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Heparan Sulfates Mediate the Interaction between Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) and the Gαq/11 Subunits of Heterotrimeric G Proteins*

Open AccessPublished:February 04, 2014DOI:https://doi.org/10.1074/jbc.M113.542514
      The endothelial cell-cell junction has emerged as a major cell signaling structure that responds to shear stress by eliciting the activation of signaling pathways. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and heterotrimeric G protein subunits Gαq and 11 (Gαq/11) are junctional proteins that have been independently proposed as mechanosensors. Our previous findings suggest that they form a mechanosensitive junctional complex that discriminates between different flow profiles. The nature of the PECAM-1·Gαq/11 interaction is still unclear although it is likely an indirect association. Here, we investigated the role of heparan sulfates (HS) in mediating this interaction and in regulating downstream signaling in response to flow. Co-immunoprecipitation studies show that PECAM-1·Gαq/11 binding is dramatically decreased by competitive inhibition with heparin, pharmacological inhibition with the HS antagonist surfen, and enzymatic removal of HS chains with heparinase III treatment as well as by site-directed mutagenesis of basic residues within the extracellular domain of PECAM-1. Using an in situ proximity ligation assay, we show that endogenous PECAM-1·Gαq/11 interactions in endothelial cells are disrupted by both competitive inhibition and HS degradation. Furthermore, we identified the heparan sulfate proteoglycan syndecan-1 in complexes with PECAM-1 that are rapidly decreased in response to flow. Finally, we demonstrate that flow-induced Akt activation is attenuated in endothelial cells in which PECAM-1 was knocked down and reconstituted with a binding mutant. Taken together, our results indicate that the PECAM-1·Gαq/11 mechanosensitive complex contains an endogenous heparan sulfate proteoglycan with HS chains that is critical for junctional complex assembly and regulating the flow response.

      Introduction

      The vascular endothelium is constantly exposed to hemodynamic forces (i.e. cyclic stretch, hydrostatic pressure, and fluid shear stress) from blood flow that act on the cells and lead to a variety of cellular responses, including cell morphology, intracellular signaling, and gene expression. With regard to fluid shear stress, these responses can be physiological or pathological depending on the type, magnitude, and direction of flow. Identification of the primary mechanosensor that enables vascular endothelial cells (ECs)
      The abbreviations used are: EC
      endothelial cell
      PECAM-1
      platelet endothelial cell adhesion molecule-1
      GAG
      glycosaminoglycan
      HSPG
      heparan sulfate (HS) proteoglycan
      HCAEC
      human coronary artery endothelial cell
      PLA
      proximity ligation assay
      BKRB2
      bradykinin receptor B2
      GPCR
      G protein-coupled receptor
      Bis-Tris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
      to discriminate between different flow profiles has been a major challenge in the field, although a number of candidate molecules, putative macromolecular complexes, and/or cell structures have been proposed (
      • Ando J.
      • Yamamoto K.
      Flow detection and calcium signalling in vascular endothelial cells.
      ,
      • Johnson B.D.
      • Mather K.J.
      • Wallace J.P.
      Mechanotransduction of shear in the endothelium. Basic studies and clinical implications.
      ).
      The endothelial cell-cell junction has been described as the region of highest tension in a continuous EC monolayer under flow (
      • Fung Y.C.
      • Liu S.Q.
      Elementary mechanics of the endothelium of blood vessels.
      ). At this location, ECs are believed to undergo rapid (within minutes) structural adaptations (i.e. inclination) in response to flow that are followed by activation of downstream signaling (
      • Melchior B.
      • Frangos J.A.
      Shear-induced endothelial cell-cell junction inclination.
      ,
      • Melchior B.
      • Frangos J.A.
      q/11-mediated intracellular calcium responses to retrograde flow in endothelial cells.
      ,
      • Melchior B.
      • Frangos J.A.
      Distinctive subcellular Akt-1 responses to shear stress in endothelial cells.
      ). Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a transmembrane glycoprotein that is abundantly expressed by ECs and primarily localized to cell-cell junctions. In response to fluid shear stress, PECAM-1 is rapidly tyrosine-phosphorylated (30 s), which was concluded to be a result of force application directly to the molecule rather than to the cell (
      • Osawa M.
