Grzegorz Sowa

Vascular Endothelial Growth Factor–Stimulated Actin Reorganization and Migration of Endothelial Cells Is Regulated via the Serine/Threonine Kinase Akt

Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding beta-galactosidase (beta-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with beta-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of beta-gal-infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.

Distinction between signaling mechanisms in lipid rafts vs. caveolae

The relative importance of lipid rafts vs. specialized rafts termed caveolae to influence signal transduction is not known. Here we show that in cells lacking caveolae, the dually acylated protein, endothelial nitric oxide synthase (eNOS), localizes to cholesterol-rich lipid raft domains of the plasma membrane. In these cells, expression of caveolin-1 (cav-1) stimulates caveolae biogenesis, promotes the interaction of cav-1 with eNOS, and the inhibition of NO release from cells. Interestingly, in cells where cav-1 does not drive caveolae assembly, despite equal levels of cav-1 and eNOS and localization of both proteins to raft domains of the plasmalemma, the physical interaction of eNOS with cav-1 is dramatically less resulting in less inhibition of NO release. Thus, cav-1 concentrated in caveolae, not in rafts, is in closer proximity to eNOS and is necessary for negative regulation of eNOS function, thereby providing the first clear example of spatial regulation of signaling in this organelle that is distinct from raft domains.

Trafficking of Endothelial Nitric-oxide Synthase in Living Cells QUANTITATIVE EVIDENCE SUPPORTING THE ROLE OF PALMITOYLATION AS A KINETIC TRAPPING MECHANISM LIMITING MEMBRANE DIFFUSION

To examine endothelial nitric-oxide synthase (eNOS) trafficking in living endothelial cells, the eNOS-deficient endothelial cell line ECV304 was stably transfected with an eNOS-green fluorescent protein (GFP) fusion construct and characterized by functional, biochemical, and microscopic analysis. eNOS-GFP was colocalized with Golgi and plasma membrane markers and produced NO in response to agonist challenge. Localization in the plasma membrane was dependent on the palmitoylation state, since the palmitoylation mutant of eNOS (C15S/C26S eNOS-GFP) was excluded from the plasma membrane and was concentrated in a diffuse perinuclear pattern. Fluorescence recovery after photobleaching (FRAP) revealed eNOS-GFP in the perinuclear region moving 3 times faster than the plasmalemmal pool, suggesting that protein-lipid or protein-protein interactions are different in these two cellular domains. FRAP of the palmitoylation mutant was two times faster than that of wild-type eNOS-GFP, indicating that palmitoylation was influencing the rate of trafficking. Interestingly, FRAP of C15S/C26S eNOS-GFP but not wild-type eNOS-GFP fit a model of protein diffusion in a lipid bilayer. These data suggest that the regulation of eNOS trafficking within the plasma membrane and Golgi are probably different mechanisms and not due to simple diffusion of the protein in a lipid bilayer.

Caveolin-1 Can Regulate Vascular Smooth Muscle Cell Fate by Switching Platelet-Derived Growth Factor Signaling From a Proliferative to an Apoptotic Pathway

Background—Caveolin-1 is a regulator of signaling events originating from plasma membrane microdomains termed caveolae. This study was performed to determine the regulatory role of caveolin-1 on the proliferative events induced by platelet-derived growth factor (PDGF) in vascular smooth muscle cells (VSMCs). Methods and Results—Treatment of VSMCs with PDGF for 24 hours resulted in a loss of caveolin-1 protein expression and plasma membrane–associated caveolae, despite a 3-fold increase in caveolin-1 mRNA. Pretreatment of VSMCs with chloroquine, an inhibitor of lysosomal function, inhibited the PDGF-induced loss of caveolin-1. These studies demonstrated that caveolin-1 was a target of PDGF signaling events. Adenoviral overexpression of caveolin-1 was associated with a switch in PDGF-induced signaling events from a proliferative response to an apoptotic response. This overexpression inhibited PDGF-induced expression of cyclin D1 in the presence of unaffected mitogen-activated protein kinase activation. Conclusions—Taken together, these studies suggest that caveolin-1 is an inhibitor of PDGF proliferative responses and might be capable of transforming PDGF-induced proliferative signals into death signals.

