Published online before print May 24, 2007, doi: 10.1161/CIRCRESAHA.106.145847
© 2007 American Heart Association, Inc.
Molecular Medicine |
RhoGDI-1 Modulation of the Activity of Monomeric RhoGTPase RhoA Regulates Endothelial Barrier Function in Mouse Lungs
From the Department of Pharmacology (M.G., R.N., J.N., S.V., D.P., V.K., D.M., A.B.M., T.V.-Y.), University of Illinois College of Medicine and Center for Lung and Vascular Biology, Chicago; the Department of Molecular Biology (J.M.), Osaka Medical Center for Cancer and Cardiovascular Diseases, and the Department of Molecular Biology and Biochemistry (Y.T.), Osaka University School of Medicine, Osaka, Japan.
Correspondence to Dr T. Voyno-Yasenetskaya, University of Illinois, Department of Pharmacology (MC 868), 835 S. Wolcott Ave, Chicago, IL 60612. E-mail tvy{at}uic.edu
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Abstract |
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Rho family GTPases have been implicated in the regulation of endothelial permeability via their actions on actin cytoskeletal organization and integrity of interendothelial junctions. In cell culture studies, activation of RhoA disrupts interendothelial junctions and increases endothelial permeability, whereas activation of Rac1 and Cdc42 enhances endothelial barrier function by promoting the formation of restrictive junctions. The primary regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), form a complex with the GDP-bound form of the Rho family of monomeric G proteins, and thus may serve as a nodal point regulating the activation state of RhoGTPases. In the present study, we addressed the in vivo role of RhoGDI-1 in regulating pulmonary microvascular permeability using RhoGDI-1–/– mice. We observed that basal endothelial permeability in lungs of RhoGDI-1–/– mice was 2-fold greater than wild-type mice. This was the result of opening of interendothelial junctions in lung microvessels which are normally sealed. The activity of RhoA (but not of Rac1 or Cdc42) was significantly increased in RhoGDI-1–/– lungs as well as in cultured endothelial cells on downregulation of RhoGDI-1 with siRNA, consistent with RhoGDI-1–mediated modulation RhoA activity. Thus, RhoGDI-1 by repressing RhoA activity regulates lung microvessel endothelial barrier function in vivo. In this regard, therapies augmenting endothelial RhoGDI-1 function may be beneficial in reestablishing the endothelial barrier and lung fluid balance in lung inflammatory diseases such as acute respiratory distress syndrome.
Key Words: RhoGDI • Rho GTPase • endothelial permeability • lung perfusion
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Introduction |
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The endothelium, the intimal lining of blood vessels, is the barrier between the blood and interstitium that regulates the extravasation of plasma proteins, fluid, and leukocytes.1 Endothelial permeability is controlled by a variety of mediators released in the blood and surrounding tissues as well as by mechanical stresses exerted by alterations in pulsatile blood flow and hydrostatic pressure.2,3 Increased lung vascular permeability induced by proinflammatory mediators such as thrombin is a major determinant of the inappropriate vascular leakage that leads to protein-rich pulmonary edema and acute lung injury.1 Increased lung vascular permeability is a consequence of increased centripetal tension created by actinomyosin-based contractility and decreased interendothelial adhesive "tethering forces".3 Rho family GTPases are key regulators of endothelial permeability induced by proinflammatory mediators as shown in cell culture studies.4–6 RhoGTPases control the state of actin cytoskeletal organization and integrity of interendothelial junctions.7,8 Activation of RhoA induced the disruption of interendothelial junctions,9–11 whereas Rac1 stabilized junctions and thereby counteracted the permeability-increasing effect of RhoA activation.6,12 Cdc42 activation was also shown to regulate the restoration of endothelial barrier function via adherens junction-dependent increase of cell adhesion.13 Despite the involvement of RhoA in signaling increased endothelial permeability shown in cultured confluent monolayers, little is known about its function in intact microvessels.