      • Masuda M.
      • Kusano K.
      • Fujiwara K.
      Evidence for a role of platelet endothelial cell adhesion molecule-1 in endothelial cell mechanosignal transduction. Is it a mechanoresponsive molecule?.
      ). Heterotrimeric G proteins are membrane-associated proteins that are activated within seconds of fluid shear stress stimulation (
      • Gudi S.R.
      • Clark C.B.
      • Frangos J.A.
      Fluid flow rapidly activates G proteins in human endothelial cells. Involvement of G proteins in mechanochemical signal transduction.
      ) and that may be direct (
      • Gudi S.
      • Nolan J.P.
      • Frangos J.A.
      Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition.
      ) or indirect via activation of G protein-coupled receptors (GPCRs) (
      • Chachisvilis M.
      • Zhang Y.L.
      • Frangos J.A.
      G protein-coupled receptors sense fluid shear stress in endothelial cells.
      ). In vivo, the G protein subunits αq and α11 (Gαq/11) are co-localized with PECAM-1 at the cell-cell junction in the straight portions of the mouse descending aorta, an area described as “atheroprotected” due to the presence of high, unidirectional laminar flow (
      • Otte L.A.
      • Bell K.S.
      • Loufrani L.
      • Yeh J.C.
      • Melchior B.
      • Dao D.N.
      • Stevens H.Y.
      • White C.R.
      • Frangos J.A.
      Rapid changes in shear stress induce dissociation of a Gαq/11-platelet endothelial cell adhesion molecule-1 complex.
      ). Interestingly, Gαq/11 is absent from the cell-cell junction at aortic branch points, which are characterized as having large temporal gradients and/or reverse flow and, therefore, “atheroprone.” Gαq/11 is also absent from the junctions in the descending aorta of the PECAM-1 knock out mouse. In vitro, PECAM-1 and Gαq/11 indeed form a complex, as demonstrated by co-immunoprecipitation experiments using primary human ECs, which is quickly dissociated in response to temporal changes in shear stress (
      • Otte L.A.
      • Bell K.S.
      • Loufrani L.
      • Yeh J.C.
      • Melchior B.
      • Dao D.N.
      • Stevens H.Y.
      • White C.R.
      • Frangos J.A.
      Rapid changes in shear stress induce dissociation of a Gαq/11-platelet endothelial cell adhesion molecule-1 complex.
      ).
      The endothelial glycocalyx layer, a complex structure composed of various proteoglycans, glycosaminoglycans (GAGs), glycoproteins, and plasma proteins that lines the apical surface of endothelial cells, has also been proposed to be the “mechanosensor” that senses fluid shear stress and transmits this force into biological responses in the cell. Evidence for this concept comes from experiments using enzymes that degrade specific glycocalyx components showing that depletion of heparan sulfates abolished shear stress-induced endothelial nitric oxide (NO) production (
      • Florian J.A.
      • Kosky J.R.
      • Ainslie K.
      • Pang Z.
      • Dull R.O.
      • Tarbell J.M.
      Heparan sulfate proteoglycan is a mechanosensor on endothelial cells.
      ) and also prevented endothelial cell alignment in response to flow (
      • Yao Y.
      • Rabodzey A.
      • Dewey Jr., C.F.
      Glycocalyx modulates the motility and proliferative response of vascular endothelium to fluid shear stress.
      ). Among the GAGs associated with the endothelial glycocalyx layer are heparan sulfate (HS), chondroitin sulfate, and hyaluronan/hyaluronic acid. These linear chains of distinct disaccharide unit repeats (a uronic acid and a hexosamine) are generally attached in varying numbers to core proteins and are collectively referred to as proteoglycans. Heparan sulfate proteoglycans (HSPGs) are the most abundant proteoglycans in the vasculature and are typically classified into three subfamilies based on their location: cell surface or membrane-bound (i.e. syndecans and glypicans), secreted extracellular matrix (i.e. perlecan, agrin, collagen XVIII), and secretory vesicle (i.e. serglycin) (
      • Sarrazin S.
      • Lamanna W.C.
      • Esko J.D.
