Trauma induced heterotopic ossification patient serum alters mitogen activated protein kinase signaling in adipose stem cells

Abstract

Post-traumatic heterotopic ossification (HO) is the formation of ectopic bone in non-osseous structures following injury. The precise mechanism for bone development following trauma is unknown; however, early onset of HO may involve the production of pro-osteogenic serum factors. Here we evaluated serum from a cohort of civilian and military patients post trauma to determine early induction gene signatures in orthopaedic trauma induced HO. To test this, human adipose derived stromal/stem cells (hASCs) were stimulated with human serum from patients who developed HO following trauma and evaluated for a gene panel with qPCR. Pathway gene analysis ontology revealed that hASCs stimulated with serum from patients who developed HO had altered gene expression in the activator protein 1 (AP1) and AP1 transcriptional targets pathways. Notably, there was a significant repression in FOS gene expression in hASCs treated with serum from individuals with HO. Furthermore, the mitogen-activated protein kinase (MAPK) signaling pathway was activated in hASCs following serum exposure from individuals with HO. Serum from both military and civilian patients with trauma induced HO had elevated downstream genes associated with the MAPK pathways. Stimulation of hASCs with known regulators of osteogenesis (BMP2, IL6, Forskolin, and WNT3A) failed to recapitulate the gene signature observed in hASCs following serum stimulation, suggesting non-canonical mechanisms for gene regulation in trauma induced HO. These findings provide new insight for the development of HO and support ongoing work linking the systemic response to injury with wound specific outcomes.

1 |. INTRODUCTION

Heterotopic ossification (HO) is the ectopic formation of bone in non-osseous structures such as fat, joints, muscle, perivasculature, and skin (Bossche & Vanderstraeten, 2005). Over the course of the Afghan and Iraq conflicts, the prevalence of high impact blast injuries among injured military personnel highlighted the importance of HO as a common clinical problem for military medicine (Alfieri, Forsberg, & Potter, 2012; Forsberg & Potter, 2010; Forsberg et al., 2009). While HO is a recognized co-morbidity in civilian patients undergoing hip and elbow surgeries electively or suffering from severe burns and traumatic brain injury (TBI), civilian orthopaedic surgeons see this complication far less frequently than their military counterparts (McCarthy & Sundaram, 2005; Rath et al., 2013). Nevertheless, when HO occurs, it presents a substantial health burden for both civilian and military orthopaedic patients. Presumably, a better understanding of the mechanisms underlying the development of HO will improve both its prevention and treatment.

To date the exact instigating cause of HO remains to be identified; however, certain correlative events precede the development of ectopic bone in HO patients. First, a precipitating event or trauma must occur, followed by release of proteins and inflammatory signals into the soft tissues. Subsequently there is infiltration of mesenchymal stem cells and transformation of non-osseous tissues into bone (Mavrogenis, Soucacos, & Papagelopoulos, 2011; McCarthy & Sundaram, 2005). Following these induction signals, HO will persist as long as there is an environment to maintain bone formation. Recent evidence from studies of military blast induced HO suggest that this environment arises from a persistently maintained hyper-inflammatory response and increased pro-osteogenic signaling factors (Evans et al., 2012, 2014; McCarthy & Sundaram, 2005). The enhanced pro-inflammatory signals manifest as an elevation in serum cytokine levels, specifically interleukin 6 (IL6), interleukin 10 (IL10), and related factors (Cavaillon & Conquy, 2006; Evans et al., 2012; Krakauer, 2001). Consistent with these observations, the healing process following orthopaedic trauma is accompanied by a surge of pro-inflammatory responses (Hughes, Hendricks, Edwards, Bastawrous, & Middleton, 2013; Sears, Strover, & Callaci, 2009). Together, this indicates that serum factors may facilitate ectopic bone formation.

