Alcohol‐induced Wnt signaling inhibition during bone fracture healing is normalized by intermittent parathyroid hormone treatment

Abstract Nearly half of orthopaedic trauma patients are intoxicated at the time of injury, and excess alcohol consumption increases the risk for fracture nonunion. Previous studies show alcohol disrupts fracture associated Wnt signaling required for normal bone fracture repair. Intermittent parathyroid hormone (PTH) promotes bone growth through canonical Wnt signaling, however, no studies have investigated the effect of PTH on alcohol‐inhibited bone fracture repair. Male C57BL/6 mice received two‐3 day alcohol binges separated by 4 days before receiving a mid‐shaft tibia fracture. Postoperatively, mice received PTH daily until euthanasia. Wnt/β‐catenin signaling was analyzed at 9 days post‐fracture. As previously observed, acute alcohol exposure resulted in a >2‐fold decrease in total and the active form of β‐catenin and a 2‐fold increase in inactive β‐catenin within the fracture callus. Intermittent PTH abrogated the effect of alcohol on β‐catenin within the fracture callus. Upstream of β‐catenin, alcohol‐treated animals had a 2‐fold decrease in total LRP6, the Wnt co‐receptor, which was restored with PTH treatment. Alcohol nor PTH had any significant effect on GSK‐3β. These data show that intermittent PTH following a tibia fracture restores normal expression of Wnt signaling proteins within the fracture callus of alcohol‐treated mice.

delayed fracture healing and reduced biomechanical strength of the callus tissue. [7][8][9] Thus, there is a need for adjunctive therapies to promote bone healing in patients deemed high risk for nonunion or those showing radiographic signs of delayed union.
Parathyroid hormone (PTH) administered intermittently promotes bone growth and increases bone mass. 10 Teriparatide, recombinant human PTH , is currently FDA approved for the treatment of osteoporosis. 11 There is also clinical interest for the use of PTH as an adjunct to fracture healing. Indeed, several animal studies have demonstrated the benefits of PTH on fracture callus size and biomechanical properties. [12][13][14] A randomized controlled trial demonstrated PTH administration lead to significantly reduced healing time and improved functional status in osteoporotic pelvic fractures. 15 However, the effect of PTH on fracture healing in alcohol-treated animals has yet to be described.
Canonical Wnt signaling is important for bone development, homeostasis and fracture repair. 16 Activation of the Wnt receptor results in the activation and stabilization of downstream effector, β-catenin. 16 Activated β-catenin translocates to the nucleus and promotes the transcription of genes involved in bone formation and regeneration. 16 Canonical Wnt/ β-catenin signaling tightly regulates the differentiation of mesenchymal stem cells into bone (osteoblasts)-and cartilage (chondrocytes)producing cells that initiate repair and make up the fracture callus. [17][18][19] Our laboratory has previously shown that alcohol exposure in rodents prior to a fracture promotes the phosphorylation of β-catenin, targeting the protein for degradation and reduces total β-catenin in the fracture callus. 20 As a result, alcohol inhibits callus formation and decreases the biomechanical strength of the fracture callus. 20 Interestingly, intermittent PTH has been shown to activate Wnt signaling and promote differentiation of mesenchymal stem cells into osteochondral lineages. 21,22 Based on this information, the goal of this study was to investigate whether intermittent PTH administration can be used as a targeted molecular therapy to counteract the deleterious effects of alcohol on fracture callus formation. We hypothesize that intermittent PTH administration following a fracture would counteract the effect of alcohol on fracture healing by restoring Wnt/β-catenin signaling during fracture repair.