      Heparan sulfate proteoglycans.
      ).
      A variety of proteins, such as growth factors, cytokines, chemokines, enzymes, enzyme inhibitors, and extracellular matrix proteins, are known to bind to HSPGs (
      • Sarrazin S.
      • Lamanna W.C.
      • Esko J.D.
      Heparan sulfate proteoglycans.
      ). It has also been described that an interaction between PECAM-1 and GAGs of the heparin/HS family exists and that the main heparin-binding site for this interaction requires both Ig domains 2 and 3 (
      • Coombe D.R.
      • Stevenson S.M.
      • Kinnear B.F.
      • Gandhi N.S.
      • Mancera R.L.
      • Osmond R.I.
      • Kett W.C.
      Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans. 2. Biochemical analyses.
      ). Coincidentally, we showed that the interaction between PECAM-1 and Gαq/11 was drastically diminished in the absence of Ig domains 2 and 3 of PECAM-1 (
      • Yeh J.C.
      • Otte L.A.
      • Frangos J.A.
      Regulation of G protein-coupled receptor activities by the platelet-endothelial cell adhesion molecule, PECAM-1.
      ). We, therefore, tested the hypothesis that GAG chains attached to a putative heparan sulfate proteoglycan are part of a mechanosensitive cell-cell junctional complex that contains PECAM-1, Gαq/11, and their respective GPCR(s). We also examined whether their presence as a mediator of physical interactions between components of this macromolecular complex is critical for the flow response.

      DISCUSSION

      Endothelial mechanotransduction is a normal physiological process by which the endothelium converts forces from blood flow into biochemical responses, but these same forces can also cause pathological responses leading to EC dysfunction and atherogenesis. Paramount to understanding how ECs sense and response to distinct flow patterns is the identification of the primary mechanosensor. Although PECAM-1 and Gαq/11 have been independently proposed as primary mechanosensors, our previous studies established that these two junctional proteins form a complex that is mechanosensitive and rapidly responds to temporal gradients in shear stress but not to steady fluid flow (
      • Otte L.A.
      • Bell K.S.
      • Loufrani L.
      • Yeh J.C.
      • Melchior B.
      • Dao D.N.
      • Stevens H.Y.
      • White C.R.
      • Frangos J.A.
      Rapid changes in shear stress induce dissociation of a Gαq/11-platelet endothelial cell adhesion molecule-1 complex.
      ,
      • Yeh J.C.
      • Otte L.A.
      • Frangos J.A.
      Regulation of G protein-coupled receptor activities by the platelet-endothelial cell adhesion molecule, PECAM-1.
      ). The exact mechanism by which PECAM-1 and Gαq/11 act together to sense and respond to early flow onset remains to be elucidated. The objective of this study was to elucidate the aspects of the immediate mechanotransduction pathway of transient shear stress, which has been shown to lead to an atherogenic phenotype both in vitro and in vivo (
      • Bao X.
      • Clark C.B.
      • Frangos J.A.
      Temporal gradient in shear-induced signaling pathway. Involvement of MAP kinase, c-fos, and connexin43.
      ,
      • Bao X.
      • Lu C.
      • Frangos J.A.
      Temporal gradient in shear but not steady shear stress induces PDGF-A and MCP-1 expression in endothelial cells. Role of NO, NFκB, and egr-1.
      ,
      • Nam D.
      • Ni C.W.
      • Rezvan A.
      • Suo J.
      • Budzyn K.
      • Llanos A.
      • Harrison D.
      • Giddens D.
      • Jo H.
      Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.
      ). To this end, we investigated the role of HS in mediating the interaction between PECAM-1 and Gαq/11 using an in vitro model system to simulate the atherogenic component of physiological flow and examined the effect that disruption of this interaction may have on shear stress-induced signaling.
      HEK293 cells are one of the most widely used cell lines for heterologous expression of proteins because they allow for high efficiency of transfection in addition to high fidelity of translation and processing of proteins (
      • Thomas P.
      • Smart T.G.
      HEK293 cell line. A vehicle for the expression of recombinant proteins.
      ). For these reasons and because these cells express high mRNA levels of numerous GPCRs (
      • Atwood B.K.
