Paraptosis-like cell death induced by yessotoxin

This study shows that BC3H1 myoblast cell lines exposed to 100 nM yessotoxin (YTX) undergo a form of programmed cell death distinct from apoptosis and with features resembling paraptosis. Morphologically, cells treated with YTX reveal extensive cytoplasmic vacuolation, mitochondrial and endoplasmic reticulum swelling, uncondensed chromatin and cytoskeletal alterations. DNA electrophoresis evidences lack of DNA fragmentation and Western blotting analysis demonstrates activation of the mitogen-activated protein kinase JNK/SAPK1. Further characterisation of this form of programmed cell death may have interest within medicine and cancer therapy.     

Specialisation of the tropomyosin composition of actin filaments provides new potential targets for chemotherapy

The actin microfilament network is important in maintaining cell shape and function in eukaryotic cells. It has a multitude of roles in cellular processes such as cell adhesion, motility, cellular signalling, intracellular trafficking and cytokinesis. Alterations in the organisation of the cytoskeleton and changes in cellular morphology, motility and adhesiveness are characteristic features of transformed cancer cells. For this reason cytoskeletal microfilaments have become promising targets for chemotherapy. In contrast to the microtubules, which have been targeted successfully with anti-tumour drugs such as Taxol-like compounds and the Vinca alkaloids, very few actin targeting drugs have been characterised. To date, no actin targeting drugs have been used in clinical trials due to their severe cytotoxicity. One reason for this cytotoxicity is that drugs such as the cytochalasins and latrunculins disrupt actin microfilaments in both non-tumour and tumour cells. To circumvent this problem, actin filament populations need to be targeted more specifically. Not all actin filaments are the same and there is growing evidence that within a cell there are different populations of actin filaments which are spatially organised into distinct cellular compartments each with a unique function. The structure and function of the actin cytoskeleton is primarily regulated by the associated actin binding proteins. Tropomyosin is an intrinsic component of most actin filaments and over 40 isoforms have been identified in non-muscle cells. Tm isoforms are spatially segregated and current evidence suggests that they can specify the functional capacity of the actin microfilaments. Therefore the composition of these functionally distinct actin filaments may be important in determining their stability and function within the cell. If actin filament populations can be discriminated and targeted based on their tropomyosin composition then this becomes a powerful approach for anticancer therapy.     

Lysosomes as the target of yessotoxin in invertebrate and vertebrate cell lines

The toxic effects of the algal polyether phycotoxin yessotoxin (YTX) are studied in the insect fat body IPLB-LdFB and the mouse fibroblast NIH3T3 cell lines. Our experiments confirm the cytotoxic action exerted by the toxin in both insect and mammalian cells, but morphological observations, TUNEL experiments and electrophoretic evalution of DNA integrity failed to evidence a clear pro-apoptotic role for YTX. In both IPLB-LdFB and NIH3T3 cell lines, neutral red and acridine orange stainings, together with evaluation of acid phosphatase activity demonstrate that YTX first damages lysosomal vesicles. This is then followed by a progressive depolymerization of actin microfilaments, as shown by phalloidin fluorescent immunostaining. Overall, our data identify in early lysosomal damage and the subsequent cytoskeletal disruption two common steps related to YTX toxicity towards metazoan cells.     

Involvement of gelsolin in cadmium-induced disruption of the mesangial cell cytoskeleton.

Cadmium (Cd2+) is known to cause a selective disruption of the filamentous actin cytoskeleton in the smooth muscle-like renal mesangial cell. We examined the effect of Cd2+ on the distribution of the actin-severing protein, gelsolin. Over 8 h, CdCl2 (10 microM) caused a progressive shift of gelsolin from a diffuse perinuclear and cytoplasmic distribution to a pattern decorating F-actin filaments. Over this time filaments were decreased in number in many cells, and membrane ruffling was initiated. Western blotting and 125I-F-actin gel overlays demonstrated an increase in actin-binding gelsolin activity in the cytoskeletal fraction of cell extracts following Cd2+ treatment. In in vitro polymerization assays, gelsolin acted as a nucleating factor and increased the rate of polymerization. Cytosolic extracts also increased the polymerization rate. Addition of Cd2+ together with gelsolin further increased the rate of polymerization. Gelsolin enhanced depolymerization of purified actin, and Cd2+ partially suppressed this effect. However, cytoskeletal extracts from Cd2+-treated cells also markedly increased depolymerization, suggesting further that Cd2+ may activate cellular component(s) such as gelsolin for actin binding. We conclude that a major effect of Cd2+ on the mesangial cell cytoskeleton is manifest through activating the association of gelsolin with actin, with gelsolin's severing properties predominating under conditions found in Cd2+-treated cells.     

