Heterotopic Ossification In Mice Research Articles

ANGPTL4 binds to the leptin receptor to regulate ectopic bone formation.

Leptin protein was thought to be unique to leptin receptor (LepR), but the phenotypes of mice with mutation in LepR [db/db (diabetes)] and leptin [ob/ob (obese)] are not identical, and the cause remains unclear. Here, we show that db/db, but not ob/ob, mice had defect in tenotomy-induced heterotopic ossification (HO), implicating alternative ligand(s) for LepR might be involved. Ligand screening revealed that ANGPTL4 (angiopoietin-like protein 4), a stress and fasting-induced factor, was elicited from brown adipose tissue after tenotomy, bound to LepR on PRRX1+ mesenchymal cells at the HO site, thus promotes chondrogenesis and HO development. Disruption of LepR in PRRX1+ cells, or lineage ablation of LepR+ cells, or deletion of ANGPTL4 impeded chondrogenesis and HO in mice. Together, these findings identify ANGPTL4 as a ligand for LepR to regulate the formation of acquired HO.

Suppression of Overactive Insulin-Like Growth Factor 1 Attenuates Trauma-Induced Heterotopic Ossification in Mice

Heterotopic ossification (HO) is the ectopic bone formation in soft tissues. Aside from hereditary HO, traumatic HO is common after orthopedic surgery, combat-related injuries, severe burns, or neurologic injuries. Recently, mTOR has been demonstrated to be involved in the chondrogenic and osteogenic processes of HO formation. However, its upstream signaling mechanism remains unknown. In this study, using an Achilles tendon puncture-induced HO model, we revealed that overactive insulin-like growth factor 1 (IGF-1) was involved in the progression of HO in mice. Micro-CT imaging showed that IGF-1 not only accelerated the rate of osteogenesis and increased ectopic bone volume but also induced spontaneous ectopic bone formation in undamaged Achilles tendons. Blocking IGF-1 activity with IGF-1 antibody or IGF-1 receptor (IGF-1R) inhibitor Picropodophyllin significantly inhibited HO formation. Mechanistically, IGF-1/IGF-1R activates PI3K/Akt signaling to promote the phosphorylation of mTOR, resulting in the chondrogenic and osteogenic differentiation of tendon-derived stem cells (TDSCs) into chondrocytes and osteoblasts in vitro and in vivo. Inhibitors of PI3K (LY294002) and mTOR (Rapamycin) both suppressed the IGF-1-stimulated mTOR signal and mitigated the formation of ectopic bones significantly. In conclusion, our results indicated that IGF-1 mediated the progression of traumatic HO through PI3K/Akt/mTOR signaling, and suppressing IGF-1 signaling cascades attenuated HO formation, providing a promising therapeutic strategy targeting HO.

Berberine and aspirin prevent traumatic heterotopic ossification by inhibition of BMP signalling pathway and osteogenic differentiation.

Heterotopic ossification (HO) is a pathological process that often occurs in soft tissues following severe trauma. There is no effective therapy for HO. The BMP signalling pathway plays an essential role in the pathogenesis of HO. Our previous study showed that AMPK negatively regulates the BMP signalling pathway and osteogenic differentiation. The present study aims to study the effect of two AMPK activators berberine and aspirin on osteogenic differentiation and HO induced by traumatic injury. The effects of two AMPK activators, berberine and aspirin, on BMP signalling and osteogenic differentiation were measured by western blot, ALP and Alizarin red S staining in C3H10T1/2 cells. A mouse model with Achilles tenotomy was employed to assess the effects of berberine and aspirin on HO using μCT and histological analysis. First, our study showed that berberine and aspirin inhibited phosphorylation of Smad1/5 induced by BMP6 and the inhibition was attributed to the down-regulation of ALK2 expression. Second, the combination of berberine and aspirin yielded more potent effects on BMP signalling. Third, we further found that there was an additive effect of berberine and aspirin combination on osteogenic differentiation. Finally, we found that berberine and aspirin blocked trauma-induced ectopic bone formation in mice, which may be through suppression of phosphorylation of Smad1/5 in injured tissues. Collectively, these findings indicate that berberine and aspirin inhibit osteogenic differentiation in C3H10T1/2 cells and traumatic HO in mice, possibly through the down-regulation of the BMP signalling pathway. Our study sheds a light on prevention and treatment of traumatic HO using AMPK pharmacological activators berberine and aspirin.

