|Home | About | Journals | Submit | Contact Us | Français|
The small GTPase Rac is critical to vascular endothelial functions, yet its regulation in endothelial cells remains unclear. Understanding the upstream pathway may delineate Rac activation mechanisms and its role in maintaining vascular endothelial barrier integrity.
By investigating P-Rex1, one of the Rac-specific guanine nucleotide exchange factors (GEFs) previously known for G protein-coupled receptor (GPCR) signaling, we sought to determine whether Rac-GEF is a nodal for signal integration and potential target for drug intervention.
Using gene deletion and siRNA silencing approach, we investigated the role of P-Rex1 in lung microvascular endothelial cells (HLMVECs). TNF-α exposure led to disruption of endothelial junctions, and silencing P-Rex1 protected junction integrity. TNF-α stimulated Rac activation and ROS production in a P-Rex1-dependent manner. Removal of P-Rex1 significantly reduced ICAM-1 expression, PMN transendothelial migration and leukocyte sequestration in TNF-α challenged mouse lungs. The P-Rex1 knockout mice were also refractory to lung vascular hyper-permeability and edema in a LPS-induced sepsis model.
These results demonstrate for the first time that P-Rex1 expressed in endothelial cells is activated downstream of TNF-α, which is not a GPCR agonist. Our data identify P-Rex1 as a critical mediator of vascular barrier disruption. Targeting P-Rex1 may effectively protect against TNF-α and LPS-induced endothelial junction disruption and vascular hyper-permeability.
Vascular endothelial cells form the lining of blood vessels and separate the underlying tissue from circulating blood. Disruption of the endothelial barrier leads to increased vascular permeability to plasma proteins and inflammatory cells, resulting in edema as seen in acute lung injury and in its more severe form, acute respiratory distress syndrome (ARDS)1. Vascular permeability can be transcellular or paracellular. Transcellular permeability involves the formation of transport vesicles whereas paracellular permeability requires disruption of the adherens junctions between two adjacent endothelial cells. VE-cadherin is an endothelial specific marker of adherens junctions2,3 and a determinant of integrity of endothelial junctions. Increased vascular permeability is associated with extravasation of leukocytes into the underlying tissue4, and VE-cadherin has been proposed to play a role in leukocyte transmigration5.
Several pro-inflammatory factors are released into the blood stream during an inflammatory response, among which thrombin, TNF-α, IL-1β and histamine are known to disrupt the endothelial barrier. TNF-α is one of the most commonly encountered proinflammatory cytokines in pathological conditions such as sepsis6. Elevated TNF-α levels are found in the bronchoalveolar fluid7 and plasma8 of ARDS patients. TNF-α increases the permeability of pulmonary microvessel endothelial barrier9,10 and causes edema in animals11. However, the mechanisms of TNF-α-induced endothelial barrier dysfunction are not clearly understood. Tyrosine phosphorylation of VE-cadherin12,13, production of reactive oxygen species (ROS)14,15, and activation of the small GTPase Rac16 have been associated with TNF-α-induced endothelial barrier dysfunction.
Rac is a monomeric GTPase of ~21 kDa17. In endothelial cells, Rac activation downstream of GPCRs, such as the thrombin receptor PAR1, induces re-annealing of endothelial junctions during endothelial barrier repair phase18. However, it was also reported that introduction of a constitutively activated Rac led to endothelial barrier dysfunction19. Similar findings were reported in endothelial cells stimulated with VEGF20 and platelet-activating factor21. In phagocytes, Rac is known for its role in NADPH oxidase activation and superoxide production22. Genetic deletion or silencing of the Rac gene showed that Rac is also essential for NADPH oxidase-dependent ROS production in endothelial cells20. Despite these observations, the mechanism by which ROS regulates vascular permeability remains unclear. TNF-α has been shown to induce ROS production and vascular permeability, but the role for Rac in ROS production downstream of the TNF-α pathway has not been established. There are approximately 70 Dbl family guanine nucleotide exchange factors (GEFs)23, yet much fewer Rho family small GTPases (to which Rac belongs), suggesting that Rho GEFs confer specificity of Rho GTPase activation in tissues where the GEFs are expressed. To date, Tiam-1 and Vav-2 are the only GEFs implicated in regulating Rac activation in endothelial cells21,24,25.
