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. 2020 Apr;24(8):4784-4790.
doi: 10.1111/jcmm.15152. Epub 2020 Mar 12.

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

Faezeh Sadeghi  1   2 , Elham Vahednia  3 , Hojjat Naderi Meshkin  4 , Mohammad Amin Kerachian  1   2
Affiliations

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

Faezeh Sadeghi et al. J Cell Mol Med. 2020 Apr.

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.

Keywords: adrenocorticotropic hormone; alpha-2-macroglobulin; avascular necrosis; mesenchymal stem cells; osteoblasts.

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Conflict of interest statement

The authors declare no conflict of interest with respect to this research.

Figures

Figure 1
Figure 1
MSCs characterization using flow cytometry. The absence of hematopoietic lineage markers CD45, CD34 and CD11b and presence the cell surface markers of MSCs
Figure 2
Figure 2
Multipotential differentiation of MSCs‐adipocyte staining: Control group (A) Differentiation to adipocyte was demonstrated by Oil Red O staining, (B)—Osteoblast staining: Control group (C) and (E), osteoblast differentiation was detected by alkaline phosphatase (D) and Alizarin Red S (F) staining
Figure 3
Figure 3
ACTH increases ALPL (fold change: 1.85), BGLAP (fold change: 1.56), A2M (fold change: 1.56), expression. Statistic: paired sample t test: *P < .05, **P < .01
Figure 4
Figure 4
ACTH effects on osteogenesis, coagulation and fibrinolysis through A2M action
See this image and copyright information in PMC

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