Phosphotyrosine protein dynamics in cell membrane rafts of sphingosine-1-phosphate-stimulated human endothelium: Role in barrier enhancement

Sphingosine-1-phosphate (S1P), a lipid growth factor, is critical to the maintenance and enhancement of vascular barrier function via processes highly dependent upon cell membrane raft-mediated signaling events. Anti-phosphotyrosine 2 dimensional gel electrophoresis (2-DE) immunoblots confirmed that disruption of membrane raft formation (via methyl-β-cyclodextrin) inhibits S1P-induced protein tyrosine phosphorylation. To explore S1P-induced dynamic changes in membrane rafts, we used 2-D techniques to define proteins within detergent-resistant cell membrane rafts which are differentially expressed in S1P-challenged (1 μM, 5 min) human pulmonary artery endothelial cells (EC), with 57 protein spots exhibiting > 3-fold change. S1P induced the recruitment of over 20 cell membrane raft proteins exhibiting increasing levels of tyrosine phosphorylation including known barrier-regulatory proteins such as focal adhesion kinase (FAK), cortactin, p85α phosphatidylinositol 3-kinase (p85αPI3K), myosin light chain kinase (nmMLCK), filamin A/C, and the non-receptor tyrosine kinase, c-Abl. Reduced expression of either FAK, MLCK, cortactin, filamin A or filamin C by siRNA transfection significantly attenuated S1P-induced EC barrier enhancement. Furthermore, S1P induced cell membrane raft components, p-caveolin-1 and glycosphingolipid (GM1), to the plasma membrane and enhanced co-localization of membrane rafts with p-caveolin-1 and p-nmMLCK. These results suggest that S1P induces both the tyrosine phosphorylation and recruitment of key actin cytoskeletal proteins to membrane rafts, resulting in enhanced human EC barrier function.     

Pulmonary endothelial cell barrier enhancement by FTY720 does not require the S1P1 receptor

Novel therapeutic strategies are needed to reverse the loss of endothelial cell (EC) barrier integrity that occurs during inflammatory disease states such as acute lung injury. We previously demonstrated potent EC barrier augmentation in vivo and in vitro by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P) via ligation of the S1P1 receptor. The S1P analogue, FTY720, similarly exerts barrier-protective vascular effects via presumed S1P1 receptor ligation. We examined the role of the S1P1 receptor in sphingolipid-mediated human lung EC barrier enhancement. Both S1P and FTY-induced sustained, dose-dependent barrier enhancement, reflected by increases in transendothelial electrical resistance (TER), which was abolished by pertussis toxin indicating Gi-coupled receptor activation. FTY-mediated increases in TER exhibited significantly delayed onset and intensity relative to the S1P response. Reduction of S1P1R expression (via siRNA) attenuated S1P-induced TER elevations whereas the TER response to FTY was unaffected. Both S1P and FTY rapidly (within 5 min) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated S1P1R phosphorylation on threonine residues. Inhibition of PI3 kinase activity attenuated S1P-mediated TER increases but failed to alter FTY-induced TER elevation. Finally, S1P, but not FTY, induced significant myosin light chain phosphorylation and dramatic actin cytoskeletal rearrangement whereas reduced expression of the cytoskeletal effectors, Rac1 and cortactin (via siRNA), attenuated S1P-, but not FTY-induced TER elevations. These results mechanistically characterize pulmonary vascular barrier regulation by FTY720, suggesting a novel barrier-enhancing pathway for modulating vascular permeability.     

Abl Tyrosine Kinase Phosphorylates Nonmuscle Myosin Light Chain Kinase to Regulate Endothelial Barrier Function

Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl–mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y²³¹, Y⁴⁶⁴, Y⁵⁵⁶, Y⁸⁴⁶) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement.     

Sphingosine-1-phosphate promotes barrier-stabilizing effects in human microvascular endothelial cells via AMPK-dependent mechanisms