In support of a humoral factor being involved in HO formation, ectopic bone is reported to form following severe trauma to the nervous system or sever burn. In some instances bone formation occurred in regions away from the primary sight of injury such as the joints or hip (Chen, Deng, & Li, 2012; Rodriquez-Carballo, Gamez, & Ventura, 2016). Current evaluation of HO in these instances strongly points to a humoral effect. The manifestation of HO can result in functional impairment, pain, loss of range of motion, and improper prosthesis fitting (Fiori, Billings, de la Pena, Kaplan, & Shore, 2006). As the primary instigating events leading to HO are currently unknown, there is no effective treatment for the prevention of HO formation. Currently therapies for the treatment of HO consist of prophylaxis through the administration of non-steroidal anti-inflammatories or radiation therapy(Fiori et al., 2006; Junttila et al., 2007; Pramanik et al., 2003). In the instance of military personnel, these are not always readily available at time of injury. Furthermore, failure to mediate HO formation results in requirement of surgery to remove ectopic bone. Ideally, the identification of pathways activated during the development of HO would lead to more viable treatment options. HO presents as a debilitating disease resulting in loss of function, pain and issues with prosthesis. Due to the evidence suggesting a humoral component to the formation of HO and the need to identify novel pathways for therapeutic intervention, we sought to evaluate the effect of serum from individuals who developed HO on human adipose derived stem cells (hASCs). The primary goal of the research performed here was to identify a serum induced signaling cascades and determine if serum from individuals with HO altered cellular proliferation and osteogenesis of hASCs.

2 |. MATERIALS AND METHODS

2.1 |. Patient populations

The exclusion criteria and demographic characteristics of the 12 patients with acute extremity injury is listed in Supplemental Table S2 and S3, respectively. Patient age ranged from 20 to 46 years (26.93 ± 2.06 years). In addition, male pooled no trauma serum obtained from blood bank donations and determined to be clinically normal were used as control group. Four groups of subjects were studied: (1) civilian injured patients that developed HO (cHO⁺); (2) civilian injured patients that did not develop HO (cHO⁻); (3) injured military service members that developed HO (mHO⁺); and (4) injured military service members that did not develop HO (mHO⁻). To account for instigating effects from trauma alone, all comparisons were made to serum from no trauma healthy age-matched volunteer serum (control). HO positivity was defined as radiographic evidence of soft-tissue bone formation within 4 months post injury. The study protocol was approved by the Institutional Review Board at Louisiana State University Health Sciences Center–New Orleans, Walter Reed National Military Medical Center, and Tulane University (Western IRB Protocol # 20130449) in compliance with all applicable Federal regulations governing the protection of human subjects.

2.2 |. Serum collection

Whole blood samples were drawn within 1–7 days after injury onset (military: first debridement 3–7 days and civilian: 24–48 hr post trauma) from either an arterial line or by peripheral venous puncture and collected in sterile Vacutainer tubes with SST Gel and Clot Activator (Becton Dickinson, Franklin Lakes, NJ). After centrifugation (1,200 rpm for 15 min at 4 °C), serum was collected, split into 200 μl aliquots and then stored at −80 °C until used for further analysis.

2.3 |. Human adipose stem/stromal cell donors

hASC were derived from subcutaneous abdominal adipose tissue donated with written informed consent under a Western Institutional Review Board approved protocol by subjects undergoing elective liposuction as previously described³⁶. All donors were female, average BMI < 25 ± SD 2.33, and had an age range of 48.33 ± SD 11.02.

2.4 |. Cell culture

Serum stimulation experiments done with pooled hASCs, n = 3 and passages of 2–4. Osteogenic qPCR and cytokine (BMP2, IL6, Forskolin, WNT3A) (R&D Systems, Minneapolis, MN) stimulation was performed with individual donors, n = 3 donors. hASCs were cultured in complete culture medium (CCM): αMEM supplemented with 10% FBS (Hyclone, Marlborough, MA), 1% antibiotic, and 2 mM L-glutamine. For osteogenic differentiation, CCM was replaced with osteogenic differentiation medium (ODM): αMEM media supplemented with 10% FBS, 1% antibiotic, 10 nM dexamethasone, 10 mM b-glycerolphosphate, and 50 μg/ml ascorbate-2-phosphate (Sigma–Aldrich, St. Louis, MO). Dextran stripped FBS supplemented media cell culture was performed for Western blot and qPCR prior to serum stimulation and cytokine stimulation. Serum starvation was performed for all experiments as follows: CCM media was removed; cells were washed with PBS and fed with 5% dextran stripped FBS in phenol free alpha-MEM for 48 hr.