The primary negative regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), complex with the GDP-bound forms of Rho family of small GTPases and inhibit their activation.14 Three human RhoGDIs have been identified: ubiquitously expressed RhoGDI (also known as GDI
or GDI-1),14,15 hematopoietic cell-selective Ly/D4GDI (also known as GDIß or GDI-2),16 and GDI-3 or RhoGDI
specifically expressed in the lung, brain, and testis.17 RhoGDI-1 was shown to bind RhoA, Rac1, and Cdc42 by in vitro analysis and in cell culture.18 The GDP-bound form of Rho in the complex with RhoGDIs cannot be activated by guanine nucleotide exchange factors (GEFs) specific for Rho family members.19 RhoGDIs dissociated from Rho GTPases to allow the consequent stimulation with RhoGEFs.19 As RhoGDI-1 functions to inhibit the activation of RhoGTPases,20 in the present study we addressed the function of RhoGDI-1 in regulating endothelial barrier function in intact microvessels using lungs from RhoGDI-1–/– mice. We found that the basal activity of RhoA in RhoGDI-1–/– lungs was significantly increased, whereas activities of Rac1 and Cdc42 were unaffected. Lung microvascular permeability of RhoGDI-1–/– mice was 2-fold greater than wild-type (WT) mice, and it was a result of open interendothelial junctions seen in the null mice. Downregulation of endogenous RhoGDI-1 with siRNA in endothelial cells resulted in dramatic augmentation of RhoA activity. These results demonstrate that RhoGDI-1 serves as a nodal point by modulating RhoA activity and thereby the RhoA-mediated increase in lung microvascular permeability.
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Methods and Materials |
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Reagents
Y-27632 was purchased from Sigma; PAR-1 peptide Ac-TFLLRNPNDK-NH2 was from Biosource; ketamine, xylazine, acepromazine were from Abbott Laboratories; anti-RhoA, anti-ZO-1, and anti–VE-cadherin antibodies were from Santa Cruz Biotechnology; anti-Cdc42 and anti-Rac1 antibodies were from BD Transduction Laboratories, anti-MLC2 and anti–phospho-MLC2 (Thr18/Ser19) were from Cell Signaling Technology; Alexa Fluor 594 phalloidin and anti-mouse Alexa Fluor 594 were from Molecular Probes.
Mice
All animal studies were approved by the Institutional Animal Care Committee of University of Illinois. The generation and characteristics of the RhoGDI-1–/– mice are described in the supplemental materials (available online at http://circres.ahajournals.org).
Determination of pulmonary microvascular permeability and lung microvessel filtration coefficient were performed as described earlier.22 Details of the procedures are given in the supplemental materials.
Details of drug infusions are given in the supplemental materials.
Tissue Preparation for Biochemical Assays
The blood was removed by a 10-minute perfusion with RPMI 1640 at RT and then the infusion of drugs was started, thereafter lungs were quickly cut free of any other tissue and snap-frozen in liquid nitrogen.
Electron microscopy and morphometric analysis are described in the supplemental materials.
Rho GTPases Activation Assay
Activation of Rho proteins in vivo was determined by using pull-down assays as described.23 Details of the procedures are given in the supplemental materials.
Transfection and culture of human umbilical vein endothelial cells (HUVECs) are described in Supplement.
Immunocytochemistry was performed as described 23. Details of the procedures are given in the supplemental materials.
Statistical Analysis
Student t test was used to compare data between 2 groups. Values are expressed as mean±1 SE. P<0.05 was considered statistically significant.
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Results |
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Basal Activation of RhoA in RhoGDI-1–/– Mice
As LPS-induced vascular injury can be attenuated by Rho kinase inhibitor, Y-27632,24 we tested whether RhoA activation can be associated with the symptoms seeing during acute lung injury in mouse lung. LPS at 20 mg/kg was injected intraperitonealy, and 6 hours later lung edema and activation of RhoA GTPases were analyzed (Figure 1A and 1B). Data showed that LPS induced lung edema formation and activation of RhoA GTPase (Figure 1A and 1B).
