Supplementary MaterialsSupp figS1

Supplementary MaterialsSupp figS1. a condition associated with low mechanical signaling, the morphology of cells is similar to the morphology of control cells on stiffer substrates, a condition that activates mechanotransduction. We further determined that cells are poised for osteogenic differentiation, expressing increased levels of chondro/osteogenic markers as compared to cells. We also identified increased YAP1 nuclear localization in cells, which can be rescued by either BMP inhibition or Rho antagonism. Our results establish Amyloid b-Peptide (10-20) (human) RhoA and YAP1 signaling as modulators of mechanotransduction in FOP and suggest that aberrant mechanical signals, combined with and as a result of the increased BMP pathway signaling through mutant ACVR1, lead to misinterpretation of the cellular microenvironment and a heightened sensitivity to mechanical stimuli that promotes commitment of progenitor cells to chondro/osteogenic lineages. mutation is influenced by disrupted mechanotransduction (8), but the specific mechanism as to how this contributes to the disease pathology of FOP is still not well understood. Here we demonstrate that YAP-associated protein (YAP1) signaling is a main contributing factor in this process. The YAP signaling pathway (18, 19) is regulated by ECM stiffness and cell geometry, and is a key regulator of cell differentiation (20C23). YAP, and its paralogue TAZ, are key factors directing MSC lineage commitment (24, 25). Phosphorylation of YAP promotes its cytoplasmic localization, preventing YAP-mediated transcriptional activation in the nucleus (20). Cytoplasmic YAP is associated with a soft surrounding ECM, cell cycle arrest, and adipogenic conditions, while translocation into the nucleus occurs in response to a stiffer ECM, proliferation, and osteogenic condition (20C23, 25). Another intracellular mechanotransductive Amyloid b-Peptide (10-20) (human) pathway, Rho GTPase, regulates downstream effectors such as Rho kinase (26), necessary for cell migration, adhesion, and differentiation (27). Rho signaling through ROCK stimulates actin polymerization, a vital part of cell contractility and cellular mechanotransduction (28). One of the Rho GTPases, RhoA, regulates ROCK to influence actin filament stability through myosin light chain (MLC) and cofilin (29, 30). Activation of RhoA in mesenchymal cells largely contributes to their chondro/osteogenic cellular identity (27, 31). Osteogenic conditions increase cell spreading, ECM production, BMP signaling, RhoA activation, and nuclear localization of YAP1(31C33). This suggests that elevated signaling by both YAP1 and BMP pathways could Amyloid b-Peptide (10-20) (human) coordinately promote the enhanced chondro/osteogenic differentiation that occurs in FOP. YAP1 responds to cell-cell contact and contractility signals mediated by Rho (34, 35), Rabbit Polyclonal to HEY2 suggesting an intersection between RhoA, YAP1, and BMP pathway signaling (35C37). Interestingly, basal activation of BMP signaling pathways, even in the absence of ligand, also regulates cell contractility in mesenchymal stem cells (38C40), further supporting that the FOP mutation could instigate aberrant mechano-signaling in FOP progenitor cells. In this study, we utilized mouse embryonic fibroblasts (MEFs) isolated from a knock-in mouse model (41, 42) that recapitulates the human disease progression to examine the YAP1 and Rho/ROCK mechano-signaling molecular pathways and investigate the ability of cells expressing the FOP Amyloid b-Peptide (10-20) (human) mutation to properly sense and respond to Amyloid b-Peptide (10-20) (human) the mechanical cues in their microenvironment. MEFs are used as an model system of mesenchymal stem cells (MSCs), including their ability to differentiate into adipogenic, chondrogenic, and osteogenic lineages (43). We previously showed increased BMP pathway signaling in FOP patient-derived stem cells from human exfoliated deciduous teeth (SHED cells) (44) and MEFs (43) as measured by phosphorylated Smad1/5/8 (pSmad1/5/8) protein levels in the presence or absence of BMP ligand. Thus, BMP pathway signaling is increased downstream due to enhanced activity of ACVR1. Our data support.

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