Intracellular generation of sphingosine 1-phosphate in human lung endothelial cells: role of lipid phosphate phosphatase-1 and sphingosine kinase 1

Sphingosine 1-phosphate (S1P) regulates diverse cellular functions through extracellular ligation to S1P receptors, and it also functions as an intracellular second messenger. Human pulmonary artery endothelial cells (HPAECs) effectively utilized exogenous S1P to generate intracellular S1P. We, therefore, examined the role of lipid phosphate phosphatase (LPP)-1 and sphingosine kinase1 (SphK1) in converting exogenous S1P to intracellular S1P. Exposure of (32)P-labeled HPAECs to S1P or sphingosine (Sph) increased the intracellular accumulation of [(32)P]S1P in a dose- and time-dependent manner. The S1P formed in the cells was not released into the medium. The exogenously added S1P did not stimulate the sphingomyelinase pathway; however, added [(3)H]S1P was hydrolyzed to [(3)H]Sph in HPAECs, and this was blocked by XY-14, an inhibitor of LPPs. HPAECs expressed LPP1-3, and overexpression of LPP-1 enhanced the hydrolysis of exogenous [(3)H]S1P to [(3)H]Sph and increased intracellular S1P production by 2-3-fold compared with vector control cells. Down-regulation of LPP-1 by siRNA decreased intracellular S1P production from extracellular S1P but had no effect on the phosphorylation of Sph to S1P. Knockdown of SphK1, but not SphK2, by siRNA attenuated the intracellular generation of S1P. Overexpression of wild type SphK1, but not SphK2 wild type, increased the accumulation of intracellular S1P after exposure to extracellular S1P. These studies provide the first direct evidence for a novel pathway of intracellular S1P generation. This involves the conversion of extracellular S1P to Sph by LPP-1, which facilitates Sph uptake, followed by the intracellular conversion of Sph to S1P by SphK1.     

Sphingosine kinase 1 and sphingosine 1-phosphate receptor 3 are functionally upregulated on astrocytes under pro-inflammatory conditions

Background

Reactive astrocytes are implicated in the development and maintenance of neuroinflammation in the demyelinating disease multiple sclerosis (MS). The sphingosine kinase 1 (SphK1)/sphingosine1-phosphate (S1P) receptor signaling pathway is involved in modulation of the inflammatory response in many cell types, but the role of S1P receptor subtype 3 (S1P₃) signaling and SphK1 in activated rat astrocytes has not been defined.

Methodology/Principal Findings

Using immunohistochemistry we observed the upregulation of S1P₃ and SphK1 expression on reactive astrocytes and SphK1 on macrophages in MS lesions. Increased mRNA and protein expression of S1P₃ and SphK1, as measured by qPCR and Western blotting respectively, was observed after treatment of rat primary astrocyte cultures with the pro-inflammatory stimulus lipopolysaccharide (LPS). Activation of SphK by LPS stimulation was confirmed by SphK activity assay and was blocked by the use of the SphK inhibitor SKI (2-(p-hydroxyanilino)-4-(p-chlorphenyl) thiazole. Treatment of astrocytes with a selective S1P₃ agonist led to increased phosphorylation of extracellular signal-regulated kinase (ERK)-1/2), which was further elevated with a LPS pre-challenge, suggesting that S1P₃ upregulation can lead to increased functionality. Moreover, astrocyte migration in a scratch assay was induced by S1P and LPS and this LPS-induced migration was sensitive to inhibition of SphK1, and independent of cell proliferation. In addition, S1P induced secretion of the potentially neuroprotective chemokine CXCL1, which was increased when astrocytes were pre-challenged with LPS. A more prominent role of S1P₃ signaling compared to S1P₁ signaling was demonstrated by the use of selective S1P₃ or S1P₁ agonists.

Conclusion/Significance

In summary, our data demonstrate that the SphK1/S1P₃ signaling axis is upregulated when astrocytes are activated by LPS. This signaling pathway appears to play a role in the establishment and maintenance of astrocyte activation. Upregulation of the pathway in MS may be detrimental, e.g. through enhancing astrogliosis, or beneficial through increased remyelination via CXCL1.     

