Elevated BMP and Mechanical Signaling Through YAP1/RhoA Poises FOP Mesenchymal Progenitors for Osteogenesis

Abstract

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by the formation of extraskeletal bone, or heterotopic ossification (HO), in soft connective tissues such as skeletal muscle. All familial and sporadic cases with a classic clinical presentation of FOP carry a gain-of-function mutation (R206H; c.617 G > A) in ACVR1, a cell surface receptor that mediates bone morphogenetic protein (BMP) signaling. The BMP signaling pathway is recognized for its chondro/osteogenic-induction potential, and HO in FOP patients forms ectopic but qualitatively normal endochondral bone tissue through misdirected cell fate decisions by tissue-resident mesenchymal stem cells. In addition to biochemical ligand-receptor signaling, mechanical cues from the physical environment are transduced to activate intracellular signaling, a process known as mechanotransduction, and can influence cell fates. Utilizing an established mesenchymal stem cell model of mouse embryonic fibroblasts (MEFs) from the Acvr1 R206H/+ mouse model that mimics the human disease, we demonstrated that activation of the mechanotransductive effectors Rho/ROCK and YAP1 are increased in Acvr1 R206H/+ cells. We show that on softer substrates, a condition associated with low mechanical signaling, the morphology of Acvr1 R206H/+ cells is similar to the morphology of control Acvr1 +/+ cells on stiffer substrates, a condition that activates mechanotransduction. We further determined that Acvr1 R206H/+ cells are poised for osteogenic differentiation, expressing increased levels of chondro/osteogenic markers compared with Acvr1 +/+ cells. We also identified increased YAP1 nuclear localization in Acvr1 R206H/+ cells, which can be rescued by either BMP inhibition or Rho antagonism. Our results establish 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 Acvr1 R206H/+ progenitor cells to chondro/osteogenic lineages. Generation of mouse embryonic fibroblasts and cell cultureKnock-in Acvr1 R206H/+ MEFs were isolated at embryonic day 13.5 (E13.5) as previously described (43) from mice described in Chakkalakal and colleagues. (41) Genotypes of cell lines (MEFs) and presence of the R206H mutation were confirmed by PCR (primers in Supplemental Table S1). Acvr1 +/+ controls were littermates that did not contain an Acvr1 R206H allele. Cells were cultured in Dulbecco's modified Eagle's medium, high glucose (DMEM, Gibco, Thermo Fisher Scientific,

ACVR1R206Hincreases osteogenic/ECM gene expression and impairs myofiber formation in human skeletal muscle stem cells

“…However, FOP iMPCs showed increased chondrogenic/osteogenic and ECM gene expression, a feature seen in other bone-related cell types in FOP (Barruet et al, 2016;Culbert et al, 2014;Lees-Shepard et al, 2018). Thus, FOP Hu-MuSCs may contribute to HO formation indirectly by modulation of the osteogenic environment such as contributing to changes in muscle stiffness, as previously reported in mice (Stanley, Heo, Mauck, Mourkioti, & Shore, 2019). In addition, while our results suggest no major differences or possibly a slight increase in the ability to form skeletal muscle progenitors, activated ACVR1 by the R206H mutation decreased (but did not abrogate) in vitro formation of mature myoblasts and in vivo muscle repair after transplant.…”

Gnas Inactivation Alters Subcutaneous Tissues in Progression to Heterotopic Ossification

“…Recent studies in mouse models of fibrodysplasia ossificans progressiva (FOP), another rare genetic disorder of HO, have established a precedent for key roles of the tissue microenvironment and biomechanical signaling in supporting and promoting HO ( Haupt et al, 2019 ; Stanley et al, 2019 ). The FOP Acvr1 R206H mutation was shown to alter the physical properties of the tissue where HO will form, producing more collagens and exhibiting increased tissue stiffness.…”

“…The FOP Acvr1 R206H mutation was shown to alter the physical properties of the tissue where HO will form, producing more collagens and exhibiting increased tissue stiffness. Further, Acvr1 R206H progenitor cells show aberrant upregulation of both RhoA and YAP/TAZ biomechanical signaling pathways, which are associated with osteogenic differentiation ( Haupt et al, 2019 ; Stanley et al, 2019 ).…”

Expression of HIF‑1α in cycling stretch‑induced osteogenic differentiation of bone mesenchymal stem cells

“…Numerous studies have examined the mechanism of mechanical stimuli. One focus in this area of research is regulating the osteogenic differentiation of stem cells (31). However, less attention has been dedicated to the consequences of cyclic equiaxial stretch (32).…”