Suilysin remodels the cytoskeletons of human brain microvascular endothelial cells by activating RhoA and Rac1 GTPase.
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ABSTRACT:
SUBMITTER: Lv Q
PROVIDER: S-EPMC3978165 | biostudies-literature | 2014 Apr
REPOSITORIES: biostudies-literature
Publications
Suilysin remodels the cytoskeletons of human brain microvascular endothelial cells by activating RhoA and Rac1 GTPase.
Lv Qingyu Q Hao Huaijie H Bi Lili L Zheng Yuling Y Zhou Xuyu X Jiang Yongqiang Y
Protein & cell 20140401 4
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Project description:ObjectiveIQ domain GTPase-activating protein 1 (IQGAP1) contributes to cytoskeletal network regulation in epithelial cells by its scaffolding properties and by binding the Rho GTPase Rac1 to maintain its activity. The functions of IQGAP1 in endothelial cells beyond angiogenesis remain unclear. We hypothesized that IQGAP1 participates in the regulation of endothelial barrier function.Methods and resultsSilencing IQGAP1 in human microvascular endothelial cells resulted in a disruption of adherens junctions, formation of interendothelial gaps, and a reduction in barrier function. Furthermore, silencing of IQGAP1 abrogated the barrier enhancement effect of angiopoietin-1 (Angpt-1) and abolished the barrier-stabilizing effect of Angpt-1 on thrombin-stimulated cells. Coimmunoprecipitation detected binding of endogenous IQGAP1 with Rac1 at baseline that was stronger when Rac1 was activated and weaker when it was deactivated. Measurement of GTP-bound Rac1 revealed that Angpt-1 failed to activate Rac1 not only if IQGAP1 was silenced but also if cells were transfected with a mutant disabled in Rac1 binding (T1050AX2). Furthermore, a dominant-active Rac1 was sufficient to completely reverse the morphological and functional changes induced by reduction in IQGAP1.ConclusionThese experiments are the first demonstration of IQGAP1 regulating barrier function in any cell type. Further, our data show that Angpt-1 requires IQGAP1 as an indispensable activator of Rac1.
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Project description:AimsEndothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed by extracellular junctions. Various mediators modulate the endothelial barrier by acting on junctional protein complexes and the therewith connected F-actin cytoskeleton. Different Rho GTPases participate in this modulation, but their mechanisms are still partly resolved. Here, we aimed to elucidate whether the opening and closure of the endothelial barrier are associated with distinct localized RhoA activities at the subcellular level.Methods and resultsLive fluorescence resonance energy transfer (FRET) microscopy revealed spatially distinct RhoA activities associated with different aspects of the regulation of endothelial monolayer integrity. Unstimulated ECs were characterized by hotspots of RhoA activity at their periphery. Thrombin receptor activation in the femoral vein of male wistar rats and in cultured ECs enhanced RhoA activity at membrane protrusions, followed by a more sustained RhoA activity associated with cytoplasmic F-actin filaments, where prolonged RhoA activity coincided with cellular contractility. Unexpectedly, thrombin-induced peripheral RhoA hotspots were not spatially correlated to the formation of large inter-endothelial gaps. Rather, spontaneous RhoA activity at membrane protrusions coincided with the closure of inter-endothelial gaps. Electrical impedance measurements showed that RhoA signalling is essential for this protrusive activity and maintenance of barrier restoration.ConclusionSpontaneous RhoA activity at membrane protrusions is spatially associated with closure, but not formation of inter-endothelial gaps, whereas RhoA activity at distant contractile filaments contributes to thrombin-induced disruption of junctional integrity. Thus, these data indicate that distinct RhoA activities are associated with disruption and re-annealing of endothelial junctions.
