scholarly journals Integrin α6Aβ1 Induces CD81-dependent Cell Motility without Engaging the Extracellular Matrix Migration Substrate

1997 ◽  
Vol 8 (11) ◽  
pp. 2253-2265 ◽  
Author(s):  
Susan Z. Domanico ◽  
Anthony J. Pelletier ◽  
Wendy L. Havran ◽  
Vito Quaranta
Keyword(s):  
Extracellular Matrix ◽  
Cell Motility ◽  
Es Cells ◽  
Embryonic Stem ◽  
Inhibitory Effect ◽  
Laminin Receptor ◽  
Cross Linking ◽  
Integrin Subunit ◽  
Dependent Cell ◽  
Underlying Mechanisms

It is well established that integrins and extracellular matrix (ECM) play key roles in cell migration, but the underlying mechanisms are poorly defined. We describe a novel mechanism whereby the integrin α6β1, a laminin receptor, can affect cell motility and induce migration onto ECM substrates with which it is not engaged. By using DNA-mediated gene transfer, we expressed the human integrin subunit α6A in murine embryonic stem (ES) cells. ES cells expressing α6A (ES6A) at the surface dimerized with endogenous β1, extended numerous filopodia and lamellipodia, and were intensely migratory in haptotactic assays on laminin (LN)-1. Transfected α6A was responsible for these effects, because cells transfected with control vector or α6B, a cytoplasmic domain α6 isoform, displayed compact morphology and no migration, like wild-type ES cells. The ES6A migratory phenotype persisted on fibronectin (Fn) and Ln-5. Adhesion inhibition assays indicated that α6β1 did not contribute detectably to adhesion to these substrates in ES cells. However, anti-α6 antibodies completely blocked migration of ES6A cells on Fn or Ln-5. Control experiments with monensin and anti-ECM antibodies indicated that this inhibition could not be explained by deposition of an α6β1 ligand (e.g., Ln-1) by ES cells. Cross-linking with secondary antibody overcame the inhibitory effect of anti-α6 antibodies, restoring migration or filopodia extension on Fn and Ln-5. Thus, to induce migration in ES cells, α6Aβ1 did not have to engage with an ECM ligand but likely participated in molecular interactions sensitive to anti-α6β1 antibody and mimicked by cross-linking. Antibodies to the tetraspanin CD81 inhibited α6Aβ1-induced migration but had no effect on ES cell adhesion. It is known that CD81 is physically associated with α6β1, therefore our results suggest a mechanism by which interactions between α6Aβ1 and CD81 may up-regulate cell motility, affecting migration mediated by other integrins.

scholarly journals The major laminin receptor of mouse embryonic stem cells is a novel isoform of the alpha 6 beta 1 integrin.

1991 ◽  
Vol 115 (3) ◽  
pp. 843-850 ◽  
Author(s):  
H M Cooper ◽  
R N Tamura ◽  
V Quaranta
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Cytoplasmic Domain ◽  
Laminin Receptor ◽  
Extracellular Matrix Protein ◽  
Cytoplasmic Domains ◽  
Alternative Mrna Splicing

