Background
Progressive population aging has contributed to the increased global prevalence of diabetes and osteoporosis. Inhibition of osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by hyperglycemia is a potential pathogenetic mechanism of osteoporosis in diabetic patients. Uncarboxylated osteocalcin (GluOC), a protein secreted by mature osteoblasts, regulates bone development as well as glucose and lipid metabolism. In our previous studies, GluOC was shown to promote osteoblastic differentiation of BMSCs; however, the underlying mechanisms are not well characterized. Tumor protein 63 (TP63), as a transcription factor, is closely related to bone development and glucose metabolism.
Results
In this study, we verified that high glucose suppressed osteogenesis and upregulated adipogenesis in BMSCs, while GluOC alleviated this phenomenon. In addition, high glucose enhanced TP63 expression while GluOC diminished it. Knock-down of TP63 by siRNA transfection restored the inhibitory effect of high glucose on osteogenic differentiation. Furthermore, we detected the downstream signaling pathway PTEN/Akt/GSK3β. We found that diminishing TP63 decreased PTEN expression and promoted the phosphorylation of Akt and GSK3β. We then applied the activator and inhibitor of Akt, and concluded that PTEN/Akt/GSK3β participated in regulating the differentiation of BMSCs.
Conclusions
Our results indicate that GluOC reduces the inhibitory effect of high glucose on osteoblast differentiation by regulating the TP63/PTEN/Akt/GSK3β pathway. TP63 is a potential novel target for the prevention and treatment of diabetic osteoporosis.
Osteoporosis is a bone disease characterized by reduced bone density, thin cortical bone and large gaps in the bone's honeycomb structure, which increases the risk of bone fragility. Uncarboxylated osteocalcin (unOC), a vitamin K-dependent bone protein, is known to regulate carbohydrate and energy metabolism. A previous study demonstrated that unOC promotes the differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs) into osteoblasts, but inhibits their differentiation into adipocytes. However, the underlying mechanism remains unknown. The present study showed that unOC regulated the differentiation potential of BMSCs via protein kinase A (PKA)/AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) signaling. SIRT1, a member of the sirtuin family with deacetylation functions, was upregulated by unOC in BMSCs. Transfection analyses with SIRT1 small interfering RNA indicated that the unOC-induced differentiation shift in BMSCs required SIRT1. Examination of SIRT1 downstream targets revealed that unOC regulated the acetylation levels of runt-related transcription factor (RUNX) 2 and peroxisome proliferator-activated receptor γ (PPARγ). Therefore, unOC inhibited adipogenic differentiation by PPARγ acetylation and promoted osteogenic differentiation by RUNX2 deacetylation. Moreover, phosphorylated PKA and AMPK protein levels increased after unOC treatment, which led to the upregulation of SIRT1. Western blot analysis with PKA and AMPK inhibitors indicated that the PKA-AMPK signaling pathway functioned upstream of SIRT1 and positively regulated SIRT1 expression. These findings led us to propose a model in which unOC regulated BMSC osteogenic differentiation through the PKA-AMPK-SIRT1 axis, giving evidence towards the therapeutic potential of unOC in osteoporosis treatment.
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