Stress Fiber Formation In Cells Research Articles

MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization

Cancer cell migration during metastasis is mediated by a highly polarized cytoskeleton. MARK2 and its invertebrate homolog Par1B are kinases that regulate the microtubule cytoskeleton to mediate polarization of neurons in mammals and embryos in invertebrates. However, the role of MARK2 in cancer cell migration is unclear. Using osteosarcoma cells, we found that in addition to its known localizations on microtubules and the plasma membrane, MARK2 also associates with the actomyosin cytoskeleton and focal adhesions. Cells depleted of MARK proteins demonstrated that MARK2 promotes phosphorylation of both myosin II and the myosin phosphatase targeting subunit MYPT1 to synergistically drive myosin II contractility and stress fiber formation in cells. Studies with isolated proteins showed that MARK2 directly phosphorylates myosin II regulatory light chain, while its effects on MYPT1 phosphorylation are indirect. Using a mutant lacking the membrane-binding domain, we found that membrane association is required for focal adhesion targeting of MARK2, where it specifically enhances cell protrusion by promoting FAK phosphorylation and formation of focal adhesions oriented in the direction of migration to mediate directionally persistent cell motility. Together, our results define MARK2 as a master regulator of the actomyosin and microtubule cytoskeletal systems and focal adhesions to mediate directional cancer cell migration.

Palmitoylation and Phosphorylation of the Na + ‐H + Exchanger Isoform 1 (NHE1) Regulate of Cell Proliferation and Stress Fiber Formation: PI3 Kinase and AKT Signaling

The Na+-H+ Exchanger Isoform 1 (NHE1) is a 12-pass transmembrane transport protein that exchanges one extracellular Na+ for one intracellular H+ with the primary function of intracellular pH regulation. Through the pH regulation NHE1 contributes to the regulation of cell proliferation and stress fiber formation. The regulation of NHE1 is extremely complex and involves protein and lipid binding sites including phosphorylation and palmitoylation sites. NHE1 transport activity is regulated by a series of signaling pathways which include kinases of the PI3 Kinase / AKT signaling pathways. Recently it was demonstrated that inhibition of either PI3 Kinase or AKT lead to an increase in the palmitoylation of NHE1. Here we assess the role of this interaction between palmitoylation and phosphorylation in the regulation of cell proliferation and stress fiber formation in PSN cells. It is hypothesized that inhibiting phosphorylation, palmitoylation, or both will differentially regulate cell proliferation and stress fiber formation in these cells. PSN cells are Chinese hamster lung fibroblast that stably express human NHE1. We evaluated cell proliferation and stress fiber formation in the presence and absence of combinations of inhibitors that alter NHE1 regulation and transport activity including, LY294002 (PI3 Kinase), MK 2206 (AKT), 2-Bromopalmitate (Palmitoylation), and Ethylisopropylamiloride (NHE1).

The short-chain free fatty acid receptor FFAR3 is expressed and potentiates contraction in human airway smooth muscle.

Emerging evidence suggests that gut microbiota-derived short-chain fatty acids (SCFAs; acetate, propionate, and butyrate) are important modulators of the inflammatory state in diseases such as asthma. However, the functional expression of the Gi protein-coupled free fatty acid receptors (FFAR2/GPR43 and FFAR3/GPR41) has not been identified on airway smooth muscle (ASM). Classically, acute activation of Gi-coupled receptors inhibits cyclic AMP (cAMP) synthesis, which impairs ASM relaxation and can also induce crosstalk between Gi- and Gq-signaling pathways, potentiating increases in intracellular Ca2+ concentration ([Ca2+]i), favoring ASM contraction. In contrast, chronic activation of Gi-coupled receptors can sensitize adenylyl cyclase resulting in increased cAMP synthesis favoring relaxation. We questioned whether the Gi-coupled FFAR2 or FFAR3 is expressed in human ASM, whether they modulate cAMP and [Ca2+]i, and whether SCFAs modulate human ASM tone. We detected the protein expression of FFAR3 but not FFAR2 in native human ASM and primary cultured human airway smooth muscle (HASM) cells. In HASM cells, acute activation of FFAR3 with SCFAs inhibited forskolin-stimulated cAMP accumulation, but chronic activation did not sensitize cAMP synthesis. SCFAs induced [Ca2+]i increases that were attenuated by pertussis toxin, gallein, U73122, or xestospongin C. Acute treatment with SCFAs potentiated acetylcholine-stimulated [Ca2+]i increases and stress fiber formation in cells and contraction of ex vivo human airway tissues. In contrast, chronic pretreatment of human ASM with propionate did not potentiate airway relaxation. Together, these findings demonstrate that FFAR3 is expressed in human ASM and contributes to ASM contraction via reduced cAMP and increased [Ca2+]i.