2.5 |. Osteogenic differentiation assay

Pooled hASC (n = 3 donors) were seeded in 96-well plates at 5 × 10³ cells/well in CCM. Once confluent, CMM was replaced with either fresh CMM, ODM, or ODM supplemented with 5% pooled patient serum. Plates were carefully aspirated and fed every 3 days with appropriate media. After 3 weeks, culture medium was aspirated and adherent cells washed with D-PBS, formalin (10%) fixed, and stained with 3% alizarin red solution (pH 4.3). Microscopic evaluation (40X) of calcium mineral deposition in osteogenic cells. Quantitative analysis of alizarin red staining in each culture, absorbance (584 nm) following de-staining with 10% cetylpyridinium chloride monohydrate (Aldrich–Sigma, St. Louis, MO) for 60 min.

2.6 |. Quantitative PCR analysis

Pooled hASCs (n = 3 donors) were seeded at 2.5 × 10⁵ cells/60 mm dish (21 cm² area) in CCM. Cells were grown to confluence and then serum starved for 48 hr and stimulated with αMEM supplemented with 5% pooled human serum and no additional supplementation of FBS, and 1% antibiotic for 4 and 24 hr. RNA was isolated using the RNeasy Kit (Qiagen, Valencia, CA) and 1 μg RNA was used to make cDNA (iScript-BioRad, Hercules, CA). qPCR was performed on an iQ5 with SYBR green (BioRad). Primer sequences are listed in Supplemental Table S4. Controls were CYCB gene and hASCs treated with serum from a no trauma healthy age-matched donor (control-nonHO).

2.7 |. Western blot analysis

Pooled hASCs (n = 3 donors, 1 × 10⁶ cells) were plated in CMM in a 10 cm² culture dish and grown to confluence prior to serum starvation for 48 hr and subsequently stimulated with 5% human serum in alpha-MEM with 1% antibiotic for 24 hr. Cells were lysed with MPER lysis buffer (Thermo Scientific, Waltham, MA) plus 1% Halt protease and phosphatase (cocktail I and II) inhibitors (Thermo Scientific). Total cell lysates were loaded on a 4–12% gel and then transferred to nitrocellulose. Blots were blocked in 5% BSA (Sigma–Aldrich) for 1 hr. Primary antibodies diluted 1:1,000 and expose to blot for 1 hr. Blots were washed 3Xs in PBS for 10 min each. Secondary fluorescent antibody (LI-COR, Lincoln, NE) diluted 1:10,000. Blot was washed 3Xs for ten minutes with PBS. Blots were imaged on LI-COR Odyssey fluorescent imager (LI-COR). Stimulation experiments were normalization to RhoGDI-α and osteogenic differentiation experiments were normalized to GAPDH. Control stimulation was serum from NO-trauma health age-matched volunteers (control-nonHO), designated as “1.” Antibodies from Cell Signaling (Beverly, MA)

2.8 |. Pathway analysis

Pathway analysis was performed through Pathway Integration Database (PID, http://pid.nci.nih.gov) (National Cancer Institute PID). The query tool was used to identify genes observed to be changed in PCR panel and the predominant molecular pathway was identified. PID is a compiled pathway integration database using reviewed journal publications and formed network maps based on known interactions. A p-value is given denoting level of confidence for association to that pathway.