Apoptotic events induced by yessotoxin in myoblast cell lines from rat and mouse.

This study reports apoptotic events after yessotoxin (YTX) exposure in L6 (rat) and BC3H1 (mouse) skeletal muscle myoblast cell lines. These cell lines are relevant targets to study the cytotoxic effect since this toxin has been reported as cardiotoxic. Mechanisms of action of YTX in multicellular organisms are not fully elucidated. Cell culture studies can contribute to find some of these mechanisms and trace the molecular pathways involved. The present work shows results from exposing cells to 100 nM purified YTX for 72 h. Morphological and biochemical changes characteristic of apoptotic cell death were evaluated in the two cell lines. Immunofluorescence and western blot techniques showed caspase-3 and caspase-9 activation. Western blot analysis of poly(ADP-ribose)-polymerase (PARP) confirmed caspase-3 activation in both cell lines. DNA fragmentation was not detected in these cell lines. This evidence reflect that oligonucleosomal DNA fragmentation is not a biochemical event that can be used as a definitive apoptotic marker in L6 and BC3H1 myoblast cell lines. The results indicate that the time-course and degree of apoptotic events induced by YTX depend on cell line sensitivity.     

Lysophosphatidic acid induces a migratory phenotype through a crosstalk between RhoA-Rock and Src-FAK signalling in colon cancer cells

Lysophosphatidic acid (LPA) acts as a potent stimulator of tumorigenesis. Cell-cell adhesion disassembly, actin cytoskeletal alterations, and increased migratory potential are initial steps of colorectal cancer progression. However, the role that LPA plays in these events in this cancer type is still unknown. We explored this question by using Caco-2 cells, as colon cancer model, and treatment with LPA or pretreatment with different cell signalling inhibitors. Changes in the location of adherent junction proteins were examined by immunofluorescence and immunoblotting. The actin cytoskeleton organisation and focal adhesion were analysed by confocal microscopy. Rho-GTPase activation was analysed by the pull-down assay, FAK and Src activation by immunoblotting, and cell migration by the wound healing technique. We show that LPA induced adherent junction disassembly, perijunctional actin cytoskeletal reorganisation, and increased cell migration. These events were dependent on Src, Rho and Rock because their chemical inhibitors PP2, toxin A and Y27632, respectively, abrogated the effects of LPA. Moreover, we showed that Src acts upstream of RhoA in this signalling cascade and that LPA induces focal adhesion formation and FAK redistribution and activation in confluent monolayers. Focal adhesion formation was also observed in the front of migrating cells in response to LPA, and Rock inhibitor abolished this effect. In conclusion, our findings show that LPA modulates adherent junction disassembly, actin cytoskeletal disorganisation, and focal adhesion formation, conferring a migratory phenotype in colon tumour cells. We suggest a functional regulatory cascade that integrates RhoA-Rock and Src-FAK signalling to control these events during colorectal cancer progression.     

Azaspiracid-1 alters the E-cadherin pool in epithelial cells.

Azaspiracids cause severe damages in the epithelium of several organs. In this study we have investigated the effects of azaspiracid-1 (AZA-1) on two epithelial cell lines. Nanomolar concentrations of AZA-1 reduced MCF-7 cell proliferation and impaired cell-cell adhesion. AZA-1 altered the cellular pool of the adhesion molecule E-cadherin by inducing a dose- and time-dependent accumulation of an E-cadherin fragment (E-cadherin-related antigen [ECRA(100)]), with a concentration inducing the half-maximal effect (EC(50)) of 0.47nM. The immunological characterization of ECRA(100) revealed that it consists of an E-cadherin molecule lacking the intracellular domain, and these data showed that the effect induced by AZA-1 in MCF-7 cells is undistinguishable from that induced by yessotoxin (YTX) in the same experimental system. A comparison of toxin effects in MCF-7 and Caco 2 cells confirmed that the effects induced by AZA-1 and YTX are undistinguishable in these cells. Treatment of fibroblasts with AZA-1 did not affect the cellular pool of N-cadherin showing that the toxin effect is cadherin-specific. A comparison of the effects induced by AZA-1, YTX, and okadaic acid on F-actin and E-cadherin in MCF-7 and Caco 2 cells showed that 1nM AZA-1 did not cause significant changes in F-actin and that accumulation of ECRA(100) did not correlate with decreased levels of F-actin under our experimental conditions. Matching our results with those available in literature, we notice that, when molecular effects induced by AZA-1 and YTX have been studied in the same in vitro systems, experimental data show that they are undistinguishable in terms of sensitive cellular parameters, effective doses, and kinetics of responses in several cell lines. The possibility that azaspiracids and YTXs might share their molecular mechanism(s) of action in defined biological settings should be considered.     

