Cortical Actin Formation Research Articles

#3760 CYTOSKELETON-ORGANELLE INTERACTION IN LIVING CELL EXPRESSING INF2 VARIANTS CAUSING FSGS AND PERIPHERAL NEUROPATHY

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a common glomerular injury and represents a leading cause of ESRD. Studies with genetic FSGS show that a majority of genes are associated with regulation of podocyte cytoskeletons. INF2 is an actin assembly factor that mediates both polymerization and depolymerization and is the most frequent cause of dominant inherited FSGS. INF2 mutations located in the proximal half of DID (residues Leu57 to Glu184) typically cause CMT with early-onset FSGS, whereas that situating in the distal half (residues Glu184 to Leu245) cause FSGS alone. Previous studies disclosed that INF2 mutations primarily disorganize actin network and alter mitochondria shapes and vesicle motility. However, it remains elusive how the cytoskeletal network is concerted with the organelle morphology and functions in the disease cells. This study aims to characterize the interaction between cytoskeleton and organelle in cells expressing INF2 FSGS or CMT/FSGS variants. Method We characterized the cytoskeletal-organelle interaction in live HeLa or COS-7 cells expressing pathogenic INF2 variants in our cohort of FSGS. We performed a triple labeling by co-transfection of GFP-INF2, cytoskeletal markers (Lifeact-mCherry or EMTB-mScarletI), and by the uptake of organelle markers (Lysotracker or Mitotracker). To clarify the detailed pattern and dynamics of cytoskeletons and organelles, we employed the Spinning Disk Confocal Microscope DragonFly, which allows real-time super-resolution imaging with 70nm resolution. Results Wild-type-INF2 (WT-INF2) cells showed an ER pattern with perinuclear, Golgi accumulation. Mild INF2 variants causing FSGS alone (T161N, N202S) preserved the subcellular distribution essential similar to WT-INF2, while showing focal irregularity of ER networks. In contrast, severe INF2 variants leading to CMT/FSGS (G73D) exhibited a diffuse coarse ER pattern with few Golgi clusters and occasional peripheral accumulation in both cell poles. In live imaging of cells coexpressing GFP-INF2 and Lifeact-mCherry, actin stress fibers were pronouncedly reduced in CMT/FSGS variant (G73D) cells, but were mildly decreased FSGS variant (T161N and N202S) cells, compared with WT-INF2 cells. The filopodia in cells expressing T161N variant were less mobile (n = 10, P< 0.01) but were longer (n = 10, P< 0.0001) than those in cells expressing WT-INF2. T161N variant reduced expression of cortactin that stabilizes actin-branching and narrowed area of cortical actin meshwork compared to the WT-INF2 (n = 11, P< 0.01), suggesting that INF2 could serve the cortical actin formation. HeLa cells coexpressing T161N variant and EMTB showed a bipolar cell elongation along the longitudinal axis (n≥30, P< 0.0001) and disarranged microtubule bundles aligned in parallel, compared with those expressing WT-INF2. A time-lapse imaging of living COS-7 cells revealed that T161N variant increases mitochondrial fragmentation as well as mitochondria-ER interfaces more prominent than WT-INF2. Moreover, T161N variant induced the mitochondria misdistribution at the cell periphery (filopodia and cell edge) in both cell poles. Such aberrant patterns frequently coexisted with disarranged microtubules. Moreover, in lysotracker-labeled living COS-7, T161N variant reduced the motility of the lysosomal vesicles in cortical actin meshwork area, compared with WT-INF2 (n = 100, P< 0.0001). These observations suggest that INF2 variants perturbate the trafficking of organelles through disorganization of actin-microtubule network. Conclusion Our results indicate that INF2 CMT/FSGS variants cause more global and severe organelle dysfunction by disrupting the organelle-cytoskeletal interaction, than INF2 FSGS variants. The microtubule disarrangement might be due to an insufficient capping mechanism by which the microtubules are stably tethered to the cortical actin meshwork. Elucidation of cell-specific factors will help better understanding why podocyte and Schwann cell are susceptible to the cytoskeletal disarrangement.

