Assembly Of Actin Stress Fibers Research Articles

An ULK1/2-PXN mechanotransduction pathway suppresses breast cancer cell migration.

The remodeling and stiffening of the extracellular matrix (ECM) is a well-recognized modulator of breast cancer progression. How changes in the mechanical properties of the ECM are converted into biochemical signals that direct tumor cell migration and metastasis remain poorly characterized. Here, we describe a new role for the autophagy-inducing serine/threonine kinases ULK1 and ULK2 in mechanotransduction. We show that ULK1/2 activity inhibits the assembly of actin stress fibers and focal adhesions (FAs) and as a consequence impedes cell contraction and migration, independent of its role in autophagy. Mechanistically, we identify PXN/paxillin, a key component of the mechanotransducing machinery, as a direct binding partner and substrate of ULK1/2. ULK-mediated phosphorylation of PXN at S32 and S119 weakens homotypic interactions and liquid-liquid phase separation of PXN, impairing FA assembly, which in turn alters the mechanical properties of breast cancer cells and their response to mechanical stimuli. ULK1/2 and the well-characterized PXN regulator, FAK/Src, have opposing functions on mechanotransduction and compete for phosphorylation of adjacent serine and tyrosine residues. Taken together, our study reveals ULK1/2 as important regulator of PXN-dependent mechanotransduction.

Increased expression of phosphodiesterase 4 in activated hepatic stellate cells promotes cytoskeleton remodeling and cell migration.

Activation and transdifferentiation of hepatic stellate cells (HSC) into migratory myofibroblasts is a key process in liver fibrogenesis. Cell migration requires an active remodeling of the cytoskeleton, which is a tightly regulated process coordinated by Rho-specific guanine nucleotide exchange factors (GEFs) and the Rho family of small GTPases. Rho-associated kinase (ROCK) promotes assembly of focal adhesions and actin stress fibers by regulating cytoskeleton organization. GEF exchange protein directly activated by cAMP 1 (EPAC1) has been implicated in modulating TGFβ1 and Rho signaling; however, its role in HSC migration has never been examined. The aim of this study was to evaluate the role of cAMP-degrading phosphodiesterase 4 (PDE4) enzymes in regulating EPAC1 signaling, HSC migration, and fibrogenesis. We show that PDE4 protein expression is increased in activated HSCs expressing alpha smooth muscle actin and active myosin light chain (MLC) in fibrotic tissues of human nonalcoholic steatohepatitis cirrhosis livers and mouse livers exposed to carbon tetrachloride. In human livers, TGFβ1 levels were highly correlated with PDE4 expression. TGFβ1 treatment of LX2 HSCs decreased levels of cAMP and EPAC1 and increased PDE4D expression. PDE4 specific inhibitor, rolipram, and an EPAC-specific agonist decreased TGFβ1-mediated cell migration in vitro. In vivo, targeted delivery of rolipram to the liver prevented fibrogenesis and collagen deposition and decreased the expression of several fibrosis-related genes, and HSC activation. Proteomic analysis of mouse liver tissues identified the regulation of actin cytoskeleton by the kinase effectors of Rho GTPases as a major pathway impacted by rolipram. Western blot analyses confirmed that PDE4 inhibition decreased active MLC and endothelin 1 levels, key proteins involved in cytoskeleton remodeling and contractility. The current study, for the first time, demonstrates that PDE4 enzymes are expressed in hepatic myofibroblasts and promote cytoskeleton remodeling and HSC migration. © 2023 The Pathological Society of Great Britain and Ireland.