Laminin is the first extracellular matrix protein expressed in the developing mouse embryo. It is known to influence morphogenesis and affect cell migration and polarization. Several laminin receptors are included in the integrin family of extracellular matrix receptors. Ligand binding by integrin heterodimers results in signal transduction events controlling cell motility. We report that the major laminin receptor on murine embryonic stem (ES) cells is the integrin heterodimer alpha 6 beta 1, an important receptor for laminin in neurons, lymphocytes, macrophages, fibroblasts, platelets and other cell types. However, the cytoplasmic domain of the ES cell alpha 6 (alpha 6 B) differs totally from the reported cytoplasmic domain amino acid sequence of alpha 6 (alpha 6 A). Comparisons of alpha 6 cDNAs from ES cells and other cells suggest that the alpha 6 A and alpha 6 B cytoplasmic domains derive from alternative mRNA splicing. Anti-peptide antibodies to alpha 6 A are unreactive with ES cells, but react with mouse melanoma cells and embryonic fibroblasts. When ES cells are cultured under conditions that permit their differentiation, they become positive for alpha 6 A, concurrent with the morphologic appearance of differentiated cell types. Thus, expression of the alpha 6 B beta 1 laminin receptor may be favored in undifferentiated, totipotent cells, while the expression of alpha 6 A beta 1 receptor occurs in committed lineages. While the functions of integrin alpha chain cytoplasmic domains are not understood, it is possible that they contribute to transferring signals to the cell interior, e.g., by delivering cytoskeleton organizing signals in response to integrin engagement with extracellular matrix ligands. It is therefore reasonable to propose that the cellular responses to laminin may vary, according to what alpha subunit isoform (alpha 6 A or alpha 6 B) is expressed as part of the alpha 6 beta 1 laminin receptor. The switch from alpha 6 B to alpha 6 A, if confirmed in early embryos, could then be of striking potential relevance to the developmental role of laminin.

scholarly journals Stem cell culture on polyvinyl alcohol hydrogels having different elasticity and immobilized with ECM-derived oligopeptides

2017 ◽  
Vol 37 (7) ◽  
pp. 647-660 ◽  
Author(s):  
Saradaprasan Muduli ◽  
Li-Hua Chen ◽  
Meng-Pei Li ◽  
Zhao-wen Heish ◽  
Cheng-Hui Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Extracellular Matrix ◽  
Stem Cell ◽  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Poly Vinyl Alcohol ◽  
Hematopoietic Stem ◽  
Stem Cell Culture

Abstract The physical characteristics of cell culture materials, such as their elasticity, affect stem cell fate with respect to cell proliferation and differentiation. We systematically investigated the morphologies and characteristics of several stem cell types, including human amniotic-derived stem cells, human hematopoietic stem cells, human induced pluripotent stem (iPS) cells, and embryonic stem (ES) cells on poly(vinyl alcohol) (PVA) hydrogels immobilized with and without extracellular matrix-derived oligopeptide. Human ES cells did not adhere well to soft PVA hydrogels immobilized with oligovitronectin, whereas they did adhere well to PVA hydrogel dishes with elasticities greater than 15 kPa. These results indicate that biomaterials such as PVA hydrogels should be designed to possess minimum elasticity to facilitate human ES cell attachment. PVA hydrogels immobilized with and without extracellular matrix-derived oligopeptides are excellent candidates of cell culture biomaterials for investigations into how cell culture biomaterial elasticity affects stem cell culture and differentiation.

scholarly journals Inhibition of Fibrinolysis by Coagulation Factor XIII

2017 ◽  
Vol 2017 ◽  
pp. 1-6 ◽  
Author(s):  
Dingeman C. Rijken ◽  
Shirley Uitte de Willige
Keyword(s):  
Factor Xiii ◽  
Coagulation Factor ◽  
Inhibitory Effect ◽  
Clot Lysis ◽  
Cross Linking ◽  
Clot Retraction ◽  
Coagulation Factor Xiii ◽  
Cross Links ◽  
Underlying Mechanisms ◽  
Insight Into

The inhibitory effect of coagulation factor XIII (FXIII) on fibrinolysis has been studied for at least 50 years. Our insight into the underlying mechanisms has improved considerably, aided in particular by the discovery that activated FXIII cross-linksα2-antiplasmin (α2AP) to fibrin. In this review, the most important effects of different cross-linking reactions on fibrinolysis are summarized. A distinction is made between fibrin-fibrin cross-links studied in purified systems and fibrin-α2AP cross-links studied in plasma or whole blood systems. While the formation ofγchain dimers in fibrin does not affect clot lysis, the formation ofαchain polymers has a weak inhibitory effect. Only strong cross-linking of fibrin, associated with high molecular weightαchain polymers and/orγchain multimers, results in a moderate inhibition fibrinolysis. The formation of fibrin-α2AP cross-links has only a weak effect on clot lysis, but this effect becomes strong when clot retraction occurs. Under these conditions, FXIII preventsα2AP being expelled from the clot and makes the clot relatively resistant to degradation by plasmin.

