Post-translational modifications of S1PR1 and endothelial barrier regulation

Abstract

Sphingosine-1-phosphate receptor-1 (S1PR1), a G-protein coupled receptor that is expressed in endothelium and activated upon ligation by the bioactive lipid sphingosine-1-phosphate (S1P), is an important vascular-barrier protective mechanism at the level of adherens junctions (AJ). Loss of endothelial barrier function is a central factor in the pathogenesis of various inflammatory conditions characterized by protein-rich lung edema formation, such as acute respiratory distress syndrome (ARDS). While several S1PR1 agonists are available, the challenge of arresting the progression of protein-rich edema formation remains to be met. In this review, we discuss the role of S1PRs, especially S1PR1, in regulating endothelial barrier function. We review recent findings showing that replenishment of the pool of cell-surface S1PR1 may be crucial to the effectiveness of S1P in repairing the endothelial barrier. In this context, we discuss the S1P generating machinery and mechanisms that regulate S1PR1 at the cell surface and their impact on endothelial barrier function.

Introduction

Sphingosine 1 phosphate receptor 1 (S1PR1), upon ligating S1P, regulates a variety of endothelial functions including barrier function, vasculogenesis, vascular tone, inflammation, lymphocyte trafficking, chemotaxis and immunity [1–4]. S1PR1 belongs to the family of seven-transmembrane-domain G-protein coupled receptors (GPCRs) [5], the largest family of membrane proteins in the human genome to transmit diverse signals from extracellular GPCR ligands [6]. So far, 5 S1PRs (S1PR1-S1PR5) (formerly termed endothelial differentiation gene (Edg) receptors) are characterized[7]. Additionally, three putative but less well-characterized S1P-GPCRs have been identified (GPR3, GPR6, and GPR12) [2, 8]. S1PRs share 40% similarity to lysophosphatidic acid (LPA)-activated receptors [9]. S1PRs 1–3 are highly expressed and distributed widely in immune, cardiovascular and nervous systems [10]. In contrast, S1PR4 and S1PR5 expression is variable within tissues and are shown to be expressed in lymphatic and nervous system, respectively, but at lower levels than S1PRs 1–3. S1PR1 predominantly couples with the G_i/o alpha subunit of heterotrimeric G-proteins, whereas S1PR2 and S1PR3 couple with G_i/o, G_q and G_12/13, and S1PR4 and S1PR5 with G_i/o and G_12/13 [11]. Thus, differential patterns of S1PR expression within tissues along with the diversity of G-proteins to which they couple dictate their varied organ functions. S1P concentrations in the circulation approach ~1 μM, while lymph contains ~100 nM S1P[12]. However, S1PRs including S1PR1 are activated by S1P concentrations within sub-nanomolar range [13]. Interestingly, as we will discuss, mechanisms exist to downregulate the receptor response to S1P by modulating the expression of S1PR1 on the endothelial cell-surface. Herein, we review the role of the S1P-S1PRs pathway with specific focus on recent advances describing the role of post-translational mechanisms in regulating S1PR1 activity and how post-translationally modified S1PR1 alters endothelial barrier function.

Endothelial Barrier

The vascular endothelium, the innermost lining of all blood vessels, allows size-dependent transport of fluids, ions, macromolecules and leukocytes across the vessel wall by dynamically regulating the opening of intercellular junctions or through vesicles such as caveolae[3, 14]. Endothelial barrier function is regulated via the transcellular and the paracellular pathways. The transcellular pathway, also known as transcytosis, is a constitutive process that transports macromolecules larger than 3 nM through caveolae and traffics albumin and other nutrients across the endothelial wall, crucial for cellular and tissue functions, in an energy dependent manner [15, 16]. The paracellular pathway is regulated by intercellular endothelial junctions known as tight junctions, adherens junctions and gap junctions [3, 14]. In endothelial cells (EC), adherens junctions (AJ) formed by vascular endothelial (VE) cadherins along with their catenins play a predominant role in creation of the endothelial barrier while allowing passive transport of solutes/ions smaller than 3 nM in diameter. Pro-inflammatory mediators secreted during tissue injury such as thrombin, histamine, platelet activating factor and vascular endothelial growth factor (VEGF) as well as cytokines-chemokines weaken AJ, leading to an increase in endothelial permeability. Persistent opening of AJ leads to the accumulation of protein-rich edema fluid in the interstitial space, a hallmark of vascular permeability disorders including atherosclerosis and acute respiratory distress syndrome (ARDS) [3, 14, 17, 18]. S1P in general enhances barrier function and also rescues endothelial barrier function after injury by inflammatory mediators [17, 19], however, it can also disrupt barrier function depending on which receptors are being ligated and activated.

