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

Abstract 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.

AMP-independent activation of AMPK has been delineated which involves lysosomes under glucose deprivation. 3,4 The activation of AMPK attenuates anabolism and stimulates catabolism, resulting in preservation of energy for acute cell survival programme and generation of more ATP to cope with stress. As AMPK activation enhances glucose uptake and insulin sensitivity, it is a well-received drug target for metabolic syndrome, type 2 diabetes and cancer. 5 Metformin, a widely used oral drug for type 2 diabetes mellitus, has been reported to have multiple functions. The action of metformin includes inhibition of hepatic gluconeogenesis and enhancement of insulin sensitivity by stimulating glucose uptake in muscle and adipose. 6 It is known that many effects of metformin are mediated by AMPK. Furthermore, pre-clinical and clinical studies have shown that metformin is a promising drug as an adjuvant or neoadjuvant agent for cancer therapy. [7][8][9] Over the last decade, metformin has been documented to regulate osteogenic differentiation through AMPK-dependent and independent manner. 10,11 AMPK is inhibited through the increased glucose uptake during the differentiation, an event that is suppressed by metformin. 12,13 Heterotopic ossification (HO) refers to aberrant formation of extra-skeletal bone in muscle, tendons and ligaments, which often occur after musculoskeletal trauma, severe burns and hip arthroplasty. 14 HO causes chronic pain and loss of joint mobility and severely impairs the quality of life. 15 At present, there is no effective management for HO, except that non-steroid anti-inflammatory drugs (NSAID) and local irradiation are used for prophylaxis. 16 The only option is to surgically resect the symptomatically established heterotopic bone. However, the risk of ectopic bone recurrence is even greater after surgical excision. 17,18 Consequently, there is unmet need for prophylaxis and treatment of HO. A critical step is to identify key targets implicated in HO and then develop intervention approaches.
The bone morphogenetic proteins (BMPs) signalling pathway plays an essential role in osteogenic differentiation and osteo-induction. 17,19 BMPs bind to the type I and type II receptor complex, triggering the type II receptor to phosphorylate and activate the type I receptor, which then elicits Smad and non-Smad signalling cascades via phosphorylation events that regulate transcription of target genes. Smad1/5/8 forms a complex with Smad4 upon phosphorylation by the type I receptor, and the complex then translocates into the nucleus and activates the transcription programme. 20 Smad6 is a negative regulator specific to the BMP signalling. It disrupts the interaction between activated type I receptor with Smad1/5/8 and/ or induces their degradation via ubiquitination pathway when it recruits Smurf1. 12,21,22 The BMP signalling pathway is also involved in many human diseases, particularly in HO formation. 23 For example, fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder characteristic of progressive HO formation, arises from mutations of the BMP type I receptor ALK2, 95% of which are R206 to H mutation resulting in a constitutive activation of the receptor and Smad1/5-mediated signalling events essential for osteogenesis. 24 Furthermore, the BMP signalling pathway is often aberrantly activated by inflammation secondary to traumatic injury and contributes to pathogenic HO. 19,25 BMP2 has also been shown to trigger HO in animal models. 25

| Cell culture and transfection
The MC3T3-E1 cell line (CRL 2593 subclone 4) was purchased from American Type Culture Collection (Manassas, USA) and cultured in Eagle's minimum essential medium alpha (α-MEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin and streptomycin at 37°C in a humidified atmosphere supplied with 5% CO2. siR-NAs were transfected into the MC3T3-E1 cells with Lipofectamine 2000 according to instructions provided by the manufacturer.

| Adenovirus preparation and cell infected
Adenovirus expressing the constitutively active or dominant negative mutant of AMPKα1 was prepared as described previously. 29 Adenovirus expressing GFP was prepared as a control. The multi-

| Osteogenic differentiation assays
Osteogenic differentiation was induced in the MSCODM, which was changed every 3 days. After 7 days, the ALP staining was performed Alizarin deposit was dissolved in solution containing 0.5 N HCL and 5% SDS and read at 405 nm absorbance. This study was performed in accordance with National Institutes of Health Guide for Animal Care. Trauma/burn induced HO model was generated by performing Achilles tenotomy plus burn injury, as previously described. 30,31 Briefly, 8 weeks old female C57BL/6 mice were subjected to a right hindlimb Achilles tenotomy under general anaesthesia with intraperitoneal injection of chloral hydrate (300mg/kg) and subsequent burn injury on the dorsum. A 60°C metal block was applied to the dorsum for 20 seconds. All mice were monitored and kept under standard conditions. Next day after surgery, mice were treated with metformin (100mg/kg, i.p.) in PBS or PBS as vehicle every 3 days for 8 weeks (vehicle = 6, metformin = 5). The health and well-being of mice were monitored during the study. The CO 2 was used to kill mice at the end of experiment.

| Micro-computerized tomography analysis
Eight weeks after Achilles tenotomy, the injured hindlimbs were col-

| Histological analysis
Eight weeks post-injury, the hindlimbs were collected after eutha-

| Protein extraction and Western Blot
Total proteins isolated from injured tissue or cells were prepared in RIPA lysis buffer plus protease inhibitors. The extracts were centrifuged at 12 000×g, 4°C for 15 minutes. Western blot was conducted using the standard protocol as described previously. 28 Equal amounts of proteins (20 μg) were resolved onto SDS-PAGE and transferred to PVDF membranes (Millipore Sigma). The membranes were incubated with primary antibodies at 4°C overnight and then HRP-conjugated secondary antibodies (Thermo Fisher Scientific) at room temperature for 1 hour. The blot signals were detected with ECL reagent.