A strategy to quantify myofibroblast activation on a continuous spectrum

Myofibroblasts are a highly secretory and contractile cell phenotype that are predominant in wound healing and fibrotic disease. Traditionally, myofibroblasts are identified by the de novo expression and assembly of alpha-smooth muscle actin stress fibers, leading to a binary classification: “activated” or “quiescent (non-activated)”. More recently, however, myofibroblast activation has been considered on a continuous spectrum, but there is no established method to quantify the position of a cell on this spectrum. To this end, we developed a strategy based on microscopy imaging and machine learning methods to quantify myofibroblast activation in vitro on a continuous scale. We first measured morphological features of over 1000 individual cardiac fibroblasts and found that these features provide sufficient information to predict activation state. We next used dimensionality reduction techniques and self-supervised machine learning to create a continuous scale of activation based on features extracted from microscopy images. Lastly, we compared our findings for mechanically activated cardiac fibroblasts to a distribution of cell phenotypes generated from transcriptomic data using single-cell RNA sequencing. Altogether, these results demonstrate a continuous spectrum of myofibroblast activation and provide an imaging-based strategy to quantify the position of a cell on that spectrum.

Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel.

Interactions between trabecular meshwork (TM) cells and their extracellular matrix (ECM) are critical for normal outflow function in the healthy eye. Multifactorial dysregulation of the TM is the principal cause of elevated intraocular pressure that is strongly associated with glaucomatous vision loss. Key characteristics of the diseased TM are pathologic contraction and actin stress fiber assembly, contributing to overall tissue stiffening. Among first-line glaucoma medications, the Rho-associated kinase inhibitor (ROCKi) netarsudil is known to directly target the stiffened TM to improve outflow function via tissue relaxation involving focal adhesion and actin stress fiber disassembly. Yet, no in vitro studies have explored the effect of netarsudil on human TM (HTM) cell contractility and actin remodeling in a 3D ECM environment. Here, we use our bioengineered HTM cell-encapsulated ECM hydrogel to investigate the efficacy of different netarsudil-family ROCKi compounds on reversing pathologic contraction and actin stress fibers. Netarsudil and all related experimental ROCKi compounds exhibited significant ROCK1/2 inhibitory and focal adhesion disruption activities. Furthermore, all ROCKi compounds displayed potent contraction-reversing effects on HTM hydrogels upon glaucomatous induction in a dose-dependent manner, relatively consistent with their biochemical/cellular inhibitory activities. At their tailored EC50 levels, netarsudil-family ROCKi compounds exhibited distinct effect signatures of reversing pathologic HTM hydrogel contraction and actin stress fibers, independent of the cell strain used. Netarsudil outperformed the experimental ROCKi compounds in support of its clinical status. In contrast, at uniform EC50-levels using netarsudil as reference, all ROCKi compounds performed similarly. Collectively, our data suggest that netarsudil exhibits high potency to rescue HTM cell pathobiology in a tissue-mimetic 3D ECM microenvironment, solidifying the utility of our bioengineered hydrogel model as a viable screening platform to further our understanding of TM pathophysiology in glaucoma.

Combinatorial effects of RhoA and Cdc42 on the actin cytoskeleton revealed by photoswitchable GEFs

Rho GTPases play crucial roles in cell motility by regulation of actin cytoskeleton dynamics. For example, Cdc42 induces the formation of filopodia, and RhoA is involved in the assembly of actin stress fibers and cell contraction. While individual roles of RhoA and Cdc42 are well studied, cellular responses to combinations of RhoA and Cdc42 activity are poorly understood. Previous methods for investigating functions of multiple proteins, such as dominant-negative mutants or siRNA, may cause secondary effects, leading to non-specific pertubations throughout the cell. Here, we co-expressed single-chain photoswitchable activators of RhoA and Cdc42, psRhoGEF and psCdc42GEF, to control ratios of RhoA and Cdc42 activity with high spatiotemporal resolutions. Utilizing this method, we discover that simultaneous activation of RhoA and Cdc42 induces strong actin fibers at the edge of the cell, with a narrow range of ratios of RhoA/Cdc42 activation promoting the response. These cortical actin fibers are mediated by a Cdc42 effector MRCK, a RhoA effector ROCK, and mDia1, a convergent target of RhoA and Cdc42. Therefore, single-chain optobiochemical systems can dissect precise functions of combinations of multiple signaling molecules, which will be critical to understanding complex and dynamic cellular processes.