Overview of S1P Receptors in regulating endothelial barrier function

A detailed view of the S1P receptors along with their agonists and antagonists have been provided in Table 1. S1PR1 is required for development of blood vessel, since global or endothelial specific deletion of S1PR1 in mice induced embryonic lethality due to severe embryonic hemorrhage [20, 21]. In addition, EC-S1PR1 prevented aberrant angiogenesis in the postnatal embryos by preventing endothelial barrier disruption by vascular endothelial growth factor receptor 2 (VEGFR2) [18, 22]. Studies showed that in the absence of EC-S1PR1, VEGFR2-mediated tyrosine phosphorylation of VE-cadherin at Y638 led to disruption of vascular barrier function enabling VEGFR2 to induce aberrant angiogenesis [22, 23]. However, EC-S1PR1 supported VEGFR2-mediated angiogenic signaling during tumor growth [24, 25]. Balaji et al showed using a xenograft model in a S1PR1 GFP-reporter mice that cancer cells induced S1PR1-Gi signaling in EC which cooperated with VEGF-VEGFR2 signaling. These authors showed that EC-S1PR1 functioned by augmenting the activity of the tyrosine kinase c-Abl1. cAbl1 phosphorylated VEGFR2 at tyrosine-951, which prolonged VEGFR2 retention on the plasmalemma to signal sustained EC migration and vascularization and thereby tumor growth. Thus, mice lacking S1PR1 in EC showed reduced tumor vascularization and stunted growth. It seems therefore that EC-S1PR1 regulates VEGFR2 signaling in a cell-context dependent manner. While S1PR1 restricts VEGFR2 angiogenic activity during development it facilitates pathological angiogenesis by co-operating with VEGFR2 [18, 22, 23, 25, 26].

Table 1.

shows S1PRs tissue distribution and documented agonists and antagonists

Receptors	Distribution	Agonists	Anatagonists
S1PR1	Most tissues, cerebellum, Plasma membrane, vesicles, cytoplasmic, nucleus/perinuclear region [27, 28]	FTY720-P [29], AUY954 [30], KRP-203 [31], RP-002 [20], SEW2871CYM5442 Arylpropionic acids RP-001 [32]	(R)-W146a or [ML056] [33, 34], VPC-23019 and VPC44116 [35]
S1PR2	Most tissues, Plasma membrane, cytoplasm [36]	ML031[37]	JTE-013 [38, 39]
S1PR3	Highest in lung, spleen, intestine and kidney, Plasma membrane [40]	FTY720-P KRP-203 ML249 [41]	VPC-23019 TY-52156 [42]
S1PR4	Lymphoid tissues and blood cells [43]	FTY720-P KRP-203 ML178 [44], ML248 [45]	ML131 [13, 45]
S1PR5	Brain, skin and NK cells expressing mostly [43, 46]	FTY720-P KRP-203 [41]

Several endothelial cell types, including pulmonary endothelial cells, typically express the S1P1, S1P2, and S1P3 receptors[10]. However, siRNA mediated depletion of S1PR1 or EC-specific loss of S1PR1 constitutively disrupted EC-barrier function [47]. Conversely, activation of S1PR1 using S1P or a S1PR1 agonist rescued endothelial barrier function following injury induced by thrombin, LPS or Pseudomonas aeriginosa [47, 48]. These findings suggest that S1PR1 is primarily required to maintain endothelial barrier function constitutively and after inflammatory insults. Consistent with this idea, EC-S1PR1 activity limited leukocyte transmigration and prevented inflammatory injury [49, 50]. However, it seems that timing of administration of S1PR1 activity may be the factor in dictating the anti-vascular injury potential of activated EC-S1PR1 since a recent study showed that SEW2871, a S1PR1 agonist, failed to attenuate inflammation-induced endothelial barrier breakdown in a sepsis model [51].

Upon ligating S1P, S1PR1 strengthens endothelial barrier by stimulating the G-protein Gαi, which in turn induces intracellular Ca²⁺ transients, activation of the small GTPases Rac1 and Cdc42 as well as phosphatidylinositol 3-kinase (PI3K), and ERK[52–55] (Fig. 1). Activated Rac1 leads to cortical actin assembly and reannealing of endothelial AJ in part by inhibiting VE-cadherin disassembly from junctions [53, 56, 57]. Activated Rac1 can also promote AJ assembly through suppressing RhoA activity at the level of junctions [56, 58]. Evidence also indicates that oxidized lipids can employ S1PR1 pathway to enhance barrier function. For example, Singleton et al showed that transactivation of S1PR1 by OxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) induced rapid recruitment of S1PR1 to caveolin-enriched microdomains, which enhanced barrier function[59].

Figure 1: Role of S1PRs signaling regulating endothelial barrier function.