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Project description:The blood-brain barrier (BBB) plays important roles in the recovery of traumatic brain injury (TBI) which is a major factor contributing to cerebral edema. Acid fibroblast growth factor (aFGF) contributes to maintain vascular integrity and restores nerve function. However, whether aFGF protects BBB following TBI remains unknown. The purpose of this study was to determine whether exogenous aFGF preserves BBB integrity by activating the PI3K-Akt-Rac1 pathway and inhibiting RhoA after TBI. BBB permeability was assessed using evans blue dye and fluorescein isothiocyanate dextran fluorescence. Neurofunctional tests, such as the garcia test, were conducted in a blinded fashion, and protein expression was evaluated via western blotting and immunofluorescence staining. Our results showed that aFGF improved neurofunctional deficits, preserved BBB integrity, and up-regulated tight junction proteins and adherens junction proteins 24 h after experimental TBI. However, the PI3K/Akt inhibitor LY294002 reversed the protective effects of aFGF on neurofunctional deficits and junction protein expression and significantly suppressed p-Akt and GTP-Rac1 activity. Furthermore, aFGF administration significantly decreased GTP-RhoA expression in the treated group compared with the vehicle group, while PI3K/Akt inhibition increased GTP-RhoA expression. Similar results were observed in vitro, as aFGF exerted protective effects on endothelial cell integrity by up-regulating junction proteins and PI3K-Akt-Rac1 pathway and down-regulating RhoA expression under oxygen-glucose deprivation/reoxygenation (OGD) conditions. These data suggest that exogenous aFGF reduces RhoA activity in part by activating the PI3K-Akt-Rac1 signaling pathway, thus improving neurofunctional deficits and preserving BBB integrity after TBI.
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Project description:Metastatic carcinoma cells exhibit at least two different phenotypes of motility and invasion - amoeboid and mesenchymal. This plasticity poses a major clinical challenge for treating metastasis, while its underlying mechanisms remain enigmatic. Transitions between these phenotypes are mediated by the Rac1/RhoA circuit that responds to external signals such as HGF/SF via c-MET pathway. Using detailed modeling of GTPase-based regulation to study the Rac1/RhoA circuit's dynamics, we found that it can operate as a three-way switch. We propose to associate the circuit's three possible states to the amoeboid, mesenchymal and amoeboid/mesenchymal hybrid phenotype. In particular, we investigated the range of existence of, and the transition between, the three states (phenotypes) in response to Grb2 and Gab1 - two downstream adaptors of c-MET. The results help to explain the regulation of metastatic cells by c-MET pathway and hence can contribute to the assessment of possible clinical interventions.
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Project description:Alpha1-chimaerin is a GTPase-activating protein (GAP) for Rac1, a member of the Rho small GTPase family, whose action leads to the inactivation of Rac1. Rac1 activity is upregulated in Alzheimer's disease, but little is known about the role of α1-chimaerin. In this study, we investigated the expression and localization of α1-chimaerin mRNA in postmortem human brains from patients with Alzheimer's disease and control subjects. In situ hybridization studies demonstrated that α1-chimaerin was expressed by neurons in the neo-cortex of the temporal lobe and the hippocampus of both controls and Alzheimer's disease cases, with the signal intensity dramatically decreased in patients with Alzheimer's disease. Real-time PCR analysis confirmed a significant reduction of α1-chimaerin mRNA expression in the temporal cortex of Alzheimer's disease cases. In contrast, α2-chimaerin mRNA levels showed no significant difference between the groups. The present study showed reduced α1-chimaerin expression in the brain of Alzheimer's disease cases, suggesting a role in the upregulation of Rac1 activity during the disease process.
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Project description:Rho GTPases are molecular switches that transmit biochemical signals in response to extracellular stimuli to elicit changes in the actin cytoskeleton. Rho GTPases cycle between an active, GTP-bound state and an inactive, GDP-bound state. These states are regulated by two distinct families of proteins-guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We studied the role of a previously uncharacterized GAP, ARHGAP18 (MacGAP). Overexpression of ARHGAP18 suppressed the activity of RhoA and disrupted stress fiber formation. Conversely, silencing of ARHGAP18 by small interfering RNA transfection-enhanced stress fiber formation and induced rounding of cells. We examined the role of ARHGAP18 in cell spreading and migration. Immunofluorescence analysis revealed that ARHGAP18 was localized to the leading edge during cell spreading and migration. ARHGAP18-knockdown cells showed impaired spreading, premature formation of stress fibers, and sustained activation of RhoA upon cell attachment. In addition, knockdown and overexpression of ARHGAP18 resulted in the inhibition and promotion of cell migration, respectively. Furthermore, ARHGAP18 was required for the polarization of cells for migration. Our results define ARHGAP18 as one of the crucial factors for the regulation of RhoA for the control of cell shape, spreading, and migration.