A deep learning approach to identify and segment alpha-smooth muscle actin stress fiber positive cells

Cardiac fibrosis is a pathological process characterized by excessive tissue deposition, matrix remodeling, and tissue stiffening, which eventually leads to organ failure. On a cellular level, the development of fibrosis is associated with the activation of cardiac fibroblasts into myofibroblasts, a highly contractile and secretory phenotype. Myofibroblasts are commonly identified in vitro by the de novo assembly of alpha-smooth muscle actin stress fibers; however, there are few methods to automate stress fiber identification, which can lead to subjectivity and tedium in the process. To address this limitation, we present a computer vision model to classify and segment cells containing alpha-smooth muscle actin stress fibers into 2 classes (α-SMA SF+ and α-SMA SF-), with a high degree of accuracy (cell accuracy: 77%, F1 score 0.79). The model combines standard image processing methods with deep learning techniques to achieve semantic segmentation of the different cell phenotypes. We apply this model to cardiac fibroblasts cultured on hyaluronic acid-based hydrogels of various moduli to induce alpha-smooth muscle actin stress fiber formation. The model successfully predicts the same trends in stress fiber identification as obtained with a manual analysis. Taken together, this work demonstrates a process to automate stress fiber identification in in vitro fibrotic models, thereby increasing reproducibility in fibroblast phenotypic characterization.

Involvement of LIMK2 in actin cytoskeleton remodeling during the definitive endoderm differentiation

LIM kinases are involved in various cellular events such as migration, cycle, and differentiation, but whether they have a role in the specification of mammalian early endoderm remains unclear. In the present study, we found that depletion of LIMK2 severely inhibited the generation of definitive endoderm (DE) from human embryonic stem cells (hESCs) and promoted an early neuroectodermal fate. Upon the silencing of LIMK2 during the endodermal differentiation, the assembly of actin stress fibers was disturbed, and the phosphorylation of cofilin was decreased. In addition, knockdown of LIMK2 during DE differentiation also interfered the upregulation of epithelial-to-mesenchymal transition (EMT)-related genes and cell migration. Collectively, the results highlight that the serine/threonine kinase LIMK2, acting as a key regulator in actin remodeling, plays a critical role in endodermal lineage determination.

Attenuation of fibrogenesis by PDE4 inhibitor via decreased cytoskeleton remodeling, activation and contractility of hepatic stellate cells