Upon activation, sphingosine kinase (SPHK1) synthesizes S1P from sphingosine (SPH). S1P is transported through the transporter, SPNS2, and ligates S1PR1, S1PR2 or S1PR3. S1PR1 stimulates Gi which in turn induces a barrier protective signaling including Ca²⁺ mobilization and PI3-kinase activity leading to activation of small GTPase Rac1. Rac1 anneals adherens junctions enhancing barrier function. S1P also ligates S1PR2 and S1PR3, which in contrast to S1PR1, induces G12/13 activity. Activated G12/G13 stimulates Rho and ROCK signaling which destabilize adherens junctions in part by counteracting Rac1 activity through Rac1GTPase (Rac1GAP) hence disrupting barrier function. S1PR3 interacts with Gq leading to induction of Ca²⁺ signaling pathway that in addition to inducing Rho/ROCK pathway activates endothelial nitric oxide synthase (e-NOS). eNOS leads to NO generation which disrupts adherens junctions by nitosylating p190RhoGAP, a GTPase that blocks RhoA activity.

Studies also showed that S1P enhanced transendothelial electrical resistance, a measure of endothelial barrier function, more in pulmonary microvascular cells than in pulmonary arterial endothelial cells, indicating that the S1PR1 expression profile may differ among EC from various vascular beds [60, 61]. Recently, Kono et al, developed a mouse model based on the “Tango system”[62], in which S1PR1 activity can be tracked by following generation of nuclear GFP upon activation of β-arrestin by S1PR1 [26, 63]. Using this murine model, Engelbrecht E et al performed single cell RNAseq of mouse aorta which revealed 8-transcriptomically distinct EC subtypes [64]. One of these arterial EC subset at vascular branch point seem to show ligand-independent S1PR1/β-arrestin coupling while the other EC subtype at non-branch point showed an inflammatory gene expression signature. Akhter et al also used this model and showed that S1PR1 activation occurs dynamically in resident EC during the repair phase of injury post-LPS challenge. Interestingly, activated S1PR1 directed resident EC to acquire a reparative lineage capable of forming vasculature de novo and repairing the damaged endothelial barrier (Akhter et al un-published data). Further, studies on subsegmental expression profiling of S1PR1 in vivo will define how S1PR1 contributes to the differences in vascular permeability in various vascular beds.

S1PR2 was one of the first S1PR to be identified as a S1P receptor and was previously also known as AGR16, H218, or lpB2 [65]. Initially, S1PR2 was shown to be important for the development of heart in zebrafish and thereby their survival by promoting migration of myocardial precursor cells[42, 66]. However, later studies showed that disruption of the S1PR2 had no effect on mouse embryonic development [67] but did decrease vascular tone in response to vasoconstrictor agents [68], indicating that S1PR2 may regulate cardiovascular function [69]. Additionally, deletion of S1PR2 interfered with formation of the vasculature in the inner ear [70]. In contrast to S1PR1, S1PR2 was shown to suppress pathological angiogenesis. Studies showed that targeted disruption of the S1PR2 in mice led to a high incidence of diffuse, large B-cell lymphoma (DLBCL) of germinal center (GC) origin [71, 72]. Moreover, S1PR2 was found to be mutated in 26% of human DLBCL [71, 72]. While mechanism remains to parse out evidence indicates that S1PR2 may prevent DLBCL by tightly confining B cells in lymphoid organs via suppressing Akt activity[72]. Activated S1PR2 signals by stimulating Gαi, Gαq and Gα12/13[73, 74]. Thus, S1P ligation of S1PR2 induces downstream signaling, some of which opposes S1PR1-mediated pathways such as induction of vascular permeability through activation of the phosphatase and tensin homologue (PTEN), RhoGTPase and its downstream effector, RHO-associated protein kinase (ROCK) [37, 75]. Consistent with these findings, S. pneumoniae infection in mice increased S1PR2 expression, which in turn induced inflammatory lung injury by pathway involving the Rho-ROCK pathway [76]. Similarly, inhibition of S1PR1 activity using VPC 23019 markedly enhanced venular leakage in response to histamine while inhibition of S1PR2 signaling by JTE-013, a specific antagonist of S1PR2, counteracted the histamine-induced microvascular permeability response [77]. Also, silencing of S1PR2 significantly attenuated EC barrier enhancement by both S1P and hepatocyte growth factor (HGF) [78]. These studies indicate that a balance between S1PR1 and S1PR2 signaling may be required to regulate vascular homeostasis.

S1PR3, earlier known as lpB3, also couples to Gαq, Gαi, and Gα12/13[73]. S1PR3 was shown to play opposite roles in arterial injury. Thus, neointimal hyperplasia following denudation of iliac-femoral arteries was suppressed in mice lacking S1PR3 [79]. However, in another study S1PR3-null mice showed increased lesion size in carotid arteries following ligation injury [80]. These discrepancies could be due to higher expression of S1PR3 expression in iliac-femoral arteries versus carotid arteries [80]. It is also possible that EC expressing S1PR3 may have interfered with the injury response in carotid arteries. Furthermore, S1PR3 expression was recently shown to be a biomarker of sepsis and mortality in intensive care patients [81]. Thus, these authors showed that S1PR3 was shed from ECs following LPS-induced lung injury. Hence, S1PR3 containing microparticles disrupted EC-barrier function. Similarly, Niessen et al have shown that mice lacking S1PR3 or treatment of mice with S1PR3 specific antagonists protected them from lethal sepsis induced by LPS [82].

