Downregulated brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 inhibits osteogenesis of BMSCs through p53 in type 2 diabetes mellitus

Abstract

The bone marrow mesenchymal stem cells (BMSCs)-mediated abnormal bone metabolism can delay and impair the bone remodeling process in type 2 diabetes mellitus (T2DM). Our previous study demonstrated that the downregulation of brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1), a circadian clock protein, inhibited the Wnt/β-catenin pathway via enhanced GSK-3β in diabetic BMSCs. In this article, we confirmed that the downregulated BMAL1 in T2DM played an inhibitory role in osteogenic differentiation of BMSCs. Upregulation of BMAL1 in the diabetic BMSCs significantly recovered the expression pattern of osteogenic marker genes and alkaline phosphatase (Alp) activity. We also observed an activation of the p53 signaling pathways, exhibited by increased p53 and p21 in diabetic BMSCs. Downregulation of p53 resulting from overexpression of BMAL1 was detected, and when we applied p53 gene silencing (shRNA) and the p53 inhibitor, pifithrin-α (PFT-α), the impaired osteogenic differentiation ability of diabetic BMSCs was greatly restored. However, there was no change in the level of expression of BMAL1. Taken together, our results first revealed that BMAL1 regulated osteogenesis of BMSCs through p53 in T2DM, providing a novel direction for further exploration of the mechanism underlying osteoporosis in diabetes.

Keywords: Bone marrow mesenchymal stem cells (BMSCs); Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 (BMAL1); Osteogenic differentiation; Type 2 diabetes mellitus (T2DM); p53.

Fig. 1.

Characteristics of BMSCs. (A) Flow cytometry analysis of the typical surface markers of BMSCs. (B) BMSCs could differentiate into osteoblasts or adipocytes after osteogenic or adipogenic induction, indicated by Alizarin Red staining and Oil Red O staining. Scale bars: 500 μm.

Fig. 2.

BMAL1 overexpression rescued the impaired osteogenic differentiation of BMSCs in T2DM. (A) Expression of the genes BMAL1, Runx2, Alp, Ocn and Osx, determined by qRT-PCR assay, in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs after 1 week of osteogenic differentiation. (B) Western blot analysis of BMAL1, Runx2, Alp, Ocn and Osx expression in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs after osteogenic differentiation for 7 days. (C) Alkaline phosphatase activity staining of WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs after 1 week of osteogenic differentiation. All data are mean±s.e.m. and representative of three independent experiments. β-actin was used as a loading control. n=3. Statistical significance was defined as *P<0.05; **P<0.01. n.s: not significant.

Fig. 3.

Decreased BMAL1 level inhibited proliferation of BMSCs in T2DM. (A) Proliferation rate of WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs as determined by CCK-8 assay, respectively. (B) Cell apoptosis analysis of WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs by flow cytometry. (C) WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs from passage 4 and passage 6 were cultured in osteogenic induction medium for 7 days before being stained for alkaline phosphatase activity. All data are mean±s.e.m. and representative of three independent experiments. n=3. Statistical significance was defined as *P<0.05; ^#P<0.03.

Fig. 4.

Downregulated BMAL1 promoted p53 expression in T2DM. (A) Protein expression of BMAL1, p53 and p21 in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs as determined by western blot. (B) qRT-PCR analysis of BMAL1, p53 and p21 in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs. (C) Immunofluorescence staining of p53 in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs. Scale bars: 100 µm. All data are mean±s.e.m. and representative of three independent experiments. n=3. β-actin was used as a loading control. Statistical significance was defined as *P<0.05; **P<0.01.

Fig. 5.

Inhibition of osteogenic differentiation of BMSCs by BMAL1 downregulation in T2DM was mediated by p53. (A) Western blot analysis of BMAL1, Runx2, Alp, Osx and p53 expression in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs with or without 20 μM PFT-α treatment after 7 days of osteogenic differentiation. (B) The mRNA expression of Runx2, Osx and Alp in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs were determined by qRT-PCR, following 7 days of osteogenic differentiation, with or without 20 μM PFT-α treatment. (C) Osteogenic differentiation ability of WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs with or without 20 μM PFT-α treatment as determined by alkaline phosphatase activity after osteogenic differentiation for 7 days. Data are from five independent experiments and are expressed as mean±s.e.m.. n=5. Statistical significance was defined as *P<0.05; ^#P<0.03. n.s: not significant.

Fig. 6.

p53 gene silencing confirmed the regulation role of BMAL1 on p53 in diabetic BMSCs' osteogenic differentiation ability. (A) Protein expression of BMAL1, Runx2, Alp, Osx and p53 in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs with or without p53 gene silencing as determined by western blot after osteogenic differentiation for 7 days. (B) The mRNA expression of Runx2, Osx and Alp in WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs were determined by qRT-PCR, following 7 days of osteogenic differentiation, with or without p53 gene silencing. (C) Osteogenic differentiation ability of WT Wistar BMSCs, diabetic GK BMSCs and GK-BMAL1 BMSCs with or without p53 gene silencing as determined by alkaline phosphatase activity after 7 days of osteogenic differentiation. Data are from five independent experiments and are expressed as mean±s.e.m. n=5. Statistical significance was defined as *P<0.05; ^#P<0.03. n.s: not significant.