protein family in this mechanism. We now intend to perform a more detailed characterization of the splicing machinery involved in the VEGF alternative splicing in tumor cells exposed to different microenvironment cues.
Importantly, in addition to alternative splicing regulation, the levels of each isoform of VEGF can also be modulated at the mRNA stability level. Pagès et al.  demonstrated that anisomycin (a strong activator of stress-activated protein kinases, SAPKs) increased VEGF mRNA stabilization through the activation of p38 kinase and JNK. This protein induce the recruitment of HuR (one of the Hu family proteins) and PAIP2 (poly(A)-binding protein-interacting protein 2) to the AU-rich elements (AREs) in the 3′-untranslated region (3′ UTR) of VEGF mRNA . Since the ARE sequence at the 3′ UTR of the VEGF mRNA is present in all the isoforms, there is a possibility that all isoforms have the same stability and that the differences observed in VEGF isoforms ratio were not due to a difference in mRNA stability. In fact, a study that correlates the VEGF144 up-regulation with glucose starvation, suggest that other mechanism apart mRNA stability must also exist to explain the dramatic increase observed (the increased stability observed for this isoform was approximately threefold but the increase in mRNA was ~400-fold) . Nevertheless, more studies have to be done to address this important question.
Additionally, the p38 pathway, which is an important stress signaling pathway that have been described to control VEGF at the mRNA expression  and stability level , was also shown to act directly or indirectly in the alternative splicing of this gene.
In conclusion, we postulate that changes of VEGF isoforms observed in acidic conditions may represent the adaptation of tumors to alterations in the microenvironment, namely by activating angiogenic signaling pathways, through different VEGF isoforms production.
Material and Methods
All reagents were obtained from Sigma, unless otherwise stated.
Cell Lines and Cell Culture Conditions
The RL95 cell line was kindly provided by Professor Steve Smith (currently Principal of the Faculty of Medicine, Imperial College, London, UK). It was cultured in 50% high glucose Dulbecco’s modified Eagle’s medium (DMEM) medium (Sigma) and 50% nutrient mixture F-12 Ham (Sigma), supplemented with 10% FBS, 100 µg/ml of streptomycin sulfate, 100 U/ml of penicillin G sodium, 2 mM of l-glutamine and 0.1 µg/ml of amphotericin B as Fungizone. Cells were cultured at 37°C in a 5% CO2 atmosphere.
Upon reaching confluency, RL95 cells were submitted to changes in pH (pH 5.5, to mimic for acidosis), oxygen, glucose (100 mM) or hormones levels. To obtain an acidic medium we used HCl and the pH 5.5 was confirmed before and after the experiment using a pH electrode. To induce hormonal changes and to mimic hypoxia, β-estradiol (100 nM)/progesterone (1 µM) and cobalt chloride (150 µM)  were used respectively.
To test the importance of the different signaling pathways in the regulation of VEGF alternative splicing, the RL95 cell line was cultured in growth medium at pH 5.5 with or without the inhibitors of p38 MAPK (SB202190, Sigma, 20 µM,) and SAPK/JNK (SP600125, Sigma, 20 µM) signaling pathways.
RNA Isolation and Real Time RT-PCR with TAQMAN or Sybergreen
Primers and probes used in real time RT-PCR for VEGF isoforms
Probes (6-FAM-5′ → 3′-TAMRA)
Primers (5′ → 3′)
165 + 165b
189 + 189b
The relative expression of each sample was calculated with respect to a standard calibration curve that represents a serial dilution of a cDNA. Each sample was analyzed in triplicate and each PCR experiment included at least one non-template control well.
ELISA, Protein Extraction and Western Blotting
Culture supernatants from RL95 in different conditions were collected and used to measure human VEGF by ELISA (Oncogene Research Products) under conditions described by the supplier.
To extract total proteins the pellets were suspended in a buffer containing 1% NP40, 10% glycerol, 50 mM Tris–HCl pH 7.5, 0.1% sodic azid and 150 mM NaCl, supplemented with protease and phosphatase inhibitors. After 30 min in ice, lysates were centrifuged for 15 min at 4°C and 12,000 rpm.
Equal proteins amount were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Blots were incubated overnight with an antibody against phosphorilated SR proteins [mouse anti-SR proteins 1 (H4) from Zymed] at a concentration of 10 µg/ml. Antibodies against P-AKT, P-ERK, P-JNK (Santa Cruz Biotechnology, Santa Cruz, CA, USA), P-p38 (cell signaling) and actin (Sigma) were used at final concentration of 100 ng/ml, 400 ng/ml, and a dilution of 1:500, 1:1,000 and 1:2,000 respectively. Western blotting was performed following conventional protocols. Blots were revealed with the ECL (Amersham) system, according to manufacturers instructions. The hybridizing signals were quantified with ImageJ software.
siRNA for SR proteins
One day before transfection, 1 × 105 RL95 cells were plated in 500 µl of growth medium without antibiotics in a 24-well culture vessel. Fifty picomoles of each siRNA (SFRS1 for SF2/ASF; SFRS3 for SRp20 and SFRS5 for SRp40 from Ambiom Company) were used. The RNA and 1 µl of Lipofectamine 2000 (Invitrogen) were diluted separately in 50 µl of Opti-MEM I reduced serum medium. After 5 min of incubation, the two dilutions were combined and incubated for 20 min at room temperature. This mixture was then added to the cells following an incubation of 6 h at 37°C and 5% CO2. After transfection, the medium was replaced. The effect of transfection was assessed after 72 h.
The splicing rainbow (Morais & Valcarcel EMBL 2002 at http://www.ebi.ac.uk/asd-srv/wb.cgi?method=8) bioinformatics software was used to find putative binding sites for SF2/ASF, SRp20 and SRp40 proteins in the VEGF sequence. From the list of binding sites received after the VEGF sequence analysis we only choose as putative binding sites the sequences with higher scores (S) that were in exons. In detail, a sequence was considered a putative binding site for SRp20, and SF2/ASF if S > 6 and for SRp40 if S > 5.
Results are expressed as mean ± standard deviation. Data were analyzed using the unpaired two-tailed Student’s t test or the one-way ANOVA with post Tukey test. p values of <0.05 were considered significant.
We are grateful to Nuno Morais (PhD student, Unidade de Biologia Celular, Instituto de Medicina Molecular, Lisbon, Portugal) for his help in the bioinformatics analysis. We also thank Professor Steve Smith (currently Principal of the Faculty of Medicine, Imperial College, London, UK) for providing the RL95 cell line, and Mr. Alex Varey (Microvascular Research Laboratories, University of Bristol) for his useful suggestions regarding the VEGFxxxb isoforms. Ana Paula Elias is a recipient of SFRH/BD/14287/2003 Fellowship (from the Portuguese Foundation for Science and Technology, FCT). This study was supported by POCTI 38391/2001 (Sérgio Dias) and by Liga Portuguesa Contra o Cancro, Nucleo Regional Sul.