Breakdown of the endothelial barrier is a critical step in the development of organ failure in severe inflammatory conditions such as sepsis. Endothelial cells from different tissues show phenotypic variations which are often neglected in endothelial research. Sphingosine-1-phosphate (S1P) and AMP-dependent kinase (AMPK) have been shown to protect the endothelium and phosphorylation of AMPK by S1P was shown in several cell types. However, the role of the S1P-AMPK interrelationship for endothelial barrier stabilization has not been investigated. To assess the role of the S1P-AMPK signalling axis in this context, we established an in vitro model allowing real-time monitoring of endothelial barrier function in human microvascular endothelial cells (HMEC-1) and murine glomerular endothelial cells (GENCs) with the electric cell-substrate impedance sensing (ECIS™) system. Following the disruption of the cell barrier by co-administration of LPS, TNF-α, IL-1ß, IFN-γ, and IL–6, we demonstrated self-recovery of the disrupted barrier in HMEC-1, while the barrier remained compromised in GENCs. Under physiological conditions we observed a rapid phosphorylation of AMPK in HMEC-1 stimulated with S1P, but not in GENCs. Consistently, S1P enhanced the basal endothelial barrier in HMEC-1 exclusively. siRNA-mediated knockdown of AMPK in HMEC-1 led to a less pronounced barrier enhancement. Thus we present evidence for a functional role of AMPK in S1P-mediated barrier stabilization in HMEC-1 and we provide insight into cell-type specific differences of the S1P-AMPK-interrelationship, which might influence the development of interventional strategies targeting endothelial barrier dysfunction.     

Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and β-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.     

The MARCKS family of phospholipid binding proteins: regulation of phospholipase D and other cellular components.

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) are essential proteins that are implicated in coordination of membrane-cytoskeletal signalling events, such as cell adhesion, migration, secretion, and phagocytosis in a variety of cell types. The most prominent structural feature of MARCKS and MRP is a central basic effector domain (ED) that binds F-actin, Ca2+-calmodulin, and acidic phospholipids; phosphorylation of key serine residues within the ED by protein kinase C (PKC) prevents the above interactions. While the precise roles of MARCKS and MRP have not been established, recent attention has focussed on the high affinity of the MARCKS ED for phosphatidylinositol 4,5-bisphosphate (PIP2), and a model has emerged in which calmodulin- or PKC-mediated regulation of these proteins at specific membrane sites could in turn control spatial availability of PIP2. The present review summarizes recent progress in this area and discusses how the above model might explain a role for MARCKS and MRP in activation of phospholipase D and other PIP2-dependent cellular processes.     

Sphingosine 1-phosphate protects human umbilical vein endothelial cells from serum-deprived apoptosis by nitric oxide production

Sphingosine 1-phosphate (S1P) can prevent endothelial cell apoptosis. We investigated the molecular mechanisms and signaling pathways by which S1P protects endothelial cells from serum deprivation-induced apoptosis. We show here that human umbilical vein endothelial cells (HUVECs) undergo apoptosis associated with increased DEVDase activity, caspase-3 activation, cytochrome c release, and DNA fragmentation after 24 h of serum deprivation. These apoptotic markers were suppressed by the addition of S1P, the NO donor S-nitroso-N-acetylpenicillamine (100 micrometer), or caspase-3 inhibitor z-VAD-fmk. The protective effects of S1P were reversed by the nitric-oxide synthase (NOS) inhibitor N-monomethyl-l-arginine, but not by the soluble guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo[4,3-a]-quanoxaline-1-one, suggesting that NO, but not cGMP, is responsible for S1P protection from apoptosis. Furthermore, S1P increased NO production by enhancing Ca(2+)-sensitive NOS activity without changes in the eNOS protein level. S1P-mediated cell survival and NO production were suppressed significantly by pretreatment with antisense oligonucleotide of EDG-1 and partially by EDG-3 antisense. S1P-mediated NO production was suppressed by the addition of pertussis toxin, an inhibitor of G(i) proteins, the specific inhibitor of phospholipase C (PLC), and the Ca(2+) chelator BAPTA-AM. These findings indicate that S1P protects HUVECs from apoptosis through the activation of eNOS activity mainly through an EDG-1 and -3/G(i)/PLC/Ca(2+) signaling pathway.     

