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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

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
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Fig2: 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

Mentions: We investigated how changes in the microenvironment might affect the pattern of VEGF alternative splicing (Fig. 1), using endometrial carcinoma cells as a model (since these cells express all VEGF-A isoforms). For this purpose, we induced changes in the culture medium (by exposing the cells to acidic pH, progesterone, β-estradiol, glucose and cobalt chloride, to mimic for hypoxia), and quantified the ratio of VEGF isoforms by real time RT-PCR (RQ-PCR). As expected, hypoxia significantly increased VEGF production, as did acidosis (Fig. 2a,b and Supplementary Fig. 1). However, a more evident shift in the pattern of VEGF isoforms produced, occurred in samples subjected to lower pH. A pH 5.5 induced a preferential VEGF121 increase (p < 0.001), suggesting it modulated the mechanisms involved in alternative splicing.Fig. 2


Microenvironment changes (in pH) affect VEGF alternative splicing.

Elias AP, Dias S - Cancer Microenviron (2008)

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
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654355&req=5

Fig2: 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
Mentions: We investigated how changes in the microenvironment might affect the pattern of VEGF alternative splicing (Fig. 1), using endometrial carcinoma cells as a model (since these cells express all VEGF-A isoforms). For this purpose, we induced changes in the culture medium (by exposing the cells to acidic pH, progesterone, β-estradiol, glucose and cobalt chloride, to mimic for hypoxia), and quantified the ratio of VEGF isoforms by real time RT-PCR (RQ-PCR). As expected, hypoxia significantly increased VEGF production, as did acidosis (Fig. 2a,b and Supplementary Fig. 1). However, a more evident shift in the pattern of VEGF isoforms produced, occurred in samples subjected to lower pH. A pH 5.5 induced a preferential VEGF121 increase (p < 0.001), suggesting it modulated the mechanisms involved in alternative splicing.Fig. 2

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