Zinc alters actin filaments in Madin-Darby canine kidney cells.

Exposure of semiconfluent cultures of Madin-Darby canine kidney cells to 10 microM zinc leads to a change in the organization of the actin filament system. Most of the stress fibers at the basal end of the cell are lost and the actin associated with the lateral membrane and junctional regions appears to retract into the cytoplasm. In addition, at the base of the cell in regions of cell-substratum contact, dense, actin-rich plaques appear. These alterations in actin filaments are associated with a change in cell shape. Microtubules were unaffected by exposure to 10 microM zinc. At zinc concentrations greater than or equal to 50 microM the microtubules depolymerized. Exposure to cadmium alters the actin filaments as well but the effect is different from the change seen with zinc. When the cells are exposed simultaneously to zinc and cadmium the cells appear the same as they would if exposed to zinc alone. Exposure of MDCK cells to either metal, individually or in combination, results in a significant and similar increase in F-actin content as determined spectrofluorometrically. The changes in organization and amount of F-actin are associated with a reduction in the ability of the cells to remain attached to the substrate, a toxic effect of these metals with regard to epithelial function. The results indicate that zinc, an essential metal, and cadmium, a highly toxic metal, interact with the actin cytoskeleton in intact cells.     

Simvastatin disrupts cytoskeleton and decreases cardiac fibroblast adhesion, migration and viability

Statins reduce the isoprenoids farnesyl and geranylgeranyl pyrophosphate, essential intermediates, which control a diversity of cellular events such as cytoskeleton integrity, adhesion, migration and viability. Cardiac fibroblasts are the major non-myocyte cell constituent in the normal heart, and play a key role in the maintenance of extracellular matrix. The effects of simvastatin on cardiac fibroblast processes previously mentioned remain unknown. Our aims were to investigate the effects of simvastatin on cytoskeleton structure and focal adhesion complex assembly and their relationships with cell adhesion, migration and viability in cultured cardiac fibroblasts. To this end, cells were treated with simvastatin for 24 h and changes in actin cytoskeleton, levels of vimentin and paxillin as well as their subcellular localization were analyzed by Western blot and immunocytochemistry, respectively. Cell adhesion to plastic or collagen coated dishes, migration in Transwell chambers, and cell viability were analyzed after simvastatin treatment. Our results show that simvastatin disrupts actin cytoskeleton and focal adhesion complex evaluated by phalloidin stain and immunocytochemistry for paxillin and vinculin. All these effects occurred by a cholesterol synthesis-independent mechanism. Simvastatin decreased cell adhesion, migration and viability in a concentration-dependent manner. Finally, simvastatin decreased angiotensin II-induced phospho-paxillin levels and cell adhesion. We concluded that simvastatin disrupts cytoskeleton integrity and focal adhesion complex assembly in cultured cardiac fibroblasts by a cholesterol-independent mechanism and consequently decreases cell migration, adhesion and viability.     

Inhibition of Rho kinases increases directional motility of microvascular endothelial cells

Rho kinases are major regulators of actin cytoskeletal organization and cell motility. Depending on the model system, inhibitors of Rho kinases (ROCK) have been reported to increase or decrease endothelial cell migration. In the present study we investigated the effect of Rho kinase inhibitors on microvascular endothelial cell migration with a special focus on the isoform ROCK2. Migration of microvascular endothelial cells was analyzed in a wound-healing, a spheroid-on-collagen migration assay and in cells embedded in collagen-1 gels. The non-selective Rho kinase inhibitor H1152 was compared to the selective ROCK2 inhibitor SLX2119 and to siRNA knock down. Non-selective inhibition of Rho kinases decreased cell-spanning F-actin fibers, loosened cell-cell contacts visualized by VE cadherin staining, and reduced cell-matrix interactions as shown by reduced Hic-5 expression in focal contacts. Rho kinase inhibitors facilitated directed migration of endothelial cells away from spheroids on fibronectin-coated plates and in collagen-1 gels. By contrast, migration of firmly attached endothelial cells, resembling intact vessels, was not promoted by Rho kinase inhibition. Selective inhibition of ROCK2 mimicked the cytoskeletal effects of H1152 and also increased cell motility, although to a lesser extent. In summary, Rho kinase inhibition enhanced the migration and cytoskeletal restructuring preferentially in freshly attached endothelial cells. ROCK2 may be a potential target to manipulate endothelial cell migration after vessel injury.     

