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.

Keywords: ACVR1; BMP SIGNALING; CELLULAR CONTRACTILITY; FIBRODYSPLASIA OSSIFICANS PROGRESSIVA; MECHANOTRANSDUCTION; RHOA; YAP1.

Figure 1.. Acvr1^R206H/+ cells misinterpret substrate rigidity through increased BMP pathway signaling.

A) Response of Acvr1^+/+ and Acvr1^R206H/+ mouse embryonic fibroblasts (MEFs) to substrate stiffness corresponding to adipogenic, myogenic, and osteogenic tissues was tested using polyacrylamide (PA) hydrogels of 5, 10, and 55 kPa. Cells were seeded at low density and stained with phalloidin (green) and DAPI (blue). On soft (5 and 10kPa) substrates, Acvr1^R206H/+ cells showed a similar morphology to Acvr1^+/+ cells on stiffer (55kPa) substrates. B) Cells were treated with 50nM of the Acvr1 inhibitor LDN-193189 (LDN) for 2 hours, which notably decreased the spread morphology of Acvr1^R206H/+ cells on softer substrates. Scale bar= 100μm. C) Cell area, analyzed as a function of matrix elasticity and cell spreading, was quantified. Without LDN, cell area scaled with substrate rigidity in Acvr1^+/+ cells but not Acvr1^R206H/+ cells. This effect is ameliorated with the addition of LDN. Graph represents mean ± SEM from 4 independent experiments (>350 cells per experiment). Statistical significance determined by 1-way ANOVA; #p<0.0001.

Figure 2.. Acvr1 ^R206H/+ cells are poised for osteogenesis.

A) Cultures of Acvr1^+/+ and Acvr1^R206H/+ cells were maintained on 10kPa or 55kPa PA gels in basal media for 24 hours (as shown in the schematic above) and expression of adipogenic and chondro/osteogenic genes was quantified by RT-PCR. Adipogenic genes were expressed similarly in Acvr1^+/+ and Acvr1^R206H/+ cells, but expression of chondro/osteogenic genes were increased significantly in Acvr1^R206H/+ cells on both 10kPa and 55kPa (n=3 biologic samples run in triplicate). B) Cells were cultured on 10kPa or 55kPa PA gels in bipotential adipogenic:osteogenic media (1:1) for 72 hours (as shown in the lower schematic) and expression of adipogenic and chondro/osteogenic genes of Acvr1^+/+ and Acvr1^R206H/+ cells was quantified by RT-PCR. Expression levels of chondro/osteogenic genes were significantly higher in Acvr1^R206H/+ cells on both 10kPa and 55kPa (n=3 biologic samples run in triplicate). Data are relative to Acvr1^+/+. Graphs represent mean ± SEM. Significance determined by two-tailed Student’s t-test, *p<0.05, **p<0.01, #p<0.0001.

Figure 3.. Acvr1^R206H/+ cells show increased nuclear YAP localization.

A) Acvr1^R206H/+ cells have more nuclear YAP on softer substrates (10kPa) compared to Acvr1^+/+ cells. Nuclear YAP1 is shown alone in monochromatic (cyan) columns, and DAPI (blue), phallodin (green), and YAP1 (red) are shown together in multicolor columns. B) Cells treated with siYAP1 have decreased nuclear YAP1 localization. Scale bar= 50μm. C) Quantification of YAP1 nuclear/cytoplasmic ratio with or without siYAP1. Quantification of cell morphology parameters with or without siYAP1 D) cell area, E) circularity, and F) aspect ratio. Circularity indicates how close an object is to a perfect circle. Aspect ratio is the proportion of the width of an object to its height. Graphs represent mean ± SEM from 4 biologic experiments (>50 cells per experiment). Statistical significance determined by 1-way ANOVA, *p<0.05, ***p<0.001, #p<0.0001.

Figure 4.. Interaction between BMP signaling and YAP1 signaling pathways in Acvr1^R206H/+ cells.