In this study, we examined phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac Exchanger 1 (P-Rex1) for its possible involvement in TNF-α-induced endothelial barrier dysfunction. P-Rex1 is a Rac-specific GEF regulated by PIP3 and G protein βγ subunits26,27. In neutrophils from P-Rex1-deficient mice, GPCR-induced activation and bactericidal functions are compromised28,29. P-Rex1 is primarily expressed in myeloid cells26 and neuronal tissue30. Its biological functions range from neuron migration and neurite differentiation30,31 to tumor metastasis32–34. In this study, we show that P-Rex1 is a key mediator of TNF-α-induced vascular permeability, which involves Rac activation and ROS production in a PI3K-dependent manner. In addition, we identified a novel role for endothelial P-Rex1 in regulating transendothelial migration of polymorphnuclear leukocytes (PMNs).
Human lung microvascular endothelial cells (HLMVEC) were obtained from Lonza (Walkersville, MD). These cells were transfected with siRNA specific for P-Rex1 or with scrambled siRNA as negative controls. Experiments with mice were conducted using procedures approved by the Institutional Animal Care and Use Committee at the University of Illinois at Chicago. A detailed, expanded Methods section can be found in the Supplemental Materials.
P-Rex1 was originally identified in neutrophils and neurons, and its function outside these cells remains unclear26,30. We examined P-Rex1 expression in endothelial cells and its potential involvement in endothelial barrier function. Using RT-PCR, P-Rex1 transcript was found in 3 different types of endothelial cells tested, including human lung microvascular endothelial cells (HLMVEC), human pulmonary artery endothelial cells (HPAEC), and human umbilical vein endothelial cells (HUVEC) (Online Figure I-A). The expression level of P-Rex1 protein in these endothelial cells was comparable to that in bone marrow-derived macrophages (Online Figure I-B).
To assess the role for P-Rex1 in endothelial barrier function, HLMVECs were treated with P-Rex1-specific siRNA to reduce P-Rex1 expression. Scrambled (sc) siRNA was used as a negative control. A ~80% reduction in P-Rex1 protein level was obtained (Online Figure I-C, I-D). The siRNA-transfected cells were then subjected to measurement of changes in transendothelial electrical resistance (TER) following TNF-α stimulation, which increases vascular permeability. As expected, the control (sc-siRNA transfected) cells showed a decrease in TER culminating 4–5 h post stimulation, suggesting barrier disruption. In comparison, P-Rex1 siRNA transfected cells showed much less barrier disruption (Figure 1A). Based on quantification of barrier disruption against absolute resistance values (Figure 1B), P-Rex1 is a necessary component for TNF-α-induced loss of TER, which reflects endothelial cell barrier dysfunction. TNF-α-induced barrier dysfunction is not a consequence of endothelial cell apoptosis, because the majority of transfected HLMVECs remained healthy after TNF-α treatment in DNA fragmentation assay (Online Figure II).
Fluorescent imaging analysis was conducted to examine barrier dysfunction of HLMVECs, characterized by intercellular gap formation14 (Figure 1C). The endothelial adherens junctions were detected with an anti-VE-cadherin antibody (green). After stimulation with TNF-α, sc-siRNA transfected endothelial cells displayed discontinuities between neighboring cells (marked with arrows). In comparison, HLMVECs receiving P-Rex1 siRNA showed minimal alteration of barrier integrity. The area of inter-endothelial gaps was quantified (Figure 1D), and the changes were significant (p<0.05). Thus, data from both TER and imaging analysis support a role for P-Rex1 in TNF-α-induced disruption of endothelial barrier.