      • Lopez J.
      • Wager-Miller J.
      • Mackie K.
      • Straiker A.
      Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis.
      ), this expression system was utilized to determine the nature of PECAM-1·Gαq/11 interactions. We found that HS are critically involved in PECAM-1·Gαq/11 association within the basal complex based on the observations that heparin, surfen, and heparinase III treatments all caused a marked decrease in their binding when co-expressed in HEK293 cells. We also examined endogenous interactions in situ using PLA in endothelial cells to validate our findings and to rule out the possibility of overexpression artifact. The strengths of this particular approach compared with traditional biochemical methods, such as co-immunoprecipitation, are that 1) it enables detection of protein interactions as they naturally occur in the cell, and 2) even weak and transient interactions are detectable.
      Although the enzymatic removal of heparan sulfates that extend from the apical surface of endothelial cells by treatment with heparinase III has been a widely used approach for determining the role of the endothelial glycocalyx layer as a primary mechanosensor, its potential effects on protein interactions mediated by HSPGs have been generally overlooked in those studies. PECAM-1 represents one such protein that has been shown to bind to HS (
      • Coombe D.R.
      • Stevenson S.M.
      • Kinnear B.F.
      • Gandhi N.S.
      • Mancera R.L.
      • Osmond R.I.
      • Kett W.C.
      Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans. 2. Biochemical analyses.
      ,
      • Watt S.M.
      • Williamson J.
      • Genevier H.
      • Fawcett J.
      • Simmons D.L.
      • Hatzfeld A.
      • Nesbitt S.A.
      • Coombe D.R.
      The heparin binding PECAM-1 adhesion molecule is expressed by CD34+ hematopoietic precursor cells with early myeloid and B-lymphoid cell phenotypes.
      ) and whose function, particularly as a critical mediator of the flow response, may be significantly altered by removal of HS. Therefore, studies using heparin and surfen were performed to complement those in which HS was enzymatically digested by heparinase III. Heparin, at a concentration range of 50–500 μg/ml, has previously been shown to inhibit PECAM-1-mediated aggregation of mouse L cell fibroblasts (
      • DeLisser H.M.
      • Yan H.C.
      • Newman P.J.
      • Muller W.A.
      • Buck C.A.
      • Albelda S.M.
      Platelet/endothelial cell adhesion molecule-1 (CD31)-mediated cellular aggregation involves cell surface glycosaminoglycans.
      ). It has also been reported that heparin at low concentrations (100 μg/ml) blocks lipoprotein binding to HSPGs (
      • Fuki I.V.
      • Blanchard N.
      • Jin W.
      • Marchadier D.H.
      • Millar J.S.
      • Glick J.M.
      • Rader D.J.
      Endogenously produced endothelial lipase enhances binding and cellular processing of plasma lipoproteins via heparan sulfate proteoglycan-mediated pathway.
      ), but higher concentrations (5–10 mg/ml) are necessary for releasing receptor-mediated binding of lipoproteins to human fibroblasts (
      • Goldstein J.L.
      • Basu S.K.
      • Brunschede G.Y.
      • Brown M.S.
      Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans.
      ). In our studies, heparin at an intermediate concentration (500 μg/ml) was used to competitively bind to PECAM-1 and prevent its binding to HS. Indeed, the presence of heparin resulted in both decreased binding of Gαq/11 to PECAM-1 when co-expressed in HEK293 and decreased association of the endogenous proteins in HCAECs. Surfen, on the other hand, was used based on its ability to bind to HS. It was utilized in this manner and shown to inhibit HS-mediated signaling of FGF2 and VEGF through their respective receptors (
      • Schuksz M.
      • Fuster M.M.
      • Brown J.R.
      • Crawford B.E.
      • Ditto D.P.
      • Lawrence R.
      • Glass C.A.
      • Wang L.
      • Tor Y.
      • Esko J.D.
      Surfen, a small molecule antagonist of heparan sulfate.
      ,
      • Xu D.
      • Fuster M.M.
      • Lawrence R.
      • Esko J.D.
      Heparan sulfate regulates VEGF165- and VEGF121-mediated vascular hyperpermeability.