TRIM40 is a pathogenic driver of inflammatory bowel disease subverting intestinal barrier integrity

The cortical actin cytoskeleton plays a critical role in maintaining intestinal epithelial integrity, and the loss of this architecture leads to chronic inflammation, as seen in inflammatory bowel disease (IBD). However, the exact mechanisms underlying aberrant actin remodeling in pathological states remain largely unknown. Here, we show that a subset of patients with IBD exhibits substantially higher levels of tripartite motif-containing protein 40 (TRIM40), a gene that is hardly detectable in healthy individuals. TRIM40 is an E3 ligase that directly targets Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), an essential kinase involved in promoting cell-cell junctions, markedly decreasing the phosphorylation of key signaling factors critical for cortical actin formation and stabilization. This causes failure of the epithelial barrier function, thereby promoting a long-lived inflammatory response. A mutant TRIM40 lacking the RING, B-box, or C-terminal domains has impaired ability to accelerate ROCK1 degradation-driven cortical actin disruption. Accordingly, Trim40-deficient male mice are highly resistant to dextran sulfate sodium (DSS)-induced colitis. Our findings highlight that aberrant upregulation of TRIM40, which is epigenetically silenced under healthy conditions, drives IBD by subverting cortical actin formation and exacerbating epithelial barrier dysfunction.

The type 3 secretion effector IpgD promotes S. flexneri dissemination.

The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery worldwide every year, resulting in more than 200,000 deaths. S. flexneri pathogenic properties rely on its ability to invade epithelial cells and spread from cell to cell within the colonic epithelium. This dissemination process relies on actin-based motility in the cytosol of infected cells and formation of membrane protrusions that project into adjacent cells and resolve into double-membrane vacuoles (DMVs) from which the pathogen escapes, thereby achieving cell-to-cell spread. S. flexneri dissemination is facilitated by the type 3 secretion system (T3SS) through poorly understood mechanisms. Here, we show that the T3SS effector IpgD facilitates the resolution of membrane protrusions into DMVs during S. flexneri dissemination. The phosphatidylinositol 4-phosphatase activity of IpgD decreases PtdIns(4,5)P2 levels in membrane protrusions, thereby counteracting de novo cortical actin formation in protrusions, a process that restricts the resolution of protrusions into DMVs. Finally, using an infant rabbit model of shigellosis, we show that IpgD is required for efficient cell-to-cell spread in vivo and contributes to the severity of dysentery.

Activation of soluble AC10 induces cytoskeletal reorganisation

The cellular monolayer of the pulmonary endothelium forms a semi-selective barrier intersecting the vascular space, the underlying interstitium, and adjacent airspace. This endothelial barrier maintains tissue homeostasis critically regulating vessel permeability. Endothelial cAMP signals regulate endothelial permeability; however, cAMP signals are highly compartmentalized, such that cAMP signals generated by transmembrane adenylyl cyclase protect the barrier, while cAMP signals generated by soluble AC disrupt the barrier. Indeed, bicarbonate stimulation of soluble AC10 disrupts the endothelial barrier increasing permeability and is required for LPS-induced lung edema. Yet, how AC10 cAMP signals coordinate endothelial barrier regulation is unclear. The endothelial cytoskeleton alters cell shape which in turn regulates endothelial permeability. Thus, we hypothesized that bicarbonate stimulation of AC10 induces cytoskeletal remodeling. PMVECs were engineered to overexpress AC10 fused to a C-terminus hemagglutinin tag (HA). Gene and protein expression were controlled using a two-tier gene expression approach in which the doxycycline inducible promoter couples with a protein destabilizing variant of FKBP12 fused to the AC10-HA construct rendering the protein unstable in the absence of Shield1. In the presence of doxycycline and Shield1, AC10-HA protein began to appear 4-hours after induction and was absent in control cells. In this system, following addition of doxycycline and Shield1 and in the presence of elevated bicarbonate, whole cell cAMP increased over the same time course as protein expression while control cells showed no additional increase above normal bicarbonate stimulation. As cAMP increases above controls, endothelial cells begin to retract inducing inter-endothelial gaps between adjacent cells. This increase in inter-endothelial gaps was consistent with decreased resistance across the endothelial monolayer. To further address cytoskeletal mediated changes during AC10-induced endothelial barrier disruption, we examined actin cytoskeleton dynamics. Significant loss of the cortical actin rim and formation of actin stress fibers progress to complete actin cytoskeletal disruption by 24 hours. Interestingly, over the same course as the restructuring of the actin cytoskeleton, we detected phosphorylation of actin binding proteins, filamin and VASP, at PKA mediated sites. Thus, it appears that AC10 mediated pulmonary endothelial barrier disruption is coupled to actin remodeling.

