ACVR1R206H FOP mutation alters mechanosensing and tissue stiffness during heterotopic ossification

Abstract

An activating bone morphogenetic proteins (BMP) type I receptor ACVR1 (ACVR1^R206H) mutation enhances BMP pathway signaling and causes the rare genetic disorder of heterotopic (extraskeletal) bone formation fibrodysplasia ossificans progressiva. Heterotopic ossification frequently occurs following injury as cells aberrantly differentiate during tissue repair. Biomechanical signals from the tissue microenvironment and cellular responses to these physical cues, such as stiffness and rigidity, are important determinants of cell differentiation and are modulated by BMP signaling. We used an Acvr1^R206H/+ mouse model of injury-induced heterotopic ossification to examine the fibroproliferative tissue preceding heterotopic bone and identified pathologic stiffening at this stage of repair. In response to microenvironment stiffness, in vitro assays showed that Acvr1^R206H/+ cells inappropriately sense their environment, responding to soft substrates with a spread morphology similar to wild-type cells on stiff substrates and to cells undergoing osteoblastogenesis. Increased activation of RhoA and its downstream effectors demonstrated increased mechanosignaling. Nuclear localization of the pro-osteoblastic factor RUNX2 on soft and stiff substrates suggests a predisposition to this cell fate. Our data support that increased BMP signaling in Acvr1^R206H/+ cells alters the tissue microenvironment and results in misinterpretation of the tissue microenvironment through altered sensitivity to mechanical stimuli that lowers the threshold for commitment to chondro/osteogenic lineages.

FIGURE 1:

Increased fibroproliferative tissue stiffness in response to skeletal muscle injury in Acvr1^R206H/+ mice. (A) Timeline of experimental procedure. The Acvr1^R206H mutation was expressed in conditional Acvr1^R206H/+ mice through doxycycline treatment 3 d prior to injection with cardiotoxin or PBS (uninjured control). Littermate controls were treated equivalently. (B) H&E staining of sections from PBS-injected or CTX-injured quadriceps showing areas of healthy muscle and fibroproliferation (arrow) 4 d post–injection of FOP mice or littermate controls. Scale bar represents 100 µm. (C) Enlarged images from insets in B. Scale bar: 50 µm. (D) Tissue stiffness was measured via AFM. Consecutive sections demonstrate increased rigidity of fibroproliferative areas (FP) in FOP lesions compared with healthy muscle (M). Graph represents mean ± SEM for N = 5–18 (in M: 5 [control] and 6 [FOP]; in FP: 10 [control] and 18 [FOP]) locations measured across three independently injured limbs. Significance was determined by two-way ANOVA (Bonferroni post test); *p < 0.05.

FIGURE 2:

Altered collagen composition in Acvr1^R206H/+ cells. In vitro analyses of collagen deposition and gene expression were conducted in immortalized MEFs. (A) Sirius red/Fast green staining demonstrated increased collagen deposition in Acvr1^R206H/+ (FOP) cells compared with control (Acvr1^+/+). Fast green counterstain was used to normalize to total protein content after extraction. Graph represents mean ± SEM. Results from one representative experiment of three independent experiments are shown. (B) Collagen type I–III mRNA expression was quantified over time by RT-PCR. Data are relative to Acvr1^+/+ controls on day 1. Acvr1^R206H/+ cells showed increased collagen type II expression (day 1: n = 5; day 2: n = 3; day 3: n = 4; day 4: n = 4; day 7: n = 3). Graphs represent mean ± SEM. Significance was determined by two-tailed Student's t test; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3:

Altered ECM composition and organization in lesions from Acvr1^R206H/+ mice. (A, B) Tissues from cardiotoxin injected quadriceps 5 d post–injection of FOP mice or littermate controls were counterstained with (A) hematoxylin after collagen types I and III immunostaining or (B) DAPI after collagen type II detection. Acvr1^R206H/+ lesions did not show qualitative differences in collagen type I or III deposition during the fibroproliferative stage, but collagen type II was more highly detected (brown stain). Scale bar represents 100 μm. (C) Representative images from SHG (blue signal) were used to quantify differences in collagen organization (D) between FOP (n = 7) and control littermates (n = 8) at the fibroproliferative stage. Significance was determined by two-tailed Student's t test; *p = 0.02.