Recent studies have shown that TNF-α, at concentrations that induce opening of inter-endothelial junctions, activates the small GTPase Rac16. However the GEF responsible for TNF-α-induced Rac activation in endothelial cells remains unidentified. To explore the signal transduction pathway downstream of TNF-α stimulation, we examined possible involvement of P-Rex1 in Rac activation. TNF-α induced a rapid and transient Rac activation in HLMVECs that peaked within 1 min and continued for 2 min before it began to decrease (Figure 2A). Based on Rac-GTP pull-down assay, TNF-α induced up to an 8-fold increase in Rac activation compared to unstimulated cells (Figure 2B). In HLMVECs receiving P-Rex1 siRNA, Rac activation was significantly reduced, indicating that P-Rex1 is required for TNF-α-induced Rac activation. In addition, the Rac inhibitor NSC23766 prevented TNF-α-induced endothelial permeability as measured by TER (Online Figure III). We also transfected HLMVECs with a dominant negative Rac (T17NRac) to exclude non-specific effects of NSC23766. The dominant negative Rac ablated TNF-α-induced barrier dysfunction (Online Figure IV). These results strongly suggest that Rac is required for TNF-α-induced endothelial barrier dysfunction and P-Rex1 is an essential Rac GEF regulating TNF–α-induced Rac activation.
A number of molecules were examined in order to exclude several possibilities that might have affected the outcome of our experiments. GEF-H1 is not only a Rac GEF but also a Rho GEF35 and has been implicated in TNF-α-induced epithelial barrier integrity36. To determine whether it plays a role in our experiments, we used siRNA to knock down GEF-H1 in HLMVECs and performed TER. Our results indicate that, unlike P-Rex1, GEF-H1 removal did not reverse barrier dysfunction induced by TNF-α (Online Figure V). We also considered potential involvement of Rho, known to be responsible for endothelial barrier dysfunction37. To exclude the involvement of Rho downstream of the TNF-α and P-Rex1 pathway, we performed Rho pull-down assay as detailed in the Methods. The absence of P-Rex1 did not alter Rho activation (Online Figure VI). Therefore, even though TNF-α has the ability to activate Rho16, it does not require P-Rex1.
NADPH oxidase (Nox) has been implicated in TNF-α-induced endothelial barrier dysfunction14,15. However, it is unclear how TNF-α regulates ROS production. In phagocytes, Rac is required for NADPH oxidase activation leading to ROS production38,39. Therefore, we determined whether P-Rex1 is involved in endothelial ROS production through Rac activation. HLMVECs plated on gelatin-coated glass dishes were treated with either sc-siRNA or P-Rex1 siRNA and then stimulated with TNF-α, ROS production was measured by dihydrorhodamine 123 (DHR123). As shown in Figure 3A and quantified in Figure 3B, siRNA-mediated silencing of P-Rex1 led to a significant reduction in TNF-α-stimulated ROS production. Diphenyleneiodonium (DPI), a flavocytochrome inhibitor, diminished TNF-α-induced ROS production, suggesting that inducible activation of the NADPH oxidase is required (Figure 3C, 3D). Likewise, NSC23766 reduced ROS production, supporting the notion that Rac is involved in TNF-α-induced NADPH oxidase activation in HLMVECs (Figure 3C, 3D). We next determined whether suppression of ROS production could alter the integrity of the HLMVEC monolayer. DPI-treated cells were refractory to TNF-α-induced decrease in TER (Figure 3E, 3F). These results demonstrate a correlation between endothelial P-Rex1 and TNF-α-induced ROS production leading to a loss of barrier function.