157 LEUKEMIA INHIBITORY FACTOR IMPROVES OOCYTE MATURATION AND DEVELOPMENTAL COMPETENCE IN PIGS

2014 ◽  
Vol 26 (1) ◽  
pp. 192 ◽  
Author(s):  
S. Haraguchi ◽  
T. Q. Dang-Nguyen ◽  
K. Kikuchi ◽  
F. Tanihara ◽  
S. Bodo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Leukemia Inhibitory Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Developmental Competence ◽  
Inhibitory Factor ◽  
Protein Levels ◽  
Underlying Mechanisms

A variety of growth factors and cytokines that are present in follicular fluid provide oocytes with a suitable environment for their maturation. One such cytokine is leukemia inhibitory factor (LIF). Although LIF-supplemented medium enhances embryo development in human, mouse, and bovine, studies investigating the effects of LIF on in vitro maturation (IVM) and subsequent embryo development are inconclusive. Additionally, the underlying mechanisms of LIF in oocyte maturation and embryo development after IVF have not been studied yet. In the present study, we examined the effect of recombinant porcine LIF (pLIF), produced in our laboratory, on porcine oocyte maturation and the mechanism of how LIF involves in oocyte maturation process at molecular level. The biological activity of pLIF was evaluated by sustenance of mouse embryonic stem (ES) cells with an undifferentiating state in ES medium supplemented with pLIF, and the final concentration (1 : 200, equivalent to 1000 U mL–1 of mouse LIF) was determined by serial dilution. Porcine cumulus–oocyte complexes (COC) were cultured in modified NCSU-37 medium supplemented with pLIF during the first 22 h [pLIF (+, –)], the latter 22 h [pLIF (–, +)], or whole 44 h [pLIF (+, +)] of IVM and the proportion of metaphase II (M-II) stage oocytes was observed. Oocyte maturation was enhanced in each group by supplementation with pLIF [pLIF (+, –): 76.1%, n = 138; pLIF (–, +): 82.1%, n = 140; pLIF (+, +): 86.6%, n = 127], when compared with control [pLIF (–, –): 69.6%, n = 112], in which a significant increase of M-II rate (P < 0.05 by ANOVA) and cumulus expansion were observed in the pLIF (+, +) group. The effect of pLIF was only seen for COC but not for denuded oocytes. When oocytes were subjected to IVF (Kikuchi et al. 2002), those matured in pLIF (+, +)-supplemented medium demonstrated higher blastocyst developmental rates (21.1% v. 16.2%; P = 0.07) with increased cell numbers (50.2 cells v. 45.0 cells; P = 0.12) compared with pLIF (–, –) on Day 6 of embryo culture (IVF = 0). Examination of transcripts and proteins of the LIF signalling pathway revealed that mRNA and protein levels of LIF, LIF receptors, and signal transducer and activator of transcription 3 (STAT3) were similar in both pLIF (–, –) and pLIF (+, +) samples. However, notable phosphorylation of STAT3 was observed in the pLIF (+, +) sample. These results suggest that the LIF/STAT3-pathway is functional during oocyte maturation in pigs. Therefore, supplementation of maturation medium with pLIF could improve the developmental competence of oocytes by activation of this pathway. This project was supported by JSPS and HAS under the Japan-Hungary Research Cooperative Program.

scholarly journals Puerarin Exerts a Delayed Inhibitory Effect on the Proliferation of Cardiomyocytes Derived from Murine ES Cells via Slowing Progression through G2/M Phase

