Mechanochemical Effects on Extracellular Signal-Regulated Kinase Dynamics in Stem Cell Differentiation.
Mechanochemical Effects on Extracellular Signal-Regulated Kinase Dynamics in Stem Cell Differentiation.
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DOI：
10.1089/ten.tea.2017.0365
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发表时间：
2018-08
影响因子：
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通讯作者：
A. Dharmarajan;M. Floren;L. Cox;Yifu Ding;Richard Johnson;W. Tan
中科院分区：
文献类型：
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作者：
A. Dharmarajan;M. Floren;L. Cox;Yifu Ding;Richard Johnson;W. Tan
了解干细胞分化过程中生化因子和物理刺激如何共同调节关键信号分子是至关重要的，但往往缺乏。由于细胞外信号调节激酶（ERK）的重要作用，本研究研究了其时间动态，以确定机械化学线索对ERK磷酸化的协同调节，以促进平滑肌细胞（SMC）分化。为了评估ERK1/2活性，将基于荧光共振能量转移的生物传感器转染到间充质干细胞中。纳米化底物、生长因子和药物对ERK活性的影响与其对SMC分化的影响有关。结果显示，纳米型底物显著增加细胞中ERK的活性，超过了生化因子对ERK的反应。经48小时生化处理后，纳米化底物降低了SMC标志物的表达，但与ERK抑制剂PD98059联合处理提高了成熟SMC标志物MYH11的表达。免疫荧光染色结果显示，黏附蛋白vinculin和zyxin在黏附蛋白簇的分布和尺寸上无显著差异，而zyxin的位置从细胞外周在无图案底物上的黏附位点转变为纳米图案底物上的肌动蛋白丝。酵素增强的应力纤维可能增强了细胞骨架张力，从而增加了ERK动力学。综上所述，物理刺激通过局点黏附改变在ERK初始信号传导和早期分化中起主导作用，实时监测信号事件的能力可用于指导细胞微环境工程。
Understanding how key signaling molecules are coregulated by biochemical agents and physical stimuli during stem cell differentiation is critical but often lacking. Due to the important role of extracellular signal-regulated kinase (ERK), this study has examined its temporal dynamics to determine the coregulation of mechanochemical cues on ERK phosphorylation for smooth muscle cell (SMC) differentiation. To assess ERK1/2 activity, a fluorescence resonance energy transfer-based biosensor was transfected into mesenchymal stem cells. The influences of nanopatterned substrates, growth factors, and drugs on ERK activities were related to their effects on SMC differentiation. Results revealed that nanopatterned substrates significantly increased ERK activity in cells, overriding ERK response from administered biochemical factors. The nanopatterned substrates reduced expression of SMC markers after a 48-h biochemical treatment, except for the combination with ERK inhibitor PD98059 treatment, which enhanced expression of mature SMC marker MYH11. Immunofluorescent staining for focal adhesion proteins, vinculin and zyxin, indicated no significant differences in vinculin cluster distribution or dimension, while the location of zyxin changed from adhesion sites of cell periphery on nonpatterned substrate to actin filaments on nanopatterned substrate. The zyxin-reinforced stress fibers likely enhanced the cytoskeletal tension to increase ERK dynamics. Collectively, results suggest that physical stimuli play a dominating role in initial ERK signaling and early-stage differentiation through focal adhesion changes, and the capability of monitoring signaling events in real time could be exploited to guide the engineering of cell microenvironment.