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

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 foun...

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Autores principales: Jeon, Won Bae, Park, Bo Hyung, Choi, Seong Kyoon, Lee, Kyeong-Min, Park, Jin-Kyu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507755/
https://www.ncbi.nlm.nih.gov/pubmed/22978264
http://dx.doi.org/10.1186/1472-6750-12-61
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author Jeon, Won Bae
Park, Bo Hyung
Choi, Seong Kyoon
Lee, Kyeong-Min
Park, Jin-Kyu
author_facet Jeon, Won Bae
Park, Bo Hyung
Choi, Seong Kyoon
Lee, Kyeong-Min
Park, Jin-Kyu
author_sort Jeon, Won Bae
collection PubMed
description 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)](20)WPC, referred to as [RGD-V(6)](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-V(6)](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-V(6)](20) was expressed in E. coli and purified by thermally-induced phase transition. N2a cell attachment to either [RGD-V(6)](20) or fibronectin followed hyperbolic binding kinetics saturating around 2 μM protein concentration. The apparent maximum cell binding to [RGD-V(6)](20) was approximately 96% of fibronectin, with half-maximal adhesion on [RGD-V(6)](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-V(6)](20) (42.9% ± 6.5%) > [V(7)](20) (15.5% ± 3.2%) > TCPS (less than 10%). The migration speed of N2a cells on [RGD-V(6)](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-V(6)](20) relative to TCPS. Moreover, by the presence of both [RGD-V(6)](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.
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spelling pubmed-35077552012-11-28 Functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting Arg-Gly-Asp peptides Jeon, Won Bae Park, Bo Hyung Choi, Seong Kyoon Lee, Kyeong-Min Park, Jin-Kyu BMC Biotechnol Research Article 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)](20)WPC, referred to as [RGD-V(6)](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-V(6)](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-V(6)](20) was expressed in E. coli and purified by thermally-induced phase transition. N2a cell attachment to either [RGD-V(6)](20) or fibronectin followed hyperbolic binding kinetics saturating around 2 μM protein concentration. The apparent maximum cell binding to [RGD-V(6)](20) was approximately 96% of fibronectin, with half-maximal adhesion on [RGD-V(6)](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-V(6)](20) (42.9% ± 6.5%) > [V(7)](20) (15.5% ± 3.2%) > TCPS (less than 10%). The migration speed of N2a cells on [RGD-V(6)](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-V(6)](20) relative to TCPS. Moreover, by the presence of both [RGD-V(6)](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. BioMed Central 2012-09-14 /pmc/articles/PMC3507755/ /pubmed/22978264 http://dx.doi.org/10.1186/1472-6750-12-61 Text en Copyright ©2012 Jeon et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Jeon, Won Bae
Park, Bo Hyung
Choi, Seong Kyoon
Lee, Kyeong-Min
Park, Jin-Kyu
Functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting Arg-Gly-Asp peptides
title Functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting Arg-Gly-Asp peptides
title_full Functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting Arg-Gly-Asp peptides
title_fullStr Functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting Arg-Gly-Asp peptides
title_full_unstemmed Functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting Arg-Gly-Asp peptides
title_short Functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting Arg-Gly-Asp peptides
title_sort functional enhancement of neuronal cell behaviors and differentiation by elastin-mimetic recombinant protein presenting arg-gly-asp peptides
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507755/
https://www.ncbi.nlm.nih.gov/pubmed/22978264
http://dx.doi.org/10.1186/1472-6750-12-61
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