Rac1 modulates sphingosine 1-phosphate-mediated activation of phosphoinositide 3-kinase/Akt signaling pathways in vascular endothelial cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that activates G protein-coupled S1P receptors and initiates a broad range of responses in vascular endothelial cells. The small GTPase Rac1 is implicated in diverse S1P-modulated cellular responses in endothelial cells, yet the molecular mechanisms involved in S1P-mediated Rac1 activation are incompletely understood. We studied the pathways involved in S1P-mediated Rac1 activation in bovine aortic endothelial cells (BAEC) and found that S1P-induced Rac1 activation is impaired following chelation of G protein betagamma subunits by transfection of betaARKct. Treatment with the Src tyrosine kinase inhibitor PP2 completely attenuated S1P-mediated Rac1 activation; however, pretreatment of BAEC with wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase, had no effect on Rac1 activation while completely blocking S1P-induced Akt phosphorylation. We used Rac1-specific small interfering RNA (siRNA) duplexes to "knock down" endogenous Rac1 expression and found that siRNA-mediated Rac1 knockdown significantly impaired basal as well as S1P-induced phosphorylation of protein kinase Akt, as well as several downstream targets of Akt including endothelial nitric-oxide synthase and glycogen synthase kinase 3beta. By contrast, S1P-induced phosphorylation of the mitogen-activated protein kinases ERK1/2 was unperturbed by siRNA-mediated Rac1 knockdown. We found that overexpression of the Rac1 guanine nucleotide exchange factor (GEF) Tiam1 markedly enhanced Rac1 activity, whereas a dominant negative Tiam1 mutant significantly attenuated S1P-mediated Rac1 activation. Taken together, these studies identify G protein betagamma subunits, Src kinase and the GEF Tiam1 as upstream modulators of S1P-mediated Rac1 activation, and establish a central role for Rac1 in S1P-mediated activation of PI 3-kinase/Akt/endothelial nitric-oxide synthase signaling in vascular endothelial cells.     

Regulation of MARCKS and MARCKS-related protein expression in BV-2 microglial cells in response to lipopolysaccharide

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) have been implicated in membrane-cytoskeletal events underlying cell adhesion, migration, secretion, and phagocytosis. In BV-2 microglial cells, lipopolysaccharide (LPS) elicited a dose-dependent increase in mRNA of both MRP (sixfold) and MARCKS (threefold) with corresponding increases in [3H]myristoylated and immunoreactive protein levels. LPS also produced significant increases in protein kinase C (PKC)-beta twofold and PKC-epsilon (1.5-fold). Pro-inflammatory cytokines produced by activated microglia (IL-1beta, IL-6, TNF-alpha) did not mimic LPS effects on MARCKS or MRP expression when added individually or in combination. LPS and IFN-gamma produced a synergistic induction of iNOS but not MARCKS or MRP. Induction of MARCKS and MRP by LPS was completely blocked by inhibitors of NF-kappaB (PDTC) and protein tyrosine kinases (herbimycin A), partially blocked by the p38 kinase inhibitor SB203580, and unaffected by the MEK inhibitor PD98059. LPS induction of iNOS was considerably more sensitive to all these inhibitors. The Src kinase inhibitor PP2 had no effect, while the closely related inhibitor PP1 actually increased LPS induction of MARCKS and MRP. Our results suggest that MARCKS and MRP may play an important role in LPS-activated microglia, but are not part of the neuroinflammatory response produced by cytokines.     

Dual roles of tight junction-associated protein, zonula occludens-1, in sphingosine 1-phosphate-mediated endothelial chemotaxis and barrier integrity

In this report, sphingosine-1-phosphate (S1P), a serum-borne bioactive lipid, is shown to activate tight-junction-associated protein Zonula Occludens-1 (ZO-1), which in turn plays a critical role in regulating endothelial chemotaxis and barrier integrity. After S1P stimulation, ZO-1 was redistributed to the lamellipodia and cell-cell junctions via the S1P1/G(i)/Akt/Rac pathway. Similarly, both endothelial barrier integrity and cell motility were significantly enhanced in S1P-treated cells through the G(i)/Akt/Rac pathway. Importantly, S1P-enhanced barrier integrity and cell migration were abrogated in ZO-1 knockdown cells, indicating ZO-1 is functionally indispensable for these processes. To investigate the underlying mechanisms, we demonstrated that cortactin plays a critical role in S1P-induced ZO-1 redistribution to the lamellipodia. In addition, S1P significantly induced the formation of endothelial tight junctions. ZO-1 and alpha-catenin polypeptides were colocalized in S1P-induced junctional structures; whereas, cortactin was not observed in these regions. Together, these results suggest that S1P induces the formation of two distinct ZO-1 complexes to regulate two different endothelial functions: ZO-1/cortactin complexes to regulate chemotactic response and ZO-1/alpha-catenin complexes to regulate endothelial barrier integrity. The concerted operation of these two ZO-1 complexes may coordinate two important S1P-mediated functions, i.e. migration and barrier integrity, in vascular endothelial cells.     