We examined the role for P-Rex1 in regulating TNF-α-induced tyrosine phosphorylation of VE-cadherin since this has been reported to be critical to the loss of vascular integrity13. In sc-siRNA transfected HLMVECs, TNF-α treatment led to phosphorylation of VE-cadherin at 10 min and peaked at 20 min (Figure 4A, 4B). TNF-α-induced VE-cadherin phosphorylation was diminished in HLMVECs transfected with P-Rex1 siRNA (Figure 4A, 4B), suggesting that VE-cadherin was phosphorylated in a P-Rex1-dependent manner. It was reported that the Src family protein tyrosine kinases undergo ROS-dependent phosphorylation that is required for VE-cadherin phosphorylation12,14. To test whether P-Rex1 is required for Src activation downstream of TNF-α stimulation, HLMVECs transfected with sc-siRNA or P-Rex1 siRNA were stimulated with TNF-α for the indicated time points, and phosphorylation of c-Src at Tyr416 was determined. HLMVECs receiving P-Rex1 siRNA displayed more than 70% reduction in phosphorylation of Tyr416 at 5 and 10 min compared to cells transfected with sc-siRNA (Figure 4C, 4D), indicating that P-Rex1 is required for TNF-α-induced Src activation.
P-Rex1 resides in the cytosol of resting neutrophils and translocates to membrane upon cell activation40. We determined whether P-Rex1 is translocated to plasma membrane in TNF-α stimulated endothelial cells. HLMVECs were stimulated with TNF-α for 0, 1 and 2 min, and P-Rex1 in the membrane and cytosolic fractions was determined. P-Rex1 underwent membrane translocation as early as 1 min and continued to increase at 2 min (Figure 5A, 5B, 5C). We also used an additional approach to test the membrane translocation of P-Rex1. HLMVECs were unstimulated or stimulated with TNF-α for 2 min. The cells were then permeabilized, fixed, and incubated with anti-P-Rex1 and an Alexafluor 488 conjugated secondary antibody. Images acquired by confocal microscopy showed accumulation of P-Rex1 to the membrane periphery after stimulation (Figure 5D). Pre-treatment of HLMVECs with the PI3K inhibitor LY294002 prevented membrane translocation of P-Rex1 (Figure 5D), suggesting that it is PI3K-dependent in TNF-α stimulated HLMVECs.
Earlier studies have shown that P-Rex1 activation downstream of GPCRs requires both PI3K and the βγ subunits of heterotrimeric G proteins26,27. However, TNF-α is not a GPCR agonist and how a non-GPCR activates P-Rex1 remains unclear. To determine whether Gβγ subunits are involved in TNF-α signaling, we pre-treated HLMVECs with Gβγ modulator II (Gallein; 3',4',5',6'-Tetrahydroxyspiro [isobenzofuran-1(3H),9'-(9H)xanthen]-3-one), which inhibits conformational changes of Gβγ subunits and blocks Gβγ-dependent activation of PI3K and Rac in HL-60 cells41. Pre-treatment with Gallein did not affect TNF-α-induced membrane translocation of P-Rex1 (Figure 5D, lower panels), whereas it significantly inhibited thrombin-induced calcium mobilization in HLMVECs (Figure 5E, 5F). These findings suggest that Gβγ is not indispensable in TNF-α-induced P-Rex1 activation, but PI3K is necessary. The requirement of PI3K for P-Rex1 activation in TNF-α-stimulated endothelial cells was also confirmed when HLMVECs pre-treated with the PI3K inhibitor LY294002 displayed reduced Rac activation by more than 50% (Online Figure VII-A, VII-B).
To determine an in vivo function of P-Rex1 in acute lung injury, WT and P-Rex1 knockout mice29 were instilled with TNF-α intra-tracheally. Changes in lung vascular permeability were evaluated based on the accumulation of Evans blue albumin (EBA) after tail vein injection. Significantly less accumulation of EBA was seen in the lungs of mice lacking P-Rex1, compared to WT controls (Figure 6A). The in vivo role for P-Rex1 in lung edema was also evaluated based on lung wet-to-dry weight ratio after intra-tracheal instillation of TNF-α. Again, the P-Rex1 knockout mice had significantly less edema compared to WT controls (Figure 6B, 6C). These results demonstrate a critical role for P-Rex1 in the dynamic regulation of lung vascular permeability in vivo.