2.9 |. Cell growth/proliferation assay

hASCs (n = 3 donors) were seeded in 96-well plates at 5 × 10³ cells/well in CCM. One plate was collected for day 0 comparison and stained with 0.1% crystal violet (CV). Cells were then collected after an addition24 hr, 48 hr, and 5 days and stained with CV stained. hASC were either grown in CCM or Osteogenic media supplemented with MAPK inhibitor or 5% human serum with no additional serum supplementation. At day of collection CV stained wells were washed with water and dried for 24 hr. Cells were lysed with 33% SDS and absorbance (570 nm) was read on a Gen5 microplate plate reader (BioTek, Winooski, VT). Normalization for each treatment was to respective day-0 measurement.

2.10 |. Statistics

Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software Inc., La Jolla, CA). Statistical significance between groups was determined by student t-test. All assays were performed in triplicate. Group means ± standard errors of the mean (SEM) were calculated for each parameter. Differences were statistically significant p-value < 0.05.

3 |. RESULTS

3.1 |. The AP1 and MAPK cascades are selectively induced in primary hASC following stimulation with serum from injured patients with heterotopic ossification

Trauma induced HO is a common co-morbidity in the military setting and less frequently reported following civilian orthopadeic surgery. Due to this our initial aim was to identify early gene signatures induced through humoral signaling in individuals in the civilian (c) and military (m) setting who developed HO. To determine this, normal non-injured hASC were stimulated for 24 hr with 5% pooled serum from either individuals who sustained injury and developed HO (HO⁺) or sustained injury but did not develop HO (HO⁻). Comparison for all trauma groups was to hASCs stimulated with uninjured/no-trauma serum (designated as “control”). Following exposure to serum, the expression of osteogenic and wound healing related genes was quantified by Quantitative RT-PCR (qPCR). Results demonstrate that hASCs stimulated with pooled patient serum from the cHO⁺ and mHO⁺ cohorts had alterations in gene expression, however an enrichment for pro-osteogenic signaling pathways was not directly indicated or evident (Supplemental Table S1). To determine if there was a correlation with altered gene expression and a specific cellular pathway, all genes observed to be altered at the 24 hr time point were evaluated for pathway correlation with the NCI Pathway Interaction Database (PID) (National Cancer Institute PID, 2016; Schaefer et al., 2009). This platform identified enriched pathways associated with “The AP1 Transcription Factor Network” “Transcriptional Targets of FRA1 and FRA2” “ALK1/2 Signaling”, “uPA and uPAR- Signaling”, and “Regulation of βCatenin Transcription” (Figure 1a). Interestingly while not traditionally considered a pro-osteogenic pathway, many bone-associated genes were identified as downstream transcriptional targets of the AP1 complex (Figure 1b). Pathway signatures observed in our gene panel array were confirmed for genes associated with AP1 (FRA1, FRA2, JUNB, FOS, FOSB) and ALK2 (SMAD1/5/7/8) signaling cascades for both 4 and 24 hr stimulation of hASCs with 5% civilian patient serum from cHO⁺ and cHO⁻ (Figures 2a and 2b). Serum from cHO⁺ individuals significantly increased FOSB, SMAD5, and SMAD8 gene expression and significantly repressed FOS gene expression in hASCs (Figures 2a and 2b). Next serum from military patients that did and did not develop HO (mHO⁺ and mHO⁻) was evaluated for AP1 family members following 24 hr stimulation. mHO⁺ serum significantly repressed AP1 family member FOS (Figure 2c). Additionally, there was a trend for increased FRA-1 and JUNB gene expression in mHO⁺ stimulated ASCs (Figure 2c). Western blot for AP1 and SMAD5 protein expression demonstrated enhanced FRA1 protein expression following stimulation of hASCs with all trauma exposed serum (cHO⁻, cHO⁺, mHO⁻, mHO⁺) serum compared to control serum, with cHO⁺ and mHO⁺ groups demonstrating the greatest increases (Figure 2d–f). Both cHO⁻ and mHO⁻ demonstrated elevated FRA1 expression levels (Figures 2d and 2f). FOS protein expression was not detectable by Western blot in any stimulation experiment (data not shown). Stimulation of p-SMAD5 with cHO⁻ and cHO⁺ serum resulted in increased phosphorylation in both cHO⁺ and cHO⁻ trauma groups compared to control; however, there was no detectable difference in p-SMAD5 levels between and cHO⁻ and cHO⁺ groups (Figure 2e). mHO⁻ and mHO⁺ serum repressed p-SMAD5 levels compared to no-trauma (control) serum (Figure 2f).

