Activation Of Small GTPase Rho Research Articles

T117: The plasticity of tumor cell migration mechanism is adaptive factor increasing metastasis efficiency

During cancer development, tumor cells gain the ability to invade and metastasize. Individual cells use alternative migration modes based on different cellular mechanisms. One of them is mesenchymal motility mode which is driven by leading edge protrusion in the form of filopodia or/and lamellipodia based on Arp2/3 dependent actin polymerization. Mesenchymal motility depends on formation of cell-substrate adhesions, activity of matrix metalloproteases (MMPs) and on activity of small GTPaseRac. Another mode is amoeboid motility, which involves formation of blebs – hollow membrane protrusions extruded from the cell surface by actin-myosin contraction. Amoeboid motility does not need both pronounced cell-substrate adhesions and MMPs activity and required increase of activity of small GTPase Rho. Fibroblasts and scattered epithelial cells migrate by mesenchymal mode, while blood cells – lymphocytes or macrophages mainly use amoeboid mode for migration. It was shown that some treatments cause transition from one motility mode to another. Switches from mesenchymal to amoeboid motility and opposite are called mesenchymal-amoeboid transition (MAT) and amoeboid -mesenchymal transition (AMT) respectively. The ability of cells for such transitions was named as plasticity of migration. We compared the plasticity of migration of normal and tumor cells. To study plasticity of mesenchymally migrated cells (MAT) we choose fibrosarcoma cells HT1080 as tumor and non-transformed subcutaneous fibroblasts 1036 as normal counterpart. To study AMT we choose a few lines of myeloid leukemia THP1, K562, KG1 in contrast to normal leukocytes obtained from healthy donors. We showed that fibrosarcoma cells in opposite to non-transformed fibroblasts could undergo MAT under treatments, which limited mesenchymal migration. Two approaches to limit mesenchymal motility of cells were used. One was decrease of substrate adhesiveness by treatment of coverslips with PolyHema solutions, which simulated the alteration of environmental conditions during cell migration. The other approach was influence on cellular pathways regulated cell motility. We used CK666, the inhibitor of Arp2/3 activity, which stopped actin polymerization and thus lamellipodia formation through Arp2/3 dependent mechanism. We showed that under both treatments the fraction of tumor cells switched from lamellipodia formation to blebbing and thus underwent MAT, while in non-transformed fibroblasts these treatments led to retraction of lamellipodia and significant failure of motility. Both leukemia cells and leucocytes of healthy donors showed blebs formation (amoeboid motility). We induced transition to mesenchymal motility by alteration of culture conditions. The first approach was the increase of substrate adhesiveness by treatment with fibronectin. Another way was to inhibit of small GTPase Rho activity. In result of both treatments, leukemia cells switched from amoeboid to mesenchymal motility (underwent AMT), but leucocytes of healthy donor could not do such transition. For the first time it was shown that AMT is features of leukemia cells but not leucocytes from healthy donors. Both MAT and AMT are reversible, meaning that cells exhibiting plasticity could change motility mode in dependence on environment. Our results demonstrate that tumor cells of different origin could transit from one mode of motility to another and normal cells could not undergo such transitions. We also investigate the effectiveness of amoeboid and mesenchymal motility during migration in different environments. It was shown that the mesenchymal motility is more effective for 2D migration, while the amoeboid motility is more effective in 3D conditions. During dissemination, cells go beyond the borders of original tissues and pass through environment with different properties. The plasticity of migration triggered by alteration of entire or internal conditions dramatically increases ability of cells to disseminate. The ability of tumor cells to plasticity permits them to choose optimal mode for migration, thus leading to metastasis development.

CAMP‐dependent Rac 1 inactivation is associated with LPS‐induced endothelial barrier breakdown

Lipopolysaccharide (LPS)‐induced breakdown of endothelial barrier functions contributes largely to the pathogenesis of sepsis. However, the underlying mechanisms are still unclear. In the present study, incubation of human dermal microvascular endothelial cells with LPS led to a breakdown of endothelial barrier functions within 2–2.5h as revealed by measurements of FITC‐dextran flux and of transendothelial electrical resistance. This was associated with the formation of intercellular gaps, stress fibers and fragmentation of VE‐cadherin immunostaining. Moreover, claudin 5 immunostaining at cell borders was reduced after LPS‐treatment. Activity of small GTPase Rho A, which has been suggested to mediate the LPS‐induced breakdown of the endothelial barrier was not increased after 2h. In contrast, activity of Rac 1, which is known to be important for maintenance of endothelial barrier functions, was significantly reduced to 65 ± 9% after 2h. All LPS‐induced changes of endothelial cells were blocked by a Forskolin/Rolipram‐mediated increase of cAMP. Consistently, LPS led to a significant decrease of intracellular cAMP‐levels to 41 ± 2% after 1h and to 46 ± 8% after 2h. In summary, we suggest that LPS disrupts endothelial barrier functions by decreasing intracellular cAMP‐levels which in turn leads to an inactivation of Rac 1‐activity. Moreover, this mechanism appears to be involved in claudin 5 regulation.

GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction

Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.

Involvement of microtubules and Rho pathway in TGF‐β1‐induced lung vascular barrier dysfunction

Transforming growth factor-beta1 (TGF-beta1) is a cytokine critically involved in acute lung injury and endothelial cell (EC) barrier dysfunction. We have studied TGF-beta1-mediated signaling pathways and examined a role of microtubule (MT) dynamics in TGF-beta1-induced actin cytoskeletal remodeling and EC barrier dysfunction. TGF-beta1 (0.1-50 ng/ml) induced dose-dependent decrease in transendothelial electrical resistance (TER) in bovine pulmonary ECs, which was linked to increased actin stress fiber formation, myosin light chain (MLC) phosphorylation, EC retraction, and gap formation. Inhibitor of TGF-beta1 receptor kinase RI (5 microM) abolished TGF-beta1-induced TER decline, whereas inhibitor of caspase-3 zVAD (10 microM) was without effect. TGF-beta1-induced EC barrier dysfunction was linked to partial dissolution of peripheral MT meshwork and decreased levels of stable (acetylated) MT pool, whereas MT stabilization by taxol (5 microM) attenuated TGF-beta1-induced barrier dysfunction and actin remodeling. TGF-beta1 induced sustained activation of small GTPase Rho and its effector Rho-kinase; phosphorylation of myosin binding subunit of myosin specific phosphatase; MLC phosphorylation; EC contraction; and gap formation, which was abolished by inhibition of Rho and Rho-kinase, and by MT stabilization with taxol. Finally, elevation of intracellular cAMP induced by forskolin (50 microM) attenuated TGF-beta1-induced barrier dysfunction, MLC phosphorylation, and protected the MT peripheral network. These results suggest a novel role for MT dynamics in the TGF-beta1-mediated Rho regulation, EC barrier dysfunction, and actin remodeling.

Alzheimer’s disease: Channeling APP to non-amyloidogenic processing

A good number of pharmacologic agents have over the years been touted as potentially beneficial in either preventing the onset or delay the progression of Alzheimer’s disease. These include compounds such as non-steroidal anti-inflammatory drugs (NSAIDs) (HMG-CoA reductase inhibitors (statins)) and flavonoids. The underlying mechanisms for the beneficial effect of these agents are by and large attributed to their ability to reduce β-amyloid (Aβ) production and amyloid load in the brain, via inhibition of amyloidogenic γ-secretase activity. Recent reports have now provided mechanistic insights as to how non-amyloidogenic processing might also be enhanced by these seemingly unrelated treatments. Intriguingly, this appears to involve the inhibition of the activity of small GTPase Rho and its effector, the Rho-associated kinase, ROCK. Dietary caloric restriction (CR) also enhances non-amyloidogenic processing of APP, and this may be part of a more general anti-aging effect of CR mediated by gene expression changes downstream of the activity of the histone deacetylase SIRT1.

Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction

Thrombin-induced barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction, and gap formation. Phosphorylation of regulatory myosin light chains (MLC) is a key mechanism of endothelial cell (EC) contraction and barrier dysfunction, which is triggered by Ca 2+/calmodulin-dependent MLC kinase (MLCK) and Rho-associated kinase (Rho-kinase). The role of MLCK in EC barrier regulation has been previously described; however, Rho-mediated pathway in thrombin-induced pulmonary EC dysfunction is not yet precisely characterized. Here, we demonstrate that thrombin-induced decreases in transendothelial electrical resistance (TER) indicating EC barrier dysfunction are universal for human and bovine pulmonary endothelium, and involve membrane translocation and direct activation of small GTPase Rho and its downstream target Rho-kinase. Transient Rho membrane translocation coincided with translocation of upstream Rho activator, guanosine nucleotide exchange factor p115-RhoGEF. Rho mediated activation of downstream target, Rho-kinase induced phosphorylation of the EC MLC phosphatase (MYPT1) at Thr 686 and Thr 850, resulting in MYPT1 inactivation, accumulation of diphospho-MLC, actin remodeling, and cell contraction. The specific Rho-kinase inhibitor, Y27632, abolished MYPT1 phosphorylation, MLC phosphorylation, significantly attenuated stress fiber formation and thrombin-induced TER decrease. Furthermore, expression of dominant-negative Rho and Rho-kinase abolished thrombin-induced stress fiber formation and MLC phosphorylation. Our data, which provide comprehensive analysis of Rho-mediated signal transduction in pulmonary EC, demonstrate involvement of guanosine nucleotide exchange factor, p115-RhoGEF, in thrombin-mediated Rho regulation, and suggest Rho, Rho-kinase, and MYPT1 as potential pharmacological and gene therapy targets critical for prevention of thrombin-induced EC barrier disruption and pulmonary edema associated with acute lung injury.