Hwang Yeo Jin, Jung Gwon-Soo, Jeon Won Bae, Lee Kyeong-Min
Division of Electronics & Information System, Daegu Gyeongbuk Institute of Science and Technology, Daegu, Republic of Korea.
New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Republic of Korea.
Heliyon. 2020 Sep 14;6(9):e04918. doi: 10.1016/j.heliyon.2020.e04918. eCollection 2020 Sep.
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.
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.
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.
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.
增强β细胞增殖在维持β细胞质量和功能以及提高移植前胰腺β细胞存活率方面发挥着重要作用。细胞外基质（ECM）成分可增加β细胞的黏附与增殖，而RGD修饰的弹性蛋白样多肽（RGD-ELP，REP）被描述为一种生物活性基质。在本研究中，我们调查了REP是否能增强β细胞的黏附与增殖，并阐明其中涉及的信号通路。
我们通过使用Rin-m细胞和大鼠胰岛的实验，研究了REP对细胞黏附、增殖和胰岛素分泌的影响。进行了结晶紫、CCK-8、BrdU检测、流式细胞术、蛋白质免疫印迹、实时定量PCR分析和胰岛素酶联免疫吸附测定。为解释相关机制，检测了Akt和细胞外信号调节激酶（Erk）的磷酸化情况。
与不含RGD基序的弹性蛋白样多肽（ELP）相比，REP更能增加Rin-m细胞的黏附、增殖和存活率。REP对β细胞增殖的增强作用与细胞周期蛋白D1、细胞周期蛋白D2和细胞周期蛋白依赖性激酶6（cdk6）增加以及p27水平降低有关。当β细胞在REP上培养时，Erk以及磷脂酰肌醇3激酶（PI3激酶）的下游效应物Akt受到刺激。用Erk通路抑制剂和PI3激酶抑制剂处理可降低REP诱导的β细胞黏附和增殖，并调节REP诱导的细胞周期蛋白。此外，REP增加了胰岛素及其转录因子PDX-1的mRNA和蛋白水平以及胰岛素分泌。
我们的结果表明，REP上调PI3K/Akt和Erk信号通路以及调节细胞周期蛋白可作为改善胰腺β细胞黏附和增殖的有效策略。
