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Arg-Gly-Asp-modified elastin-like polypeptide regulates cell proliferation and cell cycle proteins via the phosphorylation of Erk and Akt in pancreatic ?-cell.

ABSTRACT: Objective Enhancement of ?-cell proliferation plays an important role in maintaining ?-cell mass and function, and in improving pancreatic ?-cell survival before transplantation. Extracellular matrix (ECM) components increase the adhesion and proliferation of ?-cells, and the RGD-modified elastin-like polypeptide (RGD-ELP, REP) has been described as a bioactive matrix. In this study, we investigated whether REP could enhance ?-cell adhesion and proliferation and elucidated the signaling pathways involved. Methods We investigated the effect of REP on cell adhesion, proliferation and insulin secretion via assays using Rin-m and rat islets. Crystal violet, CCK-8, and BrdU assay, FACS, western blot, real time q-PCR analyses and insulin ELISA were examined. To explain the associated mechanisms, phosphorylation of Akt and extracellular signal-regulated kinase (Erk) were measured. Results REP more increased the adhesion, proliferation and survival of Rin-m cells compared to elastin-like poly peptide (ELP) without RGD-motif. The enhancement of ?-cell proliferation by REP was associated with increased cyclin D1, cyclin D2 and cdk6, and decreased p27 levels. When ?-cells were cultured on REP, Erk and the phosphatidylinositol 3-kinase (PI3-kinase) downstream effector, Akt was stimulated. Treatment with the Erk pathway inhibitor and PI3-kinase inhibitor decreased REP-induced ?-cell adhesion and proliferation, and regulated REP-induced cell cycle proteins. Additionally, REP increased the mRNA and protein levels of insulin and its transcription factor, PDX-1, and insulin secretion. Conclusions Our results demonstrate that the up-regulation of the PI3K/Akt and Erk signaling pathways and the regulation of cell cycle proteins by REP could serve as effective strategies for improving pancreatic ?-cell adhesion and proliferation. Tissue engineering; Biomedical materials; Cell biology; Biotechnology; Diabetes; Insulin; -cell adhesion; -cell proliferation; RGD-modified elastin-like polypeptide; Cell cycle

SUBMITTER: Hwang Y

PROVIDER: S-EPMC7501433 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:PurposeAdvanced glycation endproduct (AGE) formation on the basement membrane of retinal capillaries has been previously described but the impact of these adducts on capillary endothelial cell function vascular repair remains uncertain. This investigation has evaluated retinal microvascular endothelial cells (RMECs) growing on AGE-modified fibronectin (FN) and determined how this has an impact on cell-substrate interactions and downstream oxidative responses and cell survival.MethodsRMECs were grown on methylglyoxal-modified FN (AGE-FN) or native FN as a control. RMEC attachment and spreading was quantified. In a separate treatment, the AGE-FN substrate had Arg-Gly-Asp-Ser (RGDS) or scrambled peptide added before seeding. Phosphorylation of focal adhesion kinase (FAK) and alpha5beta1 integrin localization was assessed and apoptosis evaluated. In a subset of RMECs that remained attached to the AGE-FN substrate, the production of superoxide (O(2) (-)) was assayed using dihydroethidium (DHE) fluorescence or lucigenin, in the presence or absence of NADPH. The specificity of the O(2) (-) assays was confirmed by inhibition in the presence of polyethylene-glycol-superoxide dismutase (PEG-SOD). AGE-mediated changes to mRNAs encoding key basement membrane proteins and regulatory enzymes were investigated using real-time RT-PCR.ResultsAGE-FN reduced RMEC attachment and spreading when compared to FN controls (p<0.001). RGDS peptide enhanced cell attachment on AGE-FN (p<0.001), while the scrambled peptide had no effect. FAK phosphorylation in AGE-exposed RMECs was reduced in a time-dependent fashion, while alpha5beta1 integrin-immunoreactivity became focal at the basal membrane. AGE-exposure induced apoptosis, a response significantly prevented by RGDS peptide. AGE-exposure caused a significant increase in basal O(2) (-) and NADPH-stimulated production by RMECs (p<0.01), while AGE-FN also increased basement membrane associated mRNA expression (p<0.05).ConclusionsAGE substrate modifications impair the function of retinal capillary endothelium and their reparative potential in response to diabetes-related insults. Arginine-specific modifications alter vital endothelial cell interactions with the substrate. This phenomenon could play an important role in dysfunction and nonperfusion of retinal capillaries during diabetes.

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Arg-Gly-Asp-modified elastin-like polypeptide regulates cell proliferation and cell cycle proteins via the phosphorylation of Erk and Akt in pancreatic ?-cell.

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