Liver fibrosis is characterized by accumulation of extracellular matrix (ECM) proteins that occurs due to chronic liver injury. Activated hepatic stellate cells (HSCs) are main source of ECM in the liver. Rho‐associated kinase (Rho‐kinase/ROCK) promotes the assembly of focal adhesions and actin stress fibers by regulating the organization of the cytoskeleton in HSCs. Exchange protein directly regulated by cAMP 1 (EPAC1) attenuated Rho kinase signaling in HSCs and alleviated the development of liver fibrosis. We previously showed that cAMP degrading phosphodiesterase 4 (PDE4) enzymes play a critical role in the pathogenesis of liver fibrosis suggesting that hepatic cAMP signaling is compromised during fibrogenesis. Based on these observations, we hypothesize that preserving cAMP/EPAC1 signaling by inhibiting PDE4 will attenuate HSC activation and fibrotic signaling.C57Bl/6 mice were subjected to repeated carbon tetrachloride (CCl4) injection for 4 weeks to estabilesh liver fibrosis. One group of mice received PDE4 inhibitor, Rolipram (targeted to the liver) twice a week. To examine the direct effect of EPAC1/PDE4 pathway on HSCs, in vitro studies using human HSC cell line (LX2) were also performed. Activation of LX2 HSCs was achieved by using recombinant TGFβ1. Liver tissue proteomic analysis was performed. Pathway enrichment analysis was done using Clarivate analytics software, MetaCore. Sirius Red staining and hydroxyproline (HYP) assay was performed to assess collagen deposition. Real time qPCR and Western blot were performed to validate the findings from proteomic analysis. Statistical analysis was performed using ANOVA.CCl4 administration resulted in a significant accumulation of collagen confirmed by Sirius red staining and hydroxyproline (HYP) content in mice. Importantly, Rolipram prevented HYP accumulation and the expression of genes involved in collagen synthesis (Serpinh1) and crosslinking (Loxl1 and Lox). Additionally, Smad3 (central modulator of canonical TGFβ1 signaling) activation and mRNA levels of Mmp2 and Timp2 were markedly decreased by Rolipram. Pathway enrichment analysis of proteomic data identified several fibrotic pathways impacted by Rolipram, including HSC activation, integrin signaling and cytoskeleton remodeling regulated by Rho‐kinase. Western blot analyses confirmed that Rolipram significantly decreased phosphorylation of myosin light chain (MLC), target of Rho‐associated protein kinase. The effect of PDE4 inhibition on TGFβ1 inducible Smad3 and MLC phosphorylation was recapitulated in LX2 cells by using Rolipram and EPAC1 specific agonist. Moreover, Rolipram decreased Endothelin 1 (ET1) levels both in vivo and in vitro LX2 cells indicating a reduced contractility of HSCs. In LX2 cells TGFβ1 inducible nuclear translocation of Yes‐associated protein (YAP) was also inhibited by Rolipram suggesting that PDE4 inhibition promotes the crosstalk between TGFβ1 and YAP pathway to repress TGFβ1 signaling.These results demonstrate that PDE4 inhibition attenuates liver fibrosis by affecting multiple pathways involved in HSC activation and perpetuation of fibrosis.Support or Funding InformationNIH P20GM113226

TAZ target gene ITGAV regulates invasion and feeds back positively on YAP and TAZ in liver cancer cells.

The Hippo pathway effectors yes-associated protein (YAP) and WW domain containing transcription regulator 1 (TAZ/WWTR1) support tumor initiation and progression in various cancer entities including hepatocellular carcinoma (HCC). However, to which extent YAP and TAZ contribute to liver tumorigenesis via common and exclusive molecular mechanisms is poorly understood. RNAinterference (RNAi) experiments illustrate that YAP and TAZ individually support HCC cell viability and migration, while for invasion additive effects were observed. Comprehensive expression profiling revealed partly overlapping YAP/TAZ target genes as well as exclusively regulated genes. Integrin-αV (ITGAV) is a novel TAZ-specific target gene, whose overexpression in human HCC patients correlates with poor clinical outcome, TAZ expression in HCCs, and the abundance of YAP/TAZ target genes. Functionally, ITGAV contributes to actin stress fiber assembly, tumor cell migration and invasion. Perturbation of ITGAV diminishes actin fiber formation and nuclear YAP/TAZ protein levels. We describe a novel Hippo downstream mechanism in HCC cells, which is regulated by TAZ and ITGAV and that feedbacks on YAP/TAZ activity. This mechanism may represent a therapeutic target structure since it contributes to signal amplification of oncogenic YAP/TAZ in hepatocarcinogenesis.

Helical structure of actin stress fibers and its possible contribution to inducing their direction-selective disassembly upon cell shortening.