LPS-induced sepsis is a clinically relevant model of acute lung injury. We tested a potential role for P-Rex1 in this model where barrier dysfunction contributes to the pathological changes. WT and P-Rex1 knockout mice were intraperitoneally injected with LPS or PBS for 6 h, which produced septic signs such as decreases in leukocyte count and platelet count (Online Figure XI). The lungs were subjected for Kf,c measurements (Figure 6D) and EBA dye leakage measurement (Figure 6E). Both the Kf,c and EBA data showed that P-Rex1 knockout mice have significantly less lung microvascular capillary filtration and EBA leakage, indicating a role for P-Rex1 in LPS-induced barrier dysfunction during sepsis.
In acute lung injury, there is marked infiltration of PMNs and macrophages in the lungs. To determine infiltration of phagocytes into bronchoalveolar (BAL) fluid, WT and P-Rex1 knockout mice were instilled intra-tracheally with PBS or 0.5 µg of murine recombinant TNF-α. After 24 h, mice were anesthetized and BAL fluid was collected. BAL fluid obtained from P-Rex1 knockout mice showed significantly less PMNs and macrophages compared to WT mice (Figure 7A and 7B). This result indicates that P-Rex1 is necessary for transendothelial migration of these leukocytes. In a parallel experiment, WT and P-Rex1 knockout mice were intratracheally injected with murine TNF-α for 24 h followed by collection of lungs for histological analysis. Haematoxylin and Eosin staining showed significantly less cellular infiltration and interstitial tissue thickening in P-Rex1 knockout mouse lungs compared to WT lungs (Online Figure VIII).
Since the knockout approach results in a loss of P-Rex1 in all tissues, we next determined the relative contribution of P-Rex1 in endothelial cells vs PMNs to the reduced PMN transendothelial migration. Figure 7C shows the effects of eliminating P-Rex1 from endothelial cells on transendothelial migration of WT and P-Rex1−/− PMNs (a more detailed version of the experiment, with ligand controls included, was shown in Online Figure IX). HLMVECs transfected with sc-siRNA or P-Rex1 siRNA were plated on 3 µm membrane pore inserts. PMNs were isolated concurrently from both WT and P-Rex1−/− mice and applied to the HLMVEC monolayer, which received sc-siRNA (Figure 7C, filled bars) or P-Rex1 siRNA (Figure 7C, open bars) and simulated with TNF-α for 4 h. Eliminating P-Rex1 from the endothelial cells caused a significant reduction in PMN transmigration, which applies to both the WT and P-Rex1−/− PMNs (Figure 7C). In comparison, removal of P-Rex1 from PMNs does not significantly impact cell migration in this experiment (ns, Figure 7C). Based on these findings, we concluded that endothelial P-Rex1 plays an important role in PMN transendothelial migration.
We have also taken an ex vivo approach to determine the effect of P-Rex1 in PMN transmigration into the lung tissue. Lungs from WT and P-Rex1 knockout mice were perfused to remove blood cells and then exposed to murine TNF-α. Freshly isolated PMNs from WT and P-Rex1−/− mice were radiolabeled with 111Indium oxine and perfused through WT and P-Rex1−/− lungs, or vice versa. As shown in Figure 7D, the P-Rex1−/− lungs showed significantly less radioactivity accumulation than WT lungs, suggesting that absence of P-Rex1 in lung tissue could significantly reduced PMN transmigration. In contrast, no significant difference in radioactivity accumulation was observed in WT lungs perfused with either WT PMNs or P-Rex1−/− PMNs (Figure 7D). These findings suggest that endothelial P-Rex1 is highly important in PMN transmigration into the lung tissue.