The Pervasive Effects of ER Stress on a Typical Endocrine Cell: Dedifferentiation, Mesenchymal Shift and Antioxidant Response in the Thyrocyte.

The endoplasmic reticulum stress and the unfolded protein response are triggered following an imbalance between protein load and protein folding. Until recently, two possible outcomes of the unfolded protein response have been considered: life or death. We sought to substantiate a third alternative, dedifferentiation, mesenchymal shift, and activation of the antioxidant response by using typical endocrine cells, i.e. thyroid cells. The thyroid is a unique system both of endoplasmic reticulum stress (a single protein, thyroglobulin represents the majority of proteins synthesized in the endoplasmic reticulum by the thyrocyte) and of polarized epithelium (the single layer of thyrocytes delimiting the follicle). Following endoplasmic reticulum stress, in thyroid cells the folding of thyroglobulin was disrupted. The mRNAs of unfolded protein response were induced or spliced (X-box binding protein-1). Differentiation was inhibited: mRNA levels of thyroid specific genes, and of thyroid transcription factors were dramatically downregulated, at least in part, transcriptionally. The dedifferentiating response was accompanied by an upregulation of mRNAs of antioxidant genes. Moreover, cadherin-1, and the thyroid (and kidney)-specific cadherin-16 mRNAs were downregulated, vimentin, and SNAI1 mRNAs were upregulated. In addition, loss of cortical actin and stress fibers formation were observed. Together, these data indicate that ER stress in thyroid cells induces dedifferentiation, loss of epithelial organization, shift towards a mesenchymal phenotype, and activation of the antioxidant response, highlighting, at the same time, a new and wide strategy to achieve survival following ER stress, and, as a sort of the other side of the coin, a possible new molecular mechanism of decline/loss of function leading to a deficit of thyroid hormones formation.

Malaysian Tualang Honey Inhibits Hydrogen Peroxide-Induced Endothelial Hyperpermeability.

Malaysian Tualang honey (TH) is a known therapeutic honey extracted from the honeycombs of the Tualang tree (Koompassia excelsa) and has been reported for its antioxidant, anti-inflammatory, antiproliferative, and wound healing properties. However, the possible vascular protective effect of TH against oxidative stress remains unclear. In this study, the effects of TH on hydrogen peroxide- (H2O2-) elicited vascular hyperpermeability in human umbilical vein endothelial cells (HUVECs) and Balb/c mice were evaluated. Our data showed that TH concentrations ranging from 0.01% to 1.00% showed no cytotoxic effect to HUVECs. Induction with 0.5 mM H2O2 was found to increase HUVEC permeability, but the effect was significantly reversed attenuated by TH (p < 0.05), of which the permeability with the highest inhibition peaked at 0.1%. In Balb/c mice, TH (0.5 g/kg-1.5 g/kg) significantly (p < 0.05) reduced H2O2 (0.3%)-induced albumin-bound Evans blue leak, in a dose-dependent manner. Immunofluorescence staining confirmed that TH reduced actin stress fiber formation while increasing cortical actin formation and colocalization of caveolin-1 and β-catenin in HUVECs. Signaling studies showed that HUVECs pretreated with TH significantly (p < 0.05) decreased intracellular calcium release, while sustaining the level of cAMP when challenged with H2O2. These results suggested that TH could inhibit H2O2-induced vascular hyperpermeability in vitro and in vivo by suppression of adherence junction protein redistribution via calcium and cAMP, which could have a therapeutic potential for diseases related to the increase of both oxidant and vascular permeability.