Protein kinase C-epsilon regulates sphingosine 1-phosphate-mediated migration of human lung endothelial cells through activation of phospholipase D2, protein kinase C-zeta, and Rac1

The signaling pathways by which sphingosine 1-phosphate (S1P) potently stimulates endothelial cell migration and angiogenesis are not yet fully defined. We, therefore, investigated the role of protein kinase C (PKC) isoforms, phospholipase D (PLD), and Rac in S1P-induced migration of human pulmonary artery endothelial cells (HPAECs). S1P-induced migration was sensitive to S1P(1) small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S1P(1) to G(i). Overexpression of dominant negative (dn) PKC-epsilon or -zeta, but not PKC-alpha or -delta, blocked S1P-induced migration. Although S1P activated both PLD1 and PLD2, S1P-induced migration was attenuated by knocking down PLD2 or expressing dnPLD2 but not PLD1. Blocking PKC-epsilon, but not PKC-zeta, activity attenuated S1P-mediated PLD stimulation, demonstrating that PKC-epsilon, but not PKC-zeta, was upstream of PLD. Transfection of HPAECs with dnRac1 or Rac1 siRNA attenuated S1P-induced migration. Furthermore, transfection with PLD2 siRNA, infection of HPAECs with dnPKC-zeta, or treatment with myristoylated PKC-zeta peptide inhibitor abrogated S1P-induced Rac1 activation. These results establish that S1P signals through S1P(1) and G(i) to activate PKC-epsilon and, subsequently, a PLD2-PKC-zeta-Rac1 cascade. Activation of this pathway is necessary to stimulate the migration of lung endothelial cells, a key component of the angiogenic process.     

Filamin A links sphingosine kinase 1 and sphingosine-1-phosphate receptor 1 at lamellipodia to orchestrate cell migration

Sphingosine kinase 1 (SphK1) catalyzes the phosphorylation of sphingosine to produce the potent lipid mediator sphingosine-1-phosphate (S1P), which plays a critical role in cell motility via its cell surface receptors. Here, we have identified filamin A (FLNa), an actin-cross-linking protein involved in cell movement, as a bona fide SphK1-interacting protein. Heregulin stimulated SphK1 activity only in FLNa-expressing A7 melanoma cells but not in FLNa-deficient cells and induced its translocation and colocalization with FLNa at lamellipodia. SphK1 was required for heregulin-induced migration, lamellipodia formation, activation of PAK1, and subsequent FLNa phosphorylation. S1P directly stimulated PAK1 kinase, suggesting that it may be a target of intracellularly generated S1P. Heregulin also induced colocalization of S1P₁ (promotility S1P receptor) but not S1P₂, with SphK1 and FLNa at membrane ruffles. Moreover, an S1P₁ antagonist inhibited the lamellipodia formation induced by heregulin. Hence, FLNa links SphK1 and S1P₁ to locally influence the dynamics of actin cytoskeletal structures by orchestrating the concerted actions of the triumvirate of SphK1, FLNa, and PAK1, each of which requires and/or regulates the actions of the others, at lamellipodia to promote cell movement.     

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.     

Hepatocyte Growth Factor-induced Asef-IQGAP1 Complex Controls Cytoskeletal Remodeling and Endothelial Barrier

