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Class II phosphoinositide 3-kinases contribute to endothelial cells morphogenesis.

Tibolla G, Piñeiro R, Chiozzotto D, Mavrommati I, Wheeler AP, Norata GD, Catapano AL, Maffucci T, Falasca M - PLoS ONE (2013)

Bottom Line: In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma.Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration.The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2β and the class IB isoform p110γ mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2α primarily controlling EC survival.

View Article: PubMed Central - PubMed

Affiliation: Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Blizard Institute, Centre for Diabetes, Inositide Signalling Group, London, United Kingdom.

ABSTRACT
The question of whether the distinct isoforms of the family of enzymes phosphoinositide 3-kinases (PI3Ks) play redundant roles within a cell or whether they control distinct cellular processes or distinct steps within the same cellular process has gained considerable importance in the recent years due to the development of inhibitors able to selectively target individual isoforms. It is important to understand whether inhibition of one PI3K can result in compensatory effect from other isoform(s) and therefore whether strategies aimed at simultaneously blocking more than one PI3K may be needed. In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma. Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration. The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2β and the class IB isoform p110γ mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2α primarily controlling EC survival. Data further indicate that PI3K-C2β and p110γ control distinct steps involved in cell migration supporting the hypothesis that different PI3Ks regulate distinct cellular processes.

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Class II PI3Ks are involved in S1P-dependent EC migration.(A) Expression levels of PI3K-C2α and PI3K-C2β in HUVEC transfected with a scrambled siRNA or siRNAs (“Sequences 1”) specifically targeting the indicated enzymes. (B-D) Twenty four hours after transfection with the indicated siRNAs cells were serum starved overnight and S1P-mediated cell migration was determined by Transwell assays as above. Data are expressed as percentage of migration of cells transfected with scrambled siRNA and unstimulated (control) and are means ± SEM from 17 (B), 11 (C) and 6 (D) independent experiments. *p<0.05; **p<0.001.
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pone-0053808-g002: Class II PI3Ks are involved in S1P-dependent EC migration.(A) Expression levels of PI3K-C2α and PI3K-C2β in HUVEC transfected with a scrambled siRNA or siRNAs (“Sequences 1”) specifically targeting the indicated enzymes. (B-D) Twenty four hours after transfection with the indicated siRNAs cells were serum starved overnight and S1P-mediated cell migration was determined by Transwell assays as above. Data are expressed as percentage of migration of cells transfected with scrambled siRNA and unstimulated (control) and are means ± SEM from 17 (B), 11 (C) and 6 (D) independent experiments. *p<0.05; **p<0.001.

Mentions: As mentioned above, one of the features of class II isoforms PI3K-C2α and PI3K-C2β compared to class I enzymes is their peculiar resistance to treatment with classical PI3K inhibitors [1], [6]. In order to determine whether class II PI3Ks have a role in regulation of S1P- and HDL3-dependent migration in HUVEC, we decided to investigate the effect of downregulation of their protein expression on these processes using specific siRNAs. First we checked whether transfection of HUVEC affected the S1P-induced cell migration by using a non-targeting, “scrambled” siRNA as control. Transfection of HUVEC reduced the number of migrated cells compared to untransfected cells both in unstimulated and in stimulated conditions (Figure S2A), as expected from cells treated with transfection reagent. Nevertheless, transfected cells were still able to respond to S1P stimulation in a manner which was comparable to untransfected cells (Figure S2A) indicating that the transfection procedure does not affect the ability of HUVEC to migrate upon S1P stimulation. We then investigated the S1P-dependent migration in HUVEC upon PI3K-C2α and PI3K-C2β downregulation. Two distinct siRNAs were used to downregulate the expression levels of each class II PI3K isoform (Figure 2A, Figure S2B). Data revealed that downregulation of PI3K-C2β specifically reduced the S1P-mediated cell migration (Figure 2B and Figure S2C). A reduction in cell migration upon S1P stimulation was also detected in cells upon downregulation of PI3K-C2α (Figure 2C and Figure S2D). Interestingly, we observed a slight but significant inhibition of S1P-induced migration in cells treated with 1 µM of the compound A66 (Figure S2E). It has been reported that A66 is a specific inhibitor of the class IA PI3K isoform p110α (IC50 32 nM), able to reduce Akt phosphorylation in specific cancer cells at concentrations in the nanomolar range [26]. The observation that PI3K-C2β is the only other PI3K isoform to be inhibited by this compound with an IC50 in the nanomolar range (462 nM) [26] and that treatment of HUVEC with 1 µM A66 does not inhibit the S1P-induced Akt phosphorylation (described below) may suggest that the detected effect on cell migration is due to at least a partial inhibition of PI3K-C2β.


Class II phosphoinositide 3-kinases contribute to endothelial cells morphogenesis.