Mechanisms of the assembly of actin stress fibers (SFs) have been extensively studied, while those of the disassembly-particularly cell shortening-induced ones-remain unclear. Here, we show that SFs have helical structures composed of multi-subbundles, and they tend to be delaminated upon cell shortening. Specifically, we observed with atomic force microscopy delamination of helical SFs into their subbundles. We physically caught individual SFs using a pair of glass needles to observe rotational deformations during stretching as well as ATP-driven active contraction, suggesting that they deform in a manner reflecting their intrinsic helical structure. A minimal analytical model was then developed based on the Frenet-Serret formulas with force-strain measurement data to suggest that helical SFs can be delaminated into the constituent subbundles upon axial shortening. Given that SFs are large molecular clusters that bear cellular tension but must promptly disassemble upon loss of the tension, the resulting increase in their surface area due to the shortening-induced delamination may facilitate interaction with surrounding molecules to aid subsequent disintegration. Thus, our results suggest a new mechanism of the disassembly that occurs only in the specific SFs exposed to forced shortening.

The cell adhesion molecule IGPR-1 is activated by and regulates responses of endothelial cells to shear stress

Vascular endothelial cells respond to blood flow-induced shear stress. However, the mechanisms through which endothelial cells transduce mechanical signals to cellular responses remain poorly understood. In this report, using tensile-force assays, immunofluorescence and atomic force microscopy, we demonstrate that immunoglobulin and proline-rich receptor-1 (IGPR-1) responds to mechanical stimulation and increases the stiffness of endothelial cells. We observed that IGPR-1 is activated by shear stress and tensile force and that flow shear stress-mediated IGPR-1 activation modulates remodeling of endothelial cells. We found that under static conditions, IGPR-1 is present at the cell-cell contacts; however, under shear stress, it redistributes along the cell borders into the flow direction. IGPR-1 activation stimulated actin stress fiber assembly and cross-linking with vinculin. Moreover, we noted that IGPR-1 stabilizes cell-cell junctions of endothelial cells as determined by staining of cells with ZO1. Mechanistically, shear stress stimulated activation of AKT Ser/Thr kinase 1 (AKT1), leading to phosphorylation of IGPR-1 at Ser-220. Inhibition of this phosphorylation prevented shear stress-induced actin fiber assembly and endothelial cell remodeling. Our findings indicate that IGPR-1 is an important player in endothelial cell mechanosensing, insights that have important implications for the pathogenesis of common maladies, including ischemic heart diseases and inflammation.

RBM47-regulated alternative splicing of TJP1 promotes actin stress fiber assembly during epithelial-to-mesenchymal transition.

Morphological and functional changes in cells during the epithelial-mesenchymal transition (EMT) process are known to be regulated by alternative splicing. However, only a few splicing factors involved in EMT have been reported and their underlying mechanisms remain largely unknown. Here, we showed that an isoform of tight junction protein 1 (TJP1) lacking exon 20 (TJP1-α-) is predominantly expressed in tumor tissues and in A549 cells during transforming growth factor-β (TGF-β)-induced EMT. RBM47 promoted the inclusion of exon 20 of TJP1, the alternative exon encoding the α-domain, by which RBM47 recognizes to (U)GCAUG in the downstream intronic region of exon 20. We also found that the first RNA recognition motif (RRM) domain of RBM47 is critical in the regulation of alternative splicing and its recognition to pre-mRNA of TJP1. Furthermore, we demonstrated that the TJP1-α- isoform enhances the assembly of actin stress fibers, thereby promoting cellular migration in a wound healing assay. Our results suggest the regulatory mechanism for the alternative splicing of TJP1 pre-mRNA by RBM47 during EMT, providing a basis for studies related to the modulation of EMT via alternative splicing.

