Activation of the mechanosensitive ion channel PIEZO1/2 by YODA1 modulates cellular functions of human oa chondrocytes

Purpose: Piezo proteins represent large membrane proteins that form mechanically activated Ca2+-permeable non-selective cationic channels. As a mechanosensitive ion channel Piezo functions as a sensor for membrane tension and transduces these forces into biological effects. The channel protein is further known to be critical for endothelial shear stress-sensing and vascular development. In articular cartilage, chondrocytes react to mechanical stimulation, while mechanical stress can affect the chondrocyte phenotype by altering the expression of chondrocyte extracellular matrix. Therefore, mechanical forces that act on articular cartilage induce chondrocytes to produce OA-related catabolic factors; excessive stress can lead to loss of articular cartilage (1). It has been demonstrated, that in primary articular chondrocytes the mechanosenstive Piezo1/2 channel plays its role in mechanically induced changes in intracellular Ca2+. Inhibition of Piezo obviously protects cells from mechanically induced cell death (2). On the other hand, the transient receptor potential vanilloid 4 channel (TRPV4), a Ca2+-permeable osmomechano-TRP channel, is highly expressed in articular chondrocytes, and loss of TRPV4 function is associated with joint arthropathy and osteoarthritis. Agonist based activation of TRPV4 induced anabolic growth factor gene expression and inhibited proinflammatory factors. Regarding mechanical loading it has been postulated that the TRPV4 ion channel mediates low-strain mechanical loading while Piezo1/2 responses to high-strain mechanical stress. Based on these findings, it seems of great interest what activation of Piezo1/2 means for OA chondrocytes. Therefore, within this study activation of Piezo1/2 by Yoda has been implemented. Yoda1 represents a small molecule and induces activation or sensitization to membrane tension and seems particularly useable to study the regulation and function of Piezo1 (3). Since findings concerning Piezo and its involvement in the development of osteoarthritis (OA) are rare, the purpose of this study is to identify the role of Piezo in chondrocytes from OA patients and to find out, whether this ion channel and/or connected signaling can function as therapeutic target. Methods: Expression of Piezo1/2 in human OA chondrocytes was verified by immunofluorescence and qPCR, a dose response for Yoda1 was evaluated by MTT assay. Primary chondrocytes from OA patients were loaded with Fura-2 and changes in the intracellular Ca2+ concentration by Yoda1 at the concentrations of 0,1 μM, 0,3 μM, 1 μM and 3 μM were measured. To differentiate between Ca2+ influx/release cells were treated in the presence or absence of extracellular Ca2+ or SERCA was blocked by preincubation with thapsigargin. RNA isolated from chondrocytes treated with Yoda for 48h was used for cDNA synthesis and qPCR . Results: Primary human chondrocytes from OA patients responded to Yoda1 treatment with an increase in [Ca2+]I, being moderate at low massive at higher Yoda concentrations. Cells pretreated with thapsigargin still responded to perfusion with Yoda1 while depletion of Ca2+ from the bath attenuated Yoda1 evoked Ca2+ response. Treatment of cells with Yoda1 for 48 h changed the expression profile concerning MMPs, ILs and BMPs. While IL6, IL8 and IL-1β was upregulated, Yoda1 reduced MMP1, 3 and 13 expression. BMP2 was augmented while BMP7 and P2X7 were up regulated. Conclusions: Piezo1/2 is heavily expressed in OA chondrocytes and activation by Yoda1 induces Ca2+ influx rather than a release. The lowering of the MMP expression points to a chondroprotective effect whereas the observed increase in IL6, IL8 and IL-1ß indicates the opposite. Further studies are planned to clarify whether the activation of Piezo1/2 might be beneficial rather than risky for OA chondrocytes. Only then it will be possible to define this mechanosensitive ion channel as a potential therapeutic target. 1 Buckwalter et la.: “The Roles of Mechanical Stresses in the Pathogenesis of Osteoarthritis”, Cartilage, 2013, 4(4): 286-294. 2 Lee et al.: “Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosnsitivity to articular cartilage”, PNAS, 2014, E5114-E5122. 3 Syeda et al.:”Chemical activation of the mechanotransduction channel Piezo1”, eLIFE, 2015, 1-11.