A comparative study of the effect of oxidative stress on the cytoskeleton in human cortical neurons

Cytoskeleton disruption is a process by which oxidative stress disrupts cellular function. This study compares and contrasts the effect of oxidative stress on the three major cytoskeleton filaments, microfilaments (MFs), microtubule (MT), and vimentin in human cortical neuronal cell line (HCN2). HCN2 cells were treated with 100 microM tertiary butylhydroperoxide (t-BuOOH), a free radical generating neurotoxin for 1, 3, or 6 h. Cell viability studies demonstrated significant cell death although the morphology studies showed that there was a substantial loss in neurites of neurons treated with t-BuOOH for 6 h. Because the cytoskeleton plays a role in neurite outgrowth, the effect of oxidative stress on the cytoskeletal was studied. In neurons subjected to oxidative stress for 30 min or 1 h, there were no major changes in microfilament distribution though there was altered distribution of microtubule and vimentin filaments as compared to controls. However, loss and disruption of all the three cytoskeletal filaments was observed at later times (3 and 6 h), which was confirmed by Western Blot analysis. Further studies were done to measure the gene expression levels of actin, tubulin, and vimentin. Results indicated that the overall loss of the cytoskeletal proteins in neurons treated with free radical generating toxin might not be a direct result of the downregulation of the cytoskeletal genes. This study shows that free radical generation in human neurons leads to the disruption of the cytoskeleton, though there may be a difference in the susceptibility to oxidative stress among the individual components of the cytoskeletal filaments.     

Induction of apoptosis by YTX in myoblast cell lines via mitochondrial signalling transduction pathway

Yessotoxin (YTX) can induce apoptotic events in myoblast L6 and BC3H1 cell lines from rat and mouse, respectively. The present study indicates that apoptosis induced by YTX in these cell lines can occur through activation of the mitochondrial pathway indicating an intracellular response. Terminal events during mitochondrial-mediated apoptosis involve perturbations to mitochondria resulting in loss of mitochondrial membrane potential (ΔΨ_m), permeability transition pore (PTP) opening and the release of proapoptotic factors cytochrome c, smac/DIABLO into the cytosol. Results from western blotting, electron and fluorescent microscopy of YTX-treated myoblast cells provided experimental data for evaluation of cytochrome c, smac/DIABLO release and caspase-9 activation. Loss of mitochondrial membrane potential and swelling of mitochondria indicated an active role of mitochondria during the early phase of apoptosis in L6 and BC3H1 cells after YTX exposure. These observations show that YTX targets mitochondria and involve activation of a cascade of events through mitochondrial regulation.     

Alpha4‐overexpressing HL7702 cells can counteract microcystin‐LR effects on cytoskeletal structure

Our previous studies indicated that α4 was involved in the toxicity of MC‐LR on the cytoskeleton via the change of PP2A activity in HEK 293. To explore the role of α4 in MC‐LR toxicity via PP2A regulation in different cell lines, the HL7702 cell overexpressing α4 protein was exposed to MC‐LR, and the change of PP2A, cytoskeletal structure, and cytoskeleton‐related proteins were investigated. The results showed that PP2A activity was decreased, PP2A/C subunit expression and phosphorylation (Tyr307) increased significantly, but methylation (Leu 309)clearly decreased. The structure of the actin filaments and microtubules (MTs) remained unchanged, and the expression and phosphorylation of the cytoskeleton‐related proteins showed different changes. In addition, the main components of the MAPK pathway, JNK, P38, and ERK1/2, were activated together. Our results indicated that elevated α4 expression did confer some resistance to MC‐LR‐induced cytoskeletal changes, but the responses of different cell lines to MC‐LR, under the α4‐overexpression condition, are not exactly the same.     

Yessotoxin as a tool to study induction of multiple cell death pathways

This work proposes to use the marine algal toxin yessotoxin (YTX) to establish reference model experiments to explore medically valuable effects from induction of multiple cell death pathways. YTX is one of few toxins reported to make such induction. It is a small molecule compound which at low concentrations can induce apoptosis in primary cultures, many types of cells and cell lines. It can also induce a non-apoptotic form of programmed cell death in BC3H1 myoblast cell lines. The present contribution reviews arguments that this type of induction may have principal interest outside this particular example. One principal effect of medical interest may be that cancer cells will not so easily adapt to the synergistic effects from induction of more than one death pathway as compared to induction of only apoptosis.     