Targeted Treatment of Ischemic and Fibrotic Complications of Myocardial Infarction Using a Dual-Delivery Microgel Therapeutic.

Myocardial infarction (MI), commonly known as a heart attack, affects millions of people worldwide and results in significant death and disabilities. A major cause of MI is fibrin-rich thrombus formation that occludes the coronary arteries, blocking blood flow to the heart and causing fibrin deposition. In treating MI, re-establishing blood flow is critical. However, ischemia reperfusion (I/R) injury itself can also occur and contributes to cardiac fibrosis. Fibrin-specific poly( N-isopropylacrylamide) nanogels (FSNs) comprised of a core-shell colloidal hydrogel architecture are utilized in this study to design a dual-delivery system that simultaneously addresses the need to (1) re-establish blood flow and (2) inhibit cardiac fibrosis following I/R injury. These therapeutic needs are met by controlling the release of a fibrinolytic protein, tissue plasminogen activator (tPA), and a small molecule cell contractility inhibitor (Y-27632). In vitro, tPA and Y-27632-loaded FSNs rapidly degrade fibrin and decrease cardiac cell stress fiber formation and connective tissue growth factor expression, which are both upregulated in cardiac fibrosis. In vivo, FSNs localize to fibrin in injured heart tissue and, when loaded with tPA and Y-27632, showed significant improvement in left ventricular ejection fraction 2 and 4 weeks post-I/R as well as significantly decreased infarct size, α-smooth muscle actin expression, and connective tissue growth factor expression 4 weeks post-I/R. Together, these data demonstrate the feasibility of this targeted therapeutic strategy to improve cardiac function following MI.

Novel Biological Effects and Distinct Patterns of Rhoa Mutations in Adult T-Cell Leukemia/Lymphoma and Angioimmunoblastic T Cell Lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma (PTCL). ATL is initiated by immortalization of T-cells by human T-lymphotropic virus type I (HTLV-1) infection during early infancy, followed by an accumulation of multiple genetic hits to develop leukemia. However, little has been known about these genetic hits. Recently, frequent somatic mutations in RHOA, DNMT3A, IDH2 and TET2 have been reported in angioimmunoblastic T cell lymphoma (AITL) and PTCL not otherwise specified (PTCL-NOS), which are characterized by follicular helper T-cell phenotypes. Most of the RHOA mutations in AITL invariably cause an identical amino acid change (Gly17Val), which was shown to inhibit wild-type RHOA function in a dominant-negative manner. In addition, RHOA mutations have been also identified in gastric cancer and Burkitt lymphoma, with mutational hotspots including Tyr42 and Arg5. Interestingly, some of them were reported as working in a gain-of function manner.In the current study, we investigated the mutational status of RHOA, as well as TET2, IDH2, and DNMT3A in a total of 205 patients with ATL in order to reveal their role in ATL pathogenesis. In targeted deep sequencing, RHOA was mutated in 32 (15.6%) cases. In contrast to the exclusive G17V mutation found in AITL and PTCL-NOS, mutations in ATL was widely distributed within the regions responsible for GTP-binding with Cys16Arg being most frequently mutated (N = 11; 5.4%), although Gly17Val mutations were also found in 4 cases (1.9%). Being commonly located in the same amino acid with the GTP binding site, Ala161Pro and Ala161Glu mutations were exclusively found in ATL and AITL/PTCL-NOS, respectively. We found no significant differences in the RHOA mutational status among different ATL subtypes, and no significant prognostic impact of RHOA mutations on survival. TET2 mutations were very common and tightly associated with RHOA mutation in AITL, but much less common (N = 21; 10.2%) and may not associated with RHOA mutations in ATL. Mutations of IDH2 (N = 2; 1%) and DNMT3A (N = 2; 1%) were rare in ATL.RHOA encodes a ras-related GTP-binding protein that functions as a molecular switch in a variety of biological processes through cycling between an active (GTP-bound) state and an inactive (GDP-bound) state. Known biological functions of RHOA are related to actin organization, cell migration and transcriptional activation. Modeling of three-dimensional structures suggested that most of mutations identified in ATL located near nucleotide binding pocket, which affected the GTP-binding ability of RHOA protein. Indeed, we performed luciferase assay to measure transcriptional activities of wild-type and mutant RHOA. As previously reported, dominant-negative Gly17Val RHOA mutant suppressed transcriptional activity. In contrast, ATL specific RHOA mutant such as Cys16Arg or Ala161Pro enhanced transcriptional activity, similar to or even more than wild-type RHOA. In addition, when we assessed actin stress fiber formation in cells transduced with wild-type or mutant RHOA, Gly17Val or Ala161Glu RHOA attenuated actin stress fiber formations, whereas Cys16Arg or Ala161Pro RHOA induced actin stress fiber formation, similar to known gain-of-function Gly14Val RHOA mutant. These functional assays implicated Cys16Arg and Ala161Pro RHOA mutant showed different biological behaviors from Gly17Val and Ala161Glu RHOA mutant, indicating gain-of-function mechanisms of Cys16Arg or Ala161Pro mutants.In summary, RHOA mutations were also common in ATL as in AITL and PTCL-NOS. Nevertheless, mutational patterns and functional consequences of RHOA mutations identified in ATL were distinct from those in other PTCLs, suggesting differential driver roles of RHOA mutations in ATL compared to other PTCLs. DisclosuresNo relevant conflicts of interest to declare.