Inhibition of NF-κB Signaling-Mediated Crosstalk Between Macrophages and Preosteoblasts by Metformin Alleviates Trauma-Induced Heterotopic Ossification.

Heterotopic ossification (HO) is a pathological condition that occurs in soft tissues following severe trauma. The exact pathogenesis of HO remains unclear. Studies have shown that inflammation predisposes patients to the development of HO and triggers ectopic bone formation. Macrophages are crucial mediators of inflammation and are involved in HO development. The present study investigated the inhibitory effect and underlying mechanism of metformin on macrophage infiltration and traumatic HO in mice. Our results found that abundant levels of macrophages were recruited to the injury site during early HO progression and that early administration of metformin prevented traumatic HO in mice. Furthermore, we found that metformin attenuated macrophage infiltration and the NF-κB signaling pathway in injured tissue. The monocyte-to-macrophage transition in vitro was suppressed by metformin and this event was mediated by AMPK. Finally, we showed that inflammatory mediator's regulation by macrophages targeted preosteoblasts, leading to elevated BMP signaling, and osteogenic differentiation and driving HO formation, and this effect was blocked after the activation of AMPK in macrophages. Collectively, our study suggests that metformin prevents traumatic HO by inhibiting of NF-κB signaling in macrophages and subsequently attenuating BMP signaling and osteogenic differentiation in preosteoblasts. Therefore, metformin may serve as a therapeutic drug for traumatic HO by targeting NF-κB signaling in macrophages.

Ethyl caffeate inhibits macrophage polarization via SIRT1/NF-κB to attenuate traumatic heterotopic ossification in mice

Heterotopic ossification (HO) denotes the presence of mature bone tissue in soft tissues or around joints. Inflammation is a key driver of traumatic HO, and macrophages play an important role in this process. Ethyl caffeate (ECF), a critical active compound found in Petunia, exerts significant anti-inflammatory effects. Herein, we established a mouse model of HO by transection of the Achilles tendon and back burn and found abundant macrophage infiltration in the early stage of HO, which decreased with time. In vitro and in vivo experiments indicated that ECF inhibited macrophage polarization, and mechanistic studies showed that it inhibited the SIRT1/NF-κB signalling pathway, thereby suppressing the release of downstream inflammatory cytokines. ECF reduced HO in mice, and its effect was comparable to indomethacin (INDO). In vitro studies revealed that ECF did not directly affect the mineralization of mesenchymal stem cells (MSCs) or osteogenic differentiation but inhibited these processes by reducing the level of inflammatory cytokines in the conditioned medium (CM). Thus, M1 macrophages may play a crucial role in the pathogenesis of HO, and ECF is a prospective candidate for the prevention of trauma-induced HO. Data availabilityData will be made available on request

Suppression of heterotopic ossification in fibrodysplasia ossificans progressiva using AAV gene delivery

Heterotopic ossification is the most disabling feature of fibrodysplasia ossificans progressiva, an ultra-rare genetic disorder for which there is currently no prevention or treatment. Most patients with this disease harbor a heterozygous activating mutation (c.617 G > A;p.R206H) in ACVR1. Here, we identify recombinant AAV9 as the most effective serotype for transduction of the major cells-of-origin of heterotopic ossification. We use AAV9 delivery for gene replacement by expression of codon-optimized human ACVR1, ACVR1R206H allele-specific silencing by AAV-compatible artificial miRNA and a combination of gene replacement and silencing. In mouse skeletal cells harboring a conditional knock-in allele of human mutant ACVR1 and in patient-derived induced pluripotent stem cells, AAV gene therapy ablated aberrant Activin A signaling and chondrogenic and osteogenic differentiation. In Acvr1(R206H) knock-in mice treated locally in early adulthood or systemically at birth, trauma-induced endochondral bone formation was markedly reduced, while inflammation and fibroproliferative responses remained largely intact in the injured muscle. Remarkably, spontaneous heterotopic ossification also substantially decreased in in Acvr1(R206H) knock-in mice treated systemically at birth or in early adulthood. Collectively, we develop promising gene therapeutics that can prevent disabling heterotopic ossification in mice, supporting clinical translation to patients with fibrodysplasia ossificans progressiva.