Cell-Cell Adhesion and Myosin Activity Regulate Cortical Actin Assembly in Mammary Gland Epithelium on Concaved Surface

It has been demonstrated in recent studies that geometry plays an important role in cell behaviors, ranging from mucus production, cell migration and the origination and prorogation of cancer stem cells. Despite the studies on potentially curvature-sensitive proteins at the nanoscale, it is not clear how cells sense curvature and other geometric parameters at cellular and multi-cellular-level. To systematically study whether and how curvature determines cell behaviors, we developed a technique combining 3D printing and soft lithography to fabricate open channel with 60 μm radius. We found that cortical actin is 1.21-fold more enriched in EpH4-EV cells grown on curvature of 60μm−1 when compared to the flat substrate. Inhibition of myosin phosphorylation via blebbistatin and Y-27632 restored the cortical actin formation in cells cultured in channels of high curvature, suggesting myosin is involved in curvature-dependent cortical actin assembly. Furthermore, we demonstrated that cell-cell adhesion is required in the curvature-dependent cortical actin assembly. The prominent cortical actin assembly seen at 60μm−1 was not detected at low cell seeding density resulting in sparse cell-cell contact or after treating cells with β-catenin inhibitor. To summarize, our finding suggests actin cytoskeleton-adherens machinery is responsible for curvature sensing at the multi-cellular level and at the scale of tens of microns.

Chronophin activation is necessary in Doxorubicin-induced actin cytoskeleton alteration

Although doxorubicin (Dox)-induced oxidative stress is known to be associated with cytotoxicity, the precise mechanism remains unclear. Genotoxic stress not only generates free radicals, but also affects actin cytoskeleton stability. We showed that Dox-induced RhoA signaling stimulated actin cytoskeleton alterations, resulting in central stress fiber disruption at early time points and cell periphery cortical actin formation at a later stage, in HeLa cells. Interestingly, activation of a cofilin phosphatase, chronophin (CIN), was initially evoked by Dox-induced RhoA signaling, resulting in a rapid phosphorylated cofilin turnover leading to actin cytoskeleton remodeling. In addition, a novel interaction between CIN and 14-3-3ζ was detected in the absence of Dox treatment. We demonstrated that CIN activity is quite contrary to 14-3-3ζ binding, and the interaction leads to enhanced phosphorylated cofilin levels. Therefore, initial CIN activation regulation could be critical in Dox-induced actin cytoskeleton remodeling through RhoA/cofilin signaling.

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration.

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4(-/-) fibroblast, compared to Mrp4(+/+)fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

A novel high-content analysis tool reveals Rab8-driven cytoskeletal reorganization through Rho GTPases, calpain and MT1-MMP.

Rab8 is a small Ras-related GTPase that regulates polarized membrane transport to the plasma membrane. Here, we developed a high-content analysis (HCA) tool to dissect Rab8-mediated actin and focal adhesion reorganization that revealed that Rab8 activation significantly induced Rac1 and Tiam1 to mediate cortical actin polymerization and RhoA-dependent stress fibre disassembly. Rab8 activation increased Rac1 activity, whereas its depletion activated RhoA, which led to reorganization of the actin cytoskeleton. Rab8 was also associated with focal adhesions, promoting their disassembly in a microtubule-dependent manner. This Rab8 effect involved calpain, MT1-MMP (also known as MMP14) and Rho GTPases. Moreover, we demonstrate the role of Rab8 in the cell migration process. Indeed, Rab8 is required for EGF-induced cell polarization and chemotaxis, as well as for the directional persistency of intrinsic cell motility. These data reveal that Rab8 drives cell motility by mechanisms both dependent and independent of Rho GTPases, thereby regulating the establishment of cell polarity, turnover of focal adhesions and actin cytoskeleton rearrangements, thus determining the directionality of cell migration.