Much less is known about the role of S1PR4–5 in regulating endothelial barrier function. S1PR4, also known as lpC1 [83], couples to Gαi and Gα12/13 and appears to be crucial for megakaryocyte differentiation, platelet formation and activation [84]. S1PR4 activation by VPC23153 induced vasoconstriction in rat vessels [85]. S1PR4 also induced the release of IL-10 and specifically inhibited the proliferation of T cells and release of effector cytokines [86, 87], indicating T-cell immunomodulatory effect of fingolimod may in part be mediated through both S1PR1 and S1PR4 [88, 89].

S1PR5, earlier known as lpB4 or Nrg-1, couples to both Gαi and Gα12/13. Evidence indicates that S1PR5 is expressed in brain endothelial cells and suppresses the migration of oligodendrocyte precursor cells through a Rho kinase-dependent pathway, indicating that it may have a role in cellular communication during brain development and in mediating immune quiescence and integrity of blood-brain-barrier [90–92]. Consistent with these findings, A-971432, the only selective agonist of S1PR5, improved blood-brain-barrier integrity in mice and reversed age-related cognitive decline [93].

Role of S1P metabolism in regulating S1PRs function

S1P is produced by almost all cells intracellularly as an intermediate of membrane sphingolipid metabolism [94]. S1P is generated by phosphorylation of sphingosine (SPH) [95]. SPH is generated by stepwise catalysis of sphingomyelin into ceramide, the later acting as a precursor for synthesis of SPH [96]. Exported S1P can activate all S1PRs in a paracrine-autocrine manner activates S1PRs [97]. Intracellular S1P can associate with specific intracellular target proteins, such as human telomerase reverse transcriptase (hTERT), HDACs or stimulator of interferon gene (STING), to induce various cellular responses [98–100]. The half-life of S1P in the circulation is very low (~15 minutes), indicating that vascularized tissue must have high S1P synthetic activity to maintain plasma S1P. In this context, the EC and erythrocytes are the major source of S1P in plasma under physiologic conditions whereas platelets become an important source of S1P under pathological conditions [101, 102].

The conversion of sphingosine into S1P is mediated by one of the two sphingosine kinases (SPHK1 or SPHK2) [95, 103]. SPHK1 and SPHK2 are well-characterized biochemically and are synchronized in a spatial and temporal fashion by various modifications at the post-translational level and also by interaction with a particular set of proteins and lipids [104, 105]. In contrast to SPHK1, SPHK2 contains ~240 more amino acids [106]. Deletion of either SPHK1 or SPHK2 decreased plasma S1P by 50% without producing any defects in vasculature development, indicating that there is a high degree of functional redundancy between the two enzymes in generating S1P and thereby in regulation of S1PR activity [107, 108]. However, deletion of both SPHK1 and SPHK2 induced embryonic death of all homozygous pups by E13.5 due to poor development of the aorta, specifically incomplete pericyte coverage of blood vessels, severe hemorrhage of the brain (primarily mesenchymal) and to some extent spinal cord, mandible and pericardium [39, 109]. In unstimulated cells, SPHK1 is mainly present in the cytosol and can be exported through vesicles [110–112]. Upon agonist stimulation, ERK phosphorylates SPHK1 at serine 225 [113, 114]. Phosphorylated SPHK1 translocates to the plasma membrane or exported extracellularly to increase S1P generation [111–114]. Intriguingly, membrane translocation of SPHK1 was shown to be coupled with endocytic membrane trafficking [115], indicating that it has a role beyond paracrine S1P signaling [116]. In EC, SPHK1 regulation of endothelial barrier function is known to occur through paracrine signaling, since thrombin and LPS increased SPHK1 activity but decreased the intracellular S1P concentration that normally precedes the onset of barrier recovery [117].

SPHK2 is localized within membranous compartments such as endoplasmic reticulum (ER), mitochondria and nucleus [118, 119]. Thus, in contrast to SPHK1, SPHK2 induction of S1P within intracellular organs serves to regulate cellular functions directly through binding the effector proteins independent of S1PRs activation. For example, studies showed that SPHK2-induced S1P locally interacted with HDAC1 and HDAC2 in the nucleus to enhance pro-inflammatory gene transcription in epithelial cells inducing thereby lung injury [37, 120–122]. SPHK2 induced S1P also bound to hTERT allosterically promoting telomerase stability and hence telomere maintenance, cell proliferation, and tumor growth [98, 99] [37, 120]. However, a recent study showed that in macrophages SPHK2 was required for inhibiting LPS-induced inflammatory response by blocking signaling STING [100]. These authors showed that S1P directly interacted with STING and reduced IFN-β generation. Thus, S1P produced by SPHK1 enhances barrier function by stimulating S1PR1 in a paracrine manner [122] while SPHK2 induce S1P appears to act in a cell-context dependent manner and modulates gene transcription, cellular proliferation and inflammatory responses independent of S1PRs receptor activation.