Thioredoxin-Interacting Protein Is a Biomechanical Regulator of Src Activity

Rationale: Fluid shear stress differentially regulates endothelial cell stress fiber formation with decreased stress fibers in areas of disturbed flow compared with steady flow areas. Importantly, stress fibers are critical for several endothelial cell functions including cell shape, mechano-signal transduction, and endothelial cell–cell junction integrity. A key mediator of steady flow–induced stress fiber formation is Src that regulates downstream signaling mediators such as phosphorylation of cortactin, activity of focal adhesion kinase, and small GTPases. Objective: Previously, we showed that thioredoxin-interacting protein (TXNIP, also VDUP1 [vitamin D upregulated protein 1] and TBP-2 [thioredoxin binding protein 2]) was regulated by fluid shear stress; TXNIP expression was increased in disturbed flow compared with steady flow areas. Although TXNIP was originally characterized for its role in redox and metabolic cellular functions, recent reports show important scaffold functions related to its α-arrestin structure. Based on these findings, we hypothesized that TXNIP acts as a biomechanical sensor that regulates Src kinase activity and stress fiber formation. Methods and Results: Using en face immunohistochemistry of the aorta and cultured endothelial cells, we show inverse relationship between TXNIP expression and Src activity. Specifically, steady flow increased Src activity and stress fiber formation, whereas it decreased TXNIP expression. In contrast, disturbed flow had opposite effects. We studied the role of TXNIP in regulating Src homology phosphatase-2 plasma membrane localization and vascular endothelial cadherin binding because Src homology phosphatase-2 indirectly regulates dephosphorylation of Src tyrosine 527 that inhibits Src activity. Using immunohistochemistry and immunoprecipitation, we found that TXNIP prevented Src homology phosphatase-2–vascular endothelial cadherin interaction. Conclusions: In summary, these data characterize a fluid shear stress–mediated mechanism for stress fiber formation that involves a TXNIP-dependent vascular endothelial cadherin–Src homology phosphatase-2–Src pathway.

Abstract 230: Protein Kinase D1 Mediates Vegf-induced Vascular Permeability In Vitro And In Vivo By Promoting Endothelial Cell Stress Fiber Formation

