The effect of adrenocorticotropic hormone on alpha-2-macroglobulin in osteoblasts derived from human mesenchymal stem cells.

Abstract Nowadays, alpha‐2‐macroglobulin (A2M) gene has allocated escalating interest among several genes involved in the pathogenesis of avascular necrosis of the femoral head (ANFH). This molecule could interact with several osteogenic‐related proteins. It was reported that adrenocorticotropic hormone (ACTH) affects bones through its receptor located on osteoblasts, suggesting it as a potential target in ANFH treatment. In this study, the effect of ACTH on A2M expression was investigated in osteoblasts as well as during the differentiation of human mesenchymal stem cells (MSCs) into osteoblasts. In this study, MSCs derived from bone marrow were isolated and purified using Ficoll gradient and several passaging. MSCs were characterized by induction with osteogenic and adipogenic medium followed by Oil Red O, Alizarin Red and alkaline phosphatase staining. Besides, MSCs were exposed to various concentrations of ACTH to evaluate the cell variability by MTT assay. MSCs and differentiated osteoblasts were treated with 10−8 molar ACTH for 16 and 26 days, respectively. Then, the total RNA was extracted and A2M expression was quantified by real‐time qPCR. The protein expression levels of osteoblast markers including alkaline phosphatase (ALPL) and bone gamma‐carboxyglutamate protein (BGLAP) were also measured. The results showed that A2M expression in cells treated with ACTH was up‐regulated significantly compared to the control group. Similarly, the expression of osteoblast gene markers including ALPL and BGLAP was significantly increased. ACTH, as an osteoblastic differentiation enhancer, up‐regulates A2M, which promotes osteoblastic differentiation probably through TGF‐β induction.


| INTRODUC TI ON
Avascular necrosis of femoral head (ANFH) is a pathologic process, which leads to hip joint destruction as well as bone loss in femoral head. 1 It results from interruption of blood supply to the epiphyses of the femur. 2 Nearly, 50% of patients with ANFH require a major surgical procedure, for example total hip replacement (THR) for treatment. 3 Although therapies such as THR are an available procedure for controlling this problem, 4 it has its own disadvantages such as high morbidity rate. Besides, the patients have to suffer from additional major operations later in their lives. 4,5 Thus, finding novel diagnostic methods especially in early stages of the disease and modern therapeutic approaches including stem cell therapy are strongly required. 6 Mesenchymal stem cells (MSCs) have allocated a lot of interest because of their potency to repair and regenerate musculoskeletal tissues. MSCs can be easily isolated from different tissues but the main source is bone marrow called the human bone marrow mesenchymal stem cells (hBM-MSCs). These multipotent cells can differentiate into various lineages such as osteoblasts, chondrocytes and adipocytes. 7 Differentiation of osteoblasts and chondrocytes can be influenced by various substances such as adrenocorticotropic hormone (ACTH), a melanocortin peptide hormone derived from proopiomelanocortin (POMC). 8 ACTH directly increases bone markers related to expression of adrenocorticotropic receptor on the surface of MSCs and osteoblasts derived from MSCs. 9 Furthermore, it was reported that this hormone has a protective role against ANFH. 6 Thus, it could be used as a potential therapeutic agent in ANFH treatment. Its therapeutic response probably arises from an elevated osteoblastic support and the stimulation of VEGF by ACTH; the latter is largely responsible for vascularization, which surrounds highly remodelling bone. 6 Previously, a significant overexpression of alpha-2-macroglobulin gene (A2M) in a glucocorticoid-induced rat model of ANFH has been reported, 10 and in a recent study, it has been shown that the serum protein level of this protein interestingly raised in ANFH patients. 11 This protein has several roles in fibrinolysis and the coagulation cascade. 12 Besides, it is a carrier of diverse growth factors and cytokines contributing to osteogenesis. [13][14][15][16] It is feasible that ACTH influences the osteogenesis and vascularization via influencing the gene expression of A2M as a role player in ANFH. In the present article, we investigated the effects of ACTH on A2M expression in osteoblasts derived from hBM-MSCs and in the process of their differentiation. This probably could assist to have a better understanding of the pathogenesis of ANTH, discovering more effective therapeutic strategies.