CaMKK2 Regulates Mechanosensitive Assembly of Contractile Actin Stress Fibers

Stress fibers are contractile actomyosin bundles that guide cell adhesion, migration, and morphogenesis. Their assembly and alignment are under precise mechanosensitive control. Thus, stress fiber networks undergo rapid modification in response to changes in biophysical properties of the cell's surroundings. Stress fiber maturation requires mechanosensitive activation of 5'AMP-activated protein kinase (AMPK), which phosphorylates vasodilator-stimulated phosphoprotein (VASP) to inhibit actin polymerization at focal adhesions. Here, we identify Ca2+-calmodulin-dependent kinase kinase 2 (CaMKK2) as a critical upstream factor controlling mechanosensitive AMPK activation. CaMKK2 and Ca2+ influxes were enriched around focal adhesions at the ends of contractile stress fibers. Inhibition of either CaMKK2 or mechanosensitive Ca2+ channels led to defects in phosphorylation of AMPK and VASP, resulting in a loss of contractile bundles and a decrease in cell-exerted forces. These data provide evidence that Ca2+, CaMKK2, AMPK, and VASP form a mechanosensitive signaling cascade at focal adhesions that is critical for stress fiber assembly.

Vimentin intermediate filaments control actin stress fiber assembly through GEF-H1 and RhoA.

ABSTRACTThe actin and intermediate filament cytoskeletons contribute to numerous cellular processes, including morphogenesis, cytokinesis and migration. These two cytoskeletal systems associate with each other, but the underlying mechanisms of this interaction are incompletely understood. Here, we show that inactivation of vimentin leads to increased actin stress fiber assembly and contractility, and consequent elevation of myosin light chain phosphorylation and stabilization of tropomyosin-4.2 (see Geeves et al., 2015). The vimentin-knockout phenotypes can be rescued by re-expression of wild-type vimentin, but not by the non-filamentous ‘unit length form’ vimentin, demonstrating that intact vimentin intermediate filaments are required to facilitate the effects on the actin cytoskeleton. Finally, we provide evidence that the effects of vimentin on stress fibers are mediated by activation of RhoA through its guanine nucleotide exchange factor GEF-H1 (also known as ARHGEF2). Vimentin depletion induces phosphorylation of the microtubule-associated GEF-H1 on Ser886, and thereby promotes RhoA activity and actin stress fiber assembly. Taken together, these data reveal a new mechanism by which intermediate filaments regulate contractile actomyosin bundles, and may explain why elevated vimentin expression levels correlate with increased migration and invasion of cancer cells.

Activation of Prostaglandin FP and EP2 Receptors Differently Modulates Myofibroblast Transition in a Model of Adult Primary Human Trabecular Meshwork Cells.

Prostaglandin F2α analogues are the first-line medication for the treatment of ocular hypertension (OHT), and prostanoid EP2 receptor agonists are under clinical development for this indication. The goal of this study was to investigate the effects of F prostanoid (FP) and EP2 receptor activation on the myofibroblast transition of primary trabecular meshwork (TM) cells, which could be a causal mechanism of TM dysfunction in glaucoma. Human primary TM cells were treated with either latanoprost or butaprost and TGF-β2. Trabecular meshwork contraction was measured in a three-dimensional (3D) TM cell-populated collagen gel (CPCG) model. Expression of α-smooth muscle actin (α-SMA) and phosphorylation of myosin light chain (MLC) were determined by Western blot. Assembly of actin stress fibers and collagen deposition were evaluated by immunocytochemistry. Involvement of p38, extracellular signal-regulated kinase (ERK), and Rho-associated kinase (ROCK) pathways as well as matrix metalloproteinase activation was tested with specific inhibitors. In one source of validated adult TM cells, latanoprost induced cell contraction as observed by CPCG surface reduction and increased actin polymerization, α-SMA expression, and MLC phosphorylation, whereas butaprost inhibited TGF-β2-induced CPCG contraction, actin polymerization, and MLC phosphorylation. Both agonists inhibited TGF-β2-dependent collagen deposition. The latanoprost effects were mediated by p38 pathway. Latanoprost decreased TM collagen accumulation but promoted a contractile phenotype in a source of adult TM cells that could modulate the conventional outflow pathway. In contrast, butaprost attenuated both TM contraction and collagen deposition induced by TGF-β2, thereby inhibiting myofibroblast transition of TM cells. These results open new perspectives for the management of OHT.