Regulation of the micromechanical properties of pulmonary endothelium by S1P and thrombin: role of cortactin

Disruption of pulmonary endothelial cell (EC) barrier function is a critical pathophysiologic event in highly morbid inflammatory conditions such as sepsis and acute respiratory disease stress syndrome. Actin cytoskeleton, an essential regulator of endothelial permeability, is a dynamic structure whose stimuli-induced rearrangement is linked to barrier modulation. Here, we used atomic force microscopy to characterize structural and mechanical changes in the F-actin cytoskeleton of cultured human pulmonary artery EC in response to both barrier-enhancing (induced by sphingosine 1-phosphate (S1P)) and barrier-disrupting (induced by thrombin) conditions. Atomic force microscopy elasticity measurements show differential effects: for the barrier protecting molecule S1P, the elastic modulus was elevated significantly on the periphery; for the barrier-disrupting molecule thrombin, on the other hand, it was elevated significantly in the central region of the cell. The force and elasticity maps correlate with F-actin rearrangements as identified by immunofluorescence analysis. Significantly, reduced expression (via siRNA) of cortactin, an actin-binding protein essential to EC barrier regulation, resulted in a shift in the S1P-mediated elasticity pattern to more closely resemble control, unstimulated endothelium.     

Sphingosine 1-Phosphate (S1P) Carrier-dependent Regulation of Endothelial Barrier: HIGH DENSITY LIPOPROTEIN (HDL)-S1P PROLONGS ENDOTHELIAL BARRIER ENHANCEMENT AS COMPARED WITH ALBUMIN-S1P VIA EFFECTS ON LEVELS,TRAFFICKING, AND SIGNALING OF S1P1*

Sphingosine 1-phosphate (S1P) is a blood-borne lysosphingolipid that acts to promote endothelial cell (EC) barrier function. In plasma, S1P is associated with both high density lipoproteins (HDL) and albumin, but it is not known whether the carriers impart different effects on S1P signaling. Here we establish that HDL-S1P sustains EC barrier longer than albumin-S1P. We showed that the sustained barrier effects of HDL-S1P are dependent on signaling by the S1P receptor, S1P1, and involve persistent activation of Akt and endothelial NOS (eNOS), as well as activity of the downstream NO target, soluble guanylate cyclase (sGC). Total S1P1 protein levels were found to be higher in response to HDL-S1P treatment as compared with albumin-S1P, and this effect was not associated with increased S1P1 mRNA or dependent on de novo protein synthesis. Several pieces of evidence indicate that long term EC barrier enhancement activity of HDL-S1P is due to specific effects on S1P1 trafficking. First, the rate of S1P1 degradation, which is proteasome-mediated, was slower in HDL-S1P-treated cells as compared with cells treated with albumin-S1P. Second, the long term barrier-promoting effects of HDL-S1P were abrogated by treatment with the recycling blocker, monensin. Finally, cell surface levels of S1P1 and levels of S1P1 in caveolin-enriched microdomains were higher after treatment with HDL-S1P as compared with albumin-S1P. Together, the findings reveal S1P carrier-specific effects on S1P1 and point to HDL as the physiological mediator of sustained S1P1-PI3K-Akt-eNOS-sGC-dependent EC barrier function.     

Myristoylation-dependent N-terminal cleavage of the myristoylated alanine-rich C kinase substrate (MARCKS) by cellular extracts

The myristoylated alanine-rich C kinase substrate (MARCKS) has been proposed to regulate the plasticity of the actin cytoskeleton at its site of attachment to membranes. In macrophages, MARCKS is implicated in various cellular events including motility, adhesion and phagocytosis. In this report we show that macrophage extracts contain a protease which specifically cleaves human MARCKS, expressed in a cell-free system or in E. coli, between Lys-6 and Thr-7. Cleavage of MARCKS decreases its affinity for macrophage membranes by ca. one order of magnitude, highlighting the contribution of the myristoyl moiety of MARCKS to membrane binding. Importantly, cleavage requires myristoylation of MARCKS. Furthermore, MARCKS-related protein (MRP), the second member of the MARCKS family, is not digested. Since Thr-7 is lacking in MRP this suggests that Thr-7 at the P1 position is important for the recognition of lipid-modified substrates. A different product is observed when MARCKS is incubated with a calf brain cytosolic extract. This product can be remyristoylated in the presence of myristoyl-CoA and N-myristoyl transferase, demonstrating that cycles of myristoylation/demyristoylation of MARCKS can be achieved in vitro. Although the physiological relevance of these enzymes still needs to be demonstrated, our results reveal the presence of a new class of cleaving enzymes recognizing lipid-modified protein substrates.     

