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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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Effect of PI3K inhibitors on S1P- and HDL3-induced EC migration.Serum-starved HUVEC were pre-treated with 100 nM wortmannin (A,D), 25 µM LY294002 (B), 5 µM LY294002 (E) or 1 µM AS252424 (C,F) for 30 min. Cell migration induced by S1P (A–C) or HDL3 (D-F) was determined by Transwell assays. Briefly, cells resuspended in the presence or absence of the specific inhibitor (or vehicle control) were allowed to migrate in the presence of 1 µM S1P or 200 µg/ml HDL3 in the lower chamber for 4 h. Where necessary, each inhibitor was also added in the lower chamber therefore migration was performed in the continuous presence of the inhibitor to be tested. After 4 h, migrated cells were fixed with 4% paraformaldehyde, stained with 1% crystal violet and counted using a phase-contrast microscopy. Data are expressed as percentage of control (cells untreated and unstimulated) and are means ± SEM from 12 (A), 6 (B), 7 (C), 4 (D), 3 (E) and 5 (F) independent experiments. *p<0.05, **p<0.001.
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pone-0053808-g001: Effect of PI3K inhibitors on S1P- and HDL3-induced EC migration.Serum-starved HUVEC were pre-treated with 100 nM wortmannin (A,D), 25 µM LY294002 (B), 5 µM LY294002 (E) or 1 µM AS252424 (C,F) for 30 min. Cell migration induced by S1P (A–C) or HDL3 (D-F) was determined by Transwell assays. Briefly, cells resuspended in the presence or absence of the specific inhibitor (or vehicle control) were allowed to migrate in the presence of 1 µM S1P or 200 µg/ml HDL3 in the lower chamber for 4 h. Where necessary, each inhibitor was also added in the lower chamber therefore migration was performed in the continuous presence of the inhibitor to be tested. After 4 h, migrated cells were fixed with 4% paraformaldehyde, stained with 1% crystal violet and counted using a phase-contrast microscopy. Data are expressed as percentage of control (cells untreated and unstimulated) and are means ± SEM from 12 (A), 6 (B), 7 (C), 4 (D), 3 (E) and 5 (F) independent experiments. *p<0.05, **p<0.001.

Mentions: Cells were pre-treated with generic or isoform-specific PI3K inhibitors and migration was determined by Transwell assays in the continuous presence of the inhibitors. Consistent with published data, these experiments revealed that treatment with 100 nM wortmannin only partially inhibited the S1P-induced migration of HUVEC (Figure 1A). Similarly, a partial inhibition of migration was detected upon treatment with 25 µM of the reversible PI3K inhibitor LY294002 (Figure 1B). A detailed analysis of the effect of LY294002 on this process revealed that treatment with up to 5 µM did not affect migration (Figure S1A,B) whereas a slight inhibition was detected upon treatment with 10 µM LY294002 (Figure S1C). None of these treatments affected the basal migration (Figure 1A,B, Figure S1A–C). These results indicated that the S1P-dependent migration was only partially reduced by classical, generic PI3K inhibitors.


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)

Effect of PI3K inhibitors on S1P- and HDL3-induced EC migration.Serum-starved HUVEC were pre-treated with 100 nM wortmannin (A,D), 25 µM LY294002 (B), 5 µM LY294002 (E) or 1 µM AS252424 (C,F) for 30 min. Cell migration induced by S1P (A–C) or HDL3 (D-F) was determined by Transwell assays. Briefly, cells resuspended in the presence or absence of the specific inhibitor (or vehicle control) were allowed to migrate in the presence of 1 µM S1P or 200 µg/ml HDL3 in the lower chamber for 4 h. Where necessary, each inhibitor was also added in the lower chamber therefore migration was performed in the continuous presence of the inhibitor to be tested. After 4 h, migrated cells were fixed with 4% paraformaldehyde, stained with 1% crystal violet and counted using a phase-contrast microscopy. Data are expressed as percentage of control (cells untreated and unstimulated) and are means ± SEM from 12 (A), 6 (B), 7 (C), 4 (D), 3 (E) and 5 (F) independent experiments. *p<0.05, **p<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3539993&req=5

pone-0053808-g001: Effect of PI3K inhibitors on S1P- and HDL3-induced EC migration.Serum-starved HUVEC were pre-treated with 100 nM wortmannin (A,D), 25 µM LY294002 (B), 5 µM LY294002 (E) or 1 µM AS252424 (C,F) for 30 min. Cell migration induced by S1P (A–C) or HDL3 (D-F) was determined by Transwell assays. Briefly, cells resuspended in the presence or absence of the specific inhibitor (or vehicle control) were allowed to migrate in the presence of 1 µM S1P or 200 µg/ml HDL3 in the lower chamber for 4 h. Where necessary, each inhibitor was also added in the lower chamber therefore migration was performed in the continuous presence of the inhibitor to be tested. After 4 h, migrated cells were fixed with 4% paraformaldehyde, stained with 1% crystal violet and counted using a phase-contrast microscopy. Data are expressed as percentage of control (cells untreated and unstimulated) and are means ± SEM from 12 (A), 6 (B), 7 (C), 4 (D), 3 (E) and 5 (F) independent experiments. *p<0.05, **p<0.001.
Mentions: Cells were pre-treated with generic or isoform-specific PI3K inhibitors and migration was determined by Transwell assays in the continuous presence of the inhibitors. Consistent with published data, these experiments revealed that treatment with 100 nM wortmannin only partially inhibited the S1P-induced migration of HUVEC (Figure 1A). Similarly, a partial inhibition of migration was detected upon treatment with 25 µM of the reversible PI3K inhibitor LY294002 (Figure 1B). A detailed analysis of the effect of LY294002 on this process revealed that treatment with up to 5 µM did not affect migration (Figure S1A,B) whereas a slight inhibition was detected upon treatment with 10 µM LY294002 (Figure S1C). None of these treatments affected the basal migration (Figure 1A,B, Figure S1A–C). These results indicated that the S1P-dependent migration was only partially reduced by classical, generic PI3K inhibitors.

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