Background Vascular hyperpermeability is critical components of many human diseases, including inflammation, cancer and diabetic retinopathy. Vascular endothelial growth factor (VEGF) is a potent vascular permeability factor. Better understanding of the molecular mechanisms by which VEGF induces vascular permeability may provide novel therapeutic targets for these diseases. We and others have recently demonstrated that protein kinase D1 (PKD1), a newly discovered serine/threonine protein kinase, is implicated in VEGF signaling. We hypothesized that PKD1 plays a crucial role in VEGF-induced vascular permeability in vivo and vitro by promoting endothelial cell stress fiber formation. Methods and results Using Tie2-Cre transgenic mice and PKD1 flox mice, we generated endothelium-specific PKD1 knockout (PKD1-ecKO) mice. PKD1-ecKO mice were viable and born at the expected Mendelian ratio. Comparing with wild type mice, PKD1-ecKO mice exhibited the dramatic reduction of VEGF-induced vascular permeability in lung and ear with Miles assay, expressed as Evans blue leakage. Selective pharmacological PKD inhibitor also significantly attenuated in vivo vascular permeability induced by VEGF intravenously injection in mice. In cultured endothelial monolayer, down-regulation of PKD1 by siRNA, GFP-PKD1 dominant negative mutant plasmid or PKD inhibitor significantly reduced VEGF-induced endothelial permeability, measured by bovine serum albumin leakage through endothelial monolayer cultured on transwell system. Moreover, we found that the inhibition of PKD1 diminished F-actin stress fiber formation and the dismantlement of endothelial adherens junctions that lead to intercellular gap formation and leakage in response to VEGF. We further observed that PKD1 acted as a downstream mediator of Rho activation in VEGF-induced stress fiber formation. Specifically, Rho inhibitor Y27632 blocked VEGF-induced PKD1 phosphorylation, while down-regulation of PKD1 expression and activation did not affect VEGF-stimulated Rho activation in endothelial cells. Conclusion PKD1 is required for VEGF-induced vascular permeability in vivo and in vitro through promoting F-actin stress fiber formation and disrupting endothelial adherens junctions.

Downregulation of the Rho GTPase signaling pathway is involved in the microRNA‐138‐mediated inhibition of cell migration and invasion in tongue squamous cell carcinoma

Tumor metastasis is the dominant cause of death in cancer patients, including patients with oral tongue squamous cell carcinoma (TSCC). Previously, we reported that reduced miR-138 level is correlated with enhanced metastatic potential in TSCC cells. Here, we demonstrate that miR-138 suppresses TSCC cell migration and invasion by regulating 2 key genes in the Rho GTPase signaling pathway: RhoC and ROCK2. Direct targeting of miR-138 to specific sequences located in the 3'-untranslated regions of both RhoC and ROCK2 mRNAs was confirmed using luciferase reporter gene assays. Ectopic transfection of miR-138 reduced the expression of both RhoC and ROCK2 in TSCC cells. These reduced expressions, in consequence, led to the reorganization of the stress fibers and the subsequent cell morphology change to a round bleb-like shape as well as the suppression of cell migration and invasion. In contrast, knockdown of miR-138 in TSCC cells enhanced the expression of RhoC and ROCK2, which resulted in an altered, elongated cell morphology, enhanced cell stress fiber formation and accelerated cell migration and invasion. Taken together, our results suggest that miR-138 plays an important role in TSCC cell migration and invasion by concurrently targeting RhoC and ROCK2, and miR-138 may serve as a novel therapeutic target for TSCC patients at risk of metastatic disease.

The role of the sodium hydrogen exchanger in stress fiber formation in human lung cancer stimulated with urokinase plasminogen activator, lysophosphatic acid, and phenylephrine

One of cancers hallmarks is the ability of cells to spread from the tumor of origin to other areas of the body, a process called metastastis. The Sodium‐Hydrogen Exchanger (NHE) plays a role in pH regulation and cytoskeletal attachment to the plasma membrane. In studies using CCL39 lung fibroblasts, phenylephrine (PE) induced stress fibers, and caused cells to become tumor like. In wound assay studies, PE stimulated stress fiber formation in cells immediately adjacent to the wound area and in migrating cells. These data indicate that stress fiber formation has a direct involvement in cell migration. In this study, we relate data from CCL39 cells to stress fiber formation in human lung cancer. We studied stress fiber formation in H358, H460, and H1299 cells. Our data shows that in H358 cells both 50μM PE and 10nM urokinase‐type plasminogen activator (uPA) significantly increase stress fiber formation. In H460 cells, 3μM lysophosphatidic acid (LPA) and UPA significantly increase stress fiber formation, and in H1299 all three hormones significantly increase stress fiber formation. Our data also shows that using ethylisopropylamiloride to inhibit NHE decreases the level of stress fiber formation in H460 and H1229 cells. These studies indicate that NHE plays a vital role in stress fiber formation and cellular migration, metastasis, in human lung cancer cells.