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To test the role of global activation of Rho GTPases, we have used RhoGDI-1–/– mice.21 The absence of the RhoGDI-1 was confirmed at DNA and protein levels. Genomic DNA isolated from tails of wild-type, RhoGDI-1+/– and RhoGDI-1–/– mice confirmed the targeted disruption of RhoGDI-1 gene (Figure 2A). The absence of RhoGDI-1 protein in total lung lysates from RhoGDI-1–/– mice was evident by Western blotting (Figure 2B). Other RhoGDI family members are not upregulated because of the compensatory effects in RhoGDI-1–/– mice.21
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RhoGDI-1 forms 1:1 complexes in vitro with RhoA,25 Rac1,26 and Cdc42.26,27 To evaluate the effects of RhoGDI-1 deletion on activities of individual Rho GTPases, we used a pull-down assay based on the ability of specific GST-fusions of effector binding domains to capture active (GTP-bound) forms of Rho proteins in cell lysates. In the case of RhoA, we used Rho binding domain (RBD) of the RhoA effector rhotekin.28 GTP-bound Cdc42 and Rac1 levels were determined using the p21 binding domain of PAK.29 We observed in lung homogenates obtained from RhoGDI-1–/– mice that the activity of RhoA was 3-fold greater than in WT mice (Figure 2C). Rac1 was only slightly more active in RhoGDI-1–/– mice than in WT mice (Figure 2D), and Cdc42 activity was similar in WT and RhoGDI-1–/– mice (Figure 2E). Total amounts of RhoA, Rac1, and Cdc42 were similar in both WT and RhoGDI-1–/– mice.
Hyper-Phosphorylation of Myosin Light Chain-2 and Increased Lung Microvascular Permeability in RhoGDI-1–/– Mice
Phosphorylation of myosin light chain (MLC) results in increased actin-myosin cross-bridging which plays a central role in endothelial barrier deregulation.30 Thus, we analyzed the phosphorylation state of MLC in lung homogenates of WT and RhoGDI-1–/– mice using an antibody specific for MLC phosphorylated on threonine 18/serine 19. MLC was hyper-phosphorylated in lung homogenates obtained from RhoGDI-1–/– mice as compared with WT mice (Figure 3A). Perfusion of lungs of WT mice with PAR-1 peptide increased phosphorylation of MLC to levels comparable to RhoGDI-1–/– mice under basal conditions. Perfusion of PAR-1 peptide in lungs of RhoGDI-1–/– mice had no effect on the amount of phospho-MLC (Figure 3A).
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To determine the role of RhoGDI-1 in microvascular barrier function in vivo, we studied the lung microcirculation of RhoGDI-1–/– mice using the perfused lung preparation.31 To establish baseline stability of the mouse lungs, we continuously monitored lung wet weight and pulmonary arterial pressure for 120 minutes. During this period, the preparations did not gain weight and perfusion pressure was constant (data not shown).
The rate of the lung edema formation was significantly greater in RhoGDI-1–/– mice than WT mice (Figure 3B). Notably, basal Kfc of RhoGDI-1–/– mice was 2-fold greater than WT mice, indicating loss of microvascular barrier function in the absence of RhoGDI-1 protein (Figure 3C). Thrombin and the peptide agonist corresponding to the tethered ligand sequence of human PAR-1 (SFLLRN) have been shown to increase pulmonary microvascular permeability in vivo.31 Thus, using RhoGDI-1–/– mice, we determined the role of RhoGDI-1 in the mediating the PAR-1–induced increase in lung endothelial permeability. We detected a sharp increase in Kfc of WT mice (Figure 3C). The agonist-induced increase in Kfc observed in WT mice was comparable to RhoGDI-1–/– lung values under basal conditions. Perfusion of PAR-1 peptide in lungs of RhoGDI-1–/– mice had no effect on Kfc (Figure 3C), indicating that maximal barrier disruption could not be increased further by the PAR-1 peptide.