FIGURE 1.

Serum from patients with trauma induced HO activates a unique gene signature in hASCs. Confluent hASCs were stimulated with 5% human serum for 24 hr and qPCR was performed. (a) Pathway analysis with NCI pathway interaction database (PID) for all genes altered from the gene panel PCR, represented as −log(p-value) for significant pathways. (b) Gene list of top associated pathways from PID

FIGURE 2.

AP1 transcription factor network is elevated in patient with trauma induced HO. (a and b) qPCR conformation of hASCs stimulated with 5% civilian serum for (b) AP1 and (c) SMAD signaling. (c) qPCR conformation of hASCs stimulated with 5% military serum for AP1 genes. For qPCR control was NO-trauma control serum designated as “1” and CYCB, n = 4 (civilian) and n = 3 (military) individual donors, and error bar = SEM. (d and e) Western blot analysis of hASCs stimulated for 24 hr with 5% civilian serum from trauma individuals +/− HO for FRA1 and FOSB (e) and total/p-SMAD5 (f). (f) Western blot of hASCs stimulated with trauma induced 5% military serum +/− HO for 24 hr. For all Western blot experiments normalization was to Rho-GDIα and control uninjured human serum (5%). *Significantly different p < 0.05

Following interrogation of our PCR array, it is suggested that “The AP1 Transcription Factor Network” and “Transcriptional Targets of FRA1 and FRA2” would be top regulated pathways. Our qPCR results demonstrated only a significant repression of FOS in both the military and civilian cohorts (mHO⁺ and cHO⁺) compared to control serum. Expression of genes that contain an AP1 promoter can change without changes in gene expression of AP1 family members through an increase in AP1 activity. Interestingly genes FN1, PAI-1, and PLAUR all contain AP1 binding sites in their promoter regions and were observed to be regulated in “uPA and uPAR-Mediated Signaling” from our pathway data analysis. Due to this, we next evaluated transcriptional targets of the AP1 complex (FN1, PAI1, PLAUR) in hASCs following stimulation with 5% serum from cHO⁺ and mHO⁺ serum samples. Our results demonstrated that genes that contain an AP1 promoter region (PAI1 and PLAUR) trended to have higher expression levels following stimulation with both the mHO⁺ and cHO⁺ serum compared to control (Figures 3a and 3b). A significant increase in FN1 was observed in mHO⁺ serum treated hASCs (Figure 3b). To determine if these gene changes were a result of changes in upstream AP1 signaling, qPCR was used to interrogate gene expression changes in the mitogen activated protein kinase cascades (p38, ERK1/2, and JNK). Changes in gene expression associated with MAPKs upstream of AP1 were observed for the JNK pathway (JNK1 and MAP2K4), p38 pathway (MAPK12 and MAPK14), and ERK1/2 pathway (k-RAS and MEK1) following stimulation of hASCs with cHO⁺ serum compared to that of control and cHO⁻ groups (Figure 3c). Similarly, hASCs stimulated with mHO⁺ serum demonstrated changes in the MAPK components with a significant increase in MAPK14 (p38 pathway) and significant repression in MAP2K4 and MAPK12 (JNK pathway and p38 pathways, respectively) (Figure 3d). This suggests serum from individuals who develop HO preferentially enhance MAPK cascades in hASCs.

FIGURE 3.