| Statistical analysis
Data were analysed using GraphPad Prism software. All quantitative data were calculated as the mean ± standard deviation (SD).
Difference between groups was assessed with Student's t test, and P < .05 was set for significance.

| Metformin reduces BMP signalling
To explore the effect of metformin on BMP signalling pathway, MC3T3-E1 cells were treated with metformin at different doses or for different hours, and then followed with BMP6 for 30 minutes.
The results revealed that BMP-induced Smad1/5 phosphorylation was markedly offset by metformin ( Figure 1A&B), which was asso- The results clearly revealed that Smad6 was induced by metformin ( Figure 1C&D). To ascertain whether AMPK mediates the effect of metformin, we used the adenovirus expressing a constitutively active mutant of AMPK or GFP as a control. Our study revealed that the infection with the active form of AMPK progressively reduced phosphorylation of Smad1/5 induced by BMP6, concomitant with elevation of Smad6 ( Figure 1E). Next, we transfected Smad6 siRNA into MC3T3-E1 cells and found that knockdown of Smad6 blunted the inhibitory effect of metformin ( Figure 1F). Altogether, our results demonstrated that metformin via activation of AMPK up-regulated Smad6 and thus suppressed BMP-Alk2-Smad1/5 signalling event.

| Metformin suppresses osteoblast differentiation
We found that AMPK activity was inhibited during the course of osteogenic differentiation of MC3T3-E1 cells (Supplementary Figure S1). This finding together with those shown above suggests that forced AMPK activation could suppress osteogenic differentiation by either pharmacological agents or its active mutant. To test this hypothesis, we cul-  Figure 3C&D).
The partial blocking effect of the dominant negative AMPK suggests two possibilities: first, its amount was not sufficient to compete with endogenous wild-type counterpart, and second, metformin takes effect through both AMPK-dependent and independent mechanisms.
Finally, to assess whether AMPK affects mineralization, the late stage of osteoblast differentiation. As expected, the results showed that metformin significantly suppressed mineralization (Figure 4).

| Metformin inhibits trauma-induced heterotopic ossification
To determine whether metformin prevents HO induced by trauma, mice received Achilles tenotomy and burn in dorsum. One day after F I G U R E 2 AMPK activators inhibit alkaline phosphatase activity during osteogenic differentiation. MC3T3-E1 cells were incubated with osteogenic differentiation medium in the presence of different doses of metformin (Met) and Ibuprofen. After one week, the Alkaline phosphatase activity (metformin (Met) (A) and Ibuprofen(C)) was measured.
The graph represents averages of three independent experiments (mean ± SD, N = 3). *Significant difference from metformin (A) and Ibuprofen(C) versus control was assessed using Student's t test (*P < .05). Western blot (metformin (B) and Ibuprofen(D)) was also performed to study activation of AMPK and Smad1/5. NM: no differentiation medium, α-MEM complete medium all these tissues were absent in metformin-treated mice ( Figure 6A).
However, the bone mineral density and trabecular number of ectopic bone were the same in the treated and untreated groups (data not shown). In addition, we examined AMPK phosphorylation and Smad6 in the muscles around the Achilles tendon. Metformin treatment significantly increased AMPK phosphorylation and the level of Smad6 and Smurf1, concurrently with the reduction of ALK2 ( Figure 6B&C).
All these results demonstrate that metformin significantly reduces HO development following the Achilles tenotomy and burn.

| D ISCUSS I ON
In this study, we showed that metformin inhibited BMP signalling hydrolysis apyrase prevents HO through inhibition of the BMP signalling pathway. 26 Here, we showed that metformin reduced HO formation by activation of AMPK and subsequent inhibition of the BMP signalling pathway. The present findings are in line with our previous study showing that metformin inhibits Smad1/5 signalling downstream of the active mutant of ALK2 (R206H) identified in FOP patents in association with reduced osteogenesis in vitro. 28 As metformin is widely recognized as a safe and inexpensive drug, it will be attractive to repurpose its clinical application to the prevention and treatment of HO.
It has been controversial about the role of AMPK in osteogenesis and osteoclasterosis. [34][35][36] The discrepant findings might be attributable to differences in cell context and settings. Osteogenesis is a process that requires a large amount of ATP and glucose supply for anabolism, which leads to mTOR activation and AMPK inhibition, the two opposite fuel-sensing systems. 37  and BMP in mice. [56][57][58] Inhibition of MMP-9 activity by minocycline effectively prevented the HO formation in mice. 57 Several studies have showed that AMPK negatively regulates MMP-9 expression and activity. 55,59,60 Our data revealed that metformin attenuated HO in mice and induced activation of AMPK. It is possible that administration of metformin suppressed MMP9 expression via activation of AMPK, leading to prevention of HO. It will be interesting to investigate whether MMP-9 plays a role in mediating the effect of metformin/AMPK on HO.
In the end, we should point out that although we did not have direct evidence that metformin through inhibition of BMP/ALK2 attenuates HO in animal model, we observed increased expression of Smad6 and decreased abundance of ALK2 in association with AMPK activation. Therefore, we are confident that metformin through AMPK-Smad6 regulatory axis suppresses HO. Thus, our study supports the rationale to retask metformin for prevention and treatment of HO. As it is cheap and safe, it would have a great potential in clinical use for this disease. to DH) and Innovation and Entrepreneurship grant from Jiangxi Province Bureau of Foreign Experts (to ZL).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.