CONTRIBUTION OF PIEZO1 IN ECM STIFFNESS INCITED EPITHELIAL CELL REMODELING

Cells are sensitive to mechanical cues from the ECM such as, stiffness, topography, and roughness. Tissue stiffening due to ageing or diseases has been shown to affect cell proliferation, differentiation, migration, and apoptosis, but the mechanosensors that respond to ECM stiffness changes have not yet been fully understood. Recent studies on mechanosensitive Piezo1 channels have shown that these channels are sensitive to ECM cues, such as mechanical confinements implemented by micropatterns. We hypothesize that Piezo1 could serve as a force sensor for local ECM stiffness. Using a '2-well PDMS chip' consisting of substrates of different stiffness, namely, hard (~1000 kPa) and soft (~0.1 kPa), we studied the role of Piezo1 on stiffness-dependent morphology changes in epithelial cells. The results show that cells respond to substrate stiffness variations with profound cytoskeletal reorganization and moderate changes in their spreading area and shape. Cells on the hard substrates were only ~20% larger than that on soft substrates within the same culture period of 2.5 hrs and at a similar confluency. However, cells on hard substrates show abundant F-actin bundles that are reorganized to peripheral actin rings on soft substrates. Inhibition of Piezo1 with GsMTx4 or Gd³⁺, largely reduced the formation of thick actin bundles on hard substrates. Activation of Piezo1 with specific agonist, Yoda1, enhanced the formation of actin bundles. These results indicate that the epithelial cells' response to ECM stiffness is mediated by Piezo1 through its function of permeating Ca²⁺ ions. This study demonstrates that Piezo1 could be an ECM mechanosensor in epithelial cells.