Professor University of Nebraska-Lincoln Lincoln, Nebraska, United States
Introduction:: Mechanical stimuli from extracellular matrix stiffness and dynamic mechanical loading are transduced into intracellular biochemical signals through mechanotransducers such as yes-associated protein (YAP). YAP is a transcriptional co-activator acting primarily through the translocation from cytoplasm to nucleus for its activation. Nuclear translocation of YAP is involved in the regulation of cell proliferation and growth, differentiation, and resultant tissue homeostasis. The linker of the nucleoskeleton and cytoskeleton (LINC) complex at the nuclear envelope, composed of Nesprin-SUN, plays a role to transmit forces from cytoskeletons to nucleus and may regulate mechanotransduction signaling. In this study, we have identified the contribution of the LINC complex as a regulator in YAP-mediated mesenchymal stem cell (MSC) behavior under cyclic mechanical stretching loading.
Materials and Methods:: To test the role of Nesprin, C3H10T1/2 murine MSCs were treated with Nesprin-siRNA for 72 h. Then, cells were stretched using a FX-5000 cell stretching device (Flexcell) for 1 h at 10% strain and 1 Hz frequency. For immunocytochemistry, cells were incubated overnight at 4°C with anti-YAP antibody (Santa Cruz, SC-101199) and then incubated with DyLight 488-conjugated secondary antibody for 1 h. The cells were also incubated with DAPI and phalloidin, and then observed under a confocal microscope (ZEISS 800).
Results, Conclusions, and Discussions:: We investigated the effect of Nesprin knockdown on YAP activation for MSCs under physiological level stretch loading. We observed that YAP was translocated to the nucleus in MSCs by stretch loading, which was abrogated by Nesprin-siRNA (Fig. 1A with percentage of cells showing YAP nuclear localization). This could be seen in the representative immunostaining image (Fig. 1B). We also observed that stretch loading-induced actin stress fiber (F-actin) formation is impaired by Nesprin-siRNA (Fig. 1B). Considering that F-actin plays a positive regulatory role in YAP nuclear import, our data suggest a mechanism that the LINC complex, e.g., Nesprin, regulates mechanical stretch-induced YAP nuclear transport via controlling F-actin development. Ultimately, the increase in YAP residency inside nucleus will enhance MSC fate decision to the osteogenic fate, as reported in the literature, suggesting the role of LINC in MSC YAP transcriptional mechanotransduction.
Acknowledgements (Optional): : We thank the funding support given to J.Y.L. from NIH/NIGMS COBRE NPOD Project Leader Grant (P20GM104320, PI: Zempleni) and University of Nebraska Collaboration Initiative (PI: Lim).
