Department of Mechanical Engineering, University of Colorado Boulder , Boulder, Colorado.
Tissue Eng Part A. 2018 Aug;24(15-16):1179-1189. doi: 10.1089/ten.tea.2017.0365. Epub 2018 Jul 3.
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.
理解在干细胞分化过程中生化试剂和物理刺激如何共同调控关键信号分子是至关重要的，但这往往是缺乏的。由于细胞外信号调节激酶 (ERK) 的重要作用，本研究检测了其时间动态变化，以确定机械化学线索对 ERK 磷酸化的共同调控在平滑肌细胞 (SMC) 分化中的作用。为了评估 ERK1/2 的活性，将一种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。纳米图案化基底、生长因子和药物对 ERK 活性的影响与它们对 SMC 分化的影响相关。结果表明，纳米图案化基底显著增加了细胞中的 ERK 活性，从而掩盖了生化因子对 ERK 反应的影响。纳米图案化基底在经过 48 小时的生化处理后，除了与 ERK 抑制剂 PD98059 联合处理外，显著降低了 SMC 标志物的表达，而后者增强了成熟 SMC 标志物 MYH11 的表达。对粘着斑蛋白、波形蛋白和纽蛋白的免疫荧光染色表明，粘着斑蛋白簇的分布或大小没有明显差异，而纽蛋白的位置从非图案化基底细胞边缘的粘着斑转移到纳米图案化基底上的肌动蛋白丝。纽蛋白强化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。总的来说，结果表明，物理刺激通过粘着斑变化在初始 ERK 信号和早期分化中起主导作用，并且能够实时监测信号事件的能力可被利用来指导细胞微环境的工程设计。