A) Inhibition of BMP signaling with Acvr1 inhibitor LDN-193189 (LDN) in basal media decreased nuclear YAP1. Yellow dotted line outlines cell nucleus. Scale bar= 100μm. B) Quantification of nuclear/cytoplasmic YAP1 localization in the presence or absence of LDN. C) pYAP (cytoplasmic YAP1) protein was quantified in Acvr1^+/+ and Acvr1^R206H/+ cells on 10 and 55kPa substrates. Graph represents the mean from three biologic replicates ± SEM from three biologic replicates done in triplicate. D) Expression of YAP/TAZ mRNA and the YAP target gene Cyr61 in Acvr1^+/+ and Acvr1^R206H/+ cells on 10 and 55kPa substrates for 24 hours in basal media were quantified by RT-PCR. Expression of YAP1 and Cyr61 was significantly expressed in Acvr1^R206H/+ cells on 10 and 55kPa substrates. Graphs represent the mean ± SEM from three biologic replicates done in triplicate. Data are relative to Acvr1^+/+. E) Quantification of total YAP1 protein in Acvr1^+/+ and Acvr1^R206H/+ cells on 10 and 55kPa substrates. Graph represents the mean from three biologic replicates ± SEM. Statistical significance determined by 1-way ANOVA *p<0.01, **p<0.01, #p<0.0001.

Figure 5.. Cell contractility increases in Acvr1^R206H/+ cells.

A) Acvr1^+/+ and Acvr1^R206H/+ cells were cultured in basal media on 10 and 55kPa substrates and detected for paxillin (part of the focal adhesion complex). There were an increased number of focal adhesions in Acvr1^R206H/+ cells on 10 and 55kPa substrates. Cells were also treated with the B) ROCK antagonist Y272362 (Y27) or C) ROCK agonist CNO₃. Cell area as a function of substrate stiffness is decreased with Y27 addition (Bii) and increased with CNO₃ addition (Cii). Total number of focal adhesions decreased when Y27 was added (Biii) and increased with CNO₃ (Ciii). D) Cells treated with Y27 have decreased nuclear YAP1 localization. Scale bar= 100μm. Graphs represent the mean ± SEM (SD for focal adhesion number) of >50 cells analyzed from three biologic replicates. Statistical significance determined by 1-way ANOVA, **p<0.01, ***p<0.001, #p<0.0001.

Figure 6.. Acvr1^R206H/+ cells generate higher levels of contractile forces.

A) Representative traction stress vector maps for Acvr1^+/+ and Acvr1^R206H/+ cells on 10kPa substrates in basal media. Quantification shows average traction stress and average traction force per cell. Significance determined by two-tailed Student’s t-test, *p<0.05,***p<0.001. B) Representative traction stress vector maps for Acvr1^+/+ and Acvr1^R206H/+ cells on 10kPa substrates with the ROCK inhibitor Fasudil. Quantification shows relative average traction stress and average traction force per cell normalized to Acvr1^+/+ without Fasudil. Contractile forces were decreased in cells treated with Fasudil. Scale bar= 20μm. Statistical significance determined by 1-way ANOVA, **p<0.01, ***p<0.001, #p<0.0001 (n>15 cells/group, mean ± SD).

Figure 7.. Schematic for mechanotransduction and BMP signaling interactions in Acvr1^R206H/+ cells.

In Acvr1^+/+ progenitor cells on soft substrates, mechanotransduction activity is low, with lower expression of BMP target genes; on stiffer substrates, mechanotransduction activity is activated, leading to reinforcement of BMP and osteogenic gene signaling through RhoA and YAP/TAZ positive feedback. With the ACVR1^R206H mutation, cells on soft substrates have activated mechanotransduction (as indicated by red arrows), demonstrating cell mis-interpretation of substrate stiffness. Increased RhoA, YAP1, and BMP signaling converge to activate cellular contractility and chondro/osteogenic gene expression. Our data suggest that FOP Acvr1^R206H/+ cells are poised for chondro/osteogenesis, leading to endochondral ossification within skeletal muscle following injury. Asterisk (*) indicates location of the FOP ACVR1^R206H mutation.