Microvascular Research 99 (2015) 102–109
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Junctional complex and focal adhesion rearrangement mediates pulmonary endothelial barrier enhancement by FTY720 S-phosphonate Lichun Wang a, Robert Bittman b,1, Joe G.N. Garcia c, Steven M. Dudek a,⁎ a b c
Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA Department of Chemistry and Biochemistry, Queens College of The City University of New York, Flushing, New York, USA Department of Medicine, University of Arizona, Tucson, AZ, USA
a r t i c l e
i n f o
Article history: Accepted 30 March 2015 Available online 7 April 2015 Keywords: Pulmonary vascular barrier FTY720 S-phosphonate Acute lung injury VE-cadherin Rac1
a b s t r a c t Rationale: Modulation of pulmonary vascular barrier function is an important clinical goal given the devastating effects of vascular leak in acute lung injury (ALI). We previously demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine 1-phosphate (S1P) and FTY720, has more potent pulmonary barrier protective effects than these agents in vitro and in mouse models of ALI. Tys preserves expression of the barrier-promoting S1P1 receptor (S1PR1), whereas S1P and FTY720 induce its ubiquitination and degradation. Here we further characterize the novel barrier promoting effects of Tys in cultured human pulmonary endothelial cells (EC). Methods/Results: In human lung EC, Tys significantly increased peripheral redistribution of adherens junction proteins VE-cadherin and β-catenin and tight junction protein ZO-1. Inhibition of VE-cadherin with blocking antibody significantly attenuated Tys-induced transendothelial resistance (TER) elevation, while ZO-1 siRNA partially inhibited this elevation. Tys significantly increased focal adhesion formation and phosphorylation of focal adhesion kinase (FAK). Pharmacologic inhibition of FAK significantly attenuated Tys-induced TER elevation. Tys significantly increased phosphorylation and peripheral redistribution of the actin-binding protein, cortactin, while cortactin siRNA partially attenuated Tys-induced TER elevation. Although Tys significantly increased phosphorylation of Akt and GSK3β, neither PI3 kinase nor GSK3β inhibition altered Tys-induced TER elevation. Tys significantly increased Rac1 activity, while inhibition of Rac1 activity significantly attenuated Tys-induced VEcadherin redistribution and TER elevation. Conclusion: Junctional complex, focal adhesion rearrangement and Rac1 activation play critical roles in Tysmediated barrier protection in pulmonary EC. These results provide mechanistic insights into the effects of this potential ALI therapy. © 2015 Elsevier Inc. All rights reserved.
Introduction The vascular endothelium forms a semi-selective permeability barrier between the blood and the interstitial space and regulates fluid and solute exchange. Its barrier integrity is strictly and dynamically regulated by a balance between barrier-disruptive contractile forces and barrier-protective tethering forces such as cell–cell and cell–matrix adhesions (Dejana, 2004; Komarova and Malik, 2010; Wang and Dudek, 2009). Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), disrupt endothelial barrier integrity and cause a sustained increase in vascular permeability, which is associated with significant mortality (Rubenfeld et al., 2005). Thus, improving our understanding of the regulatory mechanisms involved in pulmonary
⁎ Corresponding author at: Division of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois, Chicago, COMRB 3143, MC 719, 909 S. Wolcott Ave., Chicago, IL 60612, USA. Fax: +1 312 996 4665. E-mail address: [email protected] (S.M. Dudek). 1 Posthumous authorship.
http://dx.doi.org/10.1016/j.mvr.2015.03.007 0026-2862/© 2015 Elsevier Inc. All rights reserved.
vascular permeability may help establish effective therapies for preserving or reconstituting the vascular barrier and reversing this pathophysiologic process. Sphingosine 1-phosphate (S1P) has been identified as a major and potent barrier-protective agent in the blood responsible for maintenance of vascular barrier integrity in vitro and in vivo (Garcia et al., 2001; McVerry and Garcia, 2004; McVerry et al., 2004; Peng et al., 2004). In vitro, S1P produces rapid, sustained, and dose-dependent increases in transmonolayer electrical resistance (TER) in endothelial cells (EC). S1P not only increases baseline barrier integrity but also effectively and consistently restores endothelial barrier function after disruption by edemagenic agents (Garcia et al., 2001). In vivo, intravenous administration of S1P significantly reduces LPS-induced pulmonary vascular leakage in both murine and canine models of ALI (McVerry et al., 2004; Peng et al., 2004). S1P receptor 1 (S1PR1) plays a critical role in S1P-induced barrier protection. Wild-type mice pretreated with the S1PR1 inverse agonist, SB-649146, or S1PR1+/− mice with reduced S1PR1 expression, exhibit reduced protection by S1P in LPS-Induced ALI (Sammani et al., 2010). Through S1PR1 ligation, S1P initiates a series
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of signaling pathways that result in its barrier protective effects, including rapid and dramatic cortical actin ring formation at the cell periphery, lipid raft formation, Rac1 activation, cortactin and non-muscle myosin light chain kinase (nmMLCK) translocation, peripheral myosin light chain phosphorylation, c-Abl activation, and junctional complex and focal adhesion rearrangement (Dudek et al., 2004; Garcia et al., 2001; Lee et al., 1998; Singleton et al., 2005). FTY720 (2-amino-2-[2-(4octylphenyl)ethyl]propane-1,3-diol), a structural analog of S1P, also exhibits potent barrier-enhancing properties both in vitro and in vivo under certain conditions (Dudek et al., 2007; Peng et al., 2004). We have demonstrated that c-Abl signaling and focal adhesion kinase (FAK)-driven focal adhesion rearrangement are necessary for FTY720induced barrier protection in vitro (Wang et al., 2011). Despite their impressive barrier-enhancing potential, S1P and FTY720 also produce effects that will be detrimental in ALI patients, such as bradycardia, increased airway hyperresponsiveness, barrier disruption at higher concentrations (N10 μM), S1PR1 downregulation and FTY720-induced immunosuppression (Koyrakh et al., 2005; Pelletier and Hafler, 2012; Roviezzo et al., 2007; Wang et al., 2014). Based on these limitations, we are investigating the therapeutic potential of another FTY720 analog, (S)-FTY720-phosphonate (Tys). Prior work has demonstrated that Tys has potent maximal effects on barrier function compared to S1P and FTY720 in vitro and demonstrates significant protective effects in vivo without additional immunosuppression (Camp et al., 2009). Most importantly, Tys uniquely maintains S1PR1 expression levels relative to other S1PR1 agonists both in vitro and in vivo since it fails to activate the β-arrestin/ubiquitin pathway (Wang et al., 2014). Due to its unique characteristics, Tys provides superior protection against bleomycin-induced ALI in mice compared to FTY720 (Wang et al., 2014). In this report, we further characterize the novel barrier-promoting effects of Tys on intracellular signaling and junctional assembly formation in cultured human pulmonary EC. Materials and methods Reagents Unless otherwise specified, reagents were obtained from Sigma (St. Louis, MO). (S)-FTY720-phosphonate ((3S)-3-(amino)-3-(hydroxymethyl)-5-(4′-octylphenyl)-pentylphosphonic acid, Tys) was synthesized as previously described (Lu et al., 2009). Anti-VE-cadherin (F-8), anti-VE-Cadherin (BV9) and anti-pan-Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-ZO-1, anti-β-catenin, anti-phospho-FAK (Y397), anti-pan-FAK were purchased from BD Pharmingen (San Diego, CA). Anti-phospho-FAK (Y576), p-Akt (S473) and anti-Lamin B1 were purchased from Cell Signaling Technology (Danvers, MA). Anti-phosphotyrosine 4G10, anti-cortactin (p80/85), Rac1/Cdc42 activation assay Kit, Rac1 inhibitor II, and PI3 kinase inhibitor LY294002 were purchased from EMD Millipore (Billerica, MA). PF573228 was purchased from Selleckchem (Houston, TX).