In the above experiments, we observed that TNF-α treatment of HLMVECs is necessary for PMN transmigration. A number of proteins are induced upon TNF-α stimulation of endothelial cells, among which ICAM-1 is required for efficient PMN transendothelial migration. We examined TNF-α-induced ICAM-1 expression in HLMVECs transfected with either sc-siRNA or P-Rex1 siRNA, and found that cells receiving P-Rex1 siRNA displayed significantly less ICAM-1 expression compared to cells treated with sc-siRNA (Online Figure IX). A significantly lower ICAM-1 induction may contribute to the decreased PMN transmigration across the P-Rex1 deficient HLMVEC monolayer.
The present study examines P-Rex1 expression in endothelial cells and its role in mediating TNF-α-induced increase in vascular endothelial permeability. A number of new findings were made. 1) P-Rex1 is highly expressed in vascular endothelial cells and plays important roles in these cells. 2) Our results show for the first time that P-Rex1 can be activated by a non-GPCR, in this case the TNF receptor, in endothelial cells. 3) This study reaffirms a role for Rac in TNF-α induced vascular endothelial dysfunction, which has been an unsettled issue. 4) P-Rex1 activation leads to ROS production in endothelial cells. 5) Endothelial P-Rex1 is important for PMN transmigration into the lung tissue. These findings are summarized schematically in a working model (Figure 8).
In this model, P-Rex1 is a major Rho GEF downstream of TNF-α receptor in endothelial cells. Prior to this study, P-Rex1 is mainly known for its functions in the brain and in PMNs, where it was first discovered26. Thus, the finding of P-Rex1 in various endothelial cells suggests that this Dbl family Rho GEF is more broadly expressed than previously thought. Our data demonstrates that P-Rex1 is expressed in vascular endothelial cells, and it mediates TNF-α-induced vascular permeability as well as PMN infiltration into the lung tissue. These functions of P-Rex1 require Rac activation, which leads to NADPH oxidase-dependent ROS production, c-Src activation and VE-cadherin phosphorylation in HLMVECs. Our in vitro results are corroborated by data from P-Rex1 knockout mice, which are refractory to TNF-α-induced increase in vascular permeability in the lungs as demonstrated by reduced edema. Collectively, these results demonstrate that endothelial P-Rex1 is critical to TNF-α signaling that leads to increased vascular endothelial permeability.
Our model places P-Rex1 downstream of the TNF-α receptor, whereas published reports depicts P-Rex1 as a Rac-specific GEF activated by GPCRs26. In endothelial cells, P-Rex1 can be activated by a GPCR42. Our finding that P-Rex1 is activated by TNF-α is totally unexpected since TNF-α is not known to couple to G proteins. We observed rapid membrane translocation of P-Rex1 in endothelial cells, which is characteristic of its activation40. It is also evident that TNF-α stimulates P-Rex1-dependent Rac activation in HLMVECs. Since the time course of Rac activation and P-Rex1 membrane translocation is consistent with that of GPCR signaling, we examined the requirement for P-Rex1 activation in TNF-α stimulated cells. A reported feature of P-Rex1 is its dependence on PIP3 and Gβγ for activation26,27. Our results show that TNF-α-induced Rac activation is PI3K-dependent. However, we observed no effect for the Gβγ inhibitor Gallein to affect TNF-α-induced P-Rex1 membrane translocation, thus challenging the conventional view that Gβγ is required for P-Rex1 activation. It is notable that TNF-α signaling has not been associated with activation or transactivation of heterotrimeric G proteins, although TNF-α is known for its activation of PI3K43, suggesting that TNF-α-induced PIP3 production might be sufficient to trigger P-Rex1 activation in HLMVECs.
