TNFα-dependent mTOR activity is required for tenotomy-induced ectopic ossification in mice

Ectopic ossifications often occur in skeletal muscles or tendons following local trauma or internal hemorrhage, and occasionally cause severe pain that limits activities of daily living. However, mechanisms underlying their development remain unknown. The right Achilles tendon in 8-week-old female or male mice was dissected. Some mice were injected intraperitoneally either with phosphate-buffered saline, dimethyl sulfoxide, cimetidine, rapamycin, celecoxib or loxoprofen for 10 weeks. One week after surgery, immunohistochemical analysis was performed for mTOR, TNFα or F4/80. Ten weeks after surgery, ectopic ossification at the tenotomy site was detected by 3D micro-CT. Ectopic ossification was seen at dissection sites in all wild-type mice by dissection of the Achilles tendon. mTOR activation was detected at dissection sites, and development of ectopic ossification was significantly inhibited by administration of rapamycin, an mTOR inhibitor, to wild-type mice. Moreover, administration of the histamine 2 blocker cimetidine, which reportedly inhibits ectopic ossification in tendons, was not effective in inhibiting ectopic ossification in our models. TNFα-expressing F4/80-positive macrophages accumulate at dissection sites and that ectopic ossification of the Achilles tendon dissection was significantly inhibited in TNFα-deficient mice in vivo. Ectopic ossification is significantly inhibited by administration of either celecoxib or loxoprofen, both anti-inflammatory agents, in wild-type mice. mTOR activation by Achilles tendon tenotomy is inhibited in TNFα-deficient mice. The TNFα-mTOR axis could be targeted therapeutically to prevent trauma-induced ectopic ossification in tendons.


Introduction
Ectopic ossification is frequently seen in skeletal muscle, including deltoid muscles, or in tendons of the rotator cuff in the shoulder or in the Achilles tendon [1,2].Trauma or intra-muscle bleeding has been considered a cause of ectopic ossification [3], but precise molecular mechanisms underlying these activities remain unknown.Some ectopic ossifications are asymptomatic, while others cause severe pain and limit activities of daily living.Thus, it is critical to define mechanisms underlying or governing ectopic ossification.Cimetidine, a histamine 2 (H2) blocker, is reportedly effective in preventing new ectopic ossification or reducing the size of existing ectopic ossifications in some patients [4].However, cimetidine is not effective against ectopic (Bar Harbor, ME).Mice were housed in specific pathogenfree conditions in animal facilities certified by the Keio University Institutional Animal Care and Use Committee and maintained in an environment in accordance with Institutional Guidelines on Animal Experimentation at Keio University.Mice were housed up to 5 per cage and kept on a 12-h light/dark cycle.Sterile distilled water and a standard diet (CLEA Rodent Diet CE-2, Japan) were available ad libitum.All animal experimental protocols were approved by the aforementioned committee and performed in accordance with their guidelines.This study is reported in accordance with ARRIVE guidelines.

Achilles tendon tenotomy
For surgery, 8-week-old female or male mice received a mixture of ketamine (100 mg/ kg) and xylazine (10 mg/ kg) by intraperitoneal injection as anesthesia.Then under anesthesia, the right Achilles tendon was dissected using strict aseptic technique with povidone iodine.The tendon was approached from the lateral aspect and divided at its midpoint with a gap of 5 mm from the enthesis of calcaneus.Tendon ends were not sutured, and the skin incision was closed with 5-0 nylon.Some mice were injected intraperitoneally with either phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), cimetidine (50 mg/kg), rapamycin (5 mg/kg), celecoxib (10 mg/kg) or loxoprofen (10 mg/kg) 5 days a week for 10 weeks.Ten weeks after surgery, ectopic ossification at the tenotomy site was detected by 3D micro-CT with the following parameters: field of view, 30 mm; voltage, 90 kV; current, 160 mA; scan technique, fine 3 min.Ectopically formed bone volume was evaluated using R_mCT2 (Rigaku Corporation) software.
Ectopic ossifications are also frequently seen in disease states, such as ossification of the posterior longitudinal ligament (OPLL) and fibrodysplasia ossificans progressive (FOP) [5].In OPLL, ectopic ossifications in the PLL can cause spinal canal stenosis, leading to neurological symptoms such as paralysis [5,6], as yet, mechanisms underlying ectopic ossification in OPLL patients have not been defined.On the other hand, FOP, in which ectopic ossification occurs in muscle, is reportedly promoted by a missense mutation in the gene activin receptor IA/activin-like kinase-2 (ACVRI/ ALK2) that promotes ACVRI/ALK2 constitutive activation [7,8].Constitutively active ACVRI/ALK2 then reportedly stimulates mTOR signaling, which in turn promotes ectopic ossification in muscle [9,10].The phosphatidylinositol 3-kinase (PI3K)/Akt/NF-κB pathway is reportedly stimulated by TNFα in cells such as human hepatoma cells or human embryonic kidney fibroblasts [11,12].The Akt/ mTOR complex 1 has been shown to be required in vitro for TNFα-mediated migration of human retinal pigment epithelial cells [13].Moreover, mTOR signaling reportedly plays a role in bone formation, and rapamycin treatment antagonizes osteoblast differentiation in human primary bone marrow stromal cells [14][15][16].However, it is not clear whether TNFα-mTOR signaling is activated during trauma-induced ectopic ossification in the absence of gene mutations, and the pathological mechanisms underlying ectopic bone formation after trauma have not been fully characterized.
Here, we show that dissection of the Achilles tendon promotes ectopic ossification at dissection sites in all wildtype mice evaluated.In our models, administration of cimetidine was not effective in inhibiting ectopic ossification; instead, we found that blocking the mTOR pathway by treatment with rapamycin, an mTOR inhibitor, knockout of TNFα, or administration of the anti-inflammatory agents celecoxib or loxoprofen significantly inhibited ectopic ossification induced by Achilles tendon tenotomy.We conclude that trauma-induced ectopic ossification in tendons can be prevented by targeting mTOR or TNFα or preventing inflammation.