Open Interendothelial Junctions in RhoGDI-1–/– Mice
To assess endothelial barrier structural alterations, we carried a detailed light microscopy and transmission electron microscopy analyses. At the level of magnification of light microscopy, we did not detect any difference between the structure of the lungs of wild-type and RhoGDI-1–/– mice (Figure 4A). Electron microscopy showed that interendothelial junctions were sealed in the capillary segments of lungs of WT mice, as seen by characteristic central dark elements indicative of "spot-welding" of the junctions (Figure 4B). By morphometric analysis, we did not detect the presence of open junctions in lung capillary endothelium (Table). The same analysis revealed that in postcapillary venules less than 10% of interendothelial junctions were open (Table). Endothelial cells in lungs of RhoGDI-1–/– mice appeared normal (Figure 4C and 4D); however, 6 of the 34 capillaries (
18%) (Figure 4D; Table) and 22 of 94 postcapillary venules ( 24%) (Figure 4C; Table) examined had open interendothelial junctions. In the lungs of WT mice, less than 0.2% of arteriolar junctions, 0% of the capillary junctions, and less than 11% of venular interendothelial junctions were open (Table).
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Downregulation of Endogenous RhoGDI-1 in Endothelial Monolayers Recapitulates Endothelial Phenotype and Signaling Pathways Observed in RhoGDI-1–/– Mouse Lungs
To address whether the role of RhoGDI-1 in regulating small GTPases activity and junctional integrity in lung microvessels could be recapitulated in cultured endothelial cells, studies were performed in HUVECs in which endogenous RhoGDI-1 was downregulated using siRNA. As shown in Figure 5A, siRNA targeted to RhoGDI-1 reduced the amount of endogenous RhoGDI-1x80%. Control nonsilencing siRNA did not affect endogenous RhoGDI-1 levels (Figure 5A).
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To analyze the effect of Rho-GDI downregulation on cell contacts and actin cytoskeleton, siRNA–RhoGDI-1 was cotransfected with a cDNA encoding for green fluorescent protein (GFP). Downregulation of RhoGDI-1 by siRNA–RhoGDI-1 resulted in discontinuities of the VE-cadherin distribution at cell junctions and formation of interendothelial junctional gaps (Figure 5B). By contrast, control siRNA did not modify the junctions (Figure 5B). Importantly, pretreatment of the endothelial monolayers transfected with siRNA–RhoGDI-1 with ROCK inhibitor Y-27632 for 30 minutes resulted in the restoration of the continuous VE-cadherin staining and closure of the interendothelial gaps (Figure 5B). The appearance of the interendothelial junctional gaps was further evident by staining with anti–zonula occludens-1 (ZO-1) antibody. The junctions were open in cells transfected with siRNA–RhoGDI-1 (Figure 5C), whereas cells transfected with the control siRNA displayed continuous ZO-1 staining (Figure 5C). Pretreatment of the endothelial monolayers transfected with siRNA–RhoGDI-1 with Y-27632 resulted in closure of the interendothelial gaps (Figure 5C). We also observed increased actin stress fibers in cells transfected with siRNA–RhoGDI-1 (Figure 5D) consistent with RhoA activation, whereas control siRNA had no effect (Figure 5D). In addition, Y-27632 inhibited actin stress fiber formation induced by siRNA–RhoGDI-1 (Figure 5D).
We analyzed activities of RhoA, Rac1, and Cdc42 in HUVECs transfected with siRNA–RhoGDI-1. As shown in Figure 6A, downregulation of RhoGDI-1 increased RhoA activity by 6.5-fold. Importantly, both Rac1 and Cdc42 activities were not affected (Figure 6B and 6C). In addition, MLC-2 was hyperphosphorylated on downregulation of endogenous RhoGDI-1 in HUVECs (Figure 6D).
