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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 PI3Ks downregulation on EC apoptosis.(A) Results from caspase 3 assay performed on lysates from HUVEC obtained 48 h after transfection with the indicated siRNAs. Data are means ± SEM from 3–4 independent experiments. Student’s t-Test: un-paired *p<0.05 vs cells transfected with PI3K-C2β siRNA. (B) HUVEC were transfected with the indicated siRNAs. After 48 h the percentage of apoptotic cells was determined by Annexin V staining. Annexin V positive and propidium iodide negative cells were gated. Results are expressed as means ± SEM from 3 independent experiments. *p<0.05 vs scrambled siRNA treated cells.
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pone-0053808-g006: Effect of PI3Ks downregulation on EC apoptosis.(A) Results from caspase 3 assay performed on lysates from HUVEC obtained 48 h after transfection with the indicated siRNAs. Data are means ± SEM from 3–4 independent experiments. Student’s t-Test: un-paired *p<0.05 vs cells transfected with PI3K-C2β siRNA. (B) HUVEC were transfected with the indicated siRNAs. After 48 h the percentage of apoptotic cells was determined by Annexin V staining. Annexin V positive and propidium iodide negative cells were gated. Results are expressed as means ± SEM from 3 independent experiments. *p<0.05 vs scrambled siRNA treated cells.

Mentions: Our data so far indicated that PI3K-C2α is also critical for EC morphogenesis. We therefore sought to determine the specific role of PI3K-C2α in this process. Since downregulation of PI3K-C2α had a smaller effect compared to PI3K-C2β knockdown (in the case of S1P) or no effect at all (in the case of HDL3) on migration assessed by Transwell assays, we investigated whether downregulation of PI3K-C2α affected other cellular function(s) required for EC remodelling. It has been recently reported that PI3K-C2α knockdown reduces viability [27] and augmented apoptosis of HUVEC [7]. Consistent with this, we observed that downregulation of PI3K-C2α using two distinct siRNAs induced apoptosis in HUVEC in the presence of serum as assessed by caspase 3 assay (Figure 6A, Figure S5A) and by Annexin V staining (Figure 6B) whereas no effect was observed upon silencing of PI3K-C2β (Figure 6, Figure S5A). Consistent with these data, downregulation of PI3K-C2α but not PI3K-C2β reduced the viability of HUVEC upon serum starvation (Figure S5B). Although results from caspase assay seemed to suggest a small increase in apoptosis upon downregulation of p110γ, values did not reach statistical significance (Figure 6A). Moreover no increase in the number of Annexin V-positive cells was detected in cells lacking p110γ (Figure 6B). Importantly, these data indicate that downregulation of PI3K-C2α induces apoptosis of HUVEC even in the presence of serum (Figure 6). These data suggest that PI3K-C2α but not PI3K-C2β or p110γ primarily regulates HUVEC survival, further supporting the hypothesis that the distinct PI3Ks can regulate EC morphogenesis by controlling different processes.


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 PI3Ks downregulation on EC apoptosis.(A) Results from caspase 3 assay performed on lysates from HUVEC obtained 48 h after transfection with the indicated siRNAs. Data are means ± SEM from 3–4 independent experiments. Student’s t-Test: un-paired *p<0.05 vs cells transfected with PI3K-C2β siRNA. (B) HUVEC were transfected with the indicated siRNAs. After 48 h the percentage of apoptotic cells was determined by Annexin V staining. Annexin V positive and propidium iodide negative cells were gated. Results are expressed as means ± SEM from 3 independent experiments. *p<0.05 vs scrambled siRNA treated cells.
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Related In: Results  -  Collection

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

pone-0053808-g006: Effect of PI3Ks downregulation on EC apoptosis.(A) Results from caspase 3 assay performed on lysates from HUVEC obtained 48 h after transfection with the indicated siRNAs. Data are means ± SEM from 3–4 independent experiments. Student’s t-Test: un-paired *p<0.05 vs cells transfected with PI3K-C2β siRNA. (B) HUVEC were transfected with the indicated siRNAs. After 48 h the percentage of apoptotic cells was determined by Annexin V staining. Annexin V positive and propidium iodide negative cells were gated. Results are expressed as means ± SEM from 3 independent experiments. *p<0.05 vs scrambled siRNA treated cells.
Mentions: Our data so far indicated that PI3K-C2α is also critical for EC morphogenesis. We therefore sought to determine the specific role of PI3K-C2α in this process. Since downregulation of PI3K-C2α had a smaller effect compared to PI3K-C2β knockdown (in the case of S1P) or no effect at all (in the case of HDL3) on migration assessed by Transwell assays, we investigated whether downregulation of PI3K-C2α affected other cellular function(s) required for EC remodelling. It has been recently reported that PI3K-C2α knockdown reduces viability [27] and augmented apoptosis of HUVEC [7]. Consistent with this, we observed that downregulation of PI3K-C2α using two distinct siRNAs induced apoptosis in HUVEC in the presence of serum as assessed by caspase 3 assay (Figure 6A, Figure S5A) and by Annexin V staining (Figure 6B) whereas no effect was observed upon silencing of PI3K-C2β (Figure 6, Figure S5A). Consistent with these data, downregulation of PI3K-C2α but not PI3K-C2β reduced the viability of HUVEC upon serum starvation (Figure S5B). Although results from caspase assay seemed to suggest a small increase in apoptosis upon downregulation of p110γ, values did not reach statistical significance (Figure 6A). Moreover no increase in the number of Annexin V-positive cells was detected in cells lacking p110γ (Figure 6B). Importantly, these data indicate that downregulation of PI3K-C2α induces apoptosis of HUVEC even in the presence of serum (Figure 6). These data suggest that PI3K-C2α but not PI3K-C2β or p110γ primarily regulates HUVEC survival, further supporting the hypothesis that the distinct PI3Ks can regulate EC morphogenesis by controlling different processes.

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