| Human MSCs isolation and expansion
The bone marrow aspirates were taken from femur of normal adult donors after informed consents were signed by the participants and under the protocol approved by the research ethics committee of Mashhad University of Medical Sciences (MUMS) with the ethical code of 922645. Mononuclear cells were isolated by Ficoll-Paque PLUS (GE Healthcare) and density gradient centrifugation; then, they were plated in tissue culture flasks in Dulbecco's modified Eagle's medium (Invitrogen) with 10% foetal bovine serum (Gibco) supplemented with 2 ng/mL basic fibroblast growth factor (Royan Institute) and 100 U/ mL penicillin-streptomycin (Gibco). In the following, the cells were incubated at 37°C in a humidified atmosphere with 5% CO 2 . After cell incubation for 3 days, culture medium was refreshed to remove nonadherent cells. Then, adherent cells were cultured until they reached 70%-80% confluency. After that, the cells were detached by trypsinization with 0.25% trypsin-EDTA (Gibco) followed by subculturing to new flasks. Finally, MSCs in passage 4 were used in our experiments.

| MSCs characterization
Osteogenic and adipogenic differentiation was performed in order to check MSCs characteristics. Briefly, 2 × 10 4 cells/well were seeded in 6-well plates and the medium was changed every other day with differentiation medium.

| In vitro adipogenic differentiation
The cells in passage 4 were cultured under adipogenic conditions for 16 days in order to induce adipogenic differentiation. Adipogenic medium consists of expansion medium supplemented with 100 nmol/L dexamethasone sodium phosphate and 100 µmol/L indomethacin (Sigma-Aldrich). After 16 days, cells were stained with Oil Red O (Sigma-Aldrich).

| Alkaline phosphatase staining
After culture of hBM-MSCs in dedicated time, cells were fixed with 4% paraformaldehyde and washed with phosphate buffered saline (PBS). Then, the cells were incubated with BCIP/NBT (5-bromo-4chloro-3-indolyl phosphate/nitroblue tetrazolium liquid substrate) (Sigma-Aldrich, Bornem, Belgium) for 10 minutes. Treated cells were washed with ddH 2 O and examined with invert microscopy. Positive staining was visualized as dark purple colour.

| Flow cytometric analysis
MSCs in passage 4 were grown until 100% confluency and charac- Results were analysed using Flowing Software version 2.2.

| Preparation of various concentrations of ACTH
To prepare 9 different concentrations of ACTH (10 −4 to 10 −12 molar), 0.0145 gr of ACTH was dissolved in 500 µL sodium chloride (stock concentration: 6.4 mmol/L). Required concentrations of ACTH were prepared by diluting different volumes of this solution in proper amounts of culture medium.

| MTT assay
MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay was performed to investigate the cytotoxic effects of ACTH. MTT test is dependent on cleaving the tetrazolium salt and forming purple formazan crystals. 17 To do so, cells were trypsinized after reaching 80% confluency and resuspended in culture medium. Then, 5 × 10 3 cells were plated in 96-well plates (JET BIO) and treated with various concentrations of ACTH for three con-

| ACTH treatment
MSCs were treated with differentiation medium and supplemented with 10 −8 molar ACTH for 16 days. Besides, in another group, MSCsdifferentiated osteoblasts were exposed to the same concentration of ACTH for an extra 10 days.

| RNA preparation and gene expression analysis
Total RNA was extracted using AccuZol™ reagent (Bioneer) followed

| Statistical analysis
Relative expressions were quantified using the relative Cq method with GAPDH as a reference gene. Data were represented as the Mean ± SD. To compare the outcomes, the paired sample t test was performed, and P-values < .05 were regarded statistically significant.

| RE SULTS
Identification of MSCs from other mononuclear cells was revealed by their morphology and phenotype as well as their potency to differentiate to multilineage as described later. The cells formed colonies after 28 days of expanding.