High density lipoprotein-associated sphingosine 1-phosphate promotes endothelial barrier function

High density lipoproteins (HDL) are major plasma carriers of sphingosine 1-phosphate (S1P). Here we show that HDL increases endothelial barrier integrity as measured by electric cell substrate impedance sensing. S1P was implicated as the mediator in this process through findings showing that pertussis toxin, an inhibitor of G(i)-coupled S1P receptors, as well as antagonists of the S1P receptor, S1P1, inhibited barrier enhancement by HDL. Additional findings show that HDL stimulates endothelial cell activation of Erk1/2 and Akt, signaling pathway intermediates that have been implicated in S1P-dependent endothelial barrier activity. HDL was also found to promote endothelial cell motility, a process that may also relate to endothelial barrier function in the context of a vascular injury response. The effects of HDL on endothelial cell Erk1/2 and Akt activation and motility were suppressed by pertussis toxin and S1P1 antagonists. However, both HDL-induced barrier enhancement and HDL-induced motility showed a greater dependence on Akt activation as compared with Erk1/2 activation. Together, the findings indicate that HDL has endothelial barrier promoting activities, which are attributable to its S1P component and signaling through the S1P1/Akt pathway.     

S1P induces FA remodeling in human pulmonary endothelial cells: role of Rac, GIT1, FAK, and paxillin.

Sphingosine 1-phosphate (S1P) enhances human pulmonary endothelial monolayer integrity via Rac GTPase-dependent formation of a cortical actin ring (Garcia et al. J Clin Invest 108: 689-701, 2001). The mechanisms underlying this response are not well understood but may involve rapid redistribution of focal adhesions (FA) as attachment sites for actin filaments. We evaluate the effects of S1P on the redistribution of paxillin, FA kinase (FAK), and the G protein-coupled receptor kinase-interacting proteins (GITs). S1P induced Rac GTPase activation and cortical actin ring formation at physiological concentrations (0.5 microM), whereas 5 microM S1P caused prominent stress fiber formation and activation of Rho and Rac GTPases. S1P (0.5 microM) stimulated the tyrosine phosphorylation of FAK Y(576), and paxillin was linked to FA disruption and redistribution to the cell periphery. Furthermore, S1P induced a transient association of GIT1 with paxillin and redistribution of the GIT2-paxillin complex to the cell cortical area without affecting GIT2-paxillin association. These results suggest a role of FA rearrangement in S1P-mediated barrier enhancement via Rac- and GIT-mediated processes.     

Down-regulation of MARCKS-related protein (MRP) in macrophages infected with Leishmania.

Leishmania, a protozoan parasite of macrophages, has been shown to interfere with host cell signal transduction pathways including protein kinase C (PKC)-dependent signaling. Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP, MacMARCKS) are PKC substrates in diverse cell types. MARCKS and MRP are thought to regulate the actin network and thereby participate in cellular responses involving cytoskeletal rearrangement. Because MRP is a major PKC substrate in macrophages, we examined its expression in response to infection by Leishmania. Activation of murine macrophages by cytokines increased MRP expression as determined by Western blot analysis. Infection with Leishmania promastigotes at the time of activation or up to 48 h postactivation strongly decreased MRP levels. Leishmania-dependent MRP depletion was confirmed by [3H]myristate labeling and by immunofluorescence microscopy. All species or strains of Leishmania parasites tested, including lipophosphoglycan-deficient Leishmania major L119, decreased MRP levels. MRP depletion was not obtained with other phagocytic stimuli including zymosan, latex beads, or heat-killed Streptococcus mitis, a Gram-positive bacterium. Experiments with [3H]myristate labeled proteins revealed the appearance of lower molecular weight fragments in Leishmania-infected cells suggesting that MRP depletion may be due to proteolytic degradation.     

Investigation into the mechanism regulating MRP localization

The major PKC substrates MARCKS and MacMARCKS (MRP) are membrane-binding proteins implicated in cell spreading, integrin activation and exocytosis. According to the myristoyl-electrostatic switch model the co-operation between the myristoyl moiety and the positively charged effector domain (ED) is an essential mechanism by which proteins bind to membranes. Loss of the electrostatic interaction between the ED and phospholipids, such as Ptdins(4,5)P2, results in the translocation of such proteins to the cytoplasm. While this model has been extensively tested for the binding of MARCKS far less is known about the mechanisms regulating MRP localization. We demonstrate that after phosphorylation, MRP is relocated to the intracellular membranes of late endosomes and lysosomes. MRP binds to all membranes via its myristoyl moiety, but for its localization at the plasma membrane the ED is also required. Although the ED of MRP can bind to Ptdins(4,5)P2 in vitro, this binding is not essential for its retention at or targeting to the plasma membrane. We conclude that the co-operation between the myristoyl moiety and the ED is not required for the binding to membranes in general but that it is essential for the targeting of MRP to the plasma membrane in a Ptdins(4,5)P2-independent manner.     

