UC San Diego

Human mesenchymal stem cell (hMSC) proliferation, migration, and differentiation have all been linked to extracellular matrix stiffness, yet the signaling pathway(s) that are necessary for mechanotransduction remain unproven. Vinculin has been implicated as a mechanosensor in vitro, but here we demonstrate its ability to also regulate stem cell behavior, including hMSC differentiation. RNA interference-mediated vinculin knockdown significantly decreased stiffness-induced MyoD, a muscle transcription factor, but not Runx2, an osteoblast transcription factor, and impaired stiffness- mediated migration. A kinase binding accessibility screen predicted a cryptic MAPK1 signaling site in vinculin that could regulate these behaviors. Indeed, reintroduction of vinculin domains into knocked-down cells indicated that MAPK1 binding site-containing vinculin constructs were necessary for hMSC expression of MyoD. Vinculin knockdown does not appear to interfere with focal adhesion assembly, significantly alter adhesive properties, or diminish cell traction force generation, indicating that its knockdown only adversely affected MAPK1 signaling. These data provide some of the first evidence that a force-sensitive adhesion protein can regulate stem cell fate. We build on this research by analyzing 47 different focal adhesion proteins for cryptic MAPK1 binding sites similar to that found in vinculin. Using this parameter we selected 6 candidate focal adhesion proteins for further study in a high content imaging and analysis system in which cells were treated with siRNA, plated onto a 96 well plate containing two dimensional polyacrylamide surfaces, and stained for osteogenic and myogenic differentiation markers. This is the first high throughput system specifically built to analyze stem cell differentiation as a function of substrate stiffness and the first time an siRNA screen has been applied to stem cells for the purpose of studying substrate stiffness mediated mechanotransduction