In endothelial cells stimulated with GPCR agonists such as thrombin, a reversible endothelial barrier disruption occurs. While there are multiple pathways for disrupting the endothelial barrier, Rac has been associated with re-annealing of junctions in response to GPCR activation18. Contrary to this view, there is evidence supporting a role for Rac in endothelial barrier dysfunction. For instance, van Wetering et al reported that expression of a constitutively active Rac in HUVECs could cause changes leading to increased vascular permeability19. Rac has been implicated in TNF-α and VEGF-induced increase in vascular permeability16,20. As shown in our model, GTP-bound Rac is required for TNF-α induced ROS production, and the Rac inhibitor NSC23766 abrogated TNF-α-induced vascular endothelial permeability in HLMVEC. Likewise, dominant negative RacT17N, when expressed in endothelial cells, prevented TNF-α-induced endothelial barrier dysfunction. Our data supports a role for Rac in TNF-α induced endothelial barrier dysfunction, which is mediated through P-Rex1 activation and ROS production. These findings connect a Rho GEF to previously reported functions of ROS in the regulation of vascular permeability15,19,44–46.
Disruption of endothelial barrier is a triggering factor for infiltration of PMNs into tissues4,47. We tested whether TNF-α-induced disruption of endothelial barrier aggravates PMN infiltration, and if so, whether blocking P-Rex1 expression in EC prevents PMN transmigration. We found that, in the absence of P-Rex1, PMN transmigration was significantly reduced. Much fewer PMNs and macrophages were present in BAL fluid of P-Rex1 knockout mice compared to WT mice following instillation of murine recombinant TNF-α into the airways. Silencing endothelial P-Rex1 expression resulted in significantly less PMN transmigration compared to sc-siRNA transfected endothelium. This phenomenon was also confirmed ex vivo by PMN sequestration studies where radiolabeled WT or P-Rex1 knockout PMNs were perfused into WT and P-Rex1 knockout mice respectively and vice versa. It appears that the crosstalk between PMN and EC is key to PMN transendothelial migration4. Our Western blot data showed that there is significantly less ICAM-1 expression in the absence of P-Rex1. As depicted in the model, P-Rex1 appears to play a role in the regulation of TNF-α-induced ICAM-1 expression, which involves NF-κB activation. The mechanism underlying P-Rex1 regulation of NF-κB activation has yet to be delineated. An increase in ICAM-1 expression may in turn affect PMN transendothelial migration.
Our findings strongly implicate P-Rex1 in regulating TNF-α-induced vascular permeability and lung edema. This function is mediated through the activation of Rac and generation of ROS, thus promoting endothelial barrier disruption and transendothelial PMN migration. These results demonstrate that down-regulation of P-Rex1 may affect multiple pro-inflammatory pathways, and P-Rex1 may be a new therapeutic target in controlling lung vascular injury and PMN-mediated lung inflammation.
A large number of GEFs exist for a relatively small number of the small GTPases, suggesting that GEFs confer tissue specificity. We sought to determine which GEF is responsible for Rac activation in endothelial cells. We found that in endothelial cells, GEF for Rac is regulated by P-Rex1, a PtdInsP3 and Gβγ. Using siRNA-mediated knockdown and gene deletion approaches, we identified P-Rex1 as being critical for Rac activation by TNF-α. Cells and mice lacking P-Rex1 produce less reactive oxygen species and are more resistant to TNF-α- and LPS-induced loss of endothelial barrier functions. Moreover, removal of P-Rex1 reduced ICAM-1 expression and transendothelial migration of neutrophils, thus attenuating inflammatory responses.. Our findings identify a previously unrecognized function of P-Rex1, which may be a target for therapeutic intervention aimed at reducing inflammation and tissue injury.
We would like to thank Dr. Richard Minshall and Dr. Chinnaswamy Tiruppathi for guidance to the graduate student and for sharing their knowledge in endothelial biology. We also thank Dr. Marcus Thelen for the gift of anti-P-Rex1 antibodies.
SOURCES OF FUNDING
This work was supported in part by grants R01 AI033507 (to R.D.Y.), P01 HL077806 (to A.B.M.) and P01 HL070694 and R01 HL108430 (to D.W.) from United States Public Health Services. R.D.Y. is supported in part by National Basic Research Program of China (973 Program) [Grant 2012CB518001]. R. P. N. is a recipient of an American Heart Association predoctoral fellowship (11PRE5740014).
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.