The phosphorylation of caveolin-2 on serines 23 and 36 modulates caveolin-1-dependent caveolae formation

Caveolin-1 and -2 are the two major coat proteins found in plasma membrane caveolae of most of cell types. Here, by using adenoviral transduction of either caveolin-1 or caveolin-2 or both isoforms into cells lacking both caveolins, we demonstrate that caveolin-2 positively regulates caveolin-1-dependent caveolae formation. More importantly, we show that caveolin-2 is phosphorylated in vivo at two serine residues and that the phosphorylation of caveolin-2 is necessary for its actions as a positive regulator of caveolin-1 during organelle biogenesis in prostate cancer cells. Mutation of the primary phosphorylation sites on caveolin-2, serine 23 and 36, reduces the number of plasmalemma-attached caveolae and increases the accumulation of noncoated vesicles, but does not affect trafficking of caveolin-2, interaction with caveolin-1 or its biophysical properties. Thus, the phosphorylation of caveolin-2 is a novel mechanism to regulate the dynamics of caveolae assembly.

Dynamic regulation of metabolism and respiration by endogenously produced nitric oxide protects against oxidative stress.

One of the many biological functions of nitric oxide is the ability to protect cells from oxidative stress. To investigate the potential contribution of low steady state levels of nitric oxide generated by endothelial nitric oxide synthase (eNOS) and the mechanisms of protection against H2O2, spontaneously transformed human ECV304 cells, which normally do not express eNOS, were stably transfected with a green fluorescent-tagged eNOS cDNA. The eNOS-transfected cells were found to be resistant to injury and delayed death following a 2-h exposure to H2O2 (50–150 μM). Inhibition of nitric oxide synthesis abolished the protective effect against H2O2 exposure. The ability of nitric oxide to protect cells depended on the presence of respiring mitochondria as ECV304+eNOS cells with diminished mitochondria respiration (ρ−) are injured to the same extent as nontransfected ECV304 cells and recovery of mitochondrial respiration restores the ability of nitric oxide to protect against H2O2-induced death. Nitric oxide also found to have a profound effect in cell metabolism, because ECV304+eNOS cells had lower steady state levels of ATP and higher utilization of glucose via the glycolytic pathway than ECV304 cells. However, the protective effect of nitric oxide against H2O2 exposure is not reproduced in ECV304 cells after treatment with azide and oligomycin suggesting that the dynamic regulation of respiration by nitric oxide represent a critical and unrecognized primary line of defense against oxidative stress.

Quantitative proteomics of caveolin-1-regulated proteins: characterization of polymerase i and transcript release factor/CAVIN-1 IN endothelial cells.

Caveolae are organelles abundant in the plasma membrane of many specialized cells including endothelial cells (ECs), epithelial cells, and adipocytes, and in these cells, caveolin-1 (Cav-1) is the major coat protein essential for the formation of caveolae. To identify proteins that require Cav-1 for stable incorporation into membrane raft domains, a quantitative proteomics analysis using isobaric tagging for relative and absolute quantification was performed on rafts isolated from wild-type and Cav-1-deficient mice. In three independent experiments, 117 proteins were consistently identified in membrane rafts with the largest differences in the levels of Cav-2 and in the caveola regulatory proteins Cavin-1 and Cavin-2. Because the lung is highly enriched in ECs, we validated and characterized the role of the newly described protein Cavin-1 in several cardiovascular tissues and in ECs. Cavin-1 was highly expressed in ECs lining blood vessels and in cultured ECs. Knockdown of Cavin-1 reduced the levels of Cav-1 and -2 and weakly influenced the formation of high molecular weight oligomers containing Cav-1 and -2. Cavin-1 silencing enhanced basal nitric oxide release from ECs but blocked proangiogenic phenotypes such as EC proliferation, migration, and morphogenesis in vitro. Thus, these data support an important role of Cavin-1 as a regulator of caveola function in ECs.