Several enzymes can catabolize S1P [103, 108]. S1P lyase (SPL), which is localized in the ER with the catalytic domain facing the cytosol [123], irreversibly cleaves S1P into hexadecenal and phosphoethanolamine [44, 124]. This degradation reaction represents the last step in sphingolipid catabolism. Additionally, S1P can be dephosphorylated into SPH, through two specific ER localized phosphatase namely, S1P phosphatases (SPP1 and SPP2), or through broadly-specific membrane localized lipid phosphate phosphatases (LPP1–3) [44, 124–126]. SPH can either recycle back to S1P by SPHK1 or SPHK2. SPH can also be converted into ceramide by ceramide synthase.

Deficiency of SPL led to early death due to significant developmental and functional defects, such as anemia, myeloid cell hyperplasia and multiple congenital anomalies in various organs [127, 128]. SPL^−/− lung alveoli showed increased number of alveolar macrophages and deposition of hyaline material [129]. These mice also showed increased egress of neutrophils and accumulation of pro-inflammatory cytokines such as TNF, INF-γ, MCP1 and IL-6 in the circulation [130]. However, Zhao et al showed that LPS enhanced SPL expression in mice [131]. Genetic or pharmacological alteration of SPL (SPL(+/-) in mice led to increased S1P levels in lung tissue and bronchoalveolar lavage fluids, and these mice showed reduced lung injury and inflammation. While the mechanisms by which SPL deficiency increased S1P levels in the lung remained enigmatic this study raise the possibility that SPPs and LPPs may compete with SPL to regulate S1P levels in EC [132]. More work is needed to clarify the divergent roles of SPL, SPPs and LPP in modulating S1P and regulation of barrier function.

S1P release from ECs is mediated by SPNS2. Spinster homolog 2 (SPNS2) is a twelve transmembrane domain protein that belongs to the SPNS/Spinster family (SPNS1–SPNS3 in vertebrates and Spinster in Drosophila). Global deletion of SPNS2 is embryonically lethal, as loss of Spns2 activity results in lethal defects in cardiovascular development [133], similar to the phenotypes of mice lacking both SPHK1 and SPHK2 or S1PR1^−/− mice [20]. However, EC-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global SPNS2-KO mice [134]. SPNS2 deletion in EC decreased circulating S1P to the same level as that of the global knockout [134, 135], and also had similar lymphopenic effects [136, 137]. SPNS2 overexpression led to increased secretion of S1P [133, 136], while down-regulation of SPNS2 reduced the release of both S1P and dihydro-S1P [136], thus proving that SPNS2 is a mammalian S1P transporter. In addition to endogenously produced dihydro-S1P and S1P, S1P analogues, such as FTY720 phosphate, can also be transported by SPNS2, but not the unphosphorylated sphingoid bases [136, 138]. Additionally, it was recently shown that a member of the same family of transporters as SPNS2, Mfsd2b, is responsible for releasing S1P from erythrocytes and platelets, and mice lacking Mfsd2b showed ~50% lower plasma levels [139]. It would be interesting to determine if loss of Mfs2b alters SPNS2, SPHK1 and SPHK2 and thereby barrier function.

Role of S1P chaperon in regulating S1PRs function

S1P binds to protein carriers such as albumin (≈35%), high-densitylipoprotein (HDL) (≈60%), and to a lesser extent to low-densityand very low-density lipoproteins [140]. HDL is known to have various atheroprotective functions, because of its anti-oxidative, anti-thrombotic, and anti-inflammatory properties [141, 142]. HDL contains various apolipoproteins including ApoA1-II, and ApoM [143]. ApoA and APoM suppress oxidative and inflammatory signaling [144, 145]. Hla’s group compared the effect of S1P bound to albumin versus ApoM in altering inflammatory signaling in endothelial cells [11, 146]. They showed that compared to albumin bound-S1P, ApoM-bound S1P had a higher half-life, served as a biased agonist for S1PR1 and inhibited ICAM1 and VCAM expression to a greater extent [11]. Mechanistically, these authors showed that ApoM-S1P promoted complex formation between S1PR1 and β-arrestin at the plasma membrane, which in turn limited endocytosis of S1PR1. In contrast, the albumin-bound S1P-S1PR1 endocytosed rapidly and induced markedly enhanced Gi-mediated signaling [147]. Consistent with these findings, loss of ApoM globally in mice resulted in ~50% lower S1P levels and induced lung vascular leak in an S1PR1-dependent manner [148, 149]. Obinata et al, using albumin-deficient mice, identified ApoA4 as another S1P binding protein, since recombinant ApoA4 activated multiple S1P receptors, and enhanced the barrier function of vascular endothelium [11]. Whether ApoM and ApoA4 have additional functions, including regulation of SPHK, sphingosine phosphate lyase and SPNS2, remain elusive.