Edaravone mimics sphingosine-1-phosphate-induced endothelial barrier enhancement in human microvascular endothelial cells

Edaravone is a potent scavenger of hydroxyl radicals and is quite successful in patients with acute cerebral ischemia, and several organ-protective effects have been reported. Treatment of human microvascular endothelial cells with edaravone (1.5 microM) resulted in the enhancement of transmonolayer electrical resistance coincident with cortical actin enhancement and redistribution of focal adhesion proteins and adherens junction proteins to the cell periphery. Edaravone also induced small GTPase Rac activation and focal adhesion kinase (FAK; Tyr(576)) phosphorylation associated with sphingosine-1-phosphate receptor type 1 (S1P(1)) transactivation. S1P(1) protein depletion by the short interfering RNA technique completely abolished edaravone-induced FAK (Tyr(576)) phosphorylation and Rac activation. This is the first report of edaravone-induced endothelial barrier enhancement coincident with focal adhesion remodeling and cytoskeletal rearrangement associated with Rac activation via S1P(1) transactivation. Considering the well-established endothelial barrier-protective effect of S1P, endothelial barrier enhancement as a consequence of S1P(1) transactivation may at least partly be the potent mechanisms for the organ-protective effect of edaravone and is suggestive of edaravone as a therapeutic agent against systemic vascular barrier disorder.     

Function of MRP1/ABCC1 is not dependent on cholesterol or cholesterol-stabilized lipid rafts

MRP1 (multidrug-resistance-related protein 1)/ABCC1 (ATP-binding cassette transporter C1) has been localized in cholesterol-enriched lipid rafts, which suggests a role for these lipid rafts and/or cholesterol in MRP1 function. In the present study, we have shown for the first time that nearly complete oxidation of free cholesterol in the plasma membrane of BHK-MRP1 (MRP1-expressing baby hamster kidney) cells did not affect MRP1 localization in lipid rafts or its efflux function, using 5-carboxyfluorescein diacetate as a substrate. Inhibition of cholesterol biosynthesis, using lovastatin in combination with RO 48-8071, an inhibitor of oxidosqualene cyclase, resulted in a shift of MRP1 out of lipid raft fractions, but did not affect MRP1-mediated efflux in Neuro-2a (neuroblastoma) cells. Short-term methyl-β-cyclodextrin treatment was equally effective in removing free cholesterol from Neuro-2a and BHK-MRP1 cells, but affected MRP1 function only in the latter. The kinetics of loss of both MRP1 efflux function and lipid raft association during long-term methyl-β-cyclodextrin treatment did not match the kinetics of free cholesterol removal in both cell lines. Moreover, MRP1 activity was measured in vesicles consisting of membranes isolated from BHK-MRP1 cells using the substrate cysteinyl leukotriene C4 and was not changed when the free cholesterol level of these membranes was either decreased or increased. In conclusion, MRP1 activity is not correlated with the level of free cholesterol or with localization in cholesterol-dependent lipid rafts.     

The myeloid expressed EF-hand proteins display a diverse pattern of lipid raft association

EF-hand proteins are known to translocate to membranes, suggesting that they are involved in signaling events located in the cell membrane. Many proteins involved in signaling events associate cholesterol rich membrane domains, so called lipid rafts, which serve as platforms for controlled protein-protein interaction. Here, we demonstrate that the myeloid expressed EF-hand proteins can be distinguished into three classes with respect to their membrane association. Grancalcin, a myeloid expressed penta EF-hand protein, is constitutively located in lipid rafts. S100A9 (MRP14) and S100A8 (MRP8) are translocated into detergent resistant lipid structures only after calcium activation of the neutrophils. However, the S100A9/A8 membrane association is cholesterol and sphingolipid independent. On the other hand, the association of S100A12 (EN-RAGE) and S100A6 (calcyclin) with membranes is detergent sensitive. These diverse affinities to lipid structures of the myeloid expressed EF-hand proteins most likely reflect their different functions in neutrophils.