Heterotopic ossification in mice overexpressing Bmp2 in Tie2+ lineages

Bone morphogenetic protein (Bmp) signaling is critical for organismal development and homeostasis. To elucidate Bmp2 function in the vascular/hematopoietic lineages we generated a new transgenic mouse line in which ectopic Bmp2 expression is controlled by the Tie2 promoter. Tie2CRE/+;Bmp2tg/tg mice develop aortic valve dysfunction postnatally, accompanied by pre-calcific lesion formation in valve leaflets. Remarkably, Tie2CRE/+;Bmp2tg/tg mice develop extensive soft tissue bone formation typical of acquired forms of heterotopic ossification (HO) and genetic bone disorders, such as Fibrodysplasia Ossificans Progressiva (FOP). Ectopic ossification in Tie2CRE/+;Bmp2tg/tg transgenic animals is accompanied by increased bone marrow hematopoietic, fibroblast and osteoblast precursors and circulating pro-inflammatory cells. Transplanting wild-type bone marrow hematopoietic stem cells into lethally irradiated Tie2CRE/+;Bmp2tg/tg mice significantly delays HO onset but does not prevent it. Moreover, transplanting Bmp2-transgenic bone marrow into wild-type recipients does not result in HO, but hematopoietic progenitors contribute to inflammation and ectopic bone marrow colonization rather than to endochondral ossification. Conversely, aberrant Bmp2 signaling activity is associated with fibroblast accumulation, skeletal muscle fiber damage, and expansion of a Tie2+ fibro-adipogenic precursor cell population, suggesting that ectopic bone derives from a skeletal muscle resident osteoprogenitor cell origin. Thus, Tie2CRE/+;Bmp2tg/tg mice recapitulate HO pathophysiology, and might represent a useful model to investigate therapies seeking to mitigate disorders associated with aberrant extra-skeletal bone formation.

Targeting local lymphatics to ameliorate heterotopic ossification via FGFR3-BMPR1a pathway

Acquired heterotopic ossification (HO) is the extraskeletal bone formation after trauma. Various mesenchymal progenitors are reported to participate in ectopic bone formation. Here we induce acquired HO in mice by Achilles tenotomy and observe that conditional knockout (cKO) of fibroblast growth factor receptor 3 (FGFR3) in Col2+ cells promote acquired HO development. Lineage tracing studies reveal that Col2+ cells adopt fate of lymphatic endothelial cells (LECs) instead of chondrocytes or osteoblasts during HO development. FGFR3 cKO in Prox1+ LECs causes even more aggravated HO formation. We further demonstrate that FGFR3 deficiency in LECs leads to decreased local lymphatic formation in a BMPR1a-pSmad1/5-dependent manner, which exacerbates inflammatory levels in the repaired tendon. Local administration of FGF9 in Matrigel inhibits heterotopic bone formation, which is dependent on FGFR3 expression in LECs. Here we uncover Col2+ lineage cells as an origin of lymphatic endothelium, which regulates local inflammatory microenvironment after trauma and thus influences HO development via FGFR3-BMPR1a pathway. Activation of FGFR3 in LECs may be a therapeutic strategy to inhibit acquired HO formation via increasing local lymphangiogenesis.

Activin A promotes the development of acquired heterotopic ossification and is an effective target for disease attenuation in mice.