Endothelial cells isolated from caveolin-2 knockout mice display higher proliferation rate and cell cycle progression relative to their wild-type counterparts

The goal of this study was to determine whether caveolin-2 (Cav-2) is capable of controlling endothelial cell (EC) proliferation in vitro. To realize this goal, we have directly compared proliferation rates and cell cycle-associated signaling proteins between lung ECs isolated from wild-type (WT) and Cav-2 knockout (KO) mice. Using three independent proliferation assays, we have determined that Cav-2 KO ECs proliferate by ca. 2-fold faster than their WT counterparts. Cell cycle analysis by flow cytometry of propidium iodide-stained cells showed a relatively higher percentage of Cav-2 KO ECs in S and G(2)/M and lower percentage in G(o)/G(1) phases of cell cycle relative to their WT counterparts. Furthermore, an over 2-fold increase in the percentage of S phase-associated Cav-2 KO relative to WT ECs was independently determined with bromodeoxyuridine incorporation assay. Mechanistically, the increase in proliferation/cell cycle progression of Cav-2 KO ECs correlated well with elevated expression levels of predominantly S phase- and G(2)/M phase-associated cyclin A and B1, respectively. Further mechanistic analysis of molecular events controlling cell cycle progression revealed increased level of hyperphosphorylated (inactive) form of G(1) to S phase transition inhibitor, the retinoblastoma protein in hyperproliferating Cav-2 KO ECs. Conversely, the expression level of the two cyclin-dependent kinase inhibitors p16(INK4) and p27(Kip1) was reduced in Cav-2 KO ECs. Finally, increased phosphorylation (activation) of proproliferative extracellular signal-regulated kinase 1/2 was observed in hyperproliferating Cav-2 KO ECs. Overall, our data suggest that Cav-2 negatively regulates lung EC proliferation and cell cycle progression.

Caveolin-2 is a negative regulator of anti-proliferative function and signaling of transforming growth factor-β in endothelial cells.

Using a combination of wild-type (WT) and caveolin-2 (Cav-2) knockout along with retroviral reexpression approaches, we provide the evidence for the negative role of Cav-2 in regulating anti-proliferative function and signaling of transforming growth factor β (TGF-β) in endothelial cells (ECs). Although, TGF-β had a modest inhibitory effect on WT ECs, it profoundly inhibited proliferation of Cav-2 knockout ECs. To confirm the specificity of the observed difference in response to TGF-β, we have stably reexpressed Cav-2 in Cav-2 knockout ECs using a retroviral approach. Similar to WT ECs, the anti-proliferative effect of TGF-β was dramatically reduced in the Cav-2 reexpressing ECs. The reduced anti-proliferative effect of TGF-β in Cav-2-positive cells was evidenced by three independent proliferation assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell count, and bromodeoxyuridine incorporation and correlated with a loss of TGF-β-mediated upregulation of cell cycle inhibitor p27 and subsequent reduction of the levels of hyperphosphorylated (inactive) form of the retinoblastoma protein in Cav-2 reexpressing ECs. Mechanistically, Cav-2 inhibits anti-proliferative action of TGF-β by suppressing Alk5-Smad2/3 pathway manifested by reduced magnitude and length of TGF-β-induced Smad2/3 phosphorylation as well as activation of activin receptor-like kinase-5 (Alk5)-Smad2/3 target genes plasminogen activator inhibitor-1 and collagen type I in Cav-2-positive ECs. Expression of Cav-2 does not appear to significantly change targeting of TGF-β receptors I and Smad2/3 to caveolar and lipid raft microdomains as determined by sucrose fractionation gradient. Overall, the negative regulation of TGF-β signaling and function by Cav-2 is independent of Cav-1 expression levels and is not because of changing targeting of Cav-1 protein to plasma membrane lipid raft/caveolar domains.