Mineralization regulation of MAGE-D1 on bone marrowmesenchymal stem cells in knockout mice

Abstract

Objective To investigate the effect of melanoma associated antigen D1 (Mage-D1) on mouse femoral bone mass and mineralization ability of mouse bone marrow mesenchymal cells (BMSCs) and its potential molecular mechanism. Methods Female Mage-D1 gene knockout heterozygous mice and male wild-type (WT) mice were subjected as parent mice to breed Mage-D1 gene knockout homozygous (Mage-D1 KO) mice. PCR and agarose gel electrophoresis were used to identify male Mage-D1 knockout (Mage-D1 KO) mice and littermate male wild-type (WT) mice. Micro-CT scanning was performed to observe mouse femoral bone mass, and ELISA and chemical assay were employed to detect serum levels of calcium, phosphorus, calcitonin, and parathyroid hormone in mice. After primary cultured BMSCs were identified with flow cytometry, immunofluorescence staining was utilized to detect the expression of Mage-D1 in BMSCs. BMSCs were infected by Mage-D1 silencing lentivirus, and then the cells were divided into negative control group (sh-NC) and silencing group (sh-Mage-D1). Cell scratch assay was conducted to detect the migration ability of BMSCs, and flow cytometry and CCK-8 assay were conducted to detect the cycle change and proliferation ability of BMSCs. After mineralization induction, alkaline phosphatase (ALP) staining and alizarin red staining were performed; RT-qPCR and Western blotting were used to measure the expression levels of ALP, Runx2 and Col1. RT-qPCR was used to detect mineralization-related genes p75NTR and Msx1. Results Compared with the WT mice, the femoral cortical bone thickness, cortical bone mineral content, cancellous bone mineral content, trabecular number, and cancellous bone surface density were decreased, and trabecular separation was increased in the Mage-D1 knockout homozygous mice (P<0.05). There were no significant changes in the serum levels of calcium, phosphorus, calcitonin and parathyroid hormone in mice after Mage-D1 knockout. Mage-D1 was expressed in the whole BMSCs and was highly expressed in the nucleus and perinuclear regions. Compared with the sh-NC BMSCs, the sh-Mage-D1 group had decreased proliferation ability (P<0.01), enhanced migration ability (P<0.01), and decreased expression of ALP, Runx2 and Col1 genes (P<0.05) and protein (P<0.01) after mineralization induction, milder ALP and alizarin red stain, and lower expression levels of p75NTR and Msx1. Conclusion Mage-D1 knockout can significantly reduce femur bone mass in mice. It can promote the proliferation and inhibit migration of BMSCs, and positively regulate their mineralization in vitro, and the p75NTR-Dlx1/Msx1 signaling axis may be involved in the regulation of bone metabolism by Mage-D1.

Keywords

• mage-d1
• bone mesenchymal stromal cells
• gene knockout
• mineralization regulation