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Transendothelial monolayer electrical resistance (TER) HPAEC were trypsinized and seeded in polycarbonate wells containing evaporated gold microelectrodes in EBM-2 with 2% FBS. TER measurements were performed using an electrical cell-substrate impedance sensing system (ECIS) (Applied Biophysics, Troy, NY). TER values from each microelectrode were pooled at discrete time points and plotted vs. time as the mean ± S.E.M. Immunofluorescence Confluent HPAEC grown on 35 mm glass-bottom dishes (MatTek, MA) were treated with 1 μM Tys as indicated. Cells were then fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for 5 min. After blocked with 2% BSA in PBS for 1 h, cells were incubated with primary antibodies overnight at 4 °C and incubated with secondary antibody conjugated to Alexa-488 or Alexa-568 for 1 h at room temperature. Cells were analyzed using a Nikon Eclipse TE 300 microscope and a Sony Digital Photo camera DKC 5000. Immunoprecipitation 700 μg of cell lysates (in 500 μl of RIPA buffer) was immunoprecipitated with 3 μg antibody by rotation overnight. The beads were washed with lysis buffer 3 times. The proteins were obtained by boiling beads in SDS-sample buffer at 95 °C for 5 min and separated in 4–15% SDS PAGE gels. Cell fractionation Cell fractionation was performed as described previously (Wang et al., 2011). Briefly, EC were solubilized in cytoskeleton stabilizing buffer (CSK) for 20 min at 4 °C. Cell pellets were collected and lysed in sodium dodecylsulfate (SDS) buffer as Triton X 100-insoluble fraction. Proteins were analyzed by Western blotting. Western blotting Proteins were separated in 4–15% SDS-PAGE gels and transferred onto nitrocellulose membranes. The blots were incubated with appropriate primary antibodies overnight at 4 °C or 1 h at room temperature, followed by incubation with HRP-conjugated second antibodies for 1 h at room temperature. Protein bands were detected with enhanced chemiluminescence (Pierce, Rockford, IL). Statistical analysis Student's t test was used for statistical analyses. Data are presented as ± mean SEM. Results
Cell culture
VE-cadherin complex is necessary for EC barrier enhancement by Tys
Human pulmonary artery endothelial cells (HPAEC) (Lonza, Walkersville, MD) were cultured in EBM-2 complete medium (Lonza) with 10% FBS. Passages 6–9 of EC were used for experimentation.
The VE-cadherin complex is the major structural component of adherens junctions (AJ) in EC and plays a key role in maintenance and regulation of endothelial barrier integrity (Dejana, 2004). Therefore, we first determined the effects of Tys on the distribution of VEcadherin. Confluent HPAEC monolayers were stimulated with 1 μM Tys for 10 min and immunostained for VE-cadherin and β-catenin. As shown in Fig. 1A, Tys induced substantial redistribution of VE-cadherin and β-catenin to cell–cell junctions, suggesting that Tys rapidly increases the formation of AJ. Next, the interaction of VE-cadherin with β-catenin was examined by co-immunoprecipitation. As shown in Fig. 1B, 1 μM Tys treatment increases the association of VE-cadherin with β-catenin within 10 min. Mature AJ are tightly connected to the actin cytoskeleton,
Small interference RNA (siRNA) siRNA for negative control #2, ZO-1 and cortactin, and DharmaFECT 1 transfection reagent were purchased from Dharmacon RNA Technologies (Waltham, MA). HPAEC (70% confluent) were transfected with 100 nM siRNA using DharmaFECT 1 transfection reagent per manufacturer's protocol. Silencing efficacy was assessed by Western blot after 72 h.
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Fig. 1. VE-cadherin complex is necessary for EC barrier enhancement by Tys. A) Confluent HPAEC grown on glass dishes were stimulated with vehicle control or Tys (1 μM) for 10 min. EC were then fixed and immunostained with VE-cadherin and β-catenin antibody per standard protocol. Representative figures from 3 independent experiments are shown. B) 700 μg of cell lysates was immunoprecipitated with 3 μg anti-VE-cadherin antibody. The association of β-catenin and VE-cadherin was detected by anti-β-catenin antibody. C) The amount of VEcadherin in the Triton X 100-insoluble fraction after Tys was detected by Western blot. Lamin B1 equal loading control is shown. D) HPAEC plated on gold microelectrodes were pretreated with 25 μg/ml VE-cadherin antibody F8 (control) or BV9 (functional blocking antibody) for 1 h, and then stimulated with 1 μM Tys. The TER tracing represents pooled data (± S.E.M.) from 3 independent experiments (i.e., 3 individual experiments using 3 different cell preparations on 3 separate days). Data are normalized to the starting point at time 0. Increased resistance levels in this assay correlate with decreased permeability.
A Tys 10 min
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Fig. 2. ZO-1 participates in EC barrier enhancement by Tys. A) Confluent HPAEC grown on glass bottom dishes were stimulated with vehicle control or Tys (1 μM) for 10 min. ZO-1 was immunostained per standard protocol. Representative figures from 3 independent experiments are shown. B) HPAEC transfected with ZO-1 siRNA or control were plated on gold microelectrodes and then stimulated with Tys (1 μM). The TER tracing represents pooled data (± S.E.M.) from 3 independent experiments. Also shown is a Western blot demonstrating representative downregulation of ZO-1 (inset).
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ZO-1 participates in EC barrier enhancement by Tys Tight junction (TJ) proteins such as zona occludens 1 (ZO-1) also participate in the regulation of endothelial barrier function (Vandenbroucke et al., 2008). As shown in Fig. 2A, 1 μM Tys caused increased ZO-1 accumulation at cell–cell junctions within 10 min. Although reduced ZO-1 expression by siRNA significantly decreases TER elevation induced by Tys (Fig. 2B), this effect is only modest, suggesting that ZO-1 partially modulates Tys-induced TER elevation but is not essential for barrier enhancement. Focal adhesion rearrangement is necessary for EC barrier enhancement by Tys Focal adhesions (FA) contribute to endothelial barrier function by linking EC to the underlying matrix, and focal adhesion kinase (FAK) is critical for regulating FA structure and rearrangement (Belvitch and Dudek, 2012). In confluent HPAEC stimulated by 1 μM Tys, immunostaining with FAK antibody reveals increased FA formation within 10 min (Fig. 3A). FAK activation can be assessed by phosphorylation at specific sites (Schaller, 2010; Shikata et al., 2003). As shown in Fig. 3B, Tys significantly increases FAK phosphorylation at site Y576 but not
A
Y397, a pattern consistent with observations previously reported during S1P-induced barrier enhancement (Shikata et al., 2003). Moreover, the FAK pharmacologic inhibitor PF-573228 significantly attenuates Tysinduced TER elevation (Fig. 3C), demonstrating that FAK activity is necessary for EC barrier enhancement by Tys. Rac1 activation is necessary for EC barrier enhancement by Tys Rac1 activity plays a critical role in S1P/S1PR1 signaling and subsequent S1P-mediated barrier improvement (Garcia et al., 2001). In confluent HPAEC, Tys increases Rac1 activity within 10 min (Fig. 4A). We next evaluated if this Rac1 activation is necessary for VE-cadherin redistribution induced by Tys. As shown in Figs. 4B–C, pharmacologic inhibition of Rac1 reduces peripheral VE-cadherin induced by Tys. Finally, Rac1 inhibition decreases baseline TER and significantly attenuates Tys-induced barrier enhancement (Fig. 4D). These results strongly support a critical role for Rac1 activation in EC barrier enhancement by Tys. Cortactin participates in EC barrier enhancement by Tys Expression and phosphorylation of the actin-binding protein cortactin are essential for maximal S1P-mediated EC barrier enhancement (Dudek et al., 2004). Similar to the effects of S1P, Tys increases tyrosine phosphorylation of cortactin within 5 min (Fig. 5A) and induces rapid redistribution of cortactin to the cell periphery (Fig. 5B). Although reduced cortactin expression by siRNA significantly decreases TER elevation induced by Tys (Fig. 5C), this effect is only modest, suggesting that cortactin partially modulates Tys-induced TER elevation. EC barrier enhancement by Tys does not require Akt or GSK activity Previous investigations have demonstrated that S1P-mediated EC barrier enhancement involves an intracellular PI3 kinase-Akt signaling pathway (Lee et al., 2006; Singleton et al., 2005). We therefore examined if Tys
B
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pFAK relative to control
which can be assayed by detergent fractionation (Wang et al., 2011). Therefore, HPAEC were stimulated with 1 μM Tys and fractionated by Triton X-100 solution. Tys increases VE-cadherin in the Triton insoluble fraction within 10 min, indicating that Tys increases the association of AJ with the actin cytoskeleton (Fig. 1C). Thus, Tys induces both redistribution of VE-cadherin complex to cell–cell junctions and stabilization and maturation of VE-cadherin complexes by binding with β-catenin and anchoring them to the cytoskeleton. Finally, Tys-induced TER elevation is significantly attenuated by VE-cadherin functional blocking antibody (BV9), demonstrating that VE-cadherin complexes are necessary for EC barrier enhancement by Tys (Fig. 1D).
