Class II phosphoinositide 3-kinases contribute to endothelial cells morphogenesis - PubMed (original) (raw)
Class II phosphoinositide 3-kinases contribute to endothelial cells morphogenesis
Gianpaolo Tibolla et al. PLoS One. 2013.
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.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. 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.
Figure 2. 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.
Figure 3. Class II PI3Ks are involved in HDL3-dependent EC migration.
Twenty four hours after transfection with the indicated siRNAs cells were serum starved overnight and HDL3-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 4 (A), 5 (B) and 5 (C) independent experiments. *p<0.05; **p<0.01.
Figure 4. Class II and class IB PI3Ks regulate remodelling of HUVEC.
HUVEC transfected with the indicated siRNAs were serum starved in M199 containing 0.5% FBS overnight before being detached and plated on growth factor reduced Matrigel in the presence of 1 µM S1P or 200 µg/ml HDL3. EC rearrangement was visualised after 4 to 6 h using an Axiovert200 microscope. (A) Representative images of branching points formation in HUVEC transfected with the indicated siRNAs (Sequences 1) in the absence or presence of S1P or HDL3. (B,C) Results from quantitative analysis in HUVEC transfected with the indicated siRNAs (Sequences 2) in the absence or presence of S1P or HDL3. Data indicate the total number of branching points and are means ± SEM from 4 (B) and 3 (C) independent experiments. *p<0.01; **p<0.001.
Figure 5. PI3K-C2β and p110γ play distinct role in regulation of S1P-dependent cell migration.
Random motility of HUVEC transfected with the indicated siRNAs was monitored as described in the Materials and Methods section. (A) Representative track paths throughout time of 6 cells are shown (8 h). (B) Data indicate the mean velocity/min and are means ± SEM from 3 independent experiments. **p<0.01 vs cells transfected with scrambled siRNA. (C) Results from Transwell assays performed in HUVEC transfected with the indicated siRNAs. Data are expressed as percentage of migration of cells transfected with scrambled siRNA and unstimulated (control) and are means ± SEM from 3 independent experiments. *p<0.01. Downregulation of PI3K-C2β and p110γ was confirmed by Western blotting.
Figure 6. 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.
Figure 7. Class II PI3Ks do not regulate Akt or ERK activation.
HUVEC transfected with the indicated siRNAs were serum starved in M199 containing 0.5% FBS overnight before stimulation with HDL3 or S1P for 10 min. Akt and ERK phosphorylation was assessed by Western blotting. Densitometry analysis shows means ± SEM from 3–4 (Akt) and 4 (ERK) independent experiments.
References
- Falasca M, Maffucci T (2012) Regulation and cellular functions of class II phosphoinositide 3-kinases. Biochem J 443: 587–601. - PubMed
- Vanhaesebroeck B, Guillermet-Guibert J, Graupera M, Bilanges B (2010) The emerging mechanisms of isoform-specific PI3K signalling. Nat Rev Mol Cell Biol 11: 329–341. - PubMed
- Falasca M, Maffucci T (2009) Rethinking phosphatidylinositol 3-monophosphate. Biochim Biophys Acta 1793: 1795–1803. - PubMed
- Falasca M, Hughes WE, Dominguez V, Sala G, Fostira F, et al. (2007) The role of phosphoinositide 3-kinase C2alpha in insulin signaling. J Biol Chem 282: 28226–28236. - PubMed
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