Regulation of S1PR1

Crystal structure of S1PR1 indicates that S1PR1 shares many common features with class A GPCRs including an extracellular N-terminus, seven membrane-spanning α-helices (TM), and an intracellular C-terminus. As with other GPCRs, S1PR1 is localized on the plasma membrane of endothelial cells [47, 150, 151]. Upon ligation with S1P, S1PR1 is rapidly desensitized due to depletion of the receptor from the cell surface [47, 150, 152]. S1PR1 recycles back to the surface in the next 1–2 h following S1P binding. In contrast to S1P, the phospho-FTY720, a stable S1PR1 analog, prevents S1PR1 recycling to cell-surface by promoting S1PR1 degradation [89]. Phospho-FTY720 is generated in the cell from its prodrug FTY720 (also known as Fingolimod/Gilenya). FTY720 is phosphorylated by sphingosine kinases, predominantly SPHK2 [153]. Phospho-FTY720 is then exported possibly by Spns2 to activate S1PR1 in a paracrine manner. Thus, while FTY720 in itself is a weak agonist for S1PR1 the phosphor-FTY720 binds S1PR1 irreversibly leading to S1PR1 degradation and barrier disruption [1, 154]. Since inflammatory mediators such as LPS disrupts barrier function a possibility exist that these mediator’s function by degrading S1PR1. In this scenario, resynthesis of S1PR1 and its trafficking to the cell-surface will be required to reverse the vascular injury [155, 156]. Below we discuss S1PR1 regulation by post-translational and transcriptional mechanisms.

Overview of GPCR trafficking

GPCR signaling is regulated through agonist-induced phosphorylation of the receptor followed by receptor internalization. G-protein receptor kinases, known as GRK, phosphorylate GPCRs on their C-terminus allowing uncoupling of GPCR/G-protein signaling (desensitization) [157, 158]. This is followed by the recruitment of β-arrestin1 and β-arrestin2 proteins to phosphorylated GPCRs, thus facilitating the uncoupling of the receptor from the hetereotrimeric G-protein and endocytosis of GPCRs to endosomes to allow GPCR dephosphorylation and resensitization [159, 160]. β-arrestins bind to both the clathrin heavy chain and the β2-adaptin subunit of the heterotetrameric adaptor complex to facilitate endocytosis [161]. Clathrin, AP-2, and endocytic accessory proteins assemble at the plasma membrane to form invaginating clathrin-coated pits (CCPs). CCPs then undergo a maturation process in which the pits are pinched off to form clathrin-coated vesicles (CCVs) [162]. Clathrin-independent endocytosis, which occurs through lipid rafts and a caveolin-mediated pathway, has recently emerged as another important trafficking pathway [163]. Despite the internalization pathway, it is generally thought that internalized receptors are trafficked to an early endosome, where they are sorted for degradation, recycling, or both [159, 164]. Evidence also indicates that the interaction of β-arrestins with GPCRs determines whether GPCRs are dephosphorylated, resensitized or rapidly recycled or not recycled to the plasma membrane [161, 165].

Serine-threonine phosphorylation of S1PR1 and regulation of vascular barrier function

The C-terminal tail of GPCRs contains clusters of Ser/Thr (e.g., SSS, SXSS, SSXS) which represent a barcode for formation of stable complexes between β-arrestinand GPCR [166]. In this context, Lee et al showed that deletion of the serine rich domain within the C-terminus (SRSKSDNSS) of S1PR1 inhibited receptor internalization by S1P [5, 150], indicating that S1P induces the phosphorylation of S1PR1 at the serine rich domain. While kinases inducing S1PR1 phosphorylation at the serine rich domain were not identified, Oo et al proposed that this region is a potential phosphorylation site for casein kinase II and protein kinase C [150] (Fig. 2A).

Figure 2: S1PR1 trafficking and barrier function upon serine/tyrosine phosphorylation.

S1P/phosphorylated-FTY720 (FTY720-P) activates PKC or casein kinase II (CKII) which phosphorylates S1PR1 at five-serine residues at C-terminal domain (A). Phosphorylated S1PR1 binds beta-arrestin leading to receptor internalization. In the case of S1P, serine phosphorylated receptor recycles back to cell-surface. However, FTY-720-P phosphorylated S1PR1 binds the ubiquitin ligase WWP2, which leads to degradation of receptor and loss of endothelial barrier function. B, S1P also activates pp60Src which phosphorylates S1PR1 at tyrosine 143. Y143-S1PR1 is internalized by beta-arrestin-dynamin pathway. Y143-S1PR1 recycles back to the cell-surface after transiently localizing at endoplasmic reticulum (ER) to mobilize intracellular Ca²⁺. However, in contrast to serine-phosphorylated S1PR1, tyrosine phosphorylated S1PR1 mutant (Y143D-S1PR1 mutant) is shunted from being degraded, persistently localizes at ER to sustain the increase in cytosolic Ca²⁺ in a Gi-dependent manner disrupting barrier function. Thus, finding approaches to block tyrosine phosphorylation of S1PR1 may provide novel S1PR1 barrier protective agonists.