2016 ◽  
Vol 38 (4) ◽  
pp. 1333-1342 ◽  
Author(s):  
Xueying Luo ◽  
Binlong Zhong ◽  
Xian Hong ◽  
Yurong Cui ◽  
Ying Gao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Early Stage ◽  
Es Cells ◽  
Embryonic Stem ◽  
Specific Inhibitor ◽  
Cyclin B1 ◽  
Inhibitory Effect ◽  
Cell Cycle Distribution ◽  
M Phase ◽  
Cyclin A2

Objective: Puerarin, which shows beneficial and protective effects on cardiovascular diseases, is the main isoflavone extracted from Pueraria lobata (kudzu) root. The aim of this study was to investigate the effects of puerarin on in vitro myocardial proliferation and its underlying mechanism. Methods: Myocardial differentiation of transgenic embryonic stem (ES) cells was performed by embryoid body-based differentiation method. The proliferation assay of cardiomyocytes (CMs) derived from ES cells (ES-CMs) was performed by EdU (5-Ethynyl-2'-deoxyuridine) staining. Flow cytometry was employed to determine the cell cycle distribution and apoptosis of purified ES-CMs. Quantitative real-time PCR was utilized to study the transcription of genes related to cell cycle progression. Signaling pathways relating to proliferation were studied by western blot analysis and application of specific inhibitors. Results: Puerarin exerted a delayed inhibitory effect on the proliferation of ES-CMs at the early-stage differentiation. Meanwhile, puerarin slowed progression through G2/M phase without inducing apoptosis of ES-CMs. Further assays showed that puerarin up-regulated the transcription of Cyclin A2, Cyclin B1 and Cdk1 in ES-CMs. The ERK1/2 specific inhibitor PD0325901 and the PI3K specific inhibitor Wortmannin successfully reversed puerarin-induced up-regulation of Cdk1 but not Cyclin A2 and B1. Conclusion: These findings suggest that puerarin inhibits CM proliferation via slowing progression through G2/M phase during early-stage differentiation.

scholarly journals Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

1998 ◽  
Vol 142 (4) ◽  
pp. 1121-1133 ◽  
Author(s):  
Helen Priddle ◽  
Lance Hemmings ◽  
Susan Monkley ◽  
Alison Woods ◽  
Bipin Patel ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Es Cells ◽  
Embryonic Stem ◽  
Focal Adhesions ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Integrin Subunit ◽  
Β1 Integrin ◽  
Es Cell ◽  
Wild Type

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.

Polyamines are important for attachment of IEC-6 cells to extracellular matrix

1997 ◽  
Vol 273 (1) ◽  
pp. G175-G183 ◽  
Author(s):  
M. F. Santos ◽  
M. J. Viar ◽  
S. A. McCormack ◽  
L. R. Johnson
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Cell Attachment ◽  
Specific Inhibitor ◽  
Cross Linking ◽  
Integrin Subunit ◽  
Dependent Manner ◽  
Polyamine Biosynthesis ◽  
Surface Receptors ◽  
Protein Cross Linking

The inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine in IEC-6 cells (small intestinal crypt cell line) reduces cell migration by 70%, inhibits protein cross-linking, and affects the cytoskeletal assembly. The current study examines the effects of intracellular polyamine depletion on attachment of IEC-6 cells to different matrices. Polyamine deficiency inhibited cell attachment to plastic, laminin, fibronectin, collagen IV, and Matrigel by different extents. Intracellular putrescine restored attachment to all matrices. The presence of a specific inhibitor of protein cross-linking also inhibited attachment to laminin in a dose-dependent manner. The inhibition of cell attachment to plastic and Matrigel was correlated with the inhibition of cell migration. Immunofluorescence studies showed that polyamines are essential for the correct expression of the integrin subunit alpha 2 but not for the expression of the alpha 1-subunit. This study demonstrates that polyamines are important for cell attachment and expression of the integrin alpha 2 beta 1, a putative receptor for collagen and laminin. The impairment of protein cross-linking and the inhibition of the expression of cell surface receptors that bind extracellular matrix (ECM) proteins may be part of the mechanism by which polyamine deficiency retards cell migration in the small intestine.

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