MAPK signaling is altered hASC following in vitro stimulation using serum from military patients that develop HO. (a and b) qPCR for genes associated with AP1 transcriptional targets and UPA pathway following stimulation of hASCs with 5% human serum from civilian (a) and military (b). (c and d) qPCR for MAPK associated genes following stimulation of hASC with 5% human serum from civilian (c) or military (d). For all qPCR control was NO-trauma serum designated as “1” and CYCB gene. Experiments were repeated in n = 4 (civilian) and n = 3 (military) individual donors, error bars = SEM. *Significantly different p < 0.05

3.2 |. Pro-osteogenic stimulation of hASCs alters AP1 and MAPK

To determine if AP1 and MAPK enhanced signaling occurs during normal hASC osteogenic differentiation, hASCs were grown in osteogenic differentiation media and collected at intervals (4 hr, 24 hr, 48 hr, 1, and 2 wk). mRNA and protein levels of FOS, FOSB, and FRA1 were found to be upregulated in hASCs exposed to osteogenic induction media (Figures 4a and 4b). There was no significant change in MAPKs or FN1 gene expression observed following osteogenic induction (Figure 4c). Exposure of hASCs to osteogenic stimulatory (BMP2, Forskolin, WNT3A) and anti-osteogenic (IL6) molecules did not result in a gene signatures that correlated with the observed HO⁺ serum gene changes (Figures 4d and 4e). In addition, MAPK pathways were not activated by BMP2 or IL6 stimulation (Figure 4f). Phosphorylated JNK was not detected baseline or following stimulation with either BMP2 or IL6 (data not shown). Taken together this suggests that development of HO through humoral factors may arise from non-canonical bone forming pathways outside of traditional BMP2 and cAMP cascades.

FIGURE 4.

The MAPK cascade is altered during hASC osteogenic differentiation. (a and c) hASCs were grown in osteogenic media and collected at intervals (4 hr, 24 hr, 48 hrs, 1, and 2wks) post induction. (a) qPCR and (b) Western blot analysis for AP1 genes and protein expression, respectively. (c) qPCR for MAPK genes following osteogenic differentiation. Control was maintenance media and CYCB (PCR) and GAPDH (Western blot). Error bars = SEM, n = 3. (d and e) hASCs where stimulated with BMP-2, IL6, FRSK, or WNT3A for 24 hr and qPCR was performed for (d) AP1 and (e) MAPK gene expression. Control vehicle treated hASCs and CYCB gene. Error bars = SEM. *Significantly different p < 0.05. Representative of n = 3 (f) Western blot for phosphor- levels of −pan-SMAD1/5/8, −ERK1/2, −p-38, and −STAT3 stimulation following 30 min stimulation with BMP-2 and IL6. Control was RHO-GDIα

3.3 |. MAPK signaling regulates osteogenic differentiation in hASCs

To determine if a particular MAPK signaling cascade was involved in the regulation of osteogenesis, we next induced hASCs with osteogenic media and treated with inhibitors of MAPK signaling (ERK1/2-PD184352, p38-BIRB796, SAPK/JNK-SP600125). Results demonstrated that both inhibition of JNK and ERK1/2 signaling led to a repression of osteogenesis while p38 inhibition did not alter osteogenesis (Figure 5a). To determine if the observed decrease in osteogenesis was a result of decreased cellular proliferation we next performed CV proliferation assay on hASCs with and without osteogenic differentiation media in the presence of MAPK inhibitors specific to the p38, ERK1/2, and JNK pathways. Inhibition of the JNK and ERK1/2 pathways in both culture media and during osteogenic differentiation inhibited cellular proliferation of hASCs (Figures 5b and 5c, respectively). Since serum factors from orthopaedic trauma patients who subsequently developed HO demonstrated either induce AP1 family or altered MAPK gene expression in hASCs, we next sought to determine if stimulation of hASCs grown in the presence of serum from patients with HO would augment/accelerate osteogenic growth and mineralization in vitro. hASCs were grown in osteogenic media supplemented with 2.5% serum (cHO⁺, mHO⁺, cHO⁻, mHO⁻) or osteogenic media alone. The extent of osteogenic mineralization was similar between treatment groups (Figure 5d). Evaluation of the effects of serum from patients with HO on proliferation was evaluated by CV proliferation assay of hASCs grown in the presence of 10% serum (cHO⁺, mHO⁺, cHO⁻, mHO⁻) or CCM alone. Results demonstrate there was no change in cell growth (Figure 5e).