Heterotopic ossification (HO) is a common, potentially debilitating pathology that is instigated by inflammation caused by tissue damage or other insults, which is followed by chondrogenesis, osteogenesis, and extraskeletal bone accumulation. Current remedies are not very effective and have side effects, including the risk of triggering additional HO. The TGF-β family member activin A is produced by activated macrophages and other inflammatory cells and stimulates the intracellular effectors SMAD2 and SMAD3 (SMAD2/3). Because HO starts with inflammation and because SMAD2/3 activation is chondrogenic, we tested whether activin A stimulated HO development. Using mouse models of acquired intramuscular and subdermal HO, we found that blockage of endogenous activin A by a systemically administered neutralizing antibody reduced HO development and bone accumulation. Single-cell RNA-seq analysis and developmental trajectories showed that the antibody treatment reduced the recruitment of Sox9+ skeletal progenitors, many of which also expressed the gene encoding activin A (Inhba), to HO sites. Gain-of-function assays showed that activin A enhanced the chondrogenic differentiation of progenitor cells through SMAD2/3 signaling, and inclusion of activin A in HO-inducing implants enhanced HO development in vivo. Together, our data reveal that activin A is a critical upstream signaling stimulator of acquired HO in mice and could represent an effective therapeutic target against forms of this pathology in patients.

Metformin attenuates trauma-induced heterotopic ossification via inhibition of Bone Morphogenetic Protein signalling.

AMP‐activated protein kinase (AMPK) is an intracellular sensor of energy homoeostasis that is activated under energy stress and suppressed in energy surplus. AMPK activation leads to inhibition of anabolic processes that consume ATP. Osteogenic differentiation is a process that highly demands ATP during which AMPK is inhibited. The bone morphogenetic proteins (BMPs) signalling pathway plays an essential role in osteogenic differentiation. The present study examines the inhibitory effect of metformin on BMP signalling, osteogenic differentiation and trauma‐induced heterotopic ossification. Our results showed that metformin inhibited Smad1/5 phosphorylation induced by BMP6 in osteoblast MC3T3‐E1 cells, concurrent with up‐regulation of Smad6, and this effect was attenuated by knockdown of Smad6. Furthermore, we found that metformin suppressed ALP activity and mineralization of the cells, an event that was attenuated by the dominant negative mutant of AMPK and mimicked by its constitutively active mutant. Finally, administration of metformin prevented the trauma‐induced heterotopic ossification in mice. In conjuncture, AMPK activity and Smad6 and Smurf1 expression were enhanced by metformin treatment in the muscle of injured area, concurrently with the reduction of ALK2. Collectively, our study suggests that metformin prevents heterotopic ossification via activation of AMPK and subsequent up‐regulation of Smad6. Therefore, metformin could be a potential therapeutic drug for heterotopic ossification induced by traumatic injury.

Prophylactic treatment of rapamycin ameliorates naturally developing and episode -induced heterotopic ossification in mice expressing human mutant ACVR1

BackgroundFibrodysplasia ossificans progressiva (FOP) is a rare autosomal-dominant disease characterized by heterotopic ossification (HO) in soft tissues and caused by a mutation of the ACVR1A/ALK2 gene. Activin-A is a key molecule for initiating the process of HO via the activation of mTOR, while rapamycin, an mTOR inhibitor, effectively inhibits the Activin-A-induced HO. However, few reports have verified the effect of rapamycin on FOP in clinical perspectives.MethodsWe investigated the effect of rapamycin for different clinical situations by using mice conditionally expressing human mutant ACVR1A/ALK2 gene. We also compared the effect of rapamycin between early and episode-initiated treatments for each situation.ResultsContinuous, episode-independent administration of rapamycin reduced the incidence and severity of HO in the natural course of FOP mice. Pinch-injury induced HO not only at the injured sites, but also in the contralateral limbs and provoked a prolonged production of Activin-A in inflammatory cells. Although both early and injury-initiated treatment of rapamycin suppressed HO in the injured sites, the former was more effective at preventing HO in the contralateral limbs. Rapamycin was also effective at reducing the volume of recurrent HO after the surgical resection of injury-induced HO, for which the early treatment was more effective.ConclusionOur study suggested that prophylactic treatment will be a choice of method for the clinical application of rapamycin for FOP.