Neural Wiskott-Aldrich Syndrome Protein (N-WASP)-mediated p120-Catenin Interaction with Arp2-Actin Complex Stabilizes Endothelial Adherens Junctions

Stable adherens junctions (AJs) are required for formation of restrictive endothelial barrier. Vascular endothelial cadherin from contiguous endothelial cells forms AJs, which are stabilized intracellularly by binding of p120-catenin and cortical actin. Mechanisms inducing cortical actin formation and enabling its linkage with p120-catenin remain enigmatic. We altered the function of neural Wiskott-Aldrich syndrome protein (N-WASP), which induces actin polymerization through actin-related protein 2/3 complex (Arp2/3), to address the role of N-WASP in regulating AJ stability and thereby endothelial permeability. We show that depletion of N-WASP in endothelial cells impaired AJ adhesion and favored the organization of actin from cortical actin to stress fibers, resulting thereby in formation of leaky endothelial barrier. Exposure of the N-WASP-depleted endothelial cell monolayer to the permeability-increasing mediator, thrombin, exaggerated AJ disruption and stress fiber formation, leading to an irreversible increase in endothelial permeability. We show that N-WASP binds p120-catenin through its verprolin cofilin acid (VCA) domain, induces cortical actin formation through Arp2, and links p120-catenin with cortical actin. The interaction of N-WASP with p120-catenin, actin, and Arp2 requires phosphorylation of N-WASP at the Tyr-256 residue by focal adhesion kinase. Expression of the VCA domain of N-WASP or phosphomimicking (Y256D)-N-WASP mutant in endothelial cells stabilizes AJs and facilitates barrier recovery after thrombin stimulation. Our study demonstrates that N-WASP, by mediating p120-catenin interaction with actin-polymerizing machinery, maintains AJs and mitigates disruption of endothelial barrier function by edemagenic agents, therefore representing a novel target for preventing leaky endothelial barrier syndrome.

Vascular Endothelial Adrenomedullin-RAMP2 System Is Essential for Vascular Integrity and Organ Homeostasis

Revealing the mechanisms underlying the functional integrity of the vascular system could make available novel therapeutic approaches. We previously showed that knocking out the widely expressed peptide adrenomedullin (AM) or receptor activity-modifying protein 2 (RAMP2), an AM-receptor accessory protein, causes vascular abnormalities and is embryonically lethal. Our aim was to investigate the function of the vascular AM-RAMP2 system directly. We generated endothelial cell-specific RAMP2 and AM knockout mice (E-RAMP2(-/-) and E-AM(-/-)). Most E-RAMP2(-/-) mice died perinatally. In surviving adults, vasculitis occurred spontaneously. With aging, E-RAMP2(-/-) mice showed severe organ fibrosis with marked oxidative stress and accelerated vascular senescence. Later, liver cirrhosis, cardiac fibrosis, and hydronephrosis developed. We next used a line of drug-inducible E-RAMP2(-/-) mice (DI-E-RAMP2(-/-)) to induce RAMP2 deletion in adults, which enabled us to analyze the initial causes of the aforementioned vascular and organ damage. Early after the induction, pronounced edema with enhanced vascular leakage occurred. In vitro analysis revealed the vascular leakage to be caused by actin disarrangement and detachment of endothelial cells. We found that the AM-RAMP2 system regulates the Rac1-GTP/RhoA-GTP ratio and cortical actin formation and that a defect in this system causes the disruption of actin formation, leading to vascular and organ damage at the chronic stage after the gene deletion. Our findings show that the AM-RAMP2 system is a key determinant of vascular integrity and homeostasis from prenatal stages through adulthood. Furthermore, our models demonstrate how endothelial cells regulate vascular integrity and how their dysregulation leads to organ damage.

IQGAP1 is necessary for pulmonary vascular barrier protection in murine acute lung injury and pneumonia.

