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Microenvironment changes (in pH) affect VEGF alternative splicing.

Elias AP, Dias S - Cancer Microenviron (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Angiogenesis Laboratory, Centro de Investigação em Patobiologia Molecular (CIPM), Instituto Português de Oncologia de Francisco Gentil, Lisboa, Portugal.

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.

No MeSH data available.


Related in: MedlinePlus

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)
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Fig6: 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)

Mentions: By real time RT-PCR we quantified the mRNA of different SR proteins (SF2/ASF, SRp20 and SRp40) and observed that pH 5.5 induced a significant up-regulation (p < 0.05) of SRp20 that could be partially inhibited in the presence of SB202190, the p38 MAPK inhibitor (Fig. 5b). This result indicates that p38 MAPK was not acting at the SR proteins mRNA levels and could have a role at mRNA translation or SR proteins activation (protein phosphorylation). To reveal the involvement of selective SR proteins in VEGF alternative splicing we used siRNA against each SR protein and tested the effect in the pattern of VEGF isoform production. siRNA against SF2/ASF, SRp20 or SRp40 reduced the production of the VEGF121 observed in pH 5.5 (in fact, the VEGF isoforms pattern in these conditions is equal to the control), suggesting that not only we have prevented VEGF expression but also have prevented the shift in the VEGF isoforms pattern (p > 0.05; Fig. 6b). Thus, SF2/ASF, SRp20 and SRp40 may have a role at the VEGF alternative splicing process. These three proteins might also have a role in the alternative slicing of a VEGF regulator since by its down-regulation we affected the VEGF expression.Fig. 6


Microenvironment changes (in pH) affect VEGF alternative splicing.

Elias AP, Dias S - Cancer Microenviron (2008)

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)
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Related In: Results  -  Collection

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Fig6: 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)
Mentions: By real time RT-PCR we quantified the mRNA of different SR proteins (SF2/ASF, SRp20 and SRp40) and observed that pH 5.5 induced a significant up-regulation (p < 0.05) of SRp20 that could be partially inhibited in the presence of SB202190, the p38 MAPK inhibitor (Fig. 5b). This result indicates that p38 MAPK was not acting at the SR proteins mRNA levels and could have a role at mRNA translation or SR proteins activation (protein phosphorylation). To reveal the involvement of selective SR proteins in VEGF alternative splicing we used siRNA against each SR protein and tested the effect in the pattern of VEGF isoform production. siRNA against SF2/ASF, SRp20 or SRp40 reduced the production of the VEGF121 observed in pH 5.5 (in fact, the VEGF isoforms pattern in these conditions is equal to the control), suggesting that not only we have prevented VEGF expression but also have prevented the shift in the VEGF isoforms pattern (p > 0.05; Fig. 6b). Thus, SF2/ASF, SRp20 and SRp40 may have a role at the VEGF alternative splicing process. These three proteins might also have a role in the alternative slicing of a VEGF regulator since by its down-regulation we affected the VEGF expression.Fig. 6

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Angiogenesis Laboratory, Centro de Investigação em Patobiologia Molecular (CIPM), Instituto Português de Oncologia de Francisco Gentil, Lisboa, Portugal.

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.

No MeSH data available.


Related in: MedlinePlus