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.