FIGURE 5.

MAPK signaling alters osteogenesis in hASCs. (a) Alizarin red stain for hASCs grown in osteogenic media for 2 wks in the presence of MAPK inhibitors for ERK1/2 (PD-184352), JNK (SP-600125), or p38 (BIRB-796) pathway, control was vehicle treated hASCs in osteogenic media. (b and c) Crystal violet assay for proliferation of hASCs grown in CCM media + MAPK inhibitors or (c) osteogenic media + MAPK inhibitors for 5 days. Results represent percent absorbance (570 nm) and normalized to day 0 for each treatment. (d) Alizarin red stain of hASCs grown in osteogenic media supplemented with 5% human serum form either the military or civilian cohort at end point (2 weeks) read at absorbance 570 nm. Control was osteogenic media without serum supplementation. HO, developed heterotopic ossification; E, early serum collection (24–48 hr post injury); L, late serum collection (1.5–2 month post injury). (e) Crystal violet proliferation assay of hASCs in osteogenic media + 5% human serum for 5 days. Results represent percent absorbance (570 nm) and control was day 0

4 |. DISCUSSION

The primary goal of the current research was to better define genes induced during the early phase of ectopic bone development. To achieve this we stimulated adult hASCs with serum from individuals with trauma induced HO and investigated the transcription regulation of the MAPK signaling families. While both the military and civilian cohorts demonstrated alterations to the MAPK signaling cascades, there were differences in regulated gene expression. This may provide insight to the mechanism driving the increased instances of HO in military versus civilian patients in the clinic, as the military population has reported higher levels of HO (Alfieri et al., 2012; McCarthy & Sundaram, 2005). Furthermore, differences in the two patient populations may be attributed to collection time of patient serum. There was greater variability with the military subjects’ serum collection (3–7 days post first debridement) while the period for civilian serum collection (24–48 hr post trauma) was more controlled and closer to time of initial injury. While temporal changes in the secretome were not evaluated in this study, it would be of interest to identify how serum factors change post injury over several months. The initial pathway analysis (Figure 1a) implicated signal transduction mechanisms involved in AP1, BMP2, and plasminogen, and here we suggest this is through MAPK crosstalk, a key regulator of cell proliferation. Current studies investigating differences in osteoblasts from the site of HO formation compared to no trauma sites have demonstrated that the osteoblasts obtained from sites of HO are more proliferative (Agarwal et al., 2016). In addition, MAPKs initiate non-canonical bone forming pathways through growth factors and cytokine signals (Matsushita & Murakami, 2012; Wagner, 2002). While different in cellular function, all MAPKs signal through a similar mechanism of associated proteins and contained within this are downstream transcription factors such as AP1 and the osteogenic RUNX2 and OSX (Artigas, Urena, Rodriguez-Carballo, Rosa, & Ventura, 2014; Roux & Blenis, 2004). ERK1/2 and p38 activate RUNX2 and OSX resulting in increased activity and protein stability (Artigas et al., 2014) and AP1 itself is a downstream effector of non-canonical bone forming pathway. Here we demonstrate that AP1 is altered during hASC osteogenic differentiation, consistent with previous results (Kawao et al., 2013). Serum from both civilian and military orthopaedic trauma patients who developed HO repressed hASC expression of the AP1 transcription factors FOS. Prior studies with knock out mice have demonstrated that loss of FOS results in a repression of osteoclast formation and increased bone density (Matsushita & Murakami, 2012). AP1 is a heterodimer with one FOS family member (FOS, FRA1, FRA2, FOSB) and one JUN family member (JUN, JUNB, JUND). Repression of FOS leads to increased FRA1/JUN dimerization and enhancement of a specific subset of genes. Specifically genes observed in