[GLIS family zinc finger protein 2 (GLIS2) negatively regulates the Wnt/β-catenin pathway to inhibit the osteogenic differentiation of human bone marrow mesenchymal stem cells].

Abstract

Objective To investigate how the human GLIS family zinc finger protein 2 (GLIS2) regulate the Wnt/β-catenin pathway and its influence on the differentiation of human bone marrow mesenchymal stem cells (BMMSCs). Methods Human BMMSCs were randomly divided into blank group, osteogenic induction group, GLIS2 gene overexpression (ad-GLIS2) group, ad-GLIS2 negative control group, gene knockdown (si-GLIS2) group, and si-GLIS2 negative control (si-NC) group. The expression of GLIS2 mRNA in each group was detected by reverse transcription-PCR to determine the transfection status; alkaline phosphatase (ALP) activity was detected by phenyl-p-nitrophenyl phosphate (PNPP), and calcified nodule formation was tested by alizarin red staining to determine its osteogenic properties; the activation of intracellular Wnt/β-catenin pathway was detected by T cell factor/lymphoid enhancer factor (TCF/LEF) reporter kit; the expression of GLIS2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osterix was detected by Western blot analysis. The interaction between GLIS2 and β-catenin was verified by GST-pulldown. Results Compared with the blank group, the ALP activity and calcified nodule formation of BMMSCs in the osteogenic induction group increased, and the Wnt/β-catenin pathway activity and the expression of osteogenic differentiation-related proteins increased, the osteogenic ability increased, while the expression of GLIS2 decreased. Up-regulating the expression of GLIS2 could inhibit the osteogenic differentiation of BMMSCs, while suppress the activity of the Wnt/β-catenin pathway and the expression of osteogenic differentiation-related proteins on the contrary. Down-regulating the expression of GLIS2 could promote the osteogenic differentiation of BMMSCs, and improve the activity of the Wnt/β-catenin pathway and the expression of osteogenic differentiation-related proteins. There was an interaction between β-catenin and GLIS2. Conclusion GLIS2 may negatively regulate the activation of Wnt/β-catenin pathway and affect the osteogenic differentiation of BMMSCs.