Microenvironment changes (in pH) affect VEGF alternative splicing

Abstract

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 CoCl(2) (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.

Fig. 1

VEGF isoforms that result from pre-mRNA alternative splicing. These isoforms differ in the presence or absence of exons 6 and 7 that codes for heparin-binding domains and in the presence of exon 8a or 8b that induce antagonist effects of VEGF. The localization of the probes and primers used to amplify the different isoforms of VEGF are indicated by a line or an arrow, respectively. The lines and arrows in blue represent the probes and primers used to amplify either VEGF165+VEGF165b or VEGF189+VEGF_189b

Fig. 2

VEGF isoforms expression pattern by RL95 cells in response to changes in the microenvironment. By real time RT-PCR (a) and ELISA (b), we can see an increase in VEGF production in acidic and hypoxic (mimicked by CoCl₂) conditions. A shift in the VEGF isoforms splicing pattern is more evident at pH 5.5 where VEGF121 expression is significantly different from all other isoforms (p < 0.001). In hypoxic conditions VEGF121 is also significantly different from VEGF145 and 189 (p < 0.001) and from VEGF165 (p < 0.05). In a the level of each isoform in different conditions is represented relatively to control conditions (which is considered equal to 1). In graph an = 10 and in graph bn = 3

Fig. 3

Discrimination between VEGFxxx and VEGF_xxxb isoforms expression in acidic pH. In pH 5.5 there was a significant increase in VEGF165 (p < 0.001) and 189 (p < 0.01) but the expression of VEGF165b (p < 0.001) and 189b (p < 0.001) is too low when compared respectively to the VEGF165 and 189 and do not change in acidic conditions (p > 0.05 for both isoforms). The graphs represent the mean of three experiments

Fig. 4

a Correlation between VEGF isoforms pattern inversion and the activation of stress signaling pathways in acidic conditions. After 8 h of pH 5.5 stimulation, the changes in VEGF isoform splicing pattern are more pronounced. b As shown by western blotting and densitometry quantification, at this time point, from the several signaling pathways analyzed, only the p38 MAPK (p < 0.05) and the SAPK/JNK stress pathways were activated. c SB202190 and SP600125 were used to inhibit p38 MAPK and SAPK/JNK stress pathways, respectively. In the presence of the p38 inhibitor, low pH failed to induce a shift in VEGF splicing pattern (all VEGF isoforrms are similar, p > 0.05), while the SAPK/JNK inhibitor could not prevent the shift in VEGF isoforms (VEGF121 is significantly different from VEGF165, p < 0.001). Graphs a and c represent the mean of three experiments and the blot was repeated three times

Fig. 5

a SR proteins involved in the control of the VEGF splicing pattern. Most SR proteins were up-regulated in acidic conditions both after 6 and 8 h of exposure. b Expression of the SR protein SRp20, in RL95, increased significantly (p < 0.05) in acidic conditions and was only partially inhibited in the presence of the p38 MAPK inhibitor SB202190. In graph b the error bars represent the standard deviation of three independent experiments

Fig. 6

a Effect of SR proteins down-regulation in VEGF alternative splicing in acidic conditions. siRNA for SF2/ASF, SRp20 and SRp40 (performed in triplicate) were used and its efficacy was demonstrated by real time RT-PCR. b Using siRNA for SF2/ASF, SRp20 and SRp40, there was no shift in the VEGF isoforms pattern observed in acidic conditions, since all isoforms were similar to each other (p > 0.05) and to the control (p > 0.05)

Fig. 7

Proposed model. When endometrial cancer cells are exposed to acidic medium the p38 MAPK signaling pathway is activated and an increase in SR proteins phosphorylation that will eventually regulate the VEGF alternative splicing takes place. In acidosis, not only VEGF expression is increased but also a modification of the alternative splicing occurs, where an increase in the proportion of VEGF121 versus VEGF145, 165, and 189 is observed