      ). In the case of exogenous addition of either heparin or surfen, the endothelial glycocalyx layer remains fully intact so any downstream signaling effect would be indicative of disruption of protein-GAG interactions rather than to the loss of a reputed primary mechanosensor.
      Site-specific mutations were introduced into the Ig domains 2 and 3 of PECAM-1 based on a report that predicted a cluster of basic amino acids located 20 Å apart as being critical for the interaction between PECAM-1 and heparin/HS (
      • Gandhi N.S.
      • Coombe D.R.
      • Mancera R.L.
      Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans. 1. Molecular modeling studies.
      ). The significance of this distance is that other known heparin-binding proteins, including type IV collagen, neural cell adhesion molecule (N-CAM) and apolipoprotein E, all have a similar spatial distribution of basic amino acids that accommodates a GAG pentasaccharide regardless of whether their three-dimensional structure consists of α-helices or β-strands (
      • Margalit H.
      • Fischer N.
      • Ben-Sasson S.A.
      Comparative analysis of structurally defined heparin binding sequences reveals a distinct spatial distribution of basic residues.
      ). Another important point is that the targeted amino acids in our studies here are distinct from those identified in Ig domain 1 that are required for mediating homophilic binding of PECAM-1 (
      • Newton J.P.
      • Buckley C.D.
      • Jones E.Y.
      • Simmons D.L.
      Residues on both faces of the first immunoglobulin fold contribute to homophilic binding sites of PECAM-1/CD31.
      ). By knocking down endogenous PECAM-1 in cells and reconstituting them with PECAM-1 that contains these heparin binding mutations, we are specifically targeting heterophilic interactions between PECAM-1 and HS-containing proteins without affecting homophilic interactions between PECAM-1 molecules that are known to be crucial for cell-cell junctional formation (
      • Albelda S.M.
      • Muller W.A.
      • Buck C.A.
      • Newman P.J.
      Molecular and cellular properties of PECAM-1 (endoCAM/CD31). A novel vascular cell-cell adhesion molecule.
      ,
      • Sun Q.H.
      • DeLisser H.M.
      • Zukowski M.M.
      • Paddock C.
      • Albelda S.M.
      • Newman P.J.
      Individually distinct Ig homology domains in PECAM-1 regulate homophilic binding and modulate receptor affinity.
      ). We found that this cluster of four basic amino acids was important not only for the interaction between PECAM-1 and Gαq/11 but also for the activation of Akt in response to flow. Our results regarding flow-induced Akt phosphorylation in the absence of PECAM-1 using an siRNA targeting approach is similar to those previously published using human umbilical vein endothelial cells exposed to longer periods of shear stress (
      • Fleming I.
      • Fisslthaler B.
      • Dixit M.
      • Busse R.
      Role of PECAM-1 in the shear-stress-induced activation of Akt and the endothelial nitric-oxide synthase (eNOS) in endothelial cells.
      ). However, our study differs substantially from the other in that we show experimentally that the decrease in Akt phosphorylation is due specifically to the lack of PECAM-1 function rather than to the lack of PECAM-1 protein as reconstitution of the cells with WT PECAM-1, but not mutant PECAM-1, leads to a renewed ability of cells to respond to flow changes. The decrease in flow-induced Akt phosphorylation is also quite similar to that we previously reported in human umbilical vein endothelial cells when Gαq/11 is silenced by siRNA (
      • Melchior B.
      • Frangos J.A.
      Distinctive subcellular Akt-1 responses to shear stress in endothelial cells.
      ). The incomplete restoration of Akt phosphorylation upon reconstitution of PECAM-1-silenced cells with WT PECAM-1 could be attributed to the lower levels of PECAM-1 expression compared with that present in control siRNA-transfected HCAECs (∼70% less) and/or to the relatively low transfection efficiency (up to 57%) of DNA plasmids into HCAECs by the nucleofection method. In other words, the majority of cells lack PECAM-1 due to the high efficiency of siRNA transfection, but less than half the cell population contains the WT PECAM-1 expression construct. The observed elevation in Akt phosphorylation by mutant PECAM-1-reconstituted cells compared with PECAM-1-silenced cells, albeit not significant, may be due to the inability of the quadruple amino acid substitution to completely disrupt the assembly of the putative mechanosensitive complex. Together, these results support the concept that the association of Gαq/11 with PECAM-1 at the cell-cell junction is critical for the flow response, particularly Akt signaling.