FIGURE 4:

Increased signaling through mechanosensing pathways in Acvr1^R206H/+ cells. (A) Increased activation of mechanotransduction in Acvr1^R206H/+ (FOP) cells was determined by immunoblot for Rho after pull down of the active form (n = 6). Active Rho protein was normalized to total Rho. (B, C) Detection of RhoA downstream targets cofilin by immunoblot for pCofilin (n = 5) and MLC2 by immunostaining for pMLC2 showed increased levels in Acvr1^R206H/+ cells. pCofilin was normalized to total cofilin protein. Quantification of mean intensity of pMLC2 staining from three independent experiments normalized to cell area (Acvr1^+/+ n = 61 Acvr1^R206H/+ n = 91). Scale bar represents 50 μm. Graphs represent mean ± SEM. Significance was determined by two-tailed Student's t test; *p < 0.05; ***p < 0.001.

FIGURE 5:

Increased nuclear stiffness and altered nuclear height in Acvr1^R206H/+ cells. (A) Schematic of nuclear stiffness measurement by AFM. (B) Quantification of nuclear stiffness determined by AFM indicates higher stiffness in Acvr1^R206H/+ cells compared with controls (Acvr1^+/+) (both n = 20). (C) Nuclear height in MEFs was determined by confocal microscopy, before (untreated) and after treatment with 10 μM Y-27632 (Y27), a ROCK inhibitor, for 1 h (n = 15–20 cells/group). Untreated Acvr1^R206H/+ cells show nuclear flattening (relative to control cells; i.e., negative value: Acvr1^+/+: n = 15; Acvr1^R206H/+: n = 20). The flattened morphology was rescued by reducing cellular contractility with the ROCK inhibitor Y-27632 (Acvr1^+/+: n = 18 Acvr1^R206H/+: n = 19). Scale bar represents 10 µm. Graphs represent mean ± SEM. Statistical significance was determined by two-tailed Student's t test, ***p < 0.001 (in B); or one-way ANOVA, *p < 0.05, ***p < 0.001; Bonferroni's post hoc (in C).

FIGURE 6:

Acvr1^R206H/+ cells misinterpret substrate elasticity. (A) Immortalized control and Acvr1^R206H/+ (FOP) cells on gels of various rigidities (5, 10, 15, and 55 kPa) were stained with phalloidin and DAPI. (B) Cell area and aspect ratio (AR) were measured as a function of matrix elasticity. Graphs represent mean ± SEM of >350 cells from four independent experiments. Significance was determined by two-way ANOVA (comparison between genotypes; Tukey–Kramer adjustment; ^#p < 0.05, ^###p < 0.001, NS = not significant) or one-way ANOVA (comparison between substrate stiffness; ^***p < 0.001; Bonferroni's post hoc). Scale bar represents 100 µm.

FIGURE 7:

RUNX2 nuclear localization is increased in FOP cells. (A) Immortalized Acvr1^+/+ control and Acvr1^R206H/+ cells on substrates of various rigidities (5, 10, 15, and 55 kPa) were detected for RUNX2 (green), phalloidin (red), and DAPI (blue). Nuclear localization of RUNX2 protein indicates activation of osteogenic cell fate programming. Scale bar represents 50 μm. (B) Relative nuclear localization of RUNX2 was quantified by the ratio of nuclear/cytoplasmic staining. Intensity shows that Acvr1^R206H/+ cells have more nuclear RUNX2 on softer substrates compared with Acvr1^+/+ control cells. Nuclear localization on stiffer substrates does not differ significantly between the genotypes, as expected. Graph represents mean ± SEM. Assay was repeated in three independent experiments (n = 50 cells per substrate stiffness per experiment). Statistical significance relative to Acvr1^+/+ controls at a given substrate stiffness (^**p < 0.01, ^***p < 0.001) or comparison between genotypes (^#p < 0.05, ^###p < 0.001) were determined by one-way ANOVA (Bonferroni's post hoc).