Hepatocyte growth factor (HGF) attenuates agonist-induced endothelial cell (EC) permeability and increases pulmonary endothelial barrier function via Rac-dependent enhancement of the peripheral actin cytoskeleton. However, the precise mechanisms of HGF effects on the peripheral cytoskeleton are not well understood. This study evaluated a role for Rac/Cdc42-specific guanine nucleotide exchange factor Asef and the multifunctional Rac effector, IQGAP1, in the mechanism of HGF-induced EC barrier enhancement. HGF induced Asef and IQGAP1 co-localization at the cell cortical area and stimulated formation of an Asef-IQGAP1 functional protein complex. siRNA-induced knockdown of Asef or IQGAP1 attenuated HGF-induced EC barrier enhancement. Asef knockdown attenuated HGF-induced Rac activation and Rac association with IQGAP1, and it abolished both IQGAP1 accumulation at the cell cortical layer and IQGAP1 interaction with actin cytoskeletal regulators cortactin and Arp3. Asef activation state was essential for Asef interaction with IQGAP1 and protein complex accumulation at the cell periphery. In addition to the previously reported role of the IQGAP1 RasGAP-related domain in the Rac-dependent IQGAP1 activation and interaction with its targets, we show that the IQGAP1 C-terminal domain is essential for HGF-induced IQGAP1/Asef interaction and Asef-Rac-dependent activation leading to IQGAP1 interaction with Arp3 and cortactin as a positive feedback mechanism of IQGAP1 activation. These results demonstrate a novel feedback mechanism of HGF-induced endothelial barrier enhancement via Asef/IQGAP1 interactions, which regulate the level of HGF-induced Rac activation and promote cortical cytoskeletal remodeling via IQGAP1-Arp3/cortactin interactions.     

The activation of RhoC in vascular endothelial cells is required for the S1P receptor type 2-induced inhibition of angiogenesis

Sphingosine-1-phosphate (S1P) is a multifunctional phospholipid inducing a variety of cellular responses in endothelial cells (EC). S1P responses are mediated by five G protein coupled receptors of which three types (S1P₁R–S1P₃R) have been described to be of importance in vascular endothelial cells (EC). Whereas the S1P₁R regulates endothelial barrier function by coupling to Gα_i and the monomeric GTPase Rac1, the signaling pathways involved in the S1P-induced regulation of angiogenesis are ill defined. We therefore studied the sprouting of human umbilical vein EC (HUVEC) in vitro and analyzed the activation of the RhoGTPases RhoA and RhoC. Physiological relevant concentrations of S1P (100–300 nM) induce a moderate activation of RhoA and RhoC. Inhibition or siRNA-mediated depletion of the S1P₂R preferentially decreased the activation of RhoC. Both manipulations caused an increase of sprouting in a spheroid based in vitro sprouting assay. Interestingly, a similar increase in sprouting was detected after effective siRNA-mediated knockdown of RhoC. In contrast, the depletion of RhoA had no influence on sprouting. Furthermore, suppression of the activity of G proteins of the Gα_12/13 subfamily by adenoviral overexpression of the regulator of G protein signaling domain of LSC as well as siRNA-mediated knockdown of the Rho specific guanine nucleotide exchange factor leukemia associated RhoGEF (LARG) inhibited the S1P-induced activation of RhoC and concomitantly increased sprouting of HUVEC with similar efficacy. We conclude that the angiogenic sprouting of EC is suppressed via the S1P₂R subtype. Thus, the increase in basal sprouting can be attributed to blocking of the inhibitory action of autocrine S1P stimulating the S1P₂R. This inhibitory pathway involves the activation of RhoC via Gα_12/13 and LARG, while the simultaneously occurring activation of RhoA is apparently dispensable here.     

Phospholipase D-mediated Activation of IQGAP1 through Rac1 Regulates Hyperoxia-induced p47phox Translocation and Reactive Oxygen Species Generation in Lung Endothelial Cells