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Fig. 3. Focal adhesion rearrangement is necessary for EC barrier enhancement by Tys. A) Confluent HPAEC grown on glass bottom dishes were stimulated with vehicle control or 1 μM Tys for 10 min. FAK was immunostained per standard protocol. Representative figures from 3 independent experiments are shown. B) Confluent HPAEC were stimulated with 1 μM Tys for 10 or 20 min. Phospho-FAK (Y576 or Y397) was detected by Western blot. Data are presented as mean ± SE. n = 3 per condition. *, p b 0.01 compared with control phosphorylation at Y576. C) HPAEC plated on gold microelectrodes were pretreated with 30 μM PF-573228 for 1 h, and then stimulated with 1 μM Tys. The TER tracing represents pooled data (± S.E.M.) from 3 independent experiments.
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Fig. 4. Rac1 activation is necessary for EC barrier enhancement by Tys. A) Confluent HPAEC were stimulated by 1 μM Tys for 10 or 20 min. Rac1 activation was detected by Rac1/Cdc42 activation assay as described in Methods. B) Confluent HPAEC were pretreated with 150 μM Rac1 inhibitor II for 1 h, stimulated by 1 μM Tys for 10 min, and Rac1 activation was determined. C) Confluent HPAEC grown on glass dishes were pretreated with 150 μM Rac1 inhibitor II for 1 h and stimulated with Tys for 10 min. VE-cadherin was immunostained per standard protocol. Representative figures from 3 independent experiments are shown. D) HPAEC plated on gold microelectrodes were pretreated with 150 μM Rac1 inhibitor II for 1 h, and then stimulated with 1 μM Tys. The TER tracing represents pooled data (± S.E.M.) from 3 independent experiments.
activates Akt and its downstream target GSK3β. As shown in Fig. 6A, Tys increases phosphorylation of both Akt (S473) and of GSK3β (S9) within 10 min in HPAEC. Pretreatment with the PI3 kinase inhibitor, LY
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294002, blocks the downstream phosphorylation of both Akt and GSK3β (Fig. 6B). However, in contrast to previous reports for S1Pinduced barrier enhancement (Singleton et al., 2005), LY 294002 does
B IP: Cortactin IB: p-tyrosine
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Time (hr) Fig. 5. Cortactin participates in EC barrier enhancement by Tys. A) Confluent HPAEC were stimulated by 1 μM Tys for 5–15 min. Cortactin was immunoprecipitated from cell lysates and its phosphorylation status determined by anti-phosphotyrosine antibody. B) Confluent HPAEC grown on glass dishes were stimulated with 1 μM Tys for 10 min, and cortactin was immunostained per standard protocol. Representative figures from 3 independent experiments are shown. C) HPAEC transfected with cortactin (CTTN) siRNA or control (C) were plated on gold microelectrodes and then stimulated with Tys (1 μM). The TER tracing represents pooled data (± S.E.M.) from 3 independent experiments. Shown is a Western blot demonstrating representative downregulation of cortactin (inset).
L. Wang et al. / Microvascular Research 99 (2015) 102–109
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Fig. 6. EC barrier enhancement by Tys does not require Akt or GSK activity. A) Confluent HPAEC were stimulated by 1 μM Tys for 10 or 20 min. p-Akt or p-GSK 3β was detected by Western blot. B) Confluent HPAEC were pretreated with 25 μM PI3 kinase inhibitor LY294002 for 1 h and stimulated by 1 μM Tys for 10 min. p-Akt or p-GSK 3β was detected by Western blot. C–D) HPAEC plated on gold microelectrodes were pretreated with 25 μM LY294002 (C) or 20 μM CHIR-99021 (D) for 1 h, and then stimulated with 1 μM Tys. The TER tracing represents pooled data (± S.E.M.) from 3 independent experiments.
not inhibit Tys-induced TER elevation (Fig. 6C). Furthermore, pretreatment of HPAEC with the GSK3β inhibitor, CHIR-99021, also fails to inhibit Tys-induced TER elevation (Fig. 6D). These results indicate that the PI3 kinase/Akt/GSK3β pathway is not essential for EC barrier enhancement by Tys. Discussion Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) causes severe pulmonary vascular leakage and a high mortality rate in afflicted individuals (≥30%) (Rubenfeld et al., 2005). However, effective therapies for preserving or reconstituting the vascular barrier are still lacking. A large number of studies over the past decade have demonstrated that S1P/S1PR1 signaling plays a critical role in maintaining and regulating endothelial barrier function (Garcia et al., 2001; Oo et al., 2011; Sammani et al., 2010; Wang and Dudek, 2009; Wang et al., 2014). FTY720 (S)-phosphonate (Tys) is a novel analog of S1P and FTY720 that exhibits superior endothelial barrier-promoting properties (Wang et al., 2014). S1PR1 is critical in Tys-induced barrier enhancement since either reduction of S1PR1 expression via specific siRNA or inhibition of S1PR1 by a specific inverse agonist significantly inhibited barrier enhancement by Tys (Wang et al., 2014). Similar to S1P, TER elevation induced by Tys is significantly inhibited by preincubation with methyl-cyclodextrin (lipid raft-disrupting agent), pertussis toxin (Gi protein inhibitor) or genistein (nonspecific tyrosine kinase inhibitor), suggesting that lipid raft signaling and Gi-linked receptor coupling to downstream tyrosine phosphorylation events are involved in Tys-induced signaling pathway (Camp et al., 2009). Despite these similarities to S1P, there are differences in Tys effects on EC compared to those of S1P. For example, Tys does not induce rapid (5 min) phosphorylation of myosin light chains or ERK (Camp et al., 2009) or increase intracellular calcium levels (Lu et al., 2009). Most importantly, we recently reported that unlike other S1PR1 agonists, Tys fails to induce significant ubquitination and subsequent degradation of S1PR1,
resulting in maintenance of receptor expression during prolonged stimulation, a characteristic suggesting that Tys may have superior therapeutic potential compared to S1P or FTY720 (Wang et al., 2014). As a result, the current study explores the roles of additional EC signaling pathways to better characterize the barrier regulatory effects of Tys. Endothelial barrier integrity is strictly regulated by a complex balance of intracellular contractile forces and cell–cell and cell-matrix adhesive forces (Mehta and Malik, 2006; Wang and Dudek, 2009). AJs and TJs primarily account for intercellular adhesion via formation of pericellular zipper-like structures in EC. VE-cadherin is the major structural protein of AJ in EC. The VE-cadherin cytoplasmic tail binds βcatenin or plakoglobin (γ-catenin), which stabilizes the AJ anchorage to the actin cytoskeleton. Extracellular administration of anti-VEcadherin antibodies, or intracellular disruption of binding between βcatenin and VE-cadherin, induces a marked increase in vascular permeability (Cattelino et al., 2003; Corada et al., 2001; Navarro et al., 1995). S1P significantly increases the abundance of VE-cadherin and βcatenin at cell–cell contact regions and enhances the assembly of AJs (Lee et al., 1999), while S1PR1 silencing leads to a reduction in expression of VE-cadherin (Krump-Konvalinkova et al., 2005). In the current study, we demonstrate that Tys not only increases the abundance of VE-cadherin and β-catenin at cell–cell contact regions, the association of VE-cadherin and β-catenin, and the assembly of AJs, but it also stabilizes AJs by enhancing their linkage to the underlying actin cytoskeleton (Fig. 1). Importantly, VE-cadherin functional blocking antibody significantly inhibits Tys-induced TER elevation, indicating that VE-cadherin complexes are critical to EC barrier enhancement by Tys. TJs represent approximately 20% of total junctional complexes in EC (Wang and Dudek, 2009). In contrast to AJs, the role of TJs in regulating endothelial permeability is less well characterized. Prior work has revealed that S1P activates the TJ protein ZO-1 to play an important functional role during EC barrier enhancement (Lee et al., 2006). Our current data indicate that Tys induces substantial redistribution of ZO-1 to cell to cell contacts (Fig. 2). However, reduction in ZO-1 expression siRNA results in only a