Celecoxib cannot inhibit the progression of initiated traumatic heterotopic ossification

Heterotopic ossification (HO) is a recognized sequela after trauma and arthroplasty. The purpose of this study was to evaluate the therapeutic effect of celecoxib on HO. We hypothesized that celecoxib may inhibit the progression of initiated HO. We performed a retrospective review of 37 patients who underwent elbow joint surgery between January 2014 and June 2018. Seventeen patients were prescribed orally administered celecoxib (200 mg/dose, twice daily) for 2 months after the diagnosis of HO, whereas the remaining 20 patients were administered celecoxib for 1 month starting immediately after surgery. HO progression was evaluated by plain radiographs. By use of an Achilles tendon puncture-induced HO mouse model, the curative effect of celecoxib was illustrated at different HO progression stages. The mice were assigned to 1 of 4 groups: sham group, vehicle group, group receiving celecoxib on day 1, and group receiving celecoxib in week 6. Achilles tendons were analyzed by micro-computed tomography and histochemistry after 12 weeks. Celecoxib did not inhibit the progression of initiated HO in the patients in whom HO was diagnosed, whereas those who received celecoxib after surgery had lower morbidity. Achilles tendon puncture effectively induced typical HO in mice. The ectopic bone volume was significantly reduced in the day 1 celecoxib group compared with the vehicle group; however, the difference was not statistically significant in the week 6 celecoxib group. Administration of celecoxib starting immediately after surgery can significantly inhibit the formation of HO. Once HO is visible on plain radiographs or micro-computed tomography, celecoxib cannot effectively attenuate further progression of HO in humans and mice.

Tamoxifen Inhibits the Progression of Trauma-Induced Heterotopic Ossification in Mice.

BackgroundHeterotopic ossification (HO) is a kind of abnormal mineralized bone which usually occurs in muscle, tendon, or ligament. There are currently no effective drugs for the treatment and prevention of HO. Developing effective drugs that can inhibit HO is of profound significance and would provide new strategies for clinical treatment of this disease. The present investigation evaluated the inhibitory effect of tamoxifen against HO.Material/MethodsUsing an Achilles tendon trauma-induced HO female mice model, we screened different doses of tamoxifen (1, 3, and 9 mg/kg) in mice to determine the optimal dosage on the inhibition of the HO formation. The curative effect of tamoxifen was also illustrated at different HO progression stages including inflammation, chondrogenesis, osteogenesis, and HO maturation.ResultsHeterotopic bone was formed with typical endochondral ossification in Achilles tendons 6 weeks after surgery and continued to enlarge up to 12 weeks. The formation of HO was significantly inhibited with the treatment of tamoxifen at the dosage of 9 mg/kg, whereas 1 mg/kg and 3 mg/kg did not reduce HO bone volume remarkably. The progression of HO was both attenuated by tamoxifen from Day 1 and Week 4 post-surgery, whereas no inhibitory effect was shown at the osteogenesis and maturation stages treated with tamoxifen.ConclusionsTamoxifen exerts an inhibitory effect on the heterotopic bone progression at inflammation and chondrogenesis stages, with the TGF-β signaling pathway suppressed following the increase in estrogen receptor α activity.

Inhibition of phosphatidylinositol 3-kinase α (PI3Kα) prevents heterotopic ossification.