      It was previously shown that in response to a sudden temporal onset of flow (1-s impulse), Gαq/11 is rapidly dissociated from PECAM-1 (15 and 30 s) followed by re-association at later time points (60 and 180 s) as demonstrated by co-immunoprecipitation in human umbilical vein endothelial cells (
      • Otte L.A.
      • Bell K.S.
      • Loufrani L.
      • Yeh J.C.
      • Melchior B.
      • Dao D.N.
      • Stevens H.Y.
      • White C.R.
      • Frangos J.A.
      Rapid changes in shear stress induce dissociation of a Gαq/11-platelet endothelial cell adhesion molecule-1 complex.
      ). In rapid response to a step change in shear stress, we observed a similar pattern of decreased association between Gαq/11 and PECAM-1 at early time points (7 and 15 s) in HCAECs by PLA. It is worth noting that we observed an increase in the association of the PECAM-1·Gαq/11 complex in cells starved in the presence of 1% BSA as opposed to 0.5% BSA, which supports the notion that serum albumin concentrations <1% may lead to a partial collapse of the glycocalyx (
      • Adamson R.H.
      • Clough G.
      Plasma proteins modify the endothelial cell glycocalyx of frog mesenteric microvessels.
      ) and provides further evidence that HS mediates the interaction between the two proteins.
      Using the same PLA methodology, we found that PECAM-1 is closely associated with three different HSPGs: syndecan-1, syndecan-4, and glypican-1. Although syndecan-4 remained closely associated with PECAM-1 in response to step flow, syndecan-1 rapidly dissociated (7 and 15 s) and re-associated (30 and 60 s). This pattern of dissociation and re-association is strikingly similar to that we observed for PECAM-1 and Gαq/11 suggesting that syndecan-1 is part of the same mechanosensitive complex with PECAM-1 and Gαq/11. The observation that both syndecan-1 and syndecan-4 interactions with PECAM-1 are increased at 60 s is consistent with flow-induced junctional clustering of glycocalyx components (i.e. HS and glypican-1), which has been recently reported to occur in rat fat pad endothelial cells at 30 min (
      • Zeng Y.
      • Waters M.
      • Andrews A.
      • Honarmandi P.
      • Ebong E.E.
      • Rizzo V.
      • Tarbell J.M.
      Fluid shear stress induces the clustering of heparan sulfate via mobility of glypican-1 in lipid rafts.
      ). In the case of HCAECs there seems to be a more rapid association of syndecans with the junction. In contrast, association of PECAM-1 with glypican-1 increases with early flow onset and does not appear to be localized to the cell-cell junction. Interestingly, both PECAM-1 and glypican-1 have been independently shown to be expressed in caveolin-1-containing membrane fractions (
      • Cheng F.
      • Mani K.
      • van den Born J.
      • Ding K.
      • Belting M.
      • Fransson L.A.
      Nitric oxide-dependent processing of heparan sulfate in recycling S-nitrosylated glypican-1 takes place in caveolin-1-containing endosomes.
      ,
      • Noel J.
      • Wang H.
      • Hong N.
      • Tao J.Q.
      • Yu K.
      • Sorokina E.M.
      • Debolt K.
      • Heayn M.
      • Rizzo V.
      • Delisser H.
      • Fisher A.B.
      • Chatterjee S.
      PECAM-1 and caveolae form the mechanosensing complex necessary for NOX2 activation and angiogenic signaling with stopped flow in pulmonary endothelium.
      ), with the latter believed to be involved in shear-induced NO production through a glypican-caveolae-endothelial nitric-oxide synthase mechanism (
      • Pahakis M.Y.
      • Kosky J.R.
      • Dull R.O.
      • Tarbell J.M.
      The role of endothelial glycocalyx components in mechanotransduction of fluid shear stress.