Loss of miR-203 regulates cell shape and matrix adhesion through ROBO1/Rac/FAK in response to stiffness.

Breast tumor progression is accompanied by changes in the surrounding extracellular matrix (ECM) that increase stiffness of the microenvironment. Mammary epithelial cells engage regulatory pathways that permit dynamic responses to mechanical cues from the ECM. Here, we identify a SLIT2/ROBO1 signaling circuit as a key regulatory mechanism by which cells sense and respond to ECM stiffness to preserve tensional homeostasis. We observed that Robo1 ablation in the developing mammary gland compromised actin stress fiber assembly and inhibited cell contractility to perturb tissue morphogenesis, whereas SLIT2 treatment stimulated Rac and increased focal adhesion kinase activity to enhance cell tension by maintaining cell shape and matrix adhesion. Further investigation revealed that a stiff ECM increased Robo1 levels by down-regulating miR-203. Consistently, patients whose tumor expressed a low miR-203/high Robo1 expression pattern exhibited a better overall survival prognosis. These studies show that cells subjected to stiffened environments up-regulate Robo1 as a protective mechanism that maintains cell shape and facilitates ECM adherence.

Regulation of cytoskeleton organization by sphingosine in a mouse cell model of progressive ovarian cancer.

Ovarian cancer is a multigenic disease and molecular events driving ovarian cancer progression are not well established. We have previously reported the dysregulation of the cytoskeleton during ovarian cancer progression in a syngeneic mouse cell model for progressive ovarian cancer. In the present studies, we investigated if the cytoskeleton organization is a potential target for chemopreventive treatment with the bioactive sphingolipid metabolite sphingosine. Long-term treatment with non-toxic concentrations of sphingosine but not other sphingolipid metabolites led to a partial reversal of a cytoskeleton architecture commonly associated with aggressive cancer phenotypes towards an organization reminiscent of non-malignant cell phenotypes. This was evident by increased F-actin polymerization and organization, a reduced focal adhesion kinase expression, increased α-actinin and vinculin levels which together led to the assembly of more mature focal adhesions. Downstream focal adhesion signaling, the suppression of myosin light chain kinase expression and hypophosphorylation of its targets were observed after treatment with sphingosine. These results suggest that sphingosine modulate the assembly of actin stress fibers via regulation of focal adhesions and myosin light chain kinase. The impact of these events on suppression of ovarian cancer by exogenous sphingosine and their potential as molecular markers for treatment efficacy warrants further investigation.

The Role of Myosin-II in Cell Spreading on Soft Hyaluronan-Fibronectin Substrates

Studies using polyacrylamide (PAA) substrates show that cells respond to the resistance of the substrate by modifying spread area, focal adhesions (FA) and cytoskeletal morphology. Cells on a soft substrate (E∼300 Pa) generate low traction forces, which do not support cell growth, FA formation and maturation or the assembly of actin stress fibers. We show that soft hyaluronan-fibronectin (HA-Fn) substrates support cell growth and survival similarly to rigid substrates, suggesting that the link between increased tension at the cell-substrate interface and cell growth could be altered by the substrate composition.To address this question we measured epithelial and endothelial cell growth and morphology on soft HA-Fn substrates in the presence of agents that affect the myosin II-dependent contractile mechanism, such as the ROCK inhibitor Y-27632, and using myosin II knockdown cells.Inhibition of myosin II, which decreases traction forces, causes single cells to alter morphology, decrease their adherent area, and disassemble FA and actin stress fibers. In contrast, cells in contact with one another show no significant effect.The spreading, shape and cytoskeletal morphology of single cells on soft HA-Fn substrates seems to be influenced by internal traction forces; however, this link between internal traction forces and cell spreading is eliminated when cells make cell-cell contact. These results suggest that traction forces within single cells on soft HA-Fn can be stimulated by HA as on stiffer PAA-Fn substrates which lead to similar cell spreading area and morphology response. Moreover, it is demonstrated that mechanical signaling is specific not only to substrate stiffness but also to substrate composition and cell state.

