Evidence of the Involvement of Phosphatidylinositol 3-Kinase in the Migration, Actin Stress Fiber Formation, and αvβ3-Integrin–Mediated Adherence of Human Melanoma Cells

SummaryIntegrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl2 to increase integrin affinity and monitored clustering of β1 and β3 integrins. In unstimulated HUVEC, β1 integrins were present in fibrillar adhesions, while αVβ3 was detected in peripheral focal adhesions. Clustered β1 and β3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl2-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of β3 high affinity/LIBS epitopes at focal adhesions. MnCl2-induced αVβ3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two parmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced αVβ3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl2 did not alter β1 integrin distribution or β1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl2-induced αVβ3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity αVβ3 to focal adhesions. Affinity-modulated αVβ3 clustering requires PI3-K signaling and is negatively regulate by Src.

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Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01044.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. H366-H375 ◽

Cited By ~ 22

Author(s):

MaryEllen Carlile-Klusacek ◽

Victor Rizzo

Keyword(s):

Endothelial Cells ◽

Stress Fiber ◽

Fiber Formation ◽

Signaling Cascade ◽

Membrane Rafts ◽

Actin Stress Fiber ◽

Caveolin 1 ◽

Stress Fiber Formation ◽

Mlc Phosphorylation ◽

Actin Stress Fiber Formation

The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH2 terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 μg/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.

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Paxillin controls actin stress fiber formation and migration of vascular smooth muscle cells by directly binding to the active Fyn

The FASEB Journal ◽

10.1096/fj.202101035rr ◽

2021 ◽

Vol 35 (12) ◽

Author(s):

Ying Zhang ◽

Hiroko Kishi ◽

Tomoka Morita ◽

Sei Kobayashi

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Stress Fiber ◽

Fiber Formation ◽

Actin Stress Fiber ◽

Stress Fiber Formation ◽

And Migration ◽

Actin Stress Fiber Formation

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24 PROTEIN KINASE C DELTA MEDIATES THROMBIN-INDUCED ENDOTHELIAL BARRIER DYSFUNCTION THROUGH ACTIN STRESS FIBER FORMATION

Journal of Investigative Medicine ◽

10.2310/6650.2005.00206.23 ◽

2005 ◽

Vol 53 (2) ◽

pp. S360.6-S360

Author(s):

E. T. Chiang ◽

S. M. Camp ◽

S. M. Dudek ◽

V. Natarajan ◽

J. G.N. Garcia

Keyword(s):

Protein Kinase C ◽

Stress Fiber ◽

Fiber Formation ◽

Endothelial Barrier ◽

Actin Stress Fiber ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Kinase C ◽

Stress Fiber Formation ◽

Actin Stress Fiber Formation

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Indomethacin Delays Gastric Restitution: Association with the Inhibition of Focal Adhesion Kinase and Tensin Phosphorylation and Reduced Actin Stress Fibers

Experimental Biology and Medicine ◽

10.1177/153537020222700607 ◽

2002 ◽

Vol 227 (6) ◽

pp. 412-424 ◽

Cited By ~ 20

Author(s):

Imre L. Szabó ◽

Rama Pai ◽

Michael K. Jones ◽

George R. Ehring ◽

Hirofumi Kawanaka ◽

...

Keyword(s):

Cell Migration ◽

Focal Adhesion ◽

Focal Adhesions ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Actin Stress Fiber ◽

Stress Fiber Formation ◽

Actin Stress Fiber Formation

Repair of superficial gastric mucosal injury is accomplished by the process of restitution—migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.

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Icariin Promotes the Migration of BMSCs In Vitro and In Vivo via the MAPK Signaling Pathway

Stem Cells International ◽

10.1155/2018/2562105 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 8

Author(s):

Feng Jiao ◽

Wang Tang ◽

He Huang ◽

Zhaofei Zhang ◽

Donghua Liu ◽

...

Keyword(s):

Signaling Pathway ◽

Mapk Signaling ◽

Stress Fiber ◽

Fiber Formation ◽

Mapk Signaling Pathway ◽

Actin Stress Fiber ◽

Stress Fiber Formation ◽

Actin Stress Fiber Formation

Bone marrow-derived mesenchymal stem cells (BMSCs) are widely used in tissue engineering for regenerative medicine due to their multipotent differentiation potential. However, their poor migration ability limits repair effects. Icariin (ICA), a major component of the Chinese medical herb Herba Epimedii, has been reported to accelerate the proliferation, osteogenic, and chondrogenic differentiation of BMSCs. However, it remains unknown whether ICA can enhance BMSC migration, and the possible underlying mechanisms need to be elucidated. In this study, we found that ICA significantly increased the migration capacity of BMSCs, with an optimal concentration of 1 μmol/L. Moreover, we found that ICA stimulated actin stress fiber formation in BMSCs. Our work revealed that activation of the MAPK signaling pathway was required for ICA-induced migration and actin stress fiber formation. In vivo, ICA promoted the recruitment of BMSCs to the cartilage defect region. Taken together, these results show that ICA promotes BMSC migration in vivo and in vitro by inducing actin stress fiber formation via the MAPK signaling pathway. Thus, combined administration of ICA with BMSCs has great potential in cartilage defect therapy.

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GDI-1 Phosphorylation Switch at Serine 96 Induces RhoA Activation and Increased Endothelial Permeability

Molecular and Cellular Biology ◽

10.1128/mcb.00523-07 ◽

2007 ◽

Vol 27 (18) ◽

pp. 6323-6333 ◽

Cited By ~ 46

Author(s):

Nebojsa Knezevic ◽

Arun Roy ◽

Barbara Timblin ◽

Maria Konstantoulaki ◽

Tiffany Sharma ◽

...

Keyword(s):

Endothelial Permeability ◽

Stress Fiber ◽

Fiber Formation ◽

Mutant Form ◽

Actin Stress Fiber ◽

Rhoa Activity ◽

Rhoa Activation ◽

Stress Fiber Formation ◽

C Terminus ◽

Actin Stress Fiber Formation

ABSTRACT We identified the GDI-1-regulated mechanism of RhoA activation from the Rho-GDI-1 complex and its role in mediating increased endothelial permeability. Thrombin stimulation failed to induce RhoA activation and actin stress fiber formation in human pulmonary arterial endothelial cells transduced with full-length GDI-1. Expression of a GDI-1 mutant form (C-GDI) containing the C terminus (aa 69 to 204) also prevented RhoA activation, whereas further deletions failed to alter RhoA activation. We observed that protein kinase Cα-mediated phosphorylation of the C terminus of GDI-1 at Ser96 reduced the affinity of GDI-1 for RhoA and thereby enabled RhoA activation. Rendering GDI-1 phosphodefective with a Ser96 → Ala substitution rescued the inhibitory activity of GDI-1 toward RhoA but did not alter the thrombin-induced activation of other Rho GTPases, i.e., Rac1 and Cdc42. Phosphodefective mutant GDI-1 also suppressed myosin light chain phosphorylation, actin stress fiber formation, and the increased endothelial permeability induced by thrombin. In contrast, expressing the phospho-mimicking mutant S96D-GDI-1 protein induced RhoA activity and increased endothelial permeability independently of thrombin stimulation. These results demonstrate the crucial role of the phosphorylation of the C terminus of GDI-1 at S96 in selectively activating RhoA. Inhibiting GDI-1 phosphorylation at S96 is a potential therapeutic target for modulating RhoA activity and thus preventing the increase in endothelial permeability associated with vascular inflammation.

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