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Inhibition of Rho Kinase Reverses the Increase in Microvascular Permeability in RhoGDI-1–/– Lungs
Thrombin-induced activation of RhoA stimulates Rho kinase, which inhibits myosin light chain phosphatase, thus increasing MLC phosphorylation.11,32 Thus, to test the hypothesis that activation of RhoA in RhoGDI-1–/– lungs increased vascular permeability via the Rho kinase dependent pathway, we determined whether the inhibition of Rho kinase by Rho kinase inhibitor Y-27632 would diminish the increased microvascular permeability observed in RhoGDI-1–/– mice. Lungs of WT mice were perfused for different times with Y-27632 and then stimulated with PAR-1 peptide. Monitoring phosphorylation of MLC allowed us to select the specific conditions when increased MLC phosphorylation on PAR-1 peptide treatment was abolished, whereas the basal MLC phosphorylation was unaffected (Figure 7A). Lungs of WT mice were perfused with Y-27632 for 15 minutes followed by perfusion with PAR-1 agonist peptide for 5 minutes. We observed that in WT mice pretreatment with the Y-27632 inhibited the PAR-1–induced increase in phosphorylation of MLC (Figure 7A). Importantly, phosphorylation of MLC increased by perfusion with PAR-1 agonist peptide for 5 minutes was also inhibited by perfusion with Y-27632 for 15 minutes (Figure 7A). Similarly, lungs of WT mice were perfused with Y-27632 for 15 minutes followed by perfusion with PAR-1 agonist peptide for 5 minutes. We observed that in WT mice pretreatment with the Y-27632 inhibited the PAR-1–induced increase in the endothelial permeability without affecting the basal permeability (Figure 7B). Importantly, endothelial permeability increased by perfusion with PAR-1 agonist peptide for 5 minutes was inhibited by perfusion with Y-27632 for 15 minutes (Figure 7B). Treatment of lungs of RhoGDI-1–/– mice with Y-27632 prevented the increase in lung microvascular permeability seen under basal condition (Figure 7B). Together, these data suggest that inhibition of RhoA pathway can reverse increased microvascular permeability induced by acute stimulation with PAR1 peptide or prolonged stimulation in RhoGDI-1–/– mice.
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Discussion |
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Inhibition of Rho pathway by Rho kinase inhibitor resulted in attenuation of endotoxin-induced acute lung injury.24 In addition, our data showed that endotoxin-induced lung edema is accompanied by activation of RhoA (Figure 1). Therefore, it is feasible that overactivated RhoA can contribute to endotoxin-induced acute lung injury. In the present study using RhoGDI-1–/– mice we observed that RhoGDI-1 specifically modulates RhoA activity. Basal activity of RhoA was elevated in RhoGDI-1–/– mice, whereas activities of Rac1 and Cdc42 were unaffected. Our observations in mouse lungs differ from studies in which RhoGDI-1 was shown to regulate activities of RhoA, Rac1, and Cdc42 in in vitro studies.33 RhoGDI-1 formed 1:1 complexes with RhoA,25 Rac1,26 and Cdc42.27 The binding and inhibitory activities of RhoGDI-1 toward these proteins occurred at similar nanomolar affinities.34 However, in our study, only RhoA was active in lung homogenates of RhoGDI-1–/– mice. We have provided additional data to show that downregulation of RhoGDI-1 by siRNA leads to activation of RhoA but not Cdc42 and Rac1 (Figure 6). As it was believed that RhoGDI-1 is a nonspecific inhibitor of RhoA, Rac1, and Cdc42, these are extremely important observations. Molecular mechanisms of the discrepancies obtained using in vitro and in vivo studies will be addressed in the follow-up experiments. Thus, it appears that in vivo Rac1 and Cdc42 require other RhoGDIs to regulate their activation. One possibility is that RhoGDI-1–dependent regulation of RhoGTPase activation cycle and location may be influenced by the phosphorylation status of RhoGDI-135 and RhoGTPases.36,37 We previously showed that phosphorylation of RhoGDI-1 by PKC