Thrombin stimulates stress fiber assembly in RPE cells by PKC/CPI-17-mediated MLCP inactivation

Most retinal proliferative diseases involve blood-retinal barrier (BRB) breakdown, exposing the retinal pigment epithelium (RPE) to thrombin, which triggers cell transformation, proliferation and migration through the activation of PAR-1. These processes require the assembly of contractile stress fibers containing actin and non-muscle myosin II, which allow cell movement upon phosphorylation of the myosin light chains (MLCs). PKC family of kinases promotes agonist-mediated contraction in smooth muscle and endothelial cells through the activation of its downstream target, the PKC-potentiated inhibitory protein of 17 kDa (CPI-17), which specifically inhibits MLC phosphatase. Although the participation of PKC in RPE cell transdifferentiation has been suggested, the role of PKC/CPI-17 signaling has not been investigated. The purpose of this study was to analyze the involvement of specific PKC isoenzymes and their effector protein CPI-17 in thrombin-induced MLC phosphorylation and actin stress fiber assembly in RPE cells. Rat RPE cells in primary culture were shown to respond to thrombin stimulation by activation of conventional, novel and atypical PKC isoforms and the downstream phosphorylation of CPI-17 and MLC, which in turn promoted actin stress fiber assembly. These effects were prevented by the pharmacological inhibition of conventional PKC isoenzymes (Ro-32-0432) and novel PKCδ (rottlerin and δV1-1 antagonist peptide), as well as by myristoylated pseudosubstrates specifically directed to conventional and atypical PKC isoforms. Thrombin effects were mimicked by phorbol 12-myristate 13-acetate (PMA), further confirming the involvement of diacylglycerol (DAG)-sensitive classical and novel PKC isoforms in thrombin-induced actin cytoskeleton modification. The present work shows, for the first time, the functional expression of the oncoprotein CPI-17 in RPE cells and suggests that PKC/CPI-17 signaling is involved in the control of actin cytoskeletal remodeling leading to cell motility in RPE cells exposed to thrombin, and hence could contribute to the development of proliferative eye diseases.

Apelin-13 passes through the ADMA-damaged endothelial barrier and acts on vascular smooth muscle cells

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is associated with vascular dysfunction. The polypeptide apelin mediates two major actions on blood vessels. However, their combined effects on vascular function are not fully understood. The present study aimed to determine the effect of apelin-13 on myosin light chain (MLC) phosphorylation in vascular smooth muscle cells (VSMCs) under ADMA-induced endothelial leakage conditions. To assess the increased permeability induced by ADMA, human umbilical vein endothelium cells (HUVECs) were plated in transwell dishes. The FITC-dextran flux and FITC-apelin-13 flux through the endothelial monolayer were measured. To examine the effect of leakage of apelin-13 on MLC phosphorylation in HUVSMCs, transwell dishes were used to establish a coculture system with HUVECs in upper chambers and HUVSMCs in lower chambers. Western blot was performed to assess the phospho-MLC levels. ADMA increased endothelial permeability in a concentration- and time-dependent manner, accompanied by actin stress fiber assembly and intercellular gap formation. When HUVECs were treated with ADMA, the permeability to both macromolecular dextran and micromolecular apelin-13 increased significantly. Both p38 MAPK inhibitor and NADPH oxidase inhibitor could prevent HUVECs from the increased permeability, and the changes of cytoskeleton and intercellular junction, which were induced by ADMA. Apelin-13 passed through the ADMA-stimulated endothelial monolayer and increased the expression of phospho-MLC in VSMCs. These results suggest that ADMA increases endothelial permeability, which may involve the p38 MAPK and NADPH oxidase pathway. Apelin-13 can pass through the damaged endothelial barrier, and acts directly on VSMCs to increase MLC phosphorylation.