Phosphatidic acid generated by the activation of phospholipase D (PLD) functions as a second messenger and plays a vital role in cell signaling. Here we demonstrate that PLD-dependent generation of phosphatidic acid is critical for Rac1/IQGAP1 signal transduction, translocation of p47^phox to cell periphery, and ROS production. Exposure of [³²P]orthophosphate-labeled human pulmonary artery endothelial cells (HPAECs) to hyperoxia (95% O₂ and 5% CO₂) in the presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as evidenced by accumulation of [³²P]phosphatidylbutanol. Infection of HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated hyperoxia-induced PLD activation and accumulation of /reactive oxygen species (ROS). Conversely, overexpression of catalytically inactive mutants of PLD (hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1 or PLD2 siRNA did not augment hyperoxia-induced [³²P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia caused rapid activation and redistribution of Rac1, and IQGAP1 to cell periphery, and down-regulation of Rac1, and IQGAP1 attenuated hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA, suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally, small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin tyrosine phosphorylation and abolished Src, cortactin, and p47^phox redistribution to cell periphery. These results demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1 in tyrosine phosphorylation of Src and cortactin, as well as in p47^phox translocation and ROS formation in human lung endothelial cells.Phagocytic cells of the immune system (neutrophils, eosinophils, monocytes, and macrophages) generate superoxide ()² instrumental in the killing of invading pathogens solely by NADPH oxidase (1-3). Deficiency of results in the genetically inherited disorder chronic granulomatous disease, a condition in which the affected individuals are susceptible to infection (4). Phagocytic NADPH oxidase is activated when cytosolic p47^phox, p67^phox, and Rac2 translocate to the phagosomes and plasma membrane and form a complex with integral membrane cytochrome b₅₅₈, which, in turn, is a Nox2 (gp91^phox)/p22^phox heterodimer (5, 6). Assembly of phagocytic NADPH oxidase is initiated by two signals. The first is the phosphorylation of multiple serine and tyrosine residues in the p47^phox domain, which leads to unmasking of p47^phox SH3 domains that bind to a proline-rich target in the C terminus of p22^phox (7-10). The interaction between p47^phox and p22^phox seems to be an essential requirement for the translocation of other cytosolic components of the oxidase. The second signal is the binding of GTP to Rac2, which leads to the dissociation of Rac from Rho-GDI and binding to p67^phox, followed by translocation of p67^phox/GTP-Rac2 to the membrane (11). Nonphagocytic cells express predominantly Rac1, Tiam1 (a GEF involved in Rac1 activation), Nox1-5, and most of the other cytosolic phagocytic oxidase components (12); however, the oxidative output of non-phagocytes is much smaller compared with the phagocytes. A recent study indicates that IQGAP1, an effector of Rac1, may link Nox2 to actin, thereby enhancing ROS production and contributing to cell motility in ECs (13). The one or more mechanisms responsible for differences in the oxidative burst between the phagocytic and non-phagocytic cells are yet to be defined.We have demonstrated previously that hyperoxia activates lung endothelial NADPH oxidase, which in part is mediated by ERK, p38 MAPK (14, 15), and Src (16), and hyperoxia-induced p47^phox tyrosine phosphorylation and translocation to cell periphery is dependent on Src (16). Further, tyrosine phosphorylation of cortactin mediated by Src is essential for hyperoxia-induced p47^phox translocation and /ROS generation in HPAECs (17). In addition to Src, phosphatidic acid (PA) or diacylglycerol also stimulated phosphorylation of p47^phox and p22^phox in neutrophils both in vivo and in vitro (18-20). PA is generated in mammalian cells via de novo biosynthesis or hydrolysis of membrane phospholipids catalyzed by phospholipase D (PLD) (21-25). Activation of polymorphonuclear leukocytes with formyl-Met-Leu-Phe enhanced the oxidative burst that correlated with PA accumulation, and inclusion of short-chain primary alcohols attenuated the NADPH oxidase mediated /ROS generation, suggesting a potential role for PLD in the regulation of NADPH oxidase (12, 26, 27). However, the downstream targets of PLD that signal NADPH oxidase activation have not been fully characterized.Here, we identify for the first time that activation of IQGAP1 by Rac1 is downstream of PLD in hyperoxia-induced ROS generation. In addition, we show that activation of Rac1/IQGAP1 by PLD also regulates Src-dependent tyrosine phosphorylation of cortactin and p47^phox translocation to cell periphery. Thus, our results define a novel molecular mechanism for hyperoxia-induced NADPH oxidase activation by PLD/PA-mediated p47^phox membrane translocation via Rac1/IQGAP1/Src/cortactin signaling cascade.     

