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

BMC Biotechnology

Won Bae Jeon¹  Bo Hyung Park¹  Seong Kyoon Choi¹  Kyeong-Min Lee¹  Jin-Kyu Park¹ 

[1] Laboratory of Biochemistry and Cellular Engineering, Division of NanoBio Technology, Daegu Gyeongbuk Institute of Science and Technology, Daegu 711-873, South Korea

关键词: Neuronal differentiation;    Cell migration;    Cell spreading;    Cell adhesion;    Biomimetic matrix;    Elastin-mimetic proteins;   

Others  :  1134853
DOI  :  10.1186/1472-6750-12-61

 received in 2012-06-27, accepted in 2012-09-05,  发布年份 2012

【 摘 要 】

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.

【 授权许可】

   
2012 Jeon et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Miao M, Bellingham CM, Stahl RJ, Sitarz EE, Lane CJ, Keeley FW: Sequence and structure determinants for the self-aggregation of recombinant polypeptides modeled after human elastin. J Biol Chem 2002, 278:48553-48562.
• [2]Urry DW, Parker TM, Reid MC, Gowda DC: Biocompatibility of the bioelastic materials, poly(GVGVP) and its gamma-irradiation cross-linked matrix: summary of generic biological test results. J Bioact Compat Polym 1991, 6:263-282.
• [3]Jeon WB: Contribution of lysine-containing cationic domains to thermally induced phase transition of elastin-like proteins and their sensitivity to different stimuli. BMB Rep 2011, 44:22-27.
• [4]Mie M, Mizushima Y, Kobatake E: Novel extracellular matrix for cell sheet recovery using genetically engineered elastin-like protein. J Biomed Mater Res B Appl Biomater 2008, 86:283-290.
• [5]Nicol A, Gowda DC, Urry DW: Cell adhesion and growth on synthetic elastomeric matrices containing ARG-GLY-ASP-SER-3. J Biomed Mater Res 1992, 26:393-413.
• [6]Rauvala H: Neurite outgrowth of neuroblastoma cells: dependence on adhesion surface-cell surface interactions. J Biol Chem 1984, 98:1010-1016.
• [7]Reichardt LF, Tomaselli KJ: Extracellular matrix molecules and their receptors: functions in neural development. Annu Rev Neurosci 1991, 14:531-570.
• [8]Rogers SL, Letourneau PC, Peterson BA, Furcht LT, McCarthy JB: Selective interaction of peripheral and central nervous system cells with two distinct cell-binding domains of fibronectin. J Cell Biol 1987, 105:1435-1442.
• [9]Kobatake E, Onoda K, Yanagida Y, Haruyama T, Aizawa M: Design of a thermostable cell adhesion protein. Biotech Techniq 1999, 13:23-27.
• [10]Liu JC, Heilshorn SC, Tirrell DA: Comparative cell response to artificial extracellular matrix proteins containing the RGD and CS5 cell-binding domains. Biomacromolecules 2004, 5:497-504.
• [11]Straley KS, Heilshorn SC: Design and adsorption of modular engineered proteins to prepare customized, neuron-compatible coatings. Front Neuroeng 2009, 2:1-10.
• [12]Jeon WB, Park BH, Wei J, Park RW: Stimulation of fibroblasts and neuroblasts on a biomimetic extracellular matrix consisting of tandem repeats of the elastic VGVPG domain and RGD motif. J Biomed Mater Res A 2011, 97:152-157.
• [13]Asthagiri AR, Nelson CM, Horwitz AF, Lauffenburger DA: Quantitative relationship among integrin-ligand binding, adhesion, and signaling via focal adhesion kinase and extracellular signal-regulated kinase 2. J Biol Chem 1999, 274:27119-27127.
• [14]Ma W, Fitzgerald W, Liu QY, O’Shaughnessy TJ, Maric D, Lin HJ, Alkon DL, Barkerd JL: CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels. Exp Neurol 2004, 190:276-288.
• [15]Palecek SP, Loftus JC, Ginsberg MH, Lauffenburger DA, Horwitz AF: Integrin/ligand binding properties govern cell migration speed through cell/substratum adhesiveness. Nature 1997, 385:537-540.
• [16]Yip PM, Zhao X, Montgomery AM, Siu CH: The arg-gly-asp motif in the cell adhesion molecule L1 promotes neurite outgrowth via interaction with the ανβ3 integrin. Mol Biol Cell 1998, 9:277-290.
• [17]Choung PH, Seo BM, Chung CP, Yamada KM, Jang JH: Synergistic activity of fibronectin and fibroblast growth factor receptors on neuronal adhesion and neurite extension through extracellular signal-regulated kinase pathway. Biochem Biophys Res Commun 2002, 295:898-902.
• [18]Müller U, Bossy B, Venstrom K, Reichardt LF: Integrin α8β1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin. Mol Biol Cell 1995, 6:433-448.
• [19]McPherson DT, Xu J, Urry DW: Product purification by reversible phase transition following Escherichia coli expression of genes encoding up to 251 repeats of the elastomeric pentapeptide GVGVP. Protein Expr Purif 1996, 7:51-57.
• [20]Landegren U: Measurement of cell numbers by means of endogenous enzyme hexosaminidase. Application to detction of lymphokines and cellular surface antigen. J Immunol Methods 1984, 67:379-388.
• [21]Roy DC, Wilke-Mounts SJ, Hocking DC: Chimeric fibronectin matrix mimetic as a functional growth- and migration-promoting adhesive substrate. Biomaterials 2011, 32:2077-2087.