Substrate rigidity regulates Ca2+ oscillation via RhoA pathway in stem cells

作者：Tae‐Jin Kim, Jihye Seong, Mingxing Ouyang, Jie Sun, Shaoying Lu, Jun Pyu Hong, Ning Wang, Yingxiao Wang · 发表于：Journal of Cellular Physiology · 年份：2008 · DOI：10.1002/jcp.21598 · 被引用次数：142 · 研究领域：Cellular Mechanics and Interactions、Microtubule and mitosis dynamics、Cardiomyopathy and Myosin Studies

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 downstrea...