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modest decrease in Tys-induced TER elevation, suggesting that TJs are much less important than AJs in mediating EC barrier enhancement by Tys. FAs link EC to the underlying matrix, an important determinant of cell shape and therefore paracellular permeability (Belvitch and Dudek, 2012; Wang and Dudek, 2009). Focal adhesion kinase (FAK) plays a critical role in regulating the structure and function of FAs. FAK knockout mice are embryonically lethal (Ilic et al., 1995), and EC isolated from the embryos of FAK knockout mice exhibit increased permeability compared with wild type EC (Zhao et al., 2010). A dominant negative FAK mutant inhibits AJ resealing and barrier recovery following H2O2-induced permeability (Quadri and Bhattacharya, 2007). Phosphorylation of FAK in HPAEC is associated with barrier enhancement by HGF and OxPAPC (Birukova et al., 2007). Selective phosphorylation of FAK at the Y576 site by S1P plays an important role in S1P-induced FA rearrangement and barrier enhancement (Shikata et al., 2003). However, phosphorylation and activation of FAK also involves endothelial barrier disruption via barrier-disrupting stimuli such as TGFβ (Lee et al., 2007), VEGF (Wu et al., 2003) and thrombin (Shikata et al., 2003). Thus, FAK can modulate EC barrier function both positively and negatively under different conditions. Interestingly, in this study pretreatment of lung EC with the FAK inhibitor PF-573228 induced a transient increase in barrier function (Fig. 3C). The underlining mechanism is unknown and is worthy of further investigation. One possibility is that the balance of barrier-protective and barrier-disruptive effects of FAK activity tilts toward disruption under basal conditions, leading to transient improvement in barrier function during inhibition of FAK activity by PF-573228. Similar to S1P, Tys increases phosphorylation of FAK specifically at Y576 as well as FA formation (Fig. 3). Furthermore, pharmacologic inhibition of FAK significantly inhibits Tys-induced TER elevation (Fig. 3). These results support an important role for FAK-mediated FA rearrangement in EC barrier enhancement by Tys. The Rho family of GTPases (Rho, Rac and Cdc42) consist of small (~ 21 kDa) signaling G proteins that link surface receptors to downstream effectors in regulating cytoskeletal dynamics, cell movement, and other cellular functions (Wang and Dudek, 2009). Of these, Rac1 plays a critical role in mediating S1P barrier enhancement (Dudek et al., 2004; Garcia et al., 2001). S1P activates Rac1 via S1PR1 in a Gidependent pathway that is required for S1P-induced cytoskeleton rearrangement and barrier improvement. Down-regulation of Rac expression by siRNA significantly attenuates the S1P TER response. Rac1 activity is critical for S1P-induced AJ assembly as dominative negative Rac expression in EC dramatically diminishes S1P-induced VE-cadherin and β-catenin enrichment at cell–cell junctions (Lee et al., 1999). Rac activation in EC is also associated with the peripheral translocation of cortactin, an 80/85 kD actin-binding protein that plays an important role in regulating EC barrier function (Dudek et al., 2004; Jacobson et al., 2006; Zhao et al., 2009). S1P induces a rapid increase in cortactin tyrosine phosphorylation and translocation to the EC periphery where it associates with lipid rafts and participates in structural changes that elevate the cortical elastic modulus (Arce et al., 2008). Down-regulation of cortactin expression significantly attenuates S1P- and ATP-mediated barrier enhancement in pulmonary EC (Dudek et al., 2004; Jacobson et al., 2006). Furthermore, S1P induces a rapid and significant phosphorylation of Akt that plays an important role in S1P-mediated barrier protection since either inhibition of Akt phosphorylation by the PI3 kinase inhibitor LY294002, or reduced Akt expression by siRNA, significantly decreases this effect (Lee et al., 2006; Singleton et al., 2005). Furthermore, as a well-known downstream target of the Akt pathway, GSK3β participates in hepatocyte growth factor-mediated barrier protection (Liu et al., 2002). In the current study, we characterize the effects of Tys on Rac1, cortactin, Akt, and GSK3β. Among these, only Rac1 appears to be essential for Tys-mediated barrier enhancement since pharmacologic inhibition of Rac1 significantly decreases redistribution of VE-cadherin and TER elevation by Tys (Fig. 4). Cortactin participates but only partially modulates this response as siRNA down-regulation modestly decreases
Tys-induced TER elevation (Fig. 5). However, neither inhibition of Akt nor GSK3β altered EC barrier enhancement by Tys (Fig. 6). Although Akt may be a downstream target of Rac1, it is not critical for Tysmediated barrier improvement. The failure of AKT and GSK3β inhibitors to block Tys-induced barrier enhancement (Fig. 6) is interesting given the previous reports that AKT activity is necessary for S1P-induced TER elevation and EC migration (Lee et al., 2006; Singleton et al., 2005). To our knowledge, no published study has evaluated the effects of direct GSK3β inhibition on S1P-mediated effects in EC. However, because it is a downstream mediator of AKT that has been reported to play a role in EC barrier enhancement by HGF (Liu et al., 2002), it is reasonable to speculate that GSK3β may be functionally important for S1Pinduced effects. Our current data demonstrate no functional role for either AKT or GSK3β in Tys-induced barrier enhancement (Fig. 6), indicating differential pathway involvement compared to the S1P response. Although the mechanism responsible for this differential effect is unknown, one hypothesis is that the binding of Tys to the S1PR1 results in a conformational change different from that caused by S1P binding, leading to altered downstream effects such as those previously reported for β-arrestin recruitment to S1PR1 (Wang et al., 2014). Ongoing studies are exploring this possibility. To summarize, Tys enhances pulmonary EC barrier function by rapidly inducing AJ and FA complex rearrangement. Intracellular Rac1 activation plays a critical role in Tys-mediated barrier protection. Our results indicate that this Rac1 activity and VE-cadherin redistribution are the key intracellular signaling pathways involved in Tys-mediated barrier enhancement. These results provide mechanistic insights into how this potential ALI therapy regulates EC barrier function.