Mechanisms of neurotoxicity related to selective disruption of microtubules and intermediate filaments

The neuronal response to several neurotoxic chemicals includes disruption of the cytoskeleton such as interactions with microtubules and altered distribution of neurofilaments. Methylmercury (microtubule disrupting) and acrylamide and 2,5-hexanedione (neurofilament disrupting) have been used in a cell culture (PtK2) system to distinguish the cytoskeletal targets of these compounds. Methylmercury caused disassembly of microtubules with secondary collapse of vimentin filaments (epithelial cell equivalent of neurofilaments) at higher concentrations; actin filaments were unaltered. This confirms that disruption of actin does not contribute to methylmercury-induced interference with mitosis. In contrast, both acrylamide and 2,5-hexanedione caused a perinuclear redistribution of vimentin filaments with sparing of microtubules. Biochemical studies revealed that 2,5-hexanedione treatment resulted in high molecular weight vimentin-immunoreactive species, presumably by cross-linking of proteins. Selective action of both acrylamide and 2,5-hexanedione on vimentin filaments and the similarity of effects suggest that a common mechanism of damage may occur whereby these compounds act directly on both vimentin and neurofilaments.     

The novel synthetic ether lipid inositol-C2-PAF inhibits phosphorylation of the tyrosine kinases Src and FAK independent of integrin activation in transformed skin cells

New alkyl-phospholipids that are structurally derived from platelet-activating factor are promising candidates for anticancer treatment. The mechanism of action of derivatives of the platelet-activating factor is distinctly different from that of known DNA- or tubulin-targeting anticancer agents because they are incorporated into cell membranes, where they accumulate and interfere with a wide variety of key enzymes. We recently presented evidence of a novel group of alkyl-phospholipids, glycosidated phospholipids that efficiently inhibit cell proliferation. One member of this group, inositol-C2-PAF (Ino-C2-PAF), displays high efficacy and low cytotoxicity in HaCaT-cells, an immortalized non-tumorigenic skin keratinocyte cell line.Here, we show that Ino-C2-PAF also inhibits the motility of the skin-derived transformed cell lines HaCaT and squamous cell carcinoma (SCC)-25. This decrease in motility is accompanied by an altered F-actin cytoskeleton, increased clustering of integrins, and increased cell–matrix adhesion. Despite enhanced integrin clustering and matrix adhesion, we observed less phosphorylation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and Src, key regulators of cellular motility, at focal adhesion sites. Transient transfection of constitutively active variants of FAK and Src could at least in part bybass this inhibitory effect of Ino-C2-PAF. This fact indicates that Ino-C2-PAF interferes with the fine-tuned balance between adhesion and migration. Ino-C2-PAF at least partially uncouples integrin-mediated attachment from subsequent integrin-dependent signaling steps, which inhibits migration in transformed keratinocyte cell lines.     

Interference with actin dynamics is superior to disturbance of microtubule function in the inhibition of human ovarian cancer cell motility

Cellular movement is mainly orchestrated by the actin and microtubule cytoskeleton in which Rho GTPases closely collaborate. We studied whether cytoskeleton-interfering agents at subtoxic and 50% growth-inhibiting concentrations affect motility of five unselected human ovarian cancer cell lines. Cisplatin and doxorubicin as control cytotoxic agents were not effective, the microtubule-targeting agents docetaxel, epothilone B and vinblastine only marginally inhibited cell motility, while the actin-targeting agent cytochalasin D was most potent in hampering both cell migration and invasion. Disturbance of microtubule dynamics by docetaxel did not importantly affect the cellular structures of beta-tubulin and F-actin. In contrast, hindrance of actin dynamics by cytochalasin D resulted in loss of lamellipodial extensions, induced thick layers of F-actin and disorder in cellular organization. In OVCAR-3 cells the activity of Rac1 was only slightly diminished by docetaxel, but clearly reduced by cytochalasin D. In conclusion, targeting the actin cytoskeleton might provide a means to prevent metastasis formation.     