IQGAP1 Regulates Endothelial Barrier Function via EB1-Cortactin Cross Talk

Cross talk between the actin cytoskeleton and microtubules (MT) has been implicated in the amplification of agonist-induced Rho signaling, leading to increased vascular endothelial permeability. This study tested the involvement of actin-MT cross talk in the mechanisms of barrier enhancement induced by hepatocyte growth factor (HGF) and evaluated the role of the adaptor protein IQGAP1 in integrating the MT- and actin-dependent pathways of barrier enhancement. IQGAP1 knockdown by small interfering RNA attenuated the HGF-induced increase in endothelial barrier properties and abolished HGF-activated cortical actin dynamics. IQGAP1 reduction abolished HGF-induced peripheral accumulation of Rac cytoskeletal effector cortactin and cortical actin remodeling. In addition, HGF stimulated peripheral MT growth in an IQGAP1-dependent fashion. HGF also induced Rac1-dependent IQGAP1 association with the MT fraction and the formation of a protein complex containing end-binding protein 1 (EB1), IQGAP1, and cortactin. Decreasing endogenous IQGAP1 abolished HGF-induced EB1-cortactin colocalization at the cell periphery. In turn, expression of IQGAP1ΔC (IQGAP1 lacking the C-terminal domain) attenuated the cortactin association with EB1 and suppressed HGF-induced endothelial cell peripheral actin cytoskeleton enhancement. These results demonstrate for the first time the MT-actin cross talk mechanism of HGF-induced endothelial barrier enhancement and suggest that IQGAP1 functions as a hub linking HGF-induced signaling to MT and actin remodeling via EB1-IQGAP1-cortactin interactions.     

Negative regulation of endothelial morphogenesis and angiogenesis by S1P2 receptor

We speculated that the sphingosine-1-phosphate (S1P) receptor S1P(2), which uniquely inhibits cell migration, might mediate inhibitory effects on endothelial cell migration and angiogenesis, different from S1P(1) and S1P(3). Mouse vascular endothelial cells, which endogenously express S1P(2) and S1P(3), but not S1P(1), responded to S1P and epidermal growth factor (EGF) with stimulation of Rac, migration, and the formation of tube-like structures on the Matrigel. The S1P(3)-antagonist VPC-23019 abolished S1P-induced, G(i)-dependent Rac stimulation, cell migration, and tube formation, whereas the S1P(2)-antagonist JTE-013 enhanced these S1P-induced responses, suggesting that S1P(2) exerts inhibitory effects on endothelial Rac, migration, and angiogenesis. S1P(2) overexpression markedly augmented S1P-induced, G(i)-independent inhibition of EGF-induced migration and tube formation. Finally, the blockade of S1P(2) by JTE-013 potentiated S1P-induced stimulation of angiogenesis in vivo in the Matrigel implant assay. These observations indicate that in contrast to S1P(1) and S1P(3), S1P(2) negatively regulates endothelial morphogenesis and angiogenesis most likely through down-regulating Rac.     

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.     

Essential Role of Class II Phosphatidylinositol-3-kinase-C2α in Sphingosine 1-Phosphate Receptor-1-mediated Signaling and Migration in Endothelial Cells

The phosphatidylinositol (PtdIns) 3-kinase (PI3K) family regulates diverse cellular processes, including cell proliferation, migration, and vesicular trafficking, through catalyzing 3′-phosphorylation of phosphoinositides. In contrast to class I PI3Ks, including p110α and p110β, functional roles of class II PI3Ks, comprising PI3K-C2α, PI3K-C2β, and PI3K-C2γ, are little understood. The lysophospholipid mediator sphingosine 1-phosphate (S1P) plays the important roles in regulating vascular functions, including vascular formation and barrier integrity, via the G-protein-coupled receptors S1P_1–3. We studied the roles of PI3K-C2α in S1P-induced endothelial cell (EC) migration and tube formation. S1P stimulated cell migration and activation of Akt, ERK, and Rac1, the latter of which acts as a signaling molecule essential for cell migration and tube formation, via S1P₁ in ECs. Knockdown of either PI3K-C2α or class I p110β markedly inhibited S1P-induced migration, lamellipodium formation, and tube formation, whereas that of p110α or Vps34 did not. Only p110β was necessary for S1P-iduced Akt activation, but both PI3K-C2α and p110β were required for Rac1 activation. FRET imaging showed that S1P induced Rac1 activation in both the plasma membrane and PtdIns 3-phosphate (PtdIns(3)P)-enriched endosomes. Knockdown of PI3K-C2α but not p110β markedly reduced PtdIns(3)P-enriched endosomes and suppressed endosomal Rac1 activation. Also, knockdown of PI3K-C2α but not p110β suppressed S1P-induced S1P₁ internalization into PtdIns(3)P-enriched endosomes. Finally, pharmacological inhibition of endocytosis suppressed S1P-induced S1P₁ internalization, Rac1 activation, migration, and tube formation. These observations indicate that PI3K-C2α plays the crucial role in S1P₁ internalization into the intracellular vesicular compartment, Rac1 activation on endosomes, and thereby migration through regulating vesicular trafficking in ECs.     