Heterotopic ossification (HO) is the pathological formation of ectopic endochondral bone within soft tissues. HO occurs following mechanical trauma, burns, or congenitally in patients suffering from fibrodysplasia ossificans progressiva (FOP). FOP patients carry a conserved mutation in ACVR1 that becomes neomorphic for activin A responses. Here, we demonstrate the efficacy of BYL719, a PI3Kα inhibitor, in preventing HO in mice. We found that PI3Kα inhibitors reduce SMAD, AKT, and mTOR/S6K activities. Inhibition of PI3Kα also impairs skeletogenic responsiveness to BMPs and the acquired response to activin A of the Acvr1 R206H allele. Further, the efficacy of PI3Kα inhibitors was evaluated in transgenic mice expressing Acvr1 Q207D. Mice treated daily or intermittently with BYL719 did not show ectopic bone or cartilage formation. Furthermore, the intermittent treatment with BYL719 was not associated with any substantial side effects. Therefore, this work provides evidence supporting PI3Kα inhibition as a therapeutic strategy for HO.

The metalloproteinase-proteoglycans ADAMTS7 and ADAMTS12 provide an innate, tendon-specific protective mechanism against heterotopic ossification.

Heterotopic ossification (HO) is a significant clinical problem with incompletely resolved mechanisms. Here, the secreted metalloproteinases ADAMTS7 and ADAMTS12 are shown to comprise a unique proteoglycan class that protects against a tendency toward HO in mouse hindlimb tendons, menisci, and ligaments. Adamts7 and Adamts12 mRNAs were sparsely expressed in murine forelimbs but strongly coexpressed in hindlimb tendons, skeletal muscle, ligaments, and meniscal fibrocartilage. Adamts7-/- Adamts12-/- mice, but not corresponding single-gene mutants, which demonstrated compensatory upregulation of the intact homolog mRNA, developed progressive HO in these tissues after 4 months of age. Adamts7-/- Adamts12-/- tendons had abnormal collagen fibrils, accompanied by reduced levels of the small leucine-rich proteoglycans (SLRPs) biglycan, fibromodulin, and decorin, which regulate collagen fibrillogenesis. Bgn-/0 Fmod-/- mice are known to have a strikingly similar hindlimb HO to that of Adamts7-/- Adamts12-/- mice, implicating fibromodulin and biglycan reduction as a likely mechanism underlying HO in Adamts7-/- Adamts12-/- mice. Interestingly, degenerated human biceps tendons had reduced ADAMTS7 mRNA compared with healthy biceps tendons, which expressed both ADAMTS7 and ADAMTS12. These results suggest that ADAMTS7 and ADAMTS12 drive an innate pathway protective against hindlimb HO in mice and may be essential for human tendon health.

Inhibition of overactive TGF-\u03b2 attenuates progression of heterotopic ossification in mice

Acquired heterotopic ossification (HO) is a painful and debilitating disease characterized by extraskeletal bone formation after injury. The exact pathogenesis of HO remains unknown. Here we show that TGF-β initiates and promotes HO in mice. We find that calcified cartilage and newly formed bone resorb osteoclasts after onset of HO, which leads to high levels of active TGF-β that recruit mesenchymal stromal/progenitor cells (MSPCs) in the HO microenvironment. Transgenic expression of active TGF-β in tendon induces spontaneous HO, whereas systemic injection of a TGF-β neutralizing antibody attenuates ectopic bone formation in traumatic and BMP-induced mouse HO models, and in a fibrodysplasia ossificans progressive mouse model. Moreover, inducible knockout of the TGF-β type II receptor in MSPCs inhibits HO progression in HO mouse models. Our study points toward elevated levels of active TGF-β as inducers and promoters of ectopic bone formation, and suggest that TGF-β might be a therapeutic target in HO.

Muscle and Bone: Combating the Evil Side of the Connection.

Muscle and Bone: Combating the Evil Side of the Connection.

