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Functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting Arg-Gly-Asp peptides

Functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting Arg-Gly-Asp peptides
Jeon, WB[Jeon, Won Bae]Park, BH[Park, Bo Hyung]Choi, SK[Choi, Seong Kyoon]Lee, KM[Lee, Kyeong-Min]Park, JK[Park, Jin-Kyu]
DGIST Authors
Jeon, WB[Jeon, Won Bae]; Park, BH[Park, Bo Hyung]; Choi, SK[Choi, Seong Kyoon]; Lee, KM[Lee, Kyeong-Min]; Park, JK[Park, Jin-Kyu]
Issue Date
BMC Biotechnology, 12
Article Type
AdsorptionAmino Acid SequenceAnimalsArg Gly AspsArginyl-Glycyl-Aspartic AcidBinding KineticsBioactive MatricesBiological MarkerBiomimetic MaterialsBiomimetic MatricesBiomimetic MatrixBiomimeticsCell AdhesionCell BindingCell DifferentiationCell FunctionCell Line, TumorCell MigrationCell MovementCell ProteinCell SpreadingCell StructureCell SurfacesCellsCoating ConcentrationCoatingsControlled StudyE. ColiECM ProteinsElastinElastin-Mimetic ProteinsEscherichia ColiExpression LevelsExtracellular MatricesFacile PreparationFibronectinFibronectinsFunctional EnhancementsGlial Fibrillary Acidic ProteinGlycoproteinsIn-VitroIntegrinsIsothermal AdsorptionIsothermsMessenger RNAMiceMicrotubule Associated Protein 2N2A CellsNerve Cell DifferentiationNerve Fiber GrowthNerve RegenerationNerve RepairNervous System DevelopmentNervous TissueNeural DevelopmentNeural DifferentiationsNeural Tissue RegenerationNeuroblastoma CellNeuroblastomasNeuron Specific EnolaseNeuronal CellNeuronal DifferentiationNeuronal MarkersNeuronsNucleotide SequenceOligopeptidesPeptidomimetic AgentPhase TransitionPolystyrenePolystyrenesProtein ConcentrationsProtein DomainProtein EngineeringProtein ExpressionProtein MotifProtein NF68Protein StructureProtein Tuj1Recombinant ProteinRecombinant ProteinsRGD MotifStructural DomainsTissueTissue-CultureTissue-Culture PolystyrenesTissue RegenerationTropoelastinUnclassified DrugUpregulation
Background: Integrin-mediated interaction of neuronal cells with extracellular matrix (ECM) is important for the control of cell adhesion, morphology, motility, and differentiation in both in vitro and in vivo systems. Arg-Gly-Asp (RGD) sequence is one of the most potent integrin-binding ligand found in many native ECM proteins. An elastin-mimetic recombinant protein, TGPG[VGRGD(VGVPG)6]20WPC, referred to as [RGD-V6]20, contains multiple RGD motifs to bind cell-surface integrins. This study aimed to investigate how surface-adsorbed recombinant protein can be used to modulate the behaviors and differentiation of neuronal cells in vitro. For this purpose, biomimetic ECM surfaces were prepared by isothermal adsorption of [RGD-V6]20 onto the tissue culture polystyrene (TCPS), and the effects of protein-coated surfaces on neuronal cell adhesion, spreading, migration, and differentiation were quantitatively measured using N2a neuroblastoma cells.Results: The [RGD-V6]20 was expressed in E. coli and purified by thermally-induced phase transition. N2a cell attachment to either [RGD-V6]20 or fibronectin followed hyperbolic binding kinetics saturating around 2 μM protein concentration. The apparent maximum cell binding to [RGD-V6]20 was approximately 96% of fibronectin, with half-maximal adhesion on [RGD-V6]20 and fibronectin occurring at a coating concentration of 2.4 × 10-7 and 1.4 × 10-7 M, respectively. The percentage of spreading cells was in the following order of proteins: fibronectin (84.3% ± 6.9%) > [RGD-V6]20 (42.9% ± 6.5%) > [V7]20 (15.5% ± 3.2%) > TCPS (less than 10%). The migration speed of N2a cells on [RGD-V6]20 was similar to that of cells on fibronectin. The expression of neuronal marker proteins Tuj1, MAP2, and GFAP was approximately 1.5-fold up-regulated by [RGD-V6]20 relative to TCPS. Moreover, by the presence of both [RGD-V6]20 and RA, the expression levels of NSE, TuJ1, NF68, MAP2, and GFAP were significantly elevated.Conclusion: We have shown that an elastin-mimetic protein consisting of alternating tropoelastin structural domains and cell-binding RGD motifs is able to stimulate neuronal cell behaviors and differentiation. In particular, adhesion-induced neural differentiation is highly desirable for neural development and nerve repair. In this context, our data emphasize that the combination of biomimetically engineered recombinant protein and isothermal adsorption approach allows for the facile preparation of bioactive matrix or coating for neural tissue regeneration. © 2012 Jeon et al.; licensee BioMed Central Ltd.
BioMed Central Ltd.
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