induces the activation of RhoA,38 whereas phosphorylation of RhoGDI-1 by PAK1 may induce activation of Rac1.35 It is also possible that direct phosphorylation of RhoA and Cdc42 enhances their interaction with RhoGDI-1.35,39 Although these findings do not explain differences in the specificity of RhoGDI-1 in regulating RhoA activity seen in our studies, they suggest that the phosphorylation state of RhoGDI-1 or RhoGTPases may be important in regulating the activity of RhoGDI-1 toward specific RhoGTPases. Rho family GTPases were shown to be primary signals regulating endothelial barrier function through modification of actin cytoskeletal organization and integrity of interendothelial junctions.3 Activation of RhoA induces the breakdown of interendothelial junctions,7,11 whereas Rac1 activation stabilized interendothelial junctions and counteracted the effects of RhoA.40 Cdc42 regulated the restoration of endothelial barrier function via an adherens junctional-dependent pathway.13 The mechanisms underlying the modulation of the activities of members of Rho family are unclear because it has been difficult to extrapolate findings from endothelial monolayers to the microvessel barrier in vivo.13 Therefore, a key question is whether RhoGDI-1 is a critical nodal point regulating microvascular permeability. Using RhoGDI-1–/– mice to investigate the function of RhoGDI-1 in lung endothelial barrier function in vivo, we observed that endothelial barrier was disrupted in lungs of RhoGDI-1–/– mice. This was evident by both increased microvessel filtration coefficient and rate of lung edema formation. The underlying structural basis for the increased permeability was the opening of interendothelial junctions in lungs of RhoGDI-1–/– mice. Downregulation of endogenous RhoGDI-1 in cultured endothelial cells using siRNA recapitulated the disassembly of the junctions seen in vivo in lungs.
MLC was hyperphosphorylated in the RhoGDI-1–/– mouse lungs compared with WT. Treatment of lungs of RhoGDI-1–/– mice with the Rho kinase inhibitor Y-27632 abolished the increased basal lung microvascular permeability in RhoGDI-1–/– mice. These findings support the hypothesis that deletion of RhoGDI-1 resulted in increased RhoA activity, activation of Rho kinase, and hyperphosphorylation of MLC leading to increased lung microvascular permeability. Because the deletion of RhoGDI-1 did not affect the activities of Rac1 or Cdc42 to counteract RhoA activation, it is likely that unchecked RhoA activity was responsible for signaling the increase in lung microvascular permeability in RhoGDI-1–/– mice. MLC phosphorylation was implicated in the regulation of endothelial permeability in cell cultures.41 Recently it was reported that inhibition of MLC kinase (that phosphorylates MLC) prevents endotoxin-induced acute lung injury.42 Currently, we are performing detailed analysis of the role of MLC in the regulation of microvascular permeability using MLC kinase knockout mice.
We observed that the basal increase in lung microvascular permeability seen in RhoGDI-1–/– mice was not augmented by thrombin or the PAR-1 agonist peptide challenge, as was the case in WT mice. This observation suggests that the high RhoA activity in RhoGDI-1–/– mice induced maximal endothelial contracture secondary to MLC hyperphosphorylation, thus mitigating any further increases in microvessel permeability. Although the lungs from RhoGDI-1–/– mice accumulated more water in the isogravimetric lung preparation indicative of increased barrier leakiness, there was no evidence of pulmonary edema or respiratory distress in resting mice. This could be explained by the engagement of various "safety factors" such as increased lymphatic drainage and rises in interstitial oncotic and hydrostatic pressures that would restrict fluid accumulation in presence of high basal lung vascular permeability.1
In conclusion, our findings have uncovered an important modulatory function of RhoGDI-1 in regulating basal lung endothelial barrier function in vivo. RhoGDI-1 serves as a nodal point that negatively regulates the activation state of RhoA and thereby serves to suppress Rho kinase and phosphorylation of endothelial MLC. It is conceivable therefore that therapies directed at augmenting RhoGDI-1 activity in endothelial cells may be beneficial in reestablishing the endothelial barrier and restoring lung fluid balance in inflammatory diseases such as acute respiratory distress syndrome.
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Acknowledgments |
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Sources of Funding
This work was supported by National Institutes of Health Grants GM56159, GM65160, and PO1HL606078 and by a grant from the American Heart Association (to T.V.Y.). T.V.Y. is an Established Investigator of the American Heart Association.
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this study.
Original received December 1, 2006; revision received May 15, 2007; accepted May 16, 2007.
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