      ). Additionally, it has been recently reported that PECAM-1 and caveolin-1 form a mechanosensing complex that is necessary for NAPDH oxide 2 (NOX2) and angiogenic signaling in pulmonary endothelial cells in response to abrupt cessation of flow (
      • Noel J.
      • Wang H.
      • Hong N.
      • Tao J.Q.
      • Yu K.
      • Sorokina E.M.
      • Debolt K.
      • Heayn M.
      • Rizzo V.
      • Delisser H.
      • Fisher A.B.
      • Chatterjee S.
      PECAM-1 and caveolae form the mechanosensing complex necessary for NOX2 activation and angiogenic signaling with stopped flow in pulmonary endothelium.
      ). Therefore, it is not inconceivable that a subset of PECAM-1 forms a functionally distinct mechanosensitive caveolar complex together with glypican-1.
      There is increasing evidence indicating that syndecan-1 may serve as a bridge in bringing GPCR/Gαq/11 to the endothelial cell-cell junction. First, syndecan-1 has previously been shown to interact with GPCRs, including CCR1 and CCR5 (
      • Slimani H.
      • Charnaux N.
      • Mbemba E.
      • Saffar L.
      • Vassy R.
      • Vita C.
      • Gattegno L.
      Binding of the CC-chemokine RANTES to syndecan-1 and syndecan-4 expressed on HeLa cells.
      ,
      • Slimani H.
      • Charnaux N.
      • Mbemba E.
      • Saffar L.
      • Vassy R.
      • Vita C.
      • Gattegno L.
      Interaction of RANTES with syndecan-1 and syndecan-4 expressed by human primary macrophages.
      ). Secondly, syndecan-1 has been reported to be expressed strongly along cell-cell junctions of epithelial cells (
      • Zako M.
      • Dong J.
      • Goldberger O.
      • Bernfield M.
      • Gallagher J.T.
      • Deakin J.A.
      Syndecan-1 and -4 synthesized simultaneously by mouse mammary gland epithelial cells bear heparan sulfate chains that are apparently structurally indistinguishable.
      ). Finally, syndecan-1 was observed to be co-localized with junctional PECAM-1 in HCAECs in the present study. Furthermore, because the HS chains on syndecan-1 and syndecan-4 are structurally indistinguishable and essentially identical with regard to their ligand binding affinities (
      • Zako M.
      • Dong J.
      • Goldberger O.
      • Bernfield M.
      • Gallagher J.T.
      • Deakin J.A.
      Syndecan-1 and -4 synthesized simultaneously by mouse mammary gland epithelial cells bear heparan sulfate chains that are apparently structurally indistinguishable.
      ), it is possible that syndecan-1, as opposed to syndecan-4, is the specific HSPG utilized by endothelial cells.
      It was previously demonstrated that the GPCR bradykinin receptor B2 (BKRB2) interacts with PECAM-1 and enhances the association between PECAM-1·Gαq/11 when co-transfected in HEK293 cells (
      • Yeh J.C.
      • Otte L.A.
      • Frangos J.A.
      Regulation of G protein-coupled receptor activities by the platelet-endothelial cell adhesion molecule, PECAM-1.
      ). Intriguingly, a direct interaction between BKRB2 and PECAM-1 was not established in those studies. A possible explanation for this is that BKRB2 interacts indirectly with PECAM-1 through direct associations with HSPGs. Other endogenous GPCRs besides BKRB2 are likely involved, as the PECAM-1·Gαq/11 interaction can be detected without co-expressing BKRB2, and GPCRs are well known to form heterodimers and even hetero-oligomers (
      • Jordan B.A.
      • Devi L.A.
      G-protein-coupled receptor heterodimerization modulates receptor function.
      ,
      • Rocheville M.
      • Lange D.C.
      • Kumar U.
      • Patel S.C.
      • Patel R.C.
      • Patel Y.C.
      Receptors for dopamine and somatostatin. Formation of hetero-oligomers with enhanced functional activity.
      ). It is interesting to note that the chemokine receptor CXCR4, which has previously been shown to interact with syndecan-4 (
      • Hamon M.
      • Mbemba E.
      • Charnaux N.
      • Slimani H.
      • Brule S.
      • Saffar L.
      • Vassy R.
      • Prost C.
      • Lievre N.
