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

Abstract

BackgroundIntegrin-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.ResultsThe [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.ConclusionWe 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.

Effects of Elastin-Like Peptide on Regulation of Human Mesenchymal Stem Cell Behavior

“…Since increased surface density of this binding motif has been shown to enhance cell adhesion [43], the higher concentration of RGD peptide sequences on the RGD-ELP-coated substrate compared to the FN-coated substrate may result in enhanced cell adhesion on the RGD-ELP-coated substrate. However, this may not be able to fully explain different cell adhesion between RGD-ELPand FN-coated substrates because our previous study has also shown that cells have a higher affinity to the RGD motif of FN than to that of RGD-ELP [44]. Further studies are needed to investigate how the factors of concentration and affinity of RGD affect cell adhesion between RGD-ELP-and FN-coated substrates.…”

Reduction of pluripotent gene expression in murine embryonic stem cells exposed to mechanical loading or Cyclo RGD peptide

“…The cRGDfC peptide appeared to have little effect on endodermal lineage specification. While it has been previously demonstrated that a number of neuronal cell types express αvβ3 integrins including radial glia cells [ 36 , 37 ], the role of RGD dependent integrins in ectodermal/neuronal differentiation has been suggested, but is less clear [ 38 ]. It is important to highlight, however, that the fold increases in gene expression of the tri-lineage markers were quite low and for the flow cytometry analysis the sub-population of positive cells were only identifiable in the Oct-4 negative population of the cells.…”

Integrin Activation Through the Hematopoietic Adapter Molecule ADAP Regulates Dendritic Development of Hippocampal Neurons

“…The reduction of MAP2 levels may be considered as a mere marker of reduced neuronal differentiation in ADAP knock-down cells. However, this phenomenon may also be more directly involved in the process as MAP2 is critical for dendritogenesis and dendritic outgrowth (Bernhard et al, 1985 ; Harada et al, 2002 ) and the level of MAP2 expression is controlled by integrin stimulation in developing neurons (Domingo-Espín et al, 2012 ; Jeon et al, 2012 ). Axonal growth was not significantly affected in our experiments, but might well be responsive to ADAP manipulation at an earlier time of differentiation.…”