| Binge alcohol model
A total of 23 male C57BL/6 mice 6-7 weeks of age were obtained from Jackson Laboratory (Bar Harbor, Maine) and acclimated to the facility for one week. The animal facility is a ventilated environment with a 12hour:12hour light-dark cycle at a constant temperature of 21°C and the animals had unrestricted access to food and water. Animals were then randomly assigned to one of four treatment groups: Saline/Saline (n = 6), Alcohol/Saline (n = 5), Saline/PTH (n = 6), and Alcohol/PTH (n = 6). The nomenclature for each group corresponds to the pre-fracture treatment (Saline or Alcohol) followed by the post-fracture treatment (Saline or PTH). The treatment paradigm is summarized in Figure 1. Briefly, animals received 3 consecutive daily intraperitoneal injections of a 20% (vol/vol) ethanol/ saline solution at a dose of 2 g/kg or equivalent volume of saline.
Following 4 days of abstinence, the alcohol binge regimen was repeated to simulate a binge drinking pattern. 9 At the time of fracture injury (1 hour after the last injection), blood alcohol average approximately 200 mg/dL. 23

| Post-fracture PTH administration
All animals received daily intraperitoneal injections of human  PTH (Bachem Inc, Bubendorf, Switzerland) or an equivalent volume of saline beginning at postoperative day (POD) one until euthanasia.
The dose of 40 μg/kg PTH was selected based on published rodent PTH dosing regimens. 24 Animals were humanely euthanized with CO 2 and tibia specimens collected for molecular analysis on POD 9.

| Molecular analysis
Tibia specimens were flash frozen in liquid nitrogen and stored at −80°C until use. Fracture callus tissue was separated from uninjured bone using a Dremel tool (Dremel Inc, Racine, WI, USA) and pulver-

| Statistical analysis
All data is expressed as the mean ± SEM and was analyzed using the SAS Version 9.4 (Cary, NC) statistical program. A Kruskal-Wallis test was used to assess for overall variability in the ratio among the four experimental groups for each analysis set. A P-value ≤.05 was noted as statistically significant.

| Effects of binge alcohol and parathyroid hormone on the activation state of β-catenin
Canonical Wnt signaling is propagated through downstream effector protein β-catenin. We have previously reported that alcohol-treated animals have reduced fracture callus total β-catenin compared to saline-treated animals. 20 In agreement with our previous studies, we observed callus total β-catenin decreased 50% in alcohol-treated animals as compared to saline-treated animals ( Figure 3A-B). Parathyroid receptor activation has also been reported to stabilize β-catenin and promote differentiation of mesenchymal stem cells into osteochondral lineages. 22 Postoperative administration of PTH significantly increased callus total β-catenin β-catenin is regulated by phosphorylation. Hypophosphorylated β-catenin is activated, promoting translocation of the protein to the nuclear to activate transcription, where hyperphosphorylated β-catenin is inactive and targeted for degradation through the proteasome. In agreement with our previous findings, alcohol-treated animals have a statistically significant reduction in callus hypophosphorylated β-catenin ( Figure 3C, P = .008) and a significant increase in hyperphosphorylated β-catenin as compared to saline-treated animals ( Figure 3D, P = .03). 20 Postoperative PTH treatment alone did not significantly increase hypophosphorylated β-catenin when compared to saline-treated animals ( Figure 3C), however, levels of hypophosphorylated β-catenin were restored to control levels in alcohol-treated animals with postoperative PTH (Figure 3C, P = .010). In agreement with our past studies, alcohol-treated animals had a significant increase in callus hyperphosphorylated β-catenin as compared to saline-treated animals ( Figure 3D, P = .03). 20 However, postoperative PTH did not significantly reduce hyperphosphorylated β-catenin in the fracture callus of alcohol-treated animals ( Figure 3D).

| Effects of binge alcohol and parathyroid hormone on the total LRP6
Low-density lipoprotein receptor-related protein 6 (LRP6) is a co-receptor and key component in canonical Wnt receptor activation and signal propagation. 25 We found that alcoholtreated animals had a significant reduction in callus associated LRP6 as compared to saline-treated animals ( Figure 4A P = .01).
Several studies have reported that LRP6 acts as a co-receptor for the PTH receptor (PTHr). 22,25,26 Binding of PTH to PTHr promotes the formation of PTHr/LRP6 complexes, which leads to rapid phosphorylation of LRP6 and activation of downstream β-catenin signaling. 25 To this end, we assessed whether PTH treatment restored LRP6 expression in the fracture callus of alcohol-treated animals. We found that intermittent PTH F I G U R E 3 β-catenin protein expression in callus tissue with or without intermittent parathyroid hormone treatment. (A) Representative hematoxylin and eosin stained histological samples from each treatment group. Fracture site is marked by a dashed black line. Western blot of (B) total β-catenin; (C) hypophosphorylated (active) β-catenin; and (D) hyperphosphorylated (inactive) β-catenin. Corresponding quantification is graphed underneath each plot. Data are mean ± SEM are represented as the densitometric ratio of each experimental group relative to the saline-treated animals. n = 5-6, P < .05, Kruskal-Wallis test. Groups not sharing a letter (eg a, b) are significant. Groups sharing a letter (eg a, a) are not significantly different administration rescued LRP6 expression in alcohol-treated animals.