We recently reported that integrin α(v)β(3) is necessary for vascular barrier protection in mouse models of acute lung injury and peritonitis. Here, we used mass spectrometric sequencing of integrin complexes to isolate the novel β(3)-integrin binding partner IQGAP1. Like integrin β(3), IQGAP1 localized to the endothelial cell-cell junction after sphingosine-1-phosphate (S1P) treatment, and IQGAP1 knockdown prevented cortical actin formation and barrier enhancement in response to S1P. Furthermore, knockdown of IQGAP1 prevented localization of integrin α(v)β(3) to the cell-cell junction. Similar to β(3)-null animals, IQGAP1-null mice had increased pulmonary vascular leak compared with wild-type controls 3 days after intratracheal LPS. In an Escherichia coli pneumonia model, IQGAP1 knockout mice had increased lung weights, lung water, and lung extravascular plasma equivalents of (125)I-labeled albumin compared with wild-type controls. Taken together, these experiments indicate that IQGAP1 is necessary for S1P-mediated vascular barrier protection during acute lung injury and is required for junctional localization of the barrier-protective integrin α(v)β(3).

Abstract 473: Elevated cAMP decreases migratory potential in pancreatic cancer cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers, primarily resulting from inherent, aggressive dissemination. As elevated concentrations of cAMP are known to inhibit cell movement in multiple tissues, we investigated the role of cAMP in regulating migration of model pancreatic cancer cells. Increased cAMP concentrations were induced either through forskolin, an activator of adenylyl cyclase, the cAMP analog 6-bnz-cAMP, or by two phosphodiesterase cyclic nucleotide inhibitors, cilostamide or IBMX. Transwell migration assays demonstrated cAMP elevating agents significantly decreased migration of Panc1, MiaPaCa2, and HPAFII pancreatic cancer cell lines. In Panc1 cells, inhibition was dose-dependent and not significantly different between forskolin, cilostamide, or IBMX treatment. Importantly, a significant decrease in cell migration was also observed in cells treated with 6-bnz-cAMP, a specific activator of protein kinase A. The decrease in migration did not reflect alterations in cell proliferation as cell cycle analysis demonstrated little to no change in proliferation in treated compared to control cells. Similarly, the decrease in cell migration was not a function of programmed cell death as apoptosis was not elevated in cells treated with cAMP elevating agents. Consistent with its known role in F-actin rearrangement, elevated levels of cAMP increased phosphorylated RhoA and the resultant decrease in active RhoA-GTP. As expected, fluorescence microscopy indicated a significant increase in cortical actin formation along the perimeter of forskolin or cilostamide-treated cells. Taken together these data suggest that the novel use of existing anti-inflammatory drugs that target the cAMP signaling networks have the potential to block pancreatic cancer dissemination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 473. doi:1538-7445.AM2012-473

Absence of Integrin αvβ3 Enhances Vascular Leak in Mice by Inhibiting Endothelial Cortical Actin Formation

Sepsis and acute lung injury (ALI) have devastatingly high mortality rates. Both are associated with increased vascular leak, a process regulated by complex molecular mechanisms. We hypothesized that integrin αvβ3 could be an important determinant of vascular leak and endothelial permeability in sepsis and ALI. β3 subunit knockout mice were tested for lung vascular leak after endotracheal LPS, and systemic vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. Possible contributory effects of β3 deficiency in platelets and other hematopoietic cells were excluded by bone marrow reconstitution experiments. Endothelial cells treated with αvβ3 antibodies were evaluated for sphingosine-1 phosphate (S1P)–mediated alterations in barrier function, cytoskeletal arrangement, and integrin localization. β3 knockout mice had increased vascular leak and pulmonary edema formation after endotracheal LPS, and increased vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. In endothelial cells, αvβ3 antibodies inhibited barrier-enhancing and cortical actin responses to S1P. Furthermore, S1P induced translocation of αvβ3 from discrete focal adhesions to cortically distributed sites through Gi- and Rac1-mediated pathways. Cortical αvβ3 localization after S1P was decreased by αvβ3 antibodies, suggesting that ligation of the αvβ3 with its extracellular matrix ligands is required to stabilize cortical αvβ3 focal adhesions. Our studies identify a novel mechanism by which αvβ3 mitigates increased vascular leak, a pathophysiologic function central to sepsis and ALI. These studies suggest that drugs designed to block αvβ3 may have the unexpected side effect of intensifying sepsis- and ALI-associated vascular endothelial leak.

Abl Tyrosine Kinase Phosphorylates Nonmuscle Myosin Light Chain Kinase to Regulate Endothelial Barrier Function

Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl-mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y231, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement.

