(F-217) Yes-associated protein (YAP) and piezo1 translocalization in fluid sheared MSCs

Introduction:: Yes-associated protein (YAP), a key mechanosensitive transcriptional co-activator, is an important downstream effector of the Hippo pathway. Through nuclear localization, YAP can be activated to regulate cell growth or apoptosis, proliferation, differentiation, and organ size development. Recent studies indicate that mesenchymal stem cell (MSC) fate decision is affected by dynamic mechanical loading through the activation of intracellular signaling molecules such as YAP. In addition, piezo1 is a mechanically sensitive ion channel with great influence over cell environmental sensing and mechanotransduction. Connecting these two processes which both have strong mechanically dependent linkages may be important for understanding flow shear-induced MSC differentiation. We here explore the translocalization of YAP and piezo1 in response to fluid shear.

Materials and Methods:: C3H10T1/2 murine MSCs were grown in DMEM with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were seeded on glass slides and transferred to serum starved (0.1% FBS) overnight before conducting flow experiments. Glass slides were placed in a Flexcell Streamer flow chamber and flowed at 10 dyne/cm² for 15 min, 30 min, 1 h, and 2 h, and compared to a chamber control without flow. Samples were then stained for DAPI, F-actin, and either YAP or piezo1. Immunofluorescent images were obtained with a Zeiss LSM 900 confocal microscope.

Results, Conclusions, and Discussions:: Nuclear YAP translocation was significantly increased by fluid flow in a time-dependent manner. YAP nuclear translocation peaked at 1 h of flow and decreased afterwards (Fig. 1A). Also, fluid shear greatly increased piezo1 localization to the cell membrane (Fig. 1B). These changes were accompanied by F-actin cytoskeletal remodeling with increased stressed fiber density and organization after 1 and 2 h of flow. Connecting these two fundamental processes, which are both correlated with intracellular actin stress fiber formation, may provide mechanistic data for further stem cell differentiation studies. In conclusion, fluid shear increased nuclear localization of YAP and plasma membrane localization of peizo1 in MSCs. Further understanding the crosstalk in these two pathways including the intermediate signaling cascades, such as F-actin, may provide crucial knowledge on mechanically induced MSC fate decision.

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).

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