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Project description:Bacterial meningitis is currently recognized as one of the most important life-threatening infections of the central nervous system (CNS) with high morbidity and mortality, despite the advancements in antimicrobial treatment. The disruption of blood-brain barrier (BBB) induced by meningitis bacteria is crucial for the development of bacterial meningitis. However, the complete mechanisms involving in the BBB disruption remain to be elucidated. Here, we found meningitic Escherichia coli induction of angiopoietin-like 4 (ANGPTL4) in brain microvascular endothelial cells (BMECs) contributes to BBB disruption via ARHGAP5/RhoA/MYL5 signaling cascade, by the demonstration that ANGPTL4 was significantly upregulated in meningitis E. coli infection of BMECs as well as mice, and treatment of the recombinant ANGPTL4 protein led to an increased permeability of the BBB in vitro and in vivo. Moreover, we found that ANGPTL4 did not affect the expression of tight junction proteins involved in BBB disruption, but it increased the expression of MYL5, which was found to have a negative role on the regulation of barrier function during meningitic E. coli infection, through the activation of RhoA signaling pathway. To our knowledge, this is the first report demonstrating the disruption of BBB induced by ANGPTL4 through the ARHGAP5/RhoA/MYL5 pathway, which largely supports the involvement of ANGPTL4 during meningitic E. coli invasion and further expands the theoretical basis for the mechanism of bacterial meningitis.
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Project description:Imbalanced angiogenesis is a characteristic of several diseases. Rho GTPases regulate multiple cellular processes, such as cytoskeletal rearrangement, cell movement, microtubule dynamics, signal transduction and gene expression. Among the Rho GTPases, RhoA, Rac1 and Cdc42 are best characterized. The role of endothelial Rac1 and Cdc42 in embryonic development and retinal angiogenesis has been studied, however the role of endothelial RhoA is yet to be explored. Here, we aimed to identify the role of endothelial RhoA in endothelial cell functions, in embryonic and retinal development and explored compensatory mechanisms. In vitro, RhoA is involved in cell proliferation, migration and tube formation, triggered by the angiogenesis inducers Vascular Endothelial Growth Factor (VEGF) and Sphingosine-1 Phosphate (S1P). In vivo, through constitutive and inducible endothelial RhoA deficiency we tested the role of endothelial RhoA in embryonic development and retinal angiogenesis. Constitutive endothelial RhoA deficiency, although decreased survival, was not detrimental for embryonic development, while inducible endothelial RhoA deficiency presented only mild deficiencies in the retina. The redundant role of RhoA in vivo can be attributed to potential differences in the signaling cues regulating angiogenesis in physiological versus pathological conditions and to the alternative compensatory mechanisms that may be present in the in vivo setting.
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Project description:Compromised endothelial barrier function is a hallmark of inflammation. Rho family GTPases are critical in regulating endothelial barrier function, yet their precise roles, particularly in sphingosine-1-phosphate (S1P)-induced endothelial barrier enhancement, remain elusive. Confluent cultures of human umbilical vein endothelial cells (HUVEC) or human dermal microvascular endothelial cells (HDMEC) were used to model the endothelial barrier. Barrier function was assessed by determining the transendothelial electrical resistance (TER) using an electrical cell-substrate impedance sensor (ECIS). The roles of Rac1 and RhoA were tested in S1P-induced barrier enhancement. The results show that pharmacologic inhibition of Rac1 with Z62954982 failed to block S1P-induced barrier enhancement. Likewise, expression of a dominant negative form of Rac1, or knockdown of native Rac1 with siRNA, failed to block S1P-induced elevations in TER. In contrast, blockade of RhoA with the combination of the inhibitors Rhosin and Y16 significantly reduced S1P-induced increases in TER. Assessment of RhoA activation in real time using a fluorescence resonance energy transfer (FRET) biosensor showed that S1P increased RhoA activation primarily at the edges of cells, near junctions. This was complemented by myosin light chain-2 phosphorylation at cell edges, and increased F-actin and vinculin near intercellular junctions, which could all be blocked with pharmacologic inhibition of RhoA. The results suggest that S1P causes activation of RhoA at the cell periphery, stimulating local activation of the actin cytoskeleton and focal adhesions, and resulting in endothelial barrier enhancement. S1P-induced Rac1 activation, however, does not appear to have a significant role in this process.
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