Agonist-modulated regulation of AMP-activated protein kinase (AMPK) in endothelial cells. Evidence for an AMPK -> Rac1 -> Akt -> endothelial nitric-oxide synthase pathway

The endothelial isoform of nitric-oxide synthase (eNOS), a key determinant of vascular homeostasis, is a calcium/calmodulin-dependent phosphoprotein regulated by diverse cell surface receptors. Vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) stimulate eNOS activity through Akt/phosphoinositide 3-kinase and calcium-dependent pathways. AMP-activated protein kinase (AMPK) also activates eNOS in endothelial cells; however, the molecular mechanisms linking agonist-mediated AMPK regulation with eNOS activation remain incompletely understood. We studied the role of AMPK in VEGF- and S1P-mediated eNOS activation and found that both agonists led to a striking increase in AMPK phosphorylation in pathways involving the calcium/calmodulin-dependent protein kinase kinase beta. Treatment with tyrosine kinase inhibitors or the phosphoinositide 3-kinase inhibitor wortmannin demonstrated differential effects of VEGF versus S1P. Small interfering RNA (siRNA)-mediated knockdown of AMPKalpha1or Akt1 impaired the stimulatory effects of both VEGF and S1P on eNOS activation. AMPKalpha1 knockdown impaired agonist-mediated Akt phosphorylation, whereas Akt1 knockdown did not affect AMPK activation, thus suggesting that AMPK lies upstream of Akt in the pathway leading from receptor activation to eNOS stimulation. Importantly, we found that siRNA-mediated knockdown of AMPKalpha1 abrogates agonist-mediated activation of the small GTPase Rac1. Conversely, siRNA-mediated knockdown of Rac1 decreased the agonist-mediated phosphorylation of AMPK substrates without affecting that of AMPK, implicating Rac1 as a molecular link between AMPK and Akt in agonist-mediated eNOS activation. Finally, siRNA-mediated knockdown of caveolin-1 significantly enhanced AMPK phosphorylation, suggesting that AMPK is negatively regulated by caveolin-1. Taken together, these results suggest that VEGF and S1P differentially regulate AMPK and establish a central role for an agonist-modulated AMPK --> Rac1 --> Akt axis in the control of eNOS in endothelial cells.     

Sphingosine 1-phosphate (S1P) regulates glucose-stimulated insulin secretion in pancreatic beta cells

Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS.     

Small interfering RNA-mediated down-regulation of caveolin-1 differentially modulates signaling pathways in endothelial cells

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules in endothelial cells. To explore the role of this protein in receptor-modulated signaling pathways, we transfected bovine aortic endothelial cells (BAEC) with small interfering RNA (siRNA) duplexes to down-regulate caveolin-1 expression. Transfection of BAEC with duplex siRNA targeted against caveolin-1 mRNA selectively "knocked-down" the expression of caveolin-1 by approximately 90%, as demonstrated by immunoblot analyses of BAEC lysates. We used discontinuous sucrose gradients to purify caveolin-containing lipid rafts from siRNA-treated endothelial cells. Despite the near-total down-regulation of caveolin-1 expression, the lipid raft targeting of diverse signaling proteins (including the endothelial isoform of nitric-oxide synthase, Src-family tyrosine kinases, Galphaq and the insulin receptor) was unchanged. We explored the consequences of caveolin-1 knockdown on kinase pathways modulated by the agonists sphingosine-1 phosphate (S1P) and vascular endothelial growth factor (VEGF). siRNA-mediated caveolin-1 knockdown enhanced basal as well as S1P- and VEGF-induced phosphorylation of the protein kinase Akt and did not modify the basal or agonist-induced phosphorylation of extracellular signal-regulated kinases 1/2. Caveolin-1 knock-down also significantly enhanced the basal and agonist-induced activity of the small GTPase Rac. We used siRNA to down-regulate Rac expression in BAEC, and we observed that Rac knockdown significantly reduced basal, S1P-, and VEGF-induced Akt phosphorylation, suggesting a role for Rac activation in the caveolin siRNA-mediated increase in Akt phosphorylation. By using siRNA to knockdown caveolin-1 and Rac expression in cultured endothelial cells, we have found that caveolin-1 does not seem to be required for the targeting of signaling molecules to caveolae/lipid rafts and that caveolin-1 differentially modulates specific kinase pathways in endothelial cells.     

Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of Bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1

Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P₁ is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P₁ renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P₁-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P₁-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P₁ regulation of Bim. However, S1P₁ suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P₁ in disease, analysis of tissue microarrays from ER⁺ breast cancer patients revealed a significant correlation between S1P₁ expression and tumour cell survival. In these tumours, S1P₁ expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P₁ is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context.     

Sphingosine-1-phosphate (S1P) displays sustained S1P1 receptor agonism and signaling through S1P lyase-dependent receptor recycling

The sphingosine-1-phosphate (S1P) type 1 receptor (S1P₁R) is a novel therapeutic target in lymphocyte-mediated autoimmune diseases. S1P₁ receptor desensitization caused by synthetic S1P₁ receptor agonists prevents T-lymphocyte egress from secondary lymphoid organs into the circulation. The selective S1P₁ receptor agonist ponesimod, which is in development for the treatment of autoimmune diseases, efficiently reduces peripheral lymphocyte counts and displays efficacy in animal models of autoimmune disease. Using ponesimod and the natural ligand S1P, we investigated the molecular mechanisms leading to different signaling, desensitization and trafficking behavior of S1P₁ receptors. In recombinant S1P₁ receptor-expressing cells, ponesimod and S1P triggered Gαi protein-mediated signaling and β-arrestin recruitment with comparable potency and efficiency, but only ponesimod efficiently induced intracellular receptor accumulation. In human umbilical vein endothelial cells (HUVEC), ponesimod and S1P triggered translocation of the endogenous S1P₁ receptor to the Golgi compartment. However, only ponesimod treatment caused efficient surface receptor depletion, receptor accumulation in the Golgi and degradation. Impedance measurements in HUVEC showed that ponesimod induced only short-lived Gαi protein-mediated signaling followed by resistance to further stimulation, whereas S1P induced sustained Gαi protein-mediated signaling without desensitization. Inhibition of S1P lyase activity in HUVEC rendered S1P an efficient S1P₁ receptor internalizing compound and abrogated S1P-mediated sustained signaling. This suggests that S1P lyase – by facilitating S1P1 receptor recycling – is essential for S1P-mediated sustained signaling, and that synthetic agonists are functional antagonists because they are not S1P lyase substrates.     

Sphingosine-1-phosphate receptor S1P1 is regulated by direct interactions with P-Rex1, a Rac guanine nucleotide exchange factor

Sphingosine-1-phosphate (S1P) receptors S1P₁ are emerging molecular targets for the treatment of cancer, vascular and immune diseases, due to their pivotal role in cell migration and survival of immune and endothelial cells. A therapeutic strategy to control S1P₁ function is based on agonists that promote changes on S1P₁ expression at the plasma membrane. Here, we explored the hypothesis that cell surface expression and function of S1P₁ are influenced by direct interactions with P-Rex1, a guanine nucleotide exchange factor for Rac. We demonstrate that P-Rex1-PDZ domains interact with S1P₁-carboxyl terminal tail and full length receptor monomers and dimers. Endothelial cells transfected with P-Rex1-PDZ domains show an increased migratory response to S1P. S1P₁ trafficking to intracellular compartments is diminished by coexpression of P-Rex1. We conclude that S1P₁ signaling linked to cell migration is facilitated by a functional interaction with P-Rex1 via a mechanism that involves the maintenance of S1P₁ receptors at the cell membrane.