Acknowledgments This work was supported by grants P01 HL 58064 (JGNG), R01 HL 88144 (SMD), and P01 HL 98050 (VN) from the National Heart Lung Blood Institute. This paper is dedicated to the memory of our wonderful collaborator, Dr. Robert Bittman, whose recent passing is a significant loss to his family, friends, and the scientific field.
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Contents lists available at ScienceDirect
Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
Junctional complex and focal adhesion rearrangement mediates pulmonary endothelial barrier enhancement by FTY720 S-phosphonate Lichun Wang a, Robert Bittman b,1, Joe G.N. Garcia c, Steven M. Dudek a,⁎ a b c
Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA Department of Chemistry and Biochemistry, Queens College of The City University of New York, Flushing, New York, USA Department of Medicine, University of Arizona, Tucson, AZ, USA
a r t i c l e
i n f o
Article history: Accepted 30 March 2015 Available online 7 April 2015 Keywords: Pulmonary vascular barrier FTY720 S-phosphonate Acute lung injury VE-cadherin Rac1
a b s t r a c t Rationale: Modulation of pulmonary vascular barrier function is an important clinical goal given the devastating effects of vascular leak in acute lung injury (ALI). We previously demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine 1-phosphate (S1P) and FTY720, has more potent pulmonary barrier protective effects than these agents in vitro and in mouse models of ALI. Tys preserves expression of the barrier-promoting S1P1 receptor (S1PR1), whereas S1P and FTY720 induce its ubiquitination and degradation. Here we further characterize the novel barrier promoting effects of Tys in cultured human pulmonary endothelial cells (EC). Methods/Results: In human lung EC, Tys significantly increased peripheral redistribution of adherens junction proteins VE-cadherin and β-catenin and tight junction protein ZO-1. Inhibition of VE-cadherin with blocking antibody significantly attenuated Tys-induced transendothelial resistance (TER) elevation, while ZO-1 siRNA partially inhibited this elevation. Tys significantly increased focal adhesion formation and phosphorylation of focal adhesion kinase (FAK). Pharmacologic inhibition of FAK significantly attenuated Tys-induced TER elevation. Tys significantly increased phosphorylation and peripheral redistribution of the actin-binding protein, cortactin, while cortactin siRNA partially attenuated Tys-induced TER elevation. Although Tys significantly increased phosphorylation of Akt and GSK3β, neither PI3 kinase nor GSK3β inhibition altered Tys-induced TER elevation. Tys significantly increased Rac1 activity, while inhibition of Rac1 activity significantly attenuated Tys-induced VEcadherin redistribution and TER elevation. Conclusion: Junctional complex, focal adhesion rearrangement and Rac1 activation play critical roles in Tysmediated barrier protection in pulmonary EC. These results provide mechanistic insights into the effects of this potential ALI therapy. © 2015 Elsevier Inc. All rights reserved.
Introduction The vascular endothelium forms a semi-selective permeability barrier between the blood and the interstitial space and regulates fluid and solute exchange. Its barrier integrity is strictly and dynamically regulated by a balance between barrier-disruptive contractile forces and barrier-protective tethering forces such as cell–cell and cell–matrix adhesions (Dejana, 2004; Komarova and Malik, 2010; Wang and Dudek, 2009). Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), disrupt endothelial barrier integrity and cause a sustained increase in vascular permeability, which is associated with significant mortality (Rubenfeld et al., 2005). Thus, improving our understanding of the regulatory mechanisms involved in pulmonary
⁎ Corresponding author at: Division of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois, Chicago, COMRB 3143, MC 719, 909 S. Wolcott Ave., Chicago, IL 60612, USA. Fax: +1 312 996 4665. E-mail address: [email protected] (S.M. Dudek). 1 Posthumous authorship.
http://dx.doi.org/10.1016/j.mvr.2015.03.007 0026-2862/© 2015 Elsevier Inc. All rights reserved.
vascular permeability may help establish effective therapies for preserving or reconstituting the vascular barrier and reversing this pathophysiologic process. Sphingosine 1-phosphate (S1P) has been identified as a major and potent barrier-protective agent in the blood responsible for maintenance of vascular barrier integrity in vitro and in vivo (Garcia et al., 2001; McVerry and Garcia, 2004; McVerry et al., 2004; Peng et al., 2004). In vitro, S1P produces rapid, sustained, and dose-dependent increases in transmonolayer electrical resistance (TER) in endothelial cells (EC). S1P not only increases baseline barrier integrity but also effectively and consistently restores endothelial barrier function after disruption by edemagenic agents (Garcia et al., 2001). In vivo, intravenous administration of S1P significantly reduces LPS-induced pulmonary vascular leakage in both murine and canine models of ALI (McVerry et al., 2004; Peng et al., 2004). S1P receptor 1 (S1PR1) plays a critical role in S1P-induced barrier protection. Wild-type mice pretreated with the S1PR1 inverse agonist, SB-649146, or S1PR1+/− mice with reduced S1PR1 expression, exhibit reduced protection by S1P in LPS-Induced ALI (Sammani et al., 2010). Through S1PR1 ligation, S1P initiates a series
L. Wang et al. / Microvascular Research 99 (2015) 102–109
of signaling pathways that result in its barrier protective effects, including rapid and dramatic cortical actin ring formation at the cell periphery, lipid raft formation, Rac1 activation, cortactin and non-muscle myosin light chain kinase (nmMLCK) translocation, peripheral myosin light chain phosphorylation, c-Abl activation, and junctional complex and focal adhesion rearrangement (Dudek et al., 2004; Garcia et al., 2001; Lee et al., 1998; Singleton et al., 2005). FTY720 (2-amino-2-[2-(4octylphenyl)ethyl]propane-1,3-diol), a structural analog of S1P, also exhibits potent barrier-enhancing properties both in vitro and in vivo under certain conditions (Dudek et al., 2007; Peng et al., 2004). We have demonstrated that c-Abl signaling and focal adhesion kinase (FAK)-driven focal adhesion rearrangement are necessary for FTY720induced barrier protection in vitro (Wang et al., 2011). Despite their impressive barrier-enhancing potential, S1P and FTY720 also produce effects that will be detrimental in ALI patients, such as bradycardia, increased airway hyperresponsiveness, barrier disruption at higher concentrations (N10 μM), S1PR1 downregulation and FTY720-induced immunosuppression (Koyrakh et al., 2005; Pelletier and Hafler, 2012; Roviezzo et al., 2007; Wang et al., 2014). Based on these limitations, we are investigating the therapeutic potential of another FTY720 analog, (S)-FTY720-phosphonate (Tys). Prior work has demonstrated that Tys has potent maximal effects on barrier function compared to S1P and FTY720 in vitro and demonstrates significant protective effects in vivo without additional immunosuppression (Camp et al., 2009). Most importantly, Tys uniquely maintains S1PR1 expression levels relative to other S1PR1 agonists both in vitro and in vivo since it fails to activate the β-arrestin/ubiquitin pathway (Wang et al., 2014). Due to its unique characteristics, Tys provides superior protection against bleomycin-induced ALI in mice compared to FTY720 (Wang et al., 2014). In this report, we further characterize the novel barrier-promoting effects of Tys on intracellular signaling and junctional assembly formation in cultured human pulmonary EC. Materials and methods Reagents Unless otherwise specified, reagents were obtained from Sigma (St. Louis, MO). (S)-FTY720-phosphonate ((3S)-3-(amino)-3-(hydroxymethyl)-5-(4′-octylphenyl)-pentylphosphonic acid, Tys) was synthesized as previously described (Lu et al., 2009). Anti-VE-cadherin (F-8), anti-VE-Cadherin (BV9) and anti-pan-Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-ZO-1, anti-β-catenin, anti-phospho-FAK (Y397), anti-pan-FAK were purchased from BD Pharmingen (San Diego, CA). Anti-phospho-FAK (Y576), p-Akt (S473) and anti-Lamin B1 were purchased from Cell Signaling Technology (Danvers, MA). Anti-phosphotyrosine 4G10, anti-cortactin (p80/85), Rac1/Cdc42 activation assay Kit, Rac1 inhibitor II, and PI3 kinase inhibitor LY294002 were purchased from EMD Millipore (Billerica, MA). PF573228 was purchased from Selleckchem (Houston, TX).
