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

Abstract

Objective: Enhancement of β-cell proliferation plays an important role in maintaining β-cell mass and function, and in improving pancreatic β-cell survival before transplantation. Extracellular matrix (ECM) components increase the adhesion and proliferation of β-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 β-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 β-cell proliferation by REP was associated with increased cyclin D1, cyclin D2 and cdk6, and decreased p27 levels. When β-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 β-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 β-cell adhesion and proliferation.

Keywords: Biomedical materials; Biotechnology; Cell biology; Cell cycle; Diabetes; Insulin; RGD-modified elastin-like polypeptide; Tissue engineering; β-cell adhesion; β-cell proliferation.

Figure 1

The effect of REP on β-cell adhesion and proliferation. Rin-m cells were cultured on non-coated (control, Con), 1 μM ELP- or REP- coated plate for 24 h. (A) The crystal violet assay was performed to assess cell adhesion; images are represented. (Bright field microscopy Image, ×200) ∗P < 0.01 compared with control, ∗∗P < 0.05 compared with ELP. (B) The CCK-8 assay was performed to assess the effect of REP on cell viability. ∗P < 0.01 compared with control, ∗∗P < 0.01 compared with ELP. (C) BrdU incorporation assay was investigated to evaluate the effect of REP on cell proliferation. ∗P < 0.001 compared with control, ∗∗P < 0.01 compared with ELP. All data are expressed as the mean ± SEM of three independent measurements.

Figure 2

The effect of REP on cell cycle progression in β-cells. Rin-m cells were cultured on 1 μM ELP or REP for 24h. (A) Flow cytometry analysis of cell cycle and cell distribution. (B) Western blot analysis of the protein expression of cyclin D1, cyclin D2, cdk6, p21, and p27 in Rin-m cells. Quantification of western blot analysis. ∗P < 0.05 and ∗∗P < 0.01 compared with ELP. Data are expressed as the mean ± SEM of three independent measurements. β-actin levels were 26 analyzed as an internal control.

Figure 3

The effect of REP on integrin mediated intracellular signaling. Rin-m cells were cultured on non-coated (control, Con), 1 μM ELP- or REP- coated plates for 24 h. (A) Western blot analysis of the protein expression of integrin β1, α3, and α5. (B) Western blot analysis of the phosphorylation of Fak, Erk and Akt. (C–H) Quantification of western blot analysis. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001 compared with control. #P < 0.05, ##P < 0.01 and $P < 0.001 compared with ELP. Data are expressed as the mean ± SEM of three independent measurements. β-actin levels were analyzed as an internal control.

Figure 4

Involvement of the Erk and Akt/PkB pathways in REP-induced β-cell attachment. Rin-m cells were cultured with or without indicated concentration of PD98059 (a specific Mek inhibitor) or wortmannin (PI3-kinase inhibitor) on a 1 μM REP-coated plate for 24 h. (A) Evaluation of the effects of 50 μM PD98059 or 100 nM wortmannin on REP-induced cell adhesion via crystal violet staining. (Bright field microscopy Image, ×200). Quantitative comparison of REP-induced cell adhesion treated with or without 50 μM PD98059 and 100 nM wortmannin after crystal violet staining. Data are expressed as the mean ± SEM of three independent measurements. ∗P < 0.05 and ∗∗P < 0.01 compared with REP (-). #P < 0.05 compared with REP (+). (B) CCK-8 assay showing the effects of 50 μM PD98059 or 100 nM wortmannin on REP-induced cell survival. ∗P < 0.01 and ∗∗P < 0.001 compared with REP (-). #P < 0.01 compared with REP (+). All data are expressed as the mean ± SEM of three independent measurements.

Figure 5

Involvement of the Erk pathway in REP-induced cell cycle protein regulation in β-cells. Rin-m cells were cultured with or without 50 μM PD98059 (a specific Mek inhibitor) on a 1 μM REP-coated plate for 24 h. (A) Western blot analysis of cyclin D1, cyclin D2, CDK6, p27, p-Erk and t-Erk in Rin-m cells on REP with or without Erk inhibitor. (B–F) Quantification of western blot analysis. Data are expressed as the mean ± SEM of three independent measurements. β-actin levels were analyzed as an internal control. (B–E) ∗P < 0.01 and ∗∗P < 0.05 compared with REP(-)/PD(-), #P < 0.01 compared with REP(+)/PD(-). (F) ∗P < 0.01 compared with REP(-)/PD(-), ∗∗P < 0.01 compared with REP(+)/PD(-).

Figure 6

Involvement of the Akt pathways in REP-induced cell cycle proteins in β-cell. Rin-m cells were cultured with or without 100 nM wortmannin (PI3-kinase inhibitor) on a 1 μM REP-coated plate for 24 h. (A) Western blot analysis of cyclin D1, cyclin D2, CDK6, p27, p-Akt and t-Akt in Rin-m cells on REP with or without Akt inhibitor. (B–F) Quantification of western blot analysis. Data are expressed as the mean ± SEM of three independent measurements. β-actin levels were analyzed as an internal control. (B–E) ∗P < 0.001 and ∗∗P < 0.01 compared with REP(-)/PD(-), #P < 0.01 and ##P < 0.05 compared with REP(+)/PD(-). (F) ∗P < 0.05 compared with REP(-)/PD(-), ∗∗P < 0.01 compared with REP(+)/PD(-).

Figure 7

The effect of REP on the expression of insulin and β-cell-enriched transcription factors in β-cells. Rin-m cells were cultured on 1 μM REP for 24 h (A) RT-qPCR of the mRNA expression of Insulin-1and Insulin-2 in Rin-m cells. GAPDH mRNA was used as an internal control. ∗P < 0.01 compared with REP (-). (B) Quantification of GSIS of rat islets cultured on REP-coated plate. ∗P < 0.05 compared with 3mM D-glucose. ∗∗P < 0.05 compared with REP (-). (C) RT-qPCR of the mRNA expression of PDX-1, and BETA 2 in Rin-m cells. GAPDH mRNA was used as an internal control. ∗P < 0.05 compared with REP (-). (D) Western blot analysis of the protein expression of PDX-1 and BETA 2 in Rin-m cells. β-actin levels were analyzed as an internal control. ∗P < 0.01 compared with REP (-). All data are expressed as the mean ± SEM of three independent measurements.