As mentioned above, serine-phosphorylated S1PR1 was internalized primarily by the clarthrin-dependent route [167]. In Chinese hamster ovary cells coexpressing S1PR1 and β-arrestin, activation of S1PR1 led to recruitment of β-arrestin [168], which was inhibited in cells depleted of clathrin, AP-2 or β-arrestin1 and β-arrestin2 [152]. Similarly, inhibition of dynamin, a large GTPase that promotes endocytosis of clathrin-coated pits or caveolae from the plasma membrane, inhibited S1PR1 internalization [152], indicating that S1PR1 is internalized by clathrin-dependent mechanisms. However, Oo et al also showed that S1PR1 recycled back to cell-surface after 1–2 h of S1P stimulation [150]. While the phosphatase and the recycling endosomes involved in trafficking of dephosphorylated serine-phosphorylated S1PR1 remains to be determined, a report showed that internalized S1PR1 colocalizes with clathrin and an early endosome marker, Rabaptin-5, in S1P-stimulated T cells [169] (Fig. 2A).

In contrast to S1P, FTY720P was shown to induce persistent phosphorylation of the C-terminal domain of S1PR1 at S351, S353, S355, S358 and S359 [89]. These authors showed that the serine phosphorylated S1PR1 bound the E3 ubiquitin ligase WWP2. WWP2 in turn ubiquitinated serines to lysines, thus inducing S1PR1 degradation. However, related E3 ubiquitin ligases NEDD4–1, NEDD4–2, AIP4, and Cbl were not required, supporting the specificity of S1PR1 ubiquitination. S1PR1 phosphorylation induced pulmonary vascular leak, while S5A-S1PR1 mutant mice were protected (Fig. 2A). Interestingly, a phosphodefective mutant (5Ser→5Ala) of S1PR1 or an S1PR1 mutant incapable of undergoing ubiquitination at lysine residues (S1PR1-K4R mutant), bound WWP2, indicating that neither phosphorylation nor ubiquitination of the receptor were required for S1PR1 interaction with WWP2. These studies raise an important question whether WWP2 controls the cell-surface pool of S1PR1 under basal conditions.

Tyrosine phosphorylation of S1PR1 and regulation of endothelial barrier function

In addition to serine/threonine residues, S1PR1 contains 3 tyrosine residues of unknown function. We demonstrated that S1P rapidly phosphorylates S1PR1 on tyrosine residues in EC and HEK293 cells transducing S1PR1, which precedes receptor internalization. S1PR1 recycled back to the cell surface within 1 h [47]. Mutational analysis identified Y143 as the critical residue modulating S1PR1 cell surface expression. We showed that while a phospho-defective S1PR1-mutant (S1PR1-Y143F) resisted internalization, a phospho-mimicking S1PR1 mutant (S1PR1-Y143D) showed unusually high receptor internalization (Fig. 2B). Compared to WT-S1PR1-transducing endothelial cells, EC transducing the Y143F-S1PR1 mutant showed enhanced barrier function. However, barrier function was markedly decreased in EC expressing the Y143D-S1PR1 mutant, indicating that inhibition of tyrosine phosphorylation at Y143 enhanced basal endothelial barrier function. Interestingly, phosphorylation of the S1PR1 C-terminal serine residues did not alter the role of Y143 phosphorylation in signaling S1PR1 internalization.

Chavez et al, showed that S1PR1 phosphorylation at Y143 was induced by cSrc [47]. Because S1P induces the activity of Src kinases [170–172], it is possible that S1PR1 itself recruits cSrc to dynamically regulate its responsiveness to S1P. Consistent with this notion, S1PR1 is shown to form a functional complex with PDGFRβ [172, 173], which by recruiting c-Src regulates S1PR1-Gi signaling.

The identity of the protein tyrosine phosphatase that dephosphorylates S1PR1 is unclear. SHP2, PTP-1B and VE-PTP all associate with adherens junctions in endothelial cells, and the proteins associated with AJ are known to be regulated by dephosphorylation [174, 175]. It is quite possible that these phosphatases induce S1PR1 dephosphorylation, thus reinstating endothelial cell-surface expression. Anwar et al showed that Y143D-S1PR1 mutant forms a complex with dynamin and inhibition of dynamin blocks the internalization of Y143D-S1PR1, leading to cell surface retention of the receptor [176]. These authors showed that internalized Y143D-S1PR1 was resident in endoplasmic reticulum (ER) and remained functional. In contrast to wild-type S1PR1, the mutated S1PR1 (Y143D-S1PR1) markedly enhanced increased intracellular Ca²⁺ in response to S1P in a Gi-dependent manner and disrupted EC barrier function (Fig. 2B). Thus, rapid reduction of EC surface expression of S1PR1 subsequent to serine and Y143 phosphorylation is a crucial mechanism modulating S1PR1 cell-surface expression and signaling and hence the endothelial barrier repair function of S1P. EC shows considerable heterogeneity in terms of barrier integrity in various vascular beds [3]. Whether serine versus tyrosine phosphorylation of S1PR1 occurs in a tissue specific manner to regulate their barrier property remains to parse out. Also, further studies will be required to determine why Y143D mutant localized to ER and what are its binding partners.