our qPCR experiments (FN1 and PAI1) contain AP1 sites that are preferentially bound by FRA1 heterodimers. While more studies are required it is interesting to note that many genes observed to be enhanced in tissue from HO induced models have AP1 promoter binding sites specifically favoring FRA1 based AP1 complexes over FOS complexes (MCP1, MMP9, COL2A1, COL10A1) (Evans et al., 2014). Activation of AP1 is regulated by MAPK signaling and here we have demonstrated altered gene expression associated with all three MAPK gene cascades (ERK1/2, p38, and JNK) following induction of hASCs with either cHO⁺ or mHO⁺ serum. Specifically we found that serum from military personnel who suffer trauma in combat have enhanced gene expression of the p38 isoform p38α (MAPK). p38 is known to enhance bone formation through non-canonical signaling of WNT and TGFβ (Chen et al., 2012; Rodriquez-Carballo et al., 2016; Zhao et al., 2012) and elevated p38 signaling is observed in cells of patients with fibro dysplasia ossificans, an inborn mutation leading to HO (Chen et al., 2012; Fiori et al., 2006; Rodriquez-Carballo et al., 2016; Zhao et al., 2012). Overall, the MAPK gene signature induced by serum of individuals who developed HO in the civilian setting favored the increase in genes associated with ERK1/2 signaling (k-RAS and MEK1). Independent published gain of function and loss of function studies are consistent with this observation. Knock in of mitogen extracellular kinase (MEK1), an ERK1/2 activator, into undifferentiated mesenchymal cells demonstrated that ERK1/2 signaling is involved in bone formation and constitutive activation led to increased bone formation (Matsushita & Murakami, 2012). Furthermore, knockout of MEK1 resulted in the loss of bone mineralization. Serum from both military and civilian individuals that developed HO induced FN1 gene expression in hASCs at 24 hr. It is noteworthy that considerable cross talk exists between the uPA pathway and the AP1 signaling cascade. Indeed, the uPA pathway includes the signaling molecule FN1 has previously been identified as having AP1 binding sites in its promoter region. In addition, key components of uPA pathway, such as PLG, have been identified as participating in HO formation in mouse models. Recently, a role for PLG in bone repair and ectopic bone formation was reported (Kawao et al., 2013; Yuasa et al., 2015). These mouse models demonstrated a decrease in bone healing of femoral bone defects or fracture. The mechanism of action was attributed to reduced bone vascularization and loss of VEGF expression; however, there were differences in the ability of PLG to induce ectopic bone formation in these models (Kawao et al., 2013; Yuasa et al., 2015). An additional study demonstrated that the uPA pathway is involved in spontaneous formation of ectopic bone (Suelves et al., 2007). In parallel with this report, our PCR screen detected changes to genes associated with the uPA pathway in the military HO demographic. The reciprocal crosstalk between cytokine activated MAPK signaling cascades and uPA signaling results in receptor bound FN1 enhanced MAPK signaling and in turn, uPA components are transcriptionally activated by AP1/MAPK (Smith & Marshall, 2010). Based on this we propose that ectopic bone formation may be derived from cytokine injury that leads to a feed-forward signaling mechanism between the MAPK and uPA signaling pathways that potentiates formation of ectopic bone (Figure 6). Further investigation into these interactions will be warranted in future studies. In conclusion, the current study has identified multiple pathways that may prove to be targets for drug development and potential clinical intervention to reduce or prevent HO formation. Ultimately, a successful HO therapy in trauma patients must balance the dynamic between the prevention of ectopic bone formation and healthy wound healing with sufficient angiogenesis.

FIGURE 6.

MAPK signaling feeds forward in the induction of HO. Schematic for proposed model of MAPK signaling in the development of HO following traumatic injury