Targeted stimulation of retinoic acid receptor-γ mitigates the formation of heterotopic ossification in an established blast-related traumatic injury model

Heterotopic ossification (HO) involves formation of endochondral bone at non-skeletal sites, is prevalent in severely wounded service members, and causes significant complications and delayed rehabilitation. As common prophylactic treatments such as anti-inflammatory drugs and irradiation cannot be used after multi-system combat trauma, there is an urgent need for new remedies. Previously, we showed that the retinoic acid receptor γ agonist Palovarotene inhibited subcutaneous and intramuscular HO in mice, but those models do not mimic complex combat injury. Thus, we tested Palovarotene in our validated rat trauma-induced HO model that involves blast-related limb injury, femoral fracture, quadriceps crush injury, amputation and infection with methicillin-resistant Staphylococcus aureus from combat wound infections. Palovarotene was given orally for 14days at 1mg/kg/day starting on post-operative day (POD) 1 or POD-5, and HO amount, wound dehiscence and related processes were monitored for up to 84days post injury. Compared to vehicle-control animals, Palovarotene significantly decreased HO by 50 to 60% regardless of when the treatment started and if infection was present. Histological analyses showed that Palovarotene reduced ectopic chondrogenesis, osteogenesis and angiogenesis forming at the injury site over time, while fibrotic tissue was often present in place of ectopic bone. Custom gene array data verified that while expression of key chondrogenic and osteogenic genes was decreased within soft tissues of residual limb in Palovarotene-treated rats, expression of cartilage catabolic genes was increased, including matrix metalloproteinase-9. Importantly, Palovarotene seemed to exert moderate inhibitory effects on wound healing, raising potential safety concerns related to dosing and timing. Our data show for the first time that Palovarotene significantly inhibits HO triggered by blast injury and associated complications, strongly indicating that it may prevent HO in patients at high risk such as those sustaining combat injuries and other forms of blast trauma.

Novel mouse model of spinal cord injury-induced heterotopic ossification.

Heterotopic ossification (HO) develops in about 20% to 30% of patients with spinal cord injury (SCI) and significantly impairs their rehabilitation. There is no effective prevention or treatment for this condition at this time. Our current understanding of its etiology and pathophysiology is limited partially due to the lack of clinically relevant animal models. In this study, we report a novel mouse model of SCI-induced HO by administering a subthreshold dose of bone morphogenetic protein (BMP)-2 to muscles in mice after SCI. Micro-computed tomography scanning showed that an intramuscular injection of 0.25 micrograms of BMP-2 causes significant HO in mice with SCI but not in control (sham surgery) mice. Our analysis of gene expression showed significantly increased BMP signaling in quadriceps following SCI, suggesting that BMP signaling may play a role in SCI-induced HO. Administering 0.25 micrograms of BMP-2 to the front arms of the mice with SCI also results in the development of significant HO but not in control mice. This suggests that SCI causes a systematic osteogenic effect, which is not limited to paralyzed limbs. This novel mouse model will serve as a powerful tool in exploring the molecular mechanisms of SCI-induced HO, which may lead to novel treatment for this disease.

Constitutively Active ALK2 Receptor Mutants Require Type II Receptor Cooperation

Constitutively activating mutations in receptor kinases recruit downstream effector pathways independently of upstream signaling, with consequences ranging from developmental syndromes to cancer. Classic fibrodysplasia ossificans progressiva (FOP) is a congenital syndrome resulting from highly conserved activating mutations of the glycine-serine-rich (GS) regulatory domain of ACVR1, encoding bone morphogenetic protein (BMP) type I receptor ALK2, which lead to inappropriate signaling and heterotopic ossification of soft tissues. It is unclear if constitutively active mutant ALK2 receptors (caALK2) can function independently of signaling complexes with type II receptors and ligands. We found that ablation of BmpRII and ActRIIa abrogated BMP ligand-mediated and caALK2-mediated signaling and transcription in cells and disrupted caALK2-induced heterotopic ossification in mice. Signaling via GS domain ALK2 mutants could be restored by the expression of either BMP type II receptor. The contribution of BMP type II receptors was independent of their ligand-binding or kinase function but was dependent upon an intact cytoplasmic domain. These data demonstrate that GS domain ALK2 mutants act independently of upstream signaling but may require a nonenzymatic scaffolding function provided by type II receptors to form functional, apparently ligand-independent signaling complexes. These findings define the minimal requirements for signaling of GS domain ALK2 mutants, with implications for the therapeutic targeting of their activity in disease.