| Surface markers of MSCs
The immunophenotype features of MSCs were analysed by flow cytometry. The results shown in Figure 1 revealed that MSCs were positive for CD44, CD90 and CD105; however, they were negative for CD45, CD34 and CD11b.

| Multilineage differentiation of MSCs
The hBM-MSCs were differentiated to various cell types to show their pluripotency. So, hBM-MSCs-were cultured for about 2 and 3 weeks in adipogenic and osteogenic differentiation medium, respectively. Oil Red O staining revealed droplets of fat in adipocytes ( Figure 2B). Alkaline phosphatase activity was detected in differentiated MSCs ( Figure 2D). Additionally, Alizarin Red S staining showed the calcium-rich deposits in red ( Figure 2F).

| Gene expression analysis
After induction of MSCs into osteoblasts, ACTH significantly up-regulated the expression levels of genes regarding to osteoblast maturation and function, including ALPL and osteocalcin (BGLAP). This hormone also significantly increased A2M expression (Figure 3). Besides, differentiated osteoblasts exposed to ACTH revealed a significant difference at transcriptional levels of A2M, ALPL and BGLAP in comparison with control groups, indicating a positive role of ACTH on enhancing transcriptional level of A2M as well as osteoblast markers.

| D ISCUSS I ON
Since MSCs have a great impact on bone regeneration, in the present study hBM-MSCs were isolated and characterized. Results strongly pointed that isolated hBM-MSCs revealed MSCs properties, since they formed colony and differentiated to osteogenic and adipogenic lineages.
It was shown that in vitro MSCs could attach to the plastic surface and generate colonies, and thus, they were considered as fibroblastoid colony-forming cells. 18 In vivo, MSCs at an injured bone site could proliferate and differentiate into osteoblasts. 19 Hence, The femoral head has a very high rate of bone formation and resorption. 23 The leading aetiology in destruction of femoral head called 'ANFH' is apoptosis of femoral head's bone. It has been shown that patients suffering from ANFH have obstructed blood vessels. 9,24 Reduction in shear stress due to declined blood flow could cause apoptosis of endothelial cells, which can eventually contribute to plaque erosion and thrombus formation. 25 Several genes such as A2M, which contributes to coagulation and osteogenesis, could play a role in the pathogenesis of ANFH. 10 A2M is an anti-protease that inactivates large variety of proteinase such as thrombin, contributing to coagulation, and plasmin as well as kallikrein contributing to fibrinolysis pathways. 12,26 It is able to bind to numerous growth factors and cytokines such as bone morphogenetic protein 1 (BMP-1), transforming growth factor beta (TGF-β), osteogenic growth peptide (OGP) and vascular endothelial growth factor (VEGF). 13,16 All these proteins play significant roles in osteogenesis, except TGF-β, 27 which promotes osteoblastic differentiation in the early stage and inhibits the late stage of osteogenesis. 27 It is worth noting that A2M suppresses the function of VEGF and BMP-1. 14,16 Thus, it seems that A2M has a dual effect in osteogenesis process. On the one hand, it affects VEGF and BMP-1, but on the other hand it has an effect on TGF-β ( Figure 4).

| CON CLUS ION
In summary, the results have proposed a bilateral role of ACTH as along with A2M in bone regeneration. Further investigation is required to clarify the profile expression of this marker to explain the exact role of it in ANFH pathogenesis.

ACK N OWLED G EM ENTS
We and Stem Cell Core Center, Mashhad University of Medical Sciences, Mashhad, Iran.

CO N FLI C T O F I NTE R E S T S
The authors declare no conflict of interest with respect to this research.

AUTH O R S' CO NTR I B UTI O N S
MAK supervised the study. MAK and HNM participated in study design and scientific discussion of the data. FS and EV contributed to performing the experiments.

CO N S E NT FO R PU B LI C ATI O N
All authors read and approved the final manuscript.

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 the present study are available from the corresponding author upon reasonable request.