Kim Tae-Jin, Seong Jihye, Ouyang Mingxing, Sun Jie, Lu Shaoying, Hong Jun Pyu, Wang Ning, Wang Yingxiao
Neuroscience Program, Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign, Urbana, Illinois, USA.
J Cell Physiol. 2009 Feb;218(2):285-93. doi: 10.1002/jcp.21598.
Substrate rigidity plays crucial roles in regulating cellular functions, such as cell spreading, traction forces, and stem cell differentiation. However, it is not clear how substrate rigidity influences early cell signaling events such as calcium in living cells. Using highly sensitive Ca(2+) biosensors based on fluorescence resonance energy transfer (FRET), we investigated the molecular mechanism by which substrate rigidity affects calcium signaling in human mesenchymal stem cells (HMSCs). Spontaneous Ca(2+) oscillations were observed inside the cytoplasm and the endoplasmic reticulum (ER) using the FRET biosensors targeted at subcellular locations in cells plated on rigid dishes. Lowering the substrate stiffness to 1 kPa significantly inhibited both the magnitudes and frequencies of the cytoplasmic Ca(2+) oscillation in comparison to stiffer or rigid substrate. This Ca(2+) oscillation was shown to be dependent on ROCK, a downstream effector molecule of RhoA, but independent of actin filaments, microtubules, myosin light chain kinase, or myosin activity. Lysophosphatidic acid, which activates RhoA, also inhibited the frequency of the Ca(2+) oscillation. Consistently, either a constitutive active mutant of RhoA (RhoA-V14) or a dominant negative mutant of RhoA (RhoA-N19) inhibited the Ca(2+) oscillation. Further experiments revealed that HMSCs cultured on gels with low elastic moduli displayed low RhoA activities. Therefore, our results demonstrate that RhoA and its downstream molecule ROCK may mediate the substrate rigidity-regulated Ca(2+) oscillation, which determines the physiological functions of HMSCs.
底物刚度在调节细胞功能中起着关键作用，如细胞铺展、牵引力和干细胞分化。然而，尚不清楚底物刚度如何影响活细胞中的早期细胞信号事件，如钙信号。我们使用基于荧光共振能量转移（FRET）的高灵敏度Ca(2+)生物传感器，研究了底物刚度影响人间充质干细胞（HMSC）钙信号的分子机制。使用靶向铺在刚性培养皿上细胞亚细胞位置的FRET生物传感器，在细胞质和内质网（ER）内观察到自发的Ca(2+)振荡。与较硬或刚性底物相比，将底物刚度降低到1 kPa显著抑制了细胞质Ca(2+)振荡的幅度和频率。这种Ca(2+)振荡显示依赖于RhoA的下游效应分子ROCK，但不依赖于肌动蛋白丝、微管、肌球蛋白轻链激酶或肌球蛋白活性。激活RhoA的溶血磷脂酸也抑制了Ca(2+)振荡的频率。一致地，RhoA的组成型活性突变体（RhoA-V14）或RhoA的显性负突变体（RhoA-N19）均抑制了Ca(2+)振荡。进一步的实验表明，在低弹性模量凝胶上培养的HMSC显示出低RhoA活性。因此，我们的结果表明，RhoA及其下游分子ROCK可能介导底物刚度调节的Ca(2+)振荡，这决定了HMSC的生理功能。