Statistical analysis
Results of multiple experiments are presented as means ± standard deviation (SD).Data analysis was performed using GraphPad 6.0 Software (GraphPad, San Diego, CA, USA).Statistical comparisons (P values) between two groups were calculated using Student's t test, and P values between three groups were calculated using one-way and/ or two-way analysis of variance (ANOVA).P values < 0.05 were considered statistically significant.The number of replicates and/or total number of animals are shown in figure legends or within figures.

Targeting mTOR activation inhibits ectopic ossification in tendons induced by tenotomy
To generate an ectopic ossification model in vivo in wildtype mice in the absence of mutations, we dissected the hemilateral Achilles tendon to generate a 5 mm gap at the proximal region of the enthesis of calcaneus in 8-week-old female and male mice (Fig. 1a and Fig S1).The contralateral side was sham operated (Fig. 1a).Starting at 1 week after surgery, we began analyzing ectopic bone formation over time using three-dimensional (3D) micro-CT (Fig. 1b  and c).Ectopic bone became visible by 3D micro-CT at Achilles tendon dissection sites by 3 weeks after tenotomy in both female and male mice, and those sites grew larger until at least until 10 weeks after surgery (Fig. 1b and  c and S1).As shown in Fig. 1b, ectopic bone formed at the site of tendon dissection in all mice evaluated by 3 weeks after tenotomy.Alkaline phosphatase (ALP) activity increased as did the appearance of Safranin-O-positive chondrocytes by 2 weeks after tenotomy at the site of Achilles tendon dissection (Fig. 1d), suggesting that We then analyzed mTOR signaling, since aberrant mTOR activation is reportedly required for ectopic ossification in FOP [9].Interestingly we detected significantly elevated mTOR phosphorylation indicative of tenocyte activation at resection stumps at tenotomy sites 1 week after surgery (Fig. 2a and b).We then administered the mTOR inhibitor rapamycin to mice at the time of Achilles tendon dissection and thereafter for 5 days a week for the duration of the experiment.mTOR phosphorylation at the tenotomy site was significantly reduced by administration of rapamycin 1 week after surgery (Figure S2).Ten weeks later, we assessed ectopic bone formation by 3D micro-CT (Fig. 2c and d) and observed decreased ectopic bone formation and decreased volume of bone tissue in rapamycin-treated relative to control mice (Fig. 2c and d).
Ectopic ossification is frequently seen in tendons in the rotator cuff in humans, and treatment with cimetidine, a histamine 2 (H2) receptor antagonist, is reportedly effective in inhibiting such calcification in patients [4,17].Thus, we administered cimetidine to mice with a dissected hemilateral Achilles tendon by subcutaneous injection 5 days a week starting at the time of surgery and for the duration of the experiment (Fig. 3a).Ten weeks later, we analyzed ectopic bone formation by 3D micro-CT and observed no differences in volume of newly formed bone tissue in mice treated with or without cimetidine (Fig. 3b and c).