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Transendothelial monolayer electrical resistance (TER) HPAEC were trypsinized and seeded in polycarbonate wells containing evaporated gold microelectrodes in EBM-2 with 2% FBS. TER measurements were performed using an electrical cell-substrate impedance sensing system (ECIS) (Applied Biophysics, Troy, NY). TER values from each microelectrode were pooled at discrete time points and plotted vs. time as the mean ± S.E.M. Immunofluorescence Confluent HPAEC grown on 35 mm glass-bottom dishes (MatTek, MA) were treated with 1 μM Tys as indicated. Cells were then fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for 5 min. After blocked with 2% BSA in PBS for 1 h, cells were incubated with primary antibodies overnight at 4 °C and incubated with secondary antibody conjugated to Alexa-488 or Alexa-568 for 1 h at room temperature. Cells were analyzed using a Nikon Eclipse TE 300 microscope and a Sony Digital Photo camera DKC 5000. Immunoprecipitation 700 μg of cell lysates (in 500 μl of RIPA buffer) was immunoprecipitated with 3 μg antibody by rotation overnight. The beads were washed with lysis buffer 3 times. The proteins were obtained by boiling beads in SDS-sample buffer at 95 °C for 5 min and separated in 4–15% SDS PAGE gels. Cell fractionation Cell fractionation was performed as described previously (Wang et al., 2011). Briefly, EC were solubilized in cytoskeleton stabilizing buffer (CSK) for 20 min at 4 °C. Cell pellets were collected and lysed in sodium dodecylsulfate (SDS) buffer as Triton X 100-insoluble fraction. Proteins were analyzed by Western blotting. Western blotting Proteins were separated in 4–15% SDS-PAGE gels and transferred onto nitrocellulose membranes. The blots were incubated with appropriate primary antibodies overnight at 4 °C or 1 h at room temperature, followed by incubation with HRP-conjugated second antibodies for 1 h at room temperature. Protein bands were detected with enhanced chemiluminescence (Pierce, Rockford, IL). Statistical analysis Student's t test was used for statistical analyses. Data are presented as ± mean SEM. Results
Cell culture
VE-cadherin complex is necessary for EC barrier enhancement by Tys
Human pulmonary artery endothelial cells (HPAEC) (Lonza, Walkersville, MD) were cultured in EBM-2 complete medium (Lonza) with 10% FBS. Passages 6–9 of EC were used for experimentation.
The VE-cadherin complex is the major structural component of adherens junctions (AJ) in EC and plays a key role in maintenance and regulation of endothelial barrier integrity (Dejana, 2004). Therefore, we first determined the effects of Tys on the distribution of VEcadherin. Confluent HPAEC monolayers were stimulated with 1 μM Tys for 10 min and immunostained for VE-cadherin and β-catenin. As shown in Fig. 1A, Tys induced substantial redistribution of VE-cadherin and β-catenin to cell–cell junctions, suggesting that Tys rapidly increases the formation of AJ. Next, the interaction of VE-cadherin with β-catenin was examined by co-immunoprecipitation. As shown in Fig. 1B, 1 μM Tys treatment increases the association of VE-cadherin with β-catenin within 10 min. Mature AJ are tightly connected to the actin cytoskeleton,
Small interference RNA (siRNA) siRNA for negative control #2, ZO-1 and cortactin, and DharmaFECT 1 transfection reagent were purchased from Dharmacon RNA Technologies (Waltham, MA). HPAEC (70% confluent) were transfected with 100 nM siRNA using DharmaFECT 1 transfection reagent per manufacturer's protocol. Silencing efficacy was assessed by Western blot after 72 h.
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Fig. 1. VE-cadherin complex is necessary for EC barrier enhancement by Tys. A) Confluent HPAEC grown on glass dishes were stimulated with vehicle control or Tys (1 μM) for 10 min. EC were then fixed and immunostained with VE-cadherin and β-catenin antibody per standard protocol. Representative figures from 3 independent experiments are shown. B) 700 μg of cell lysates was immunoprecipitated with 3 μg anti-VE-cadherin antibody. The association of β-catenin and VE-cadherin was detected by anti-β-catenin antibody. C) The amount of VEcadherin in the Triton X 100-insoluble fraction after Tys was detected by Western blot. Lamin B1 equal loading control is shown. D) HPAEC plated on gold microelectrodes were pretreated with 25 μg/ml VE-cadherin antibody F8 (control) or BV9 (functional blocking antibody) for 1 h, and then stimulated with 1 μM Tys. The TER tracing represents pooled data (± S.E.M.) from 3 independent experiments (i.e., 3 individual experiments using 3 different cell preparations on 3 separate days). Data are normalized to the starting point at time 0. Increased resistance levels in this assay correlate with decreased permeability.
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Fig. 2. ZO-1 participates in EC barrier enhancement by Tys. A) Confluent HPAEC grown on glass bottom dishes were stimulated with vehicle control or Tys (1 μM) for 10 min. ZO-1 was immunostained per standard protocol. Representative figures from 3 independent experiments are shown. B) HPAEC transfected with ZO-1 siRNA or control were plated on gold microelectrodes and then stimulated with Tys (1 μM). The TER tracing represents pooled data (± S.E.M.) from 3 independent experiments. Also shown is a Western blot demonstrating representative downregulation of ZO-1 (inset).