Tibolla G, Piñeiro R, Chiozzotto D, Mavrommati I, Wheeler AP, Norata GD, Catapano AL, Maffucci T, Falasca M - PLoS ONE (2013)

Class II PI3Ks are involved in S1P-dependent EC migration.(A) Expression levels of PI3K-C2α and PI3K-C2β in HUVEC transfected with a scrambled siRNA or siRNAs (“Sequences 1”) specifically targeting the indicated enzymes. (B-D) Twenty four hours after transfection with the indicated siRNAs cells were serum starved overnight and S1P-mediated cell migration was determined by Transwell assays as above. Data are expressed as percentage of migration of cells transfected with scrambled siRNA and unstimulated (control) and are means ± SEM from 17 (B), 11 (C) and 6 (D) independent experiments. *p<0.05; **p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3539993&req=5

pone-0053808-g002: Class II PI3Ks are involved in S1P-dependent EC migration.(A) Expression levels of PI3K-C2α and PI3K-C2β in HUVEC transfected with a scrambled siRNA or siRNAs (“Sequences 1”) specifically targeting the indicated enzymes. (B-D) Twenty four hours after transfection with the indicated siRNAs cells were serum starved overnight and S1P-mediated cell migration was determined by Transwell assays as above. Data are expressed as percentage of migration of cells transfected with scrambled siRNA and unstimulated (control) and are means ± SEM from 17 (B), 11 (C) and 6 (D) independent experiments. *p<0.05; **p<0.001.
Mentions: As mentioned above, one of the features of class II isoforms PI3K-C2α and PI3K-C2β compared to class I enzymes is their peculiar resistance to treatment with classical PI3K inhibitors [1], [6]. In order to determine whether class II PI3Ks have a role in regulation of S1P- and HDL3-dependent migration in HUVEC, we decided to investigate the effect of downregulation of their protein expression on these processes using specific siRNAs. First we checked whether transfection of HUVEC affected the S1P-induced cell migration by using a non-targeting, “scrambled” siRNA as control. Transfection of HUVEC reduced the number of migrated cells compared to untransfected cells both in unstimulated and in stimulated conditions (Figure S2A), as expected from cells treated with transfection reagent. Nevertheless, transfected cells were still able to respond to S1P stimulation in a manner which was comparable to untransfected cells (Figure S2A) indicating that the transfection procedure does not affect the ability of HUVEC to migrate upon S1P stimulation. We then investigated the S1P-dependent migration in HUVEC upon PI3K-C2α and PI3K-C2β downregulation. Two distinct siRNAs were used to downregulate the expression levels of each class II PI3K isoform (Figure 2A, Figure S2B). Data revealed that downregulation of PI3K-C2β specifically reduced the S1P-mediated cell migration (Figure 2B and Figure S2C). A reduction in cell migration upon S1P stimulation was also detected in cells upon downregulation of PI3K-C2α (Figure 2C and Figure S2D). Interestingly, we observed a slight but significant inhibition of S1P-induced migration in cells treated with 1 µM of the compound A66 (Figure S2E). It has been reported that A66 is a specific inhibitor of the class IA PI3K isoform p110α (IC50 32 nM), able to reduce Akt phosphorylation in specific cancer cells at concentrations in the nanomolar range [26]. The observation that PI3K-C2β is the only other PI3K isoform to be inhibited by this compound with an IC50 in the nanomolar range (462 nM) [26] and that treatment of HUVEC with 1 µM A66 does not inhibit the S1P-induced Akt phosphorylation (described below) may suggest that the detected effect on cell migration is due to at least a partial inhibition of PI3K-C2β.

Bottom Line: In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma.Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration.The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2β and the class IB isoform p110γ mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2α primarily controlling EC survival.

View Article: PubMed Central - PubMed

Affiliation: Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Blizard Institute, Centre for Diabetes, Inositide Signalling Group, London, United Kingdom.

ABSTRACT
The question of whether the distinct isoforms of the family of enzymes phosphoinositide 3-kinases (PI3Ks) play redundant roles within a cell or whether they control distinct cellular processes or distinct steps within the same cellular process has gained considerable importance in the recent years due to the development of inhibitors able to selectively target individual isoforms. It is important to understand whether inhibition of one PI3K can result in compensatory effect from other isoform(s) and therefore whether strategies aimed at simultaneously blocking more than one PI3K may be needed. In this study we investigated the relative contribution of distinct PI3K isoforms to endothelial cells (EC) functions specifically regulated by the sphingolipid sphingosine-1-phosphate (S1P) and by high density lipoproteins (HDL), the major carrier of S1P in human plasma. Here we show that a co-ordinated action of different PI3Ks is required to tightly regulate remodelling of EC on Matrigel, a process dependent on cell proliferation, apoptosis and migration. The contribution of each isoform to this process appears to be distinct, with the class II enzyme PI3K-C2β and the class IB isoform p110γ mainly regulating the S1P- and HDL-dependent EC migration and PI3K-C2α primarily controlling EC survival. Data further indicate that PI3K-C2β and p110γ control distinct steps involved in cell migration supporting the hypothesis that different PI3Ks regulate distinct cellular processes.

Show MeSH
Related in: MedlinePlus