Transcriptional mechanisms inducing S1PR1 synthesis

Besides acute GPCR internalization-recycling signaling, evidence suggests that S1PR1 is also synthesized following EC barrier dysfunction. Simvastatin, an HMG-CoA reductase inhibitor, is a well-known anti-inflammatory and vaso-protective reagent in murine and human inflammatory diseases [177, 178]. Like S1P, simvastatin also enhanced endothelial barrier function [179]. Sun et al showed that simvastatin activates the transcriptional activity of Kruppel-like factor 2 (KLF2) [156]. Mechanism by which simvastatin induce KLF2 expression is not clear but may involve ERK activity [180, 181]. KLF2 contains a conserved myocyte enhancer factor 2c (MEF2c)-binding site [180, 181]. Evidence indicates that ERK increases the transcriptional activity of MEF2c [182]. Thus, statin by inducing ERK activity may increase KLF2 synthesis by MEF2c. KLF2 is a zinc-finger transcription factor that directly promotes expression of the genes encoding sphingosine-1 phosphate receptor 1 (S1PR1) [183–185]. Consequently, EC-specific KLF2-null mice showed decreased barrier function [156, 185, 186]. We have shown that KLF2 induces S1PR1 expression downstream of focal adhesion kinase (FAK) [187, 188]. Loss of FAK in endothelium spontaneously induces vascular leak in mice, recapitulating the findings in EC-S1PR1-null mice [189]. We showed that FAK forms the endothelial barrier by maintaining S1PR1 expression. Thus, depletion of FAK significantly reduces the expression of S1PR1 both at the mRNA and protein level without changing the expression of other S1PRs [187]. Intriguingly, we found that depletion of FAK led to hypermethylation of KLF2 by DNA methylases [188].

Cai et al showed that FOXF1, the forkhead transcription factor known to regulate embryonic lung development, bound the S1PR1 promoter and thereby increased S1PR1 synthesis [155]. Thus, loss of FOXF1 in EC impairs S1PR1 synthesis, disrupting lung vascular homeostasis. Akhter et al (un-published data) also showed that S1PR1 activates STAT3, which in turn induces S1PR1 synthesis [190]. Together these studies indicate that new synthesis of S1PR1 can rescue S1PR1 cell-surface pool and thereby EC-barrier function.

Summary and Concluding Remarks

This review describes recent developments in the area of endothelial barrier function. We particularly focused on regulation of endothelial barrier function by S1PR1. As such, we describe herein the critical role of S1P generation and transport by SPHK1/SPNS2 in regulating S1PR1 activity, which in turn maintains endothelial barrier integrity. Intriguingly, data also show a key role of serine versus tyrosine phosphorylation of S1PR1 in regulating the EC-surface expression. However, several questions remain to be addressed, such as 1) how inflammatory mediators selectively alter the serine versus tyrosine phosphorylation of S1PR1 in EC?, 2) what is the pool of S1PR1 that is phosphorylated on serine versus tyrosine residues and does it occur in a vascular bed specific manner, 3) whether tyrosine-phosphorylated S1PR1 interacts differentially with barrier disrupting or barrier forming mechanisms (RhoA versus Rac1) to alter endothelial permeability?, 4) what is the fate of tyrosine phosphorylated S1PR1 in ER? and 5) would development of antagonists that prevent S1PR1 phosphorylation at Y143 be beneficial in blocking unwanted vascular leak? To address these and other questions outlined in this review it will be necessary to use a combination of physiological approaches in knockout mouse models along with cell-based studies under normal and inflammatory conditions.

Highlights.

• Sphingosine-1-phosphate receptor-1 (S1PR1), a high affinity G-protein coupled receptor expressed at the endothelial cell (EC) surface, upon ligating its ligand S1P protects endothelial barrier by signaling adherens junction assembly.

• Loss of S1PR1 from EC-surface disrupts endothelial barrier function leading to inflammatory conditions, such as acute lung injury and respiratory distress syndrome (ARDS).

• Post-translational modification of S1PR1 at serine and tyrosine-143 residues play an important role in modulating functional pool of S1PR1 at the EC-surface.

• New synthesis of S1PR1 can rescue S1PR1 EC-surface pool during injury for restoring EC-barrier function.