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ZO-1 participates in EC barrier enhancement by Tys Tight junction (TJ) proteins such as zona occludens 1 (ZO-1) also participate in the regulation of endothelial barrier function (Vandenbroucke et al., 2008). As shown in Fig. 2A, 1 μM Tys caused increased ZO-1 accumulation at cell–cell junctions within 10 min. Although reduced ZO-1 expression by siRNA significantly decreases TER elevation induced by Tys (Fig. 2B), this effect is only modest, suggesting that ZO-1 partially modulates Tys-induced TER elevation but is not essential for barrier enhancement. Focal adhesion rearrangement is necessary for EC barrier enhancement by Tys Focal adhesions (FA) contribute to endothelial barrier function by linking EC to the underlying matrix, and focal adhesion kinase (FAK) is critical for regulating FA structure and rearrangement (Belvitch and Dudek, 2012). In confluent HPAEC stimulated by 1 μM Tys, immunostaining with FAK antibody reveals increased FA formation within 10 min (Fig. 3A). FAK activation can be assessed by phosphorylation at specific sites (Schaller, 2010; Shikata et al., 2003). As shown in Fig. 3B, Tys significantly increases FAK phosphorylation at site Y576 but not
A
Y397, a pattern consistent with observations previously reported during S1P-induced barrier enhancement (Shikata et al., 2003). Moreover, the FAK pharmacologic inhibitor PF-573228 significantly attenuates Tysinduced TER elevation (Fig. 3C), demonstrating that FAK activity is necessary for EC barrier enhancement by Tys. Rac1 activation is necessary for EC barrier enhancement by Tys Rac1 activity plays a critical role in S1P/S1PR1 signaling and subsequent S1P-mediated barrier improvement (Garcia et al., 2001). In confluent HPAEC, Tys increases Rac1 activity within 10 min (Fig. 4A). We next evaluated if this Rac1 activation is necessary for VE-cadherin redistribution induced by Tys. As shown in Figs. 4B–C, pharmacologic inhibition of Rac1 reduces peripheral VE-cadherin induced by Tys. Finally, Rac1 inhibition decreases baseline TER and significantly attenuates Tys-induced barrier enhancement (Fig. 4D). These results strongly support a critical role for Rac1 activation in EC barrier enhancement by Tys. Cortactin participates in EC barrier enhancement by Tys Expression and phosphorylation of the actin-binding protein cortactin are essential for maximal S1P-mediated EC barrier enhancement (Dudek et al., 2004). Similar to the effects of S1P, Tys increases tyrosine phosphorylation of cortactin within 5 min (Fig. 5A) and induces rapid redistribution of cortactin to the cell periphery (Fig. 5B). Although reduced cortactin expression by siRNA significantly decreases TER elevation induced by Tys (Fig. 5C), this effect is only modest, suggesting that cortactin partially modulates Tys-induced TER elevation. EC barrier enhancement by Tys does not require Akt or GSK activity Previous investigations have demonstrated that S1P-mediated EC barrier enhancement involves an intracellular PI3 kinase-Akt signaling pathway (Lee et al., 2006; Singleton et al., 2005). We therefore examined if Tys
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which can be assayed by detergent fractionation (Wang et al., 2011). Therefore, HPAEC were stimulated with 1 μM Tys and fractionated by Triton X-100 solution. Tys increases VE-cadherin in the Triton insoluble fraction within 10 min, indicating that Tys increases the association of AJ with the actin cytoskeleton (Fig. 1C). Thus, Tys induces both redistribution of VE-cadherin complex to cell–cell junctions and stabilization and maturation of VE-cadherin complexes by binding with β-catenin and anchoring them to the cytoskeleton. Finally, Tys-induced TER elevation is significantly attenuated by VE-cadherin functional blocking antibody (BV9), demonstrating that VE-cadherin complexes are necessary for EC barrier enhancement by Tys (Fig. 1D).
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Fig. 3. Focal adhesion rearrangement is necessary for EC barrier enhancement by Tys. A) Confluent HPAEC grown on glass bottom dishes were stimulated with vehicle control or 1 μM Tys for 10 min. FAK was immunostained per standard protocol. Representative figures from 3 independent experiments are shown. B) Confluent HPAEC were stimulated with 1 μM Tys for 10 or 20 min. Phospho-FAK (Y576 or Y397) was detected by Western blot. Data are presented as mean ± SE. n = 3 per condition. *, p b 0.01 compared with control phosphorylation at Y576. C) HPAEC plated on gold microelectrodes were pretreated with 30 μM PF-573228 for 1 h, and then stimulated with 1 μM Tys. The TER tracing represents pooled data (± S.E.M.) from 3 independent experiments.
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Fig. 4. Rac1 activation is necessary for EC barrier enhancement by Tys. A) Confluent HPAEC were stimulated by 1 μM Tys for 10 or 20 min. Rac1 activation was detected by Rac1/Cdc42 activation assay as described in Methods. B) Confluent HPAEC were pretreated with 150 μM Rac1 inhibitor II for 1 h, stimulated by 1 μM Tys for 10 min, and Rac1 activation was determined. C) Confluent HPAEC grown on glass dishes were pretreated with 150 μM Rac1 inhibitor II for 1 h and stimulated with Tys for 10 min. VE-cadherin was immunostained per standard protocol. Representative figures from 3 independent experiments are shown. D) HPAEC plated on gold microelectrodes were pretreated with 150 μM Rac1 inhibitor II for 1 h, and then stimulated with 1 μM Tys. The TER tracing represents pooled data (± S.E.M.) from 3 independent experiments.
activates Akt and its downstream target GSK3β. As shown in Fig. 6A, Tys increases phosphorylation of both Akt (S473) and of GSK3β (S9) within 10 min in HPAEC. Pretreatment with the PI3 kinase inhibitor, LY
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L. Wang et al. / Microvascular Research 99 (2015) 102–109
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Fig. 6. EC barrier enhancement by Tys does not require Akt or GSK activity. A) Confluent HPAEC were stimulated by 1 μM Tys for 10 or 20 min. p-Akt or p-GSK 3β was detected by Western blot. B) Confluent HPAEC were pretreated with 25 μM PI3 kinase inhibitor LY294002 for 1 h and stimulated by 1 μM Tys for 10 min. p-Akt or p-GSK 3β was detected by Western blot. C–D) HPAEC plated on gold microelectrodes were pretreated with 25 μM LY294002 (C) or 20 μM CHIR-99021 (D) for 1 h, and then stimulated with 1 μM Tys. The TER tracing represents pooled data (± S.E.M.) from 3 independent experiments.
not inhibit Tys-induced TER elevation (Fig. 6C). Furthermore, pretreatment of HPAEC with the GSK3β inhibitor, CHIR-99021, also fails to inhibit Tys-induced TER elevation (Fig. 6D). These results indicate that the PI3 kinase/Akt/GSK3β pathway is not essential for EC barrier enhancement by Tys. Discussion Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) causes severe pulmonary vascular leakage and a high mortality rate in afflicted individuals (≥30%) (Rubenfeld et al., 2005). However, effective therapies for preserving or reconstituting the vascular barrier are still lacking. A large number of studies over the past decade have demonstrated that S1P/S1PR1 signaling plays a critical role in maintaining and regulating endothelial barrier function (Garcia et al., 2001; Oo et al., 2011; Sammani et al., 2010; Wang and Dudek, 2009; Wang et al., 2014). FTY720 (S)-phosphonate (Tys) is a novel analog of S1P and FTY720 that exhibits superior endothelial barrier-promoting properties (Wang et al., 2014). S1PR1 is critical in Tys-induced barrier enhancement since either reduction of S1PR1 expression via specific siRNA or inhibition of S1PR1 by a specific inverse agonist significantly inhibited barrier enhancement by Tys (Wang et al., 2014). Similar to S1P, TER elevation induced by Tys is significantly inhibited by preincubation with methyl-cyclodextrin (lipid raft-disrupting agent), pertussis toxin (Gi protein inhibitor) or genistein (nonspecific tyrosine kinase inhibitor), suggesting that lipid raft signaling and Gi-linked receptor coupling to downstream tyrosine phosphorylation events are involved in Tys-induced signaling pathway (Camp et al., 2009). Despite these similarities to S1P, there are differences in Tys effects on EC compared to those of S1P. For example, Tys does not induce rapid (5 min) phosphorylation of myosin light chains or ERK (Camp et al., 2009) or increase intracellular calcium levels (Lu et al., 2009). Most importantly, we recently reported that unlike other S1PR1 agonists, Tys fails to induce significant ubquitination and subsequent degradation of S1PR1,