PAK1 Negatively Regulates the Activity of the Rho Exchange Factor NET1

Rho family small G-protein activity is controlled by guanine nucleotide exchange factors that stimulate the release of GDP, thus allowing GTP binding. Once activated, Rho proteins control cell signaling through interactions with downstream effector proteins, leading to changes in cytoskeletal organization and gene expression. The ability of Rho family members to modulate the activity of other Rho proteins is also intrinsic to these processes. In this work we show that the Rac/Cdc42hs-regulated protein kinase PAK1 down-regulates the activity of the RhoA-specific guanine nucleotide exchange factor NET1. Specifically, PAK1 phosphorylates NET1 on three sites in vitro: serines 152, 153, and 538. Replacement of serines 152 and 153 with glutamate residues down-regulates the activity of NET1 as an exchange factor in vitro and its ability to stimulate actin stress fiber formation in cells. Using a phospho-specific antibody that recognizes NET1 phosphorylated on serine 152, we show that PAK1 phosphorylates NET1 on this site in cells and that Rac1 stimulates serine 152 phosphorylation in a PAK1-dependent manner. Furthermore, coexpression of constitutively active PAK1 inhibits the ability of NET1 to stimulate actin polymerization only when serines 152 and 153 are present. These data provide a novel mechanism for the control of RhoA activity by Rac1 through the PAK-dependent phosphorylation of NET1 to reduce its activity as a guanine nucleotide exchange factor.

P116Rip Decreases Myosin II Phosphorylation by Activating Myosin Light Chain Phosphatase and by Inactivating RhoA

p116Rip was originally found to be a RhoA-binding protein, but its function has been unknown. Here, we clarify the function of p116Rip. Two critical findings were made. First, we found that p116Rip activated the GTPase activity of RhoA in vitro and that p116Rip overexpression in cells consistently diminished the epidermal growth factor-induced increase in GTP-bound RhoA. Second, p116Rip activated the myosin light chain phosphatase (MLCP) activity of the holoenzyme. p116Rip did not activate the catalytic subunit alone, indicating that the activation is due to the binding of p116Rip to the myosin phosphatase targeting subunit MYPT1. Interestingly, the activation of phosphatase was specific to myosin as substrate, and p116Rip directly bound to myosin, thus facilitating myosin/MLCP interaction. The gene silencing of p116Rip consistently and significantly increased myosin phosphorylation as well as stress fiber formation in cells. Based upon these findings, we propose that p116Rip is an important regulatory component that controls the RhoA signaling pathway, thus regulating MLCP activity and myosin phosphorylation in cells.

CGMP-Dependent Protein Kinase Phosphorylates and Inactivates RhoA

Small GTPase Rho and cGMP/cGMP-dependent protein kinase (cGK) pathways exert opposing effects in specific systems such as vascular contraction and growth. However, the direct interaction between these pathways has remained elusive. We demonstrate that cGK phosphorylates RhoA in vitro at Ser188, the same residue phosphorylated by cAMP-dependent protein kinase. In HeLa cells transfected with constitutively active cGK (C-cGK), stress fiber formation induced by lysophosphatidic acid or V14RhoA was blocked. By contrast, C-cGK failed to inhibit stress fiber formation in cells transfected with mutant RhoA with substitution of Ser188 to Ala. C-cGK did not affect actin reorganization induced by Rac1 or Rho-associated kinase, one of the effectors for RhoA. Furthermore, C-cGK expression inhibited the membrane translocation of RhoA. Collectively, our findings suggest that cGK phosphorylates RhoA at Ser188 and inactivates RhoA signaling. The physiological relevance of the direct interaction between RhoA and cGK awaits further investigation.

Protein Kinase C Inhibitors Prevent Cultured Human Endothelial Cell Stress Fiber Formation, but not Heightened Endocytosis

In order to study the signal transduction mechanism of endothelial perturbation, the effects of phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), both protein kinase C (PKC) activators, on cultured human endothelial cell (EC) hydrogen peroxide (H2O2) generation, endocytotic activity, and cytoskeletal structure have been investigated. EC were incubated with 1-100 nM PMA, or PDBu, and cellular H2O2 generation and endocytotic activity measured. PMA and PDBu exposure caused dose-dependent rises in EC H2O2 production. Likewise, EC incubated with PMA and PDBu had dose-related endocytosis increases. Cytoskeletal inspection of 10 nM PMA-perturbed EC revealed structural remodeling with stress fiber formation. Similar cellular functional changes occur in EC exposed to high low-density lipoprotein (LDL) concentrations. Protein kinase C (PKC) inhibition with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) prevented cytoskeletal remodeling in PMA-stimulated EC. In differenc...