Ectopic ossification induced by tenotomy is significantly inhibited by blocking TNFα
The inflammatory factor TNFα reportedly functions upstream of mTOR [11,18].Thus, we analyzed TNFα expression at the site of Achilles tendon dissection 1 week after surgery in wild-type mice using immunohistochemistry (Fig. 4).We detected TNFα expression by F4/80-positive macrophages at the site of tenotomy, based on overlapping immunostaining patterns (Fig. 4a and b).We also confirmed TNFα expression in CD68-positive macrophages at tenotomy sites (Figure S3).We did not observe TNFα expression in either CD3-positive T cells or CD20-positive B cells (Figure S3), suggesting migration of TNFα-expressing inflammatory cells to sites of tendon dissection.In fact, we detected TNFα-expressing cells at sites of or surrounding pmTOR-positive cells (Figure S4).We also detected induction of mTOR phosphorylation following TNFα stimulation in NIH3T3 fibroblastic cells in vitro (Figure S5).Thus, we created an Achilles tendon tenotomy-induced ectopic ossification model in TNFα-deficient (TNFα KO) mice (Fig. 4c and d) and observed significantly decreased volume of ectopic bone by 10 weeks after surgery in TNFα-deficient relative to wild-type mice (Fig. 4c  and d).These findings strongly suggest that TNFα signaling is required for ectopic bone formation in this mouse model.

Administration of anti-inflammatory drugs effectively inhibits ectopic ossification induced by Achilles tendon dissection
We next asked whether treatment with the anti-inflammatory drugs celecoxib or loxoprofen would inhibit ectopic ossification by tenotomy, given that ectopic bone formation was significantly inhibited by blocking TNFα (Fig. 5).To do so, we administered celecoxib, loxoprofen or PBS/ vehicle to wild-type mice 5 days a week, starting at the time of tendon dissection and continuing for 10 weeks.We observed that relative to controls, the volume of ectopically formed bone following tenotomy was significantly Fig. 3 Cimetidine does not suppress ectopic ossification seen following Achilles tendon tenotomy in wild-type mice.a Achilles tendon tenotomy was established in 8-week-old wild-type female mice.Mice were also injected intraperitoneally with either PBS (control) or cimetidine (50 mg/kg) and thereafter for 5 days a week for the duration of the experiment.Ten weeks later, when mice were 18 weeks old, ectopic bone formation was analyzed by 3D micro-CT (b), and bone volume was measured (c).Data represent mean ectopic bone volumes of cimetidine relative to PBS-injected mice ± SD (control, n = 11; cimetidine, n = 11).Representative data of at least two independent experiments are shown reduced by celecoxib or loxoprofen treatment (Fig. 5a  and b).Also, starting at the time of tenotomy, we found that seven daily injections of either rapamycin, celecoxib or loxoprofen significantly inhibited ectopic ossification (Figure S6).
Finally, to determine whether TNFα is upstream of mTOR in this model, we performed tenotomy in wildtype mice, treated mice with vehicle or rapamycin to block mTOR, and evaluated TNFα expression by immunohistochemistry.We observed equivalent TNFα expression at the tenotomy site in control and rapamycin-treated mice (Fig. 6a and b).We then performed tenotomy in TNFα KO and wild-type mice and found that mTOR activation at the tenotomy site, as analyzed by immunohistochemistry 1 week after surgery, was significantly inhibited in TNFα KO mice (Fig. 6c and d).We also created tenotomy in wild-type mice and treated them with celecoxib or loxoprofen (Fig. 6e  and f).In that analysis, fluorescence intensity of phosphorylated mTOR (pmTOR) at the dissection site 1 week after surgery was significantly inhibited in mice treated with either of agent (Fig. 6e and f).We conclude mTOR activation by TNFα or other inflammatory factors is required for ectopic ossification.