      • Starzec A.
      • Gattegno L.
      A syndecan-4/CXCR4 complex expressed on human primary lymphocytes and macrophages and HeLa cell line binds the CXC chemokine stromal cell-derived factor-1 (SDF-1).
      ), is highly expressed in HEK293 cells (
      • Atwood B.K.
      • Lopez J.
      • Wager-Miller J.
      • Mackie K.
      • Straiker A.
      Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis.
      ). However, HEK293 also express mRNAs of at least 75 other GPCRs (
      • Atwood B.K.
      • Lopez J.
      • Wager-Miller J.
      • Mackie K.
      • Straiker A.
      Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis.
      ) for which endogenous interactions with HSPGs have not yet been described and, therefore, cannot be excluded from consideration.
      It has been recently reported that the chemokine receptor CXCR7 is co-localized with PECAM-1 in tumor endothelial cells of bladder tissue, and its overexpression causes increased Akt phosphorylation in the bladder carcinoma cell line HT1376 (
      • Yates T.J.
      • Knapp J.
      • Gosalbez M.
      • Lokeshwar S.D.
      • Gomez C.S.
      • Benitez A.
      • Ekwenna O.O.
      • Young E.E.
      • Manoharan M.
      • Lokeshwar V.B.
      C-X-C chemokine receptor 7. A functionally associated molecular marker for bladder cancer.
      ). Our results here extend those findings in that we show that association of CXCR7 with PECAM-1 occurs primarily at the cell-cell junctions in HCAECs. Additionally, CXCR4 and CXCR7 are known to form heterodimers, which have been shown to enhance CXCL12-induced signaling (
      • Sierro F.
      • Biben C.
      • Martínez-Muñoz L.
      • Mellado M.
      • Ransohoff R.M.
      • Li M.
      • Woehl B.
      • Leung H.
      • Groom J.
      • Batten M.
      • Harvey R.P.
      • Martínez-A C.
      • Mackay C.R.
      • Mackay F.
      Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7.
      ,
      • Levoye A.
      • Balabanian K.
      • Baleux F.
      • Bachelerie F.
      • Lagane B.
      CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling.
      ). Therefore, CXCR7 either alone or together with CXCR4 may be the putative GPCRs that are directly bound to syndecans, which in turn are bound to junctional PECAM-1. Our results from studies in HEK293 cells and HCAECs support a model in which Gαq/11 is coupled to hetero-oligomerized GPCRs that are localized at the junction through HS-mediated interactions with PECAM-1. These GPCRs may include BKRB2, CXCR7, and CXCR4, the latter of which has been shown to be coupled to Gαq/11 contrary to the notion that it only signals through Gαi/o activation (
      • Maghazachi A.A.
      Role of the heterotrimeric G proteins in stromal-derived factor-1α-induced natural killer cell chemotaxis and calcium mobilization.
      ). Future studies directed at identifying which these endogenous GPCR(s) in endothelial cells interacts specifically with syndecan-1 should shed more light not only on the composition of the junctional mechanosensitive complex but also on the mechanism by which temporal changes in flow leads to activation of downstream signaling pathways.
      Collectively, our data suggest that a mechanosensitive complex containing PECAM-1, GPCR/Gαq/11, and HSPG(s), presumably syndecan-1, resides at the endothelial cell-cell junction during quiescence and is primed for activation upon sensing temporal changes in shear stress. Interactions between the proteins in this complex appear to be mediated by HS as its targeted disruption leads to dissociation of Gαq/11 from PECAM-1 and to an abrogated flow response. To our knowledge this is the first study to demonstrate interplay between the junctional complex containing PECAM-1 and Gαq/11 and components of the glycocalyx, providing a unifying model for endothelial mechanosensing in response to temporal changes in shear stress. In light of our findings, uncoupling of PECAM-1 from Gαq/11 at the EC cell-cell junction through heparin mimetics or specific targeting of HSPGs such as syndecan-1 may represent a therapeutic approach for treating flow-induced vascular diseases.

      Acknowledgments

      We thank Dr. Jiunn-Chern Yeh for preliminary studies and Erika Duggan for technical support with flow cytometry experiments.

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