resulting in maintenance of receptor expression during prolonged stimulation, a characteristic suggesting that Tys may have superior therapeutic potential compared to S1P or FTY720 (Wang et al., 2014). As a result, the current study explores the roles of additional EC signaling pathways to better characterize the barrier regulatory effects of Tys. Endothelial barrier integrity is strictly regulated by a complex balance of intracellular contractile forces and cell–cell and cell-matrix adhesive forces (Mehta and Malik, 2006; Wang and Dudek, 2009). AJs and TJs primarily account for intercellular adhesion via formation of pericellular zipper-like structures in EC. VE-cadherin is the major structural protein of AJ in EC. The VE-cadherin cytoplasmic tail binds βcatenin or plakoglobin (γ-catenin), which stabilizes the AJ anchorage to the actin cytoskeleton. Extracellular administration of anti-VEcadherin antibodies, or intracellular disruption of binding between βcatenin and VE-cadherin, induces a marked increase in vascular permeability (Cattelino et al., 2003; Corada et al., 2001; Navarro et al., 1995). S1P significantly increases the abundance of VE-cadherin and βcatenin at cell–cell contact regions and enhances the assembly of AJs (Lee et al., 1999), while S1PR1 silencing leads to a reduction in expression of VE-cadherin (Krump-Konvalinkova et al., 2005). In the current study, we demonstrate that Tys not only increases the abundance of VE-cadherin and β-catenin at cell–cell contact regions, the association of VE-cadherin and β-catenin, and the assembly of AJs, but it also stabilizes AJs by enhancing their linkage to the underlying actin cytoskeleton (Fig. 1). Importantly, VE-cadherin functional blocking antibody significantly inhibits Tys-induced TER elevation, indicating that VE-cadherin complexes are critical to EC barrier enhancement by Tys. TJs represent approximately 20% of total junctional complexes in EC (Wang and Dudek, 2009). In contrast to AJs, the role of TJs in regulating endothelial permeability is less well characterized. Prior work has revealed that S1P activates the TJ protein ZO-1 to play an important functional role during EC barrier enhancement (Lee et al., 2006). Our current data indicate that Tys induces substantial redistribution of ZO-1 to cell to cell contacts (Fig. 2). However, reduction in ZO-1 expression siRNA results in only a
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modest decrease in Tys-induced TER elevation, suggesting that TJs are much less important than AJs in mediating EC barrier enhancement by Tys. FAs link EC to the underlying matrix, an important determinant of cell shape and therefore paracellular permeability (Belvitch and Dudek, 2012; Wang and Dudek, 2009). Focal adhesion kinase (FAK) plays a critical role in regulating the structure and function of FAs. FAK knockout mice are embryonically lethal (Ilic et al., 1995), and EC isolated from the embryos of FAK knockout mice exhibit increased permeability compared with wild type EC (Zhao et al., 2010). A dominant negative FAK mutant inhibits AJ resealing and barrier recovery following H2O2-induced permeability (Quadri and Bhattacharya, 2007). Phosphorylation of FAK in HPAEC is associated with barrier enhancement by HGF and OxPAPC (Birukova et al., 2007). Selective phosphorylation of FAK at the Y576 site by S1P plays an important role in S1P-induced FA rearrangement and barrier enhancement (Shikata et al., 2003). However, phosphorylation and activation of FAK also involves endothelial barrier disruption via barrier-disrupting stimuli such as TGFβ (Lee et al., 2007), VEGF (Wu et al., 2003) and thrombin (Shikata et al., 2003). Thus, FAK can modulate EC barrier function both positively and negatively under different conditions. Interestingly, in this study pretreatment of lung EC with the FAK inhibitor PF-573228 induced a transient increase in barrier function (Fig. 3C). The underlining mechanism is unknown and is worthy of further investigation. One possibility is that the balance of barrier-protective and barrier-disruptive effects of FAK activity tilts toward disruption under basal conditions, leading to transient improvement in barrier function during inhibition of FAK activity by PF-573228. Similar to S1P, Tys increases phosphorylation of FAK specifically at Y576 as well as FA formation (Fig. 3). Furthermore, pharmacologic inhibition of FAK significantly inhibits Tys-induced TER elevation (Fig. 3). These results support an important role for FAK-mediated FA rearrangement in EC barrier enhancement by Tys. The Rho family of GTPases (Rho, Rac and Cdc42) consist of small (~ 21 kDa) signaling G proteins that link surface receptors to downstream effectors in regulating cytoskeletal dynamics, cell movement, and other cellular functions (Wang and Dudek, 2009). Of these, Rac1 plays a critical role in mediating S1P barrier enhancement (Dudek et al., 2004; Garcia et al., 2001). S1P activates Rac1 via S1PR1 in a Gidependent pathway that is required for S1P-induced cytoskeleton rearrangement and barrier improvement. Down-regulation of Rac expression by siRNA significantly attenuates the S1P TER response. Rac1 activity is critical for S1P-induced AJ assembly as dominative negative Rac expression in EC dramatically diminishes S1P-induced VE-cadherin and β-catenin enrichment at cell–cell junctions (Lee et al., 1999). Rac activation in EC is also associated with the peripheral translocation of cortactin, an 80/85 kD actin-binding protein that plays an important role in regulating EC barrier function (Dudek et al., 2004; Jacobson et al., 2006; Zhao et al., 2009). S1P induces a rapid increase in cortactin tyrosine phosphorylation and translocation to the EC periphery where it associates with lipid rafts and participates in structural changes that elevate the cortical elastic modulus (Arce et al., 2008). Down-regulation of cortactin expression significantly attenuates S1P- and ATP-mediated barrier enhancement in pulmonary EC (Dudek et al., 2004; Jacobson et al., 2006). Furthermore, S1P induces a rapid and significant phosphorylation of Akt that plays an important role in S1P-mediated barrier protection since either inhibition of Akt phosphorylation by the PI3 kinase inhibitor LY294002, or reduced Akt expression by siRNA, significantly decreases this effect (Lee et al., 2006; Singleton et al., 2005). Furthermore, as a well-known downstream target of the Akt pathway, GSK3β participates in hepatocyte growth factor-mediated barrier protection (Liu et al., 2002). In the current study, we characterize the effects of Tys on Rac1, cortactin, Akt, and GSK3β. Among these, only Rac1 appears to be essential for Tys-mediated barrier enhancement since pharmacologic inhibition of Rac1 significantly decreases redistribution of VE-cadherin and TER elevation by Tys (Fig. 4). Cortactin participates but only partially modulates this response as siRNA down-regulation modestly decreases
Tys-induced TER elevation (Fig. 5). However, neither inhibition of Akt nor GSK3β altered EC barrier enhancement by Tys (Fig. 6). Although Akt may be a downstream target of Rac1, it is not critical for Tysmediated barrier improvement. The failure of AKT and GSK3β inhibitors to block Tys-induced barrier enhancement (Fig. 6) is interesting given the previous reports that AKT activity is necessary for S1P-induced TER elevation and EC migration (Lee et al., 2006; Singleton et al., 2005). To our knowledge, no published study has evaluated the effects of direct GSK3β inhibition on S1P-mediated effects in EC. However, because it is a downstream mediator of AKT that has been reported to play a role in EC barrier enhancement by HGF (Liu et al., 2002), it is reasonable to speculate that GSK3β may be functionally important for S1Pinduced effects. Our current data demonstrate no functional role for either AKT or GSK3β in Tys-induced barrier enhancement (Fig. 6), indicating differential pathway involvement compared to the S1P response. Although the mechanism responsible for this differential effect is unknown, one hypothesis is that the binding of Tys to the S1PR1 results in a conformational change different from that caused by S1P binding, leading to altered downstream effects such as those previously reported for β-arrestin recruitment to S1PR1 (Wang et al., 2014). Ongoing studies are exploring this possibility. To summarize, Tys enhances pulmonary EC barrier function by rapidly inducing AJ and FA complex rearrangement. Intracellular Rac1 activation plays a critical role in Tys-mediated barrier protection. Our results indicate that this Rac1 activity and VE-cadherin redistribution are the key intracellular signaling pathways involved in Tys-mediated barrier enhancement. These results provide mechanistic insights into how this potential ALI therapy regulates EC barrier function.
Acknowledgments This work was supported by grants P01 HL 58064 (JGNG), R01 HL 88144 (SMD), and P01 HL 98050 (VN) from the National Heart Lung Blood Institute. This paper is dedicated to the memory of our wonderful collaborator, Dr. Robert Bittman, whose recent passing is a significant loss to his family, friends, and the scientific field.
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