FTY720 inhibits S1P-mediated endothelial healing: Relationship to S1P 1-receptor surface expression

The phosphorylated derivative of the immunosuppressant FTY720 interacts with and modulates the function of sphingosine-1-phosphate (S1P)-receptors. We observed a significant reduction of endothelial surface binding of a S1P 1-specific antibody after FTY720 treatment of 6 h and longer, which was associated with a reduced healing after mechanic injury, impaired angiogenesis and enhanced adhesion molecule expression. FTY720 (5 h) had no impact on the expression of S1P 1- or S1P 3-encoding transcripts. Notably, pre-treatment of cells with FTY720 for only 30 min, which did not reduce S1P 1 surface expression, inhibited the rapid S1P- and SEW2871- (a S1P 1 agonist) induced cortical actin formation and cell migration. FTY720 was effective at concentrations as low as 5 nM. FTY720 at therapeutic concentrations may be harmful by impairing important endothelial functions. Interestingly, FTY720 inhibited endothelial actin remodelling and cell migration without decreasing S1P 1 surface expression.

PI-3-kinase-dependent membrane recruitment of centaurin-α2 is essential for its effect on ARF6-mediated actin cytoskeleton reorganisation

GTPase activating proteins (GAPs) of the centaurin family regulate the actin cytoskeleton and vesicle trafficking through inactivation of the ADP-ribosylation factor (ARF) family of small GTP-binding proteins. We report the functional characterisation of centaurin-alpha(2), which is structurally related to the centaurin-alpha(1) ARF6 GAP. centaurin-alpha(2) contains an N-terminal GAP domain followed by two pleckstrin homology (PH) domains (N-PH and C-PH). In vitro, GFP-centaurin-alpha(2) specifically binds the phosphatidylinositol (PI) 3-kinase lipid products, PI 3,4-P(2) and PI 3,4,5-P(3) (PIP(3)), through its C-terminal PH domain. In agreement with this observation, GFP-centaurin-alpha(2) was recruited to the plasma membrane from the cytosol in EGF-stimulated cells in a PI-3-kinase-dependent manner. Moreover, the C-PH domain is sufficient and necessary for membrane recruitment of centaurin-alpha(2). centaurin-alpha(2) shows sustained kinetics of PI-3-kinase-mediated membrane recruitment in EGF-stimulated cells, owing to its binding to PI 3,4-P(2). centaurin-alpha(2) prevents ARF6 translocation to, and cortical actin formation at, the plasma membrane, which are phenotypic indications for ARF6 activation in EGF-stimulated cells. Moreover, the constitutively active mutant of ARF6 reverses the effect of centaurin-alpha(2) on cortical actin formation. The membrane targeted centaurin-alpha(2) is constitutively active. Together, these studies indicate that centaurin-alpha(2) is recruited in a sustained manner to the plasma membrane through binding to PI 3,4-P(2) and thereby regulates actin reorganisation via ARF6.

Role of PKA as a negative regulator of PCP signaling pathway during Xenopus gastrulation movements

Convergent extension (CE) movements in gastrulation are essential for the establishment of the body axis during early vertebrate development. Although the precise molecular mechanisms of CE movements are not clearly understood, noncanonical Wnt pathway is known to be important for the control of CE movements. Here, we present evidence that PKA is implicated in noncanonical Wnt pathway. Overexpression and specific depletion of PKA inhibit CE movements. PKA depletion also disrupts cell morphology, protrusive activity, and cortical actin formation in dorsal mesodermal cells. Moreover, PKA activity is negatively regulated by major components of planar cell polarity (PCP) pathway. In line with this, overexpression of PKA can rescue the inhibition of CE movements caused by overexpression of these molecules. We also demonstrate that this regulation of PKA activity is dependent upon Gαi signaling. As a negative component of PCP signaling, PKA inhibits not only the activation of RhoA and JNK but also the Dsh-Daam1-RhoA complex formation which is essential for the regulation of RhoA activity. Together, our study suggests a molecular pathway from Wnt/Dsh/PKA signaling to Rho activation in PCP signaling.