Discussion
Physiological calcium deposition is limited to bone tissues, and calcified bone, which supports body weight and motion, also functions in calcium storage.When calcium homeostasis is disrupted, calcium deposition begins in soft tissues where physiological calcium deposition does not normally occur.Some mutations disrupt calcium homeostasis, as seen in FOP patients [7].OPLL patients also exhibit mutations identified by genome-wide association studies associated with ectopic ossification in ligaments [19].In addition to genetic factors, organ dysfunction such as renal failure can cause ectopic calcification in various tissues [20,21].
Here we demonstrate that local inflammation and subsequent mTOR activation following tenotomy promote ectopic ossification in the absence of mutations or organ dysfunction.Our findings are in accord with observations that ectopic ossification can be induced by local minor trauma or bleeding in humans [3].Indeed, ectopic ossifications are seen in athletes following sports-associated trauma or in middle-aged to elderly people with degenerative tendon/ligament tears but who lack mutations or organ dysfunction [22].We detected TNFα expression at sites of and in the areas surrounding pmTOR-positive cells after tenotomy (Figure S4).We also show that TNFα was expressed in F4/80-positive macrophages at tenotomy sites after surgery (Fig. 6a).These findings suggest that macrophages stimulate mTOR signaling via TNFα at sites of tenotomy to promote ectopic bone formation.Indeed, mTOR signaling in fibroblastic cells was stimulated by TNFα in vitro (Figure S5).Since prostaglandins (PGs), including PGE2, are reportedly required for bone formation during fracture healing [23], the cyclooxygenase enzyme/PG pathway may also play a role in ectopic bone formation following tenotomy.Indeed, we demonstrate that inhibition of that pathway by NSAIDs inhibited ectopic bone formation after tenotomy (Figs.6e, f and S6).
Endochondral ossification is reportedly activated in ectopic bone in PLL in OPLL patients [6].Klotho mice, which show pre-mature aging phenotypes, exhibit Möncheberg's vascular calcification in the tunica media [24,25].In this process, vascular smooth muscle cells undergo phenotypic trans-differentiation into osteoblasts to induce biological calcification in vessels [26].In this case, mechanisms underlying ectopic ossification by the trauma caused by tendon tear are unclear.We observed mTOR phosphorylation in tenocytes at resection stumps of tenotomy sites followed by the appearance of Safranin-O-positive chondrocytes and increased ALP activity, suggesting that mTOR activity stimulates tenocyte transdifferentiation into osteoblastic cells in manner similar to endochondral ossification.
In our study, we show that inflammation-related mTOR signaling promotes ectopic ossification and demonstrate that this activity is attenuated by blocking inflammation or mTOR signaling.We conclude that treatment with antiinflammatory agents could decrease ectopic ossification caused by tendon or ligament tear and benefit athletes or middle-aged or elderly people after injury.

Fig. 1
Fig. 1 Achilles tendon dissection promotes ectopic ossification at tenotomy sites in wild-type mice.a Real image and scheme showing tenotomy procedure for Achilles tendons performed in in 8-week-old wild-type female mice.The right Achilles tendon was dissected at the distal end to create a gap of 5 mm.The left side was sham operated.b and c At 1, 2, 3, 5 and 10 weeks after tenotomy, ectopic ossification was analyzed by 3D micro-CT.Ectopic ossification was detected at tenotomy sites by 3D micro-CT starting at 3 weeks after tenotomy.Tenotomy sites are highlighted by arrowheads.Data represent mean

Fig. 2
Fig. 2 Rapamycin suppresses ectopic ossification in vivo.a Achilles tendon tenotomy was performed in 8-week-old wild-type female mice.One week later, mice were sacrificed and both lower legs were removed, and tissue sections were stained with rabbit anti-phosphorylated mTOR antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG and observed under a fluorescence microscope.Scale bar = 100 μm.Dashed box indicates Achilles tendon dissection site.Yellow boxes indicate higher magnification images of dashed boxes.b The ratios of pmTOR-positive to DAPI-positive cells in red boxed areas were calculated.Data represent mean pmTOR-positive cells/DAPI-positive cells of rapamycin relative to DMSO-injected

Fig. 4
Fig. 4 Ectopic ossification seen following Achilles tendon tenotomy is suppressed in TNFα KO mice.a Achilles tendon tenotomy (tenotomy) or sham surgery (non-tenotomy) was performed in 8-weekold wild-type female mice.One week later, mice were sacrificed, and both lower legs were removed, and tissue sections were stained with rabbit anti-TNFα antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG, and rat anti-F4/80 antibody followed by Alexa Fluor 546-conjugated goat anti-rat IgG for analysis by fluorescence microscopy.Dashed circles indicate dissected sites of Achilles tendon.Scale bar = 100 μm.b The ratio of TNFα-positive to

Fig. 5
Fig. 5 Celecoxib or loxoprofen suppresses ectopic ossification by Achilles tendon tenotomy in vivo.a and b Achilles tendon tenotomy (tenotomy) or sham surgery (non-tenotomy) was performed in 8-week-old wild-type female mice, which were at the same time injected intraperitoneally with DMSO, celecoxib (10 mg/kg) or loxoprofen (10 mg/kg) for 5 days a week for 10 weeks.Ten weeks after surgery, when mice were 18 weeks old, 3D micro-CT analysis was performed (a) and ectopically formed ossifications were quantified (b).Data represent mean ectopic bone volumes of celecoxib-or loxoprofen-injected relative to DMSO-injected mice ± SD (each n = 5, p* < 0.01 versus DMSO group)