Downregulation of DNA methyltransferase-3a ameliorates the osteogenic differentiation ability of adipose-derived stem cells in diabetic osteoporosis via Wnt/β-catenin signaling pathway

Abstract

Background Diabetes-related osteoporosis (DOP) is a chronic disease caused by the high glucose environment that induces a metabolic disorder of osteocytes and osteoblast-associated mesenchymal stem cells. The processes of bone defect repair and regeneration become extremely difficult with DOP. Adipose-derived stem cells (ASCs), as seed cells in bone tissue engineering technology, provide a promising therapeutic approach for bone regeneration in DOP patients. The osteogenic ability of ASCs is lower in a DOP model than that of control ASCs. DNA methylation, as a mechanism of epigenetic regulation, may be involved in DNA methylation of various genes, thereby participating in biological behaviors of various cells. Emerging evidence suggests that increased DNA methylation levels are associated with activation of Wnt/β-catenin signaling pathway. The purpose of this study was to investigate the influence of the diabetic environment on the osteogenic potential of ASCs, to explore the role of DNA methylation on osteogenic differentiation of DOP-ASCs via Wnt/β-catenin signaling pathway, and to improve the osteogenic differentiation ability of ASCs with DOP. Methods DOP-ASCs and control ASCs were isolated from DOP C57BL/6 and control mice, respectively. The multipotency of DOP-ASCs was confirmed by Alizarin Red-S, Oil Red-O, and Alcian blue staining. Real-time polymerase chain reaction (RT-PCR), immunofluorescence, and western blotting were used to analyze changes in markers of osteogenic differentiation, DNA methylation, and Wnt/β-catenin signaling. Alizarin Red-S staining was also used to confirm changes in the osteogenic ability. DNMT small interfering RNA (siRNA), shRNA-Dnmt3a, and LVRNA-Dnmt3a were used to assess the role of Dnmt3a in osteogenic differentiation of control ASCs and DOP-ASCs. Micro-computed tomography, hematoxylin and eosin staining, and Masson staining were used to analyze