Cell growth inhibition and actin cytoskeleton disorganization induced by azaspiracid-1 structure-activity studies

Azaspiracid-1 (AZA-1) is a marine toxin discovered 10 years ago. Since then, toxicologic studies have demonstrated that AZA-1 targets several organs in vivo, including the intestine, lymphoid tissues, lungs, and nervous system; however, the mechanism of action of AZA-1 remains unknown. Studies in vitro suggest that AZA-1 affects the actin cytoskeleton in nonadherent cells. We characterized the effects of AZA-1 on the cytoskeleton of adherent cells and on cell growth, an adhesion-dependent process in many cell types, and analyzed the structure dependency of this toxicity. Confocal and TIRF imaging of fluorescently labeled cytosketon showed that AZA-1 induced the rearrangement of stress fibers (actin filament bundles) and the loss of focal adhesion points in neuroblastoma and Caco-2 cells, without affecting the amount of polymerized actin. AZA-1 did not seem to alter the microtubule cytoskeleton, but it changed the cell shape and internal morphology observed by phase contrast imaging. Cell growth of lung carcinoma and neuroblastoma cells was inhibited by the toxin, as measured by a sulforhodamine B assay and BrdU incorporation to newly synthesized DNA. Fifteen different fragments and/or stereoisomers of AZA-1 were tested for cytoskeletal rearrangement and cell growth inhibition. Results showed that no fragment or stereoisomer had any activity, except for ABCD-epi-AZA-1, which conserved toxicity. AZA-1-induced reorganization of the actin cytoskeleton concurred with detachment and growth inhibition, three events that are probably related.     

Involvement of Rho signaling in PAR2-mediated regulation of neutrophil adhesion to lung epithelial cells

Protease-activated receptor 2 (PAR2) has been implicated in the pathogenesis of airway inflammation. We report that epithelial PAR2 stimulation with trypsin (0.05-1 U/ml) or an agonist peptide (SLIGKV-NH2, 1-100 microM) for 0.5-3 h dose- and time-dependently enhanced neutrophil adhesion to alveolar type II epithelial cells (A549 cells) and that this stimulation also induced the formation of epithelial actin filaments. Both responses in neutrophil adhesion and epithelial actin reorganization were reduced by a Rho inhibitor, mevastatin and by a Rho-associated kinase (ROCK) inhibitor, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide). Neutrophil adherence was also inhibited by an inhibitor of actin polymerization, cytochalasin D and a tyrosine kinase inhibitor, genistein. Further, the PAR2-mediated tyrosine phosphorylation of focal adhesion kinase (FAK), a major cytoskeleton protein, was detected, and this response was inhibited by mevastatin or Y-27632. These results suggest that PAR2 stimulation of alveolar epithelial cells enhances neutrophil adhesion presumably at least in part through Rho/ROCK signal-mediated actin cytoskeleton reorganization associated with the tyrosine phosphorylation of FAK.     

Protein kinase signaling in the modulation of microvascular permeability

The permeability of exchange microvessels is regulated through complex interactions between signaling molecules and structural proteins in the endothelium. Endothelial barrier integrity is maintained by adhesive interactions occurring at the cell-cell and cell-matrix contacts via junctional proteins and focal adhesion complexes that are anchored to the cytoskeleton. Cyclic AMP (cAMP) and cAMP-dependent kinase counteract with the nitric oxide (NO)-cyclic GMP (cGMP) pathway to protect the basal barrier function. Upon stimulation by physical stress, growth factors, or inflammatory agents, endothelial cells undergo a series of intracellular signaling reactions involving activation of protein kinase C (PKC), protein kinase G (PKG), mitogen-activated protein kinases (MAPK), and/or protein tyrosine kinases. The phosphorylation cascades trigger biochemical and conformational changes in the barrier structure and ultimately lead to an opening of the paracellular pathway. In particular, myosin light chain kinase (MLCK) activation and subsequent myosin light chain (MLC) phosphorylation in endothelial cells directly result in cell contraction and shape changes. The phosphorylation of beta-catenin may cause disorganization of adherens junctions or dissociation of vascular endothelial (VE)-cadherin-catenin complex from its cytoskeletal anchor, leading to loose or opened intercellular junctions. Additionally, focal adhesion kinase (FAK) phosphorylation-coupled focal adhesion assembly and redistribution provide an anchorage support for the conformational changes occurring in the cells and at the cell junctions. The Src family tyrosine kinases may serve as common signals that coordinate these molecular events to facilitate the paracellular transport of macromolecules. The critical roles of protein kinases in endothelial hyperpermeability implicate the therapeutic significance of protein kinase inhibitors in the prevention and treatment of diseases and injuries that are associated with microvascular barrier dysfunction.