| D ISCUSS I ON
Alcohol abuse has been implicated as a major risk factor for delayed fracture healing and nonunion in fracture repair. 4,8,28,29 This is of particular importance, as it is estimated that between 25%-50% of orthopaedic trauma patients present acutely intoxicated. 5,6 Current options for treating fracture nonunion include internal and external fixation and autogenous or allogenic bone graft, however, each option has serious limitations. Thus, there is a significant need for adjunct therapeutics to surgical intervention which can support and promote fracture healing in individuals with an increased risk for nonunion. Intermittent PTH treatment has been reported to be well tolerated in animal and clinical studies, with supporting evidence for its role in fracture healing. [30][31][32][33][34] The current investigation explored whether intermittent PTH could counteract the negative effects of alcohol on canonical Wnt/β-catenin signaling in a binge alcohol model of deficient fracture repair. The Wnt/β-catenin signaling pathway plays a pivotal role in fracture healing as it is required for the differentiation of mesenchymal stem cells into cartilage-and boneproducing cells. 35 Furthermore, it has been reported that β-catenin signaling is tightly regulated in the early phases of fracture healing and alterations to β-catenin signaling can have positive or negative effects on fracture healing. 35 In this study, we found that intermittent PTH rescued several key components of callus associated canonical Wnt/β-catenin signaling that were perturbed in alcohol-treated animals. F I G U R E 4 LRP6 and GSK-3β protein expression in callus tissue with or without intermittent parathyroid hormone treatment. Western blot of (A) low-density lipoprotein receptor-related protein 6 (LRP6); (B) total Glycogen synthase kinase 3 beta (GSK-3β); (C) inactive GSK-3β (phospho-S9); and (D) active GSK-3β (phospho-Y216). Corresponding quantification is graphed underneath each plot. Data are mean ± SEM are represented as the densitometric ratio of each experimental group relative to the saline-treated animals. n = 5-6, P < .05, Kruskal-Wallis test. Groups not sharing a letter (eg a, b) are significant. Groups sharing a letter (eg a, a) are not significantly different Parathyroid hormone acts as an anabolic bone agent when delivered intermittently and several randomized controlled trials have been performed to determine its effect on fracture healing and spinal arthrodesis. 15,[36][37][38][39][40] The results thus far have been equivocal. formation. 20,[42][43][44] Based on this information, we hypothesized that intermittent PTH treatment would normalize the effects of alcohol on Wnt signaling during fracture healing. We carried out our analysis on postoperative day 9 fracture callus tissue, which has been determined to be peak cartilaginous callus formation. 45,46 In agreement with our previous findings, we found that alcohol decreased fracture callus hypophosphorylated β-catenin and increased hyperphosphorylated β-catenin. 9,20,47 These comparisons were used to experimental validate our present study. Alcohol-treated animals receiving PTH had 65-fold increase in hypophosphorylated, active β-catenin as compared with animals treated with alcohol alone. These results are in agreement with prior studies that show PTH activates βcatenin, and suggests that intermittent PTH stimulation can prevent the negative effects of alcohol on Wnt/β-catenin signaling. 21 When our alcohol-treated mice were given PTH, we observed a recovery of total and hypophosphorylated β-catenin expression in the fracture callus. These data suggests that the administration of PTH ameliorates the effect of alcohol on β-catenin signaling in fracture callus of alcohol-treated animals.
Administration of intermittent PTH has been found to stimulate bone formation. 24,31 Binding of PTH to the PTHr leads to PTHr and LRP6 complex formation on the cell surface. 22,25,26 Previous studies have found that complex formation between PTHr and LRP6 leads to rapid activation of LRP6 and activation of downstream βcatenin. 25 Due to its role in stabilizing β-catenin, LRP6 is of particular importance in the fracture repair pathway. 48 Importantly, phosphorylated LRP6 has been found to be upregulated acutely during fracture healing. 35 In our investigation we found a statistically significant decrease in the amount of LRP6 in alcohol-treated animals as compared to the saline-treated animals. To date, no prior studies have described the effect of alcohol on LRP6 expression within the fracture callus. We have previously reported that alcohol decreases LRP5 in the bones of alcohol-treated mice. 49 Combined with our current findings, our observations suggest that alcohol downregulates LRP5/6, which is elevated acutely in a fracture, inhibiting Wnt/ β-catenin signaling. When alcohol-treated animals are administered intermittent PTH, we observed a 2-fold increase in callus LRP6 as compared to alcohol-treated animals. These findings suggest that one mechanism by which intermittent PTH might rescue β-catenin signaling within the fracture callus of alcohol-treated animals is by upregulation of LRP6 expression. The observation that intermittent PTH does not further increase LRP6 expression in saline-treated animals may suggest that LRP6 is already maximally expressed within the fracture callus during normal fracture healing.
β-catenin is negatively regulated by the APC, axin and GSK-3β complex by hyperphosphorylation, which targets β-catenin to the proteasome for degradation. 50 Within that complex, active GSK-3β (phospho-Y216) phosphorylates β-catenin. Phosphorylated LRP6 has been shown to bind GSK-3 complexes, inhibiting GSK-3 phosphorylation of β-catenin. 51 We have previously reported that active GSK-3β (phospho-Y216) is increased in the fracture callus of alcohol-treated animals, and that these effects were mitigated with post-operative treatment with GSK-3β inhibitor lithium chloride. 47 We observed a similar trend in levels of active GSK-3β (phospho-Y216, p=0.07)* in our alcohol-treated animals as compared to saline-treated animals. Further studies need to be done to determine if intermittent PTH reduces active GSK-3β (phospho-Y216) within the fracture callus of alcohol-treated animals.
In a process that resembles endochondral ossification, mesen- callus that forms during fracture healing. Additionally, this study was limited by the relatively small sample size of mice, leading to some of the variability in the dataset. Finally, the results of all the analyses are at a single time point in the complex process of fracture healing. Previous research show β-catenin expression remains elevated for weeks after fracture, however, levels in a fracture callus reach maximal expression on post-injury day 9. 35 Having multiple post-injury time points would have allowed for a temporal analysis into the effects of intermittent PTH on the fracture callus of alcohol-treated animals.
In summary, the current work adds new information about the effects of intermittent PTH on Wnt/β-catenin signaling within the fracture callus of alcohol-treated animals. Intermittent PTH should continue to be investigated as a useful adjunct to fracture union given the scarcity of other clinically available systemic therapies.

| CON CLUS IONS
Alcohol exposure inhibits Wnt signaling within the fracture callus.
Administering intermittent PTH following a tibia fracture in alcoholtreated mice restores normal expression of Wnt signaling proteins within the fracture callus.

ACK N OWLED G EM ENTS
Research reported in this publication was supported by the National Institute on Alcohol Abuse and Alcoholism of the National Institutes of Health under award numbers R21AA025551 (R21) to JJC and T32AA013527 (T32) to JME, and an AO North America Resident Research grant (AONAR) to TJF. R21 funding provided the location for experiments and animal feeding. The R21,T32 and AONAR provided the fund for design of the study and collection, analysis and interpretation of data. All of these funding supported the writing of the manuscript.

AUTH O R CO NTR I B UTI O N S
EMK and TJR performed and analyzed experiments. EMK, TJR, AT and JME analyzed data and participated in manuscript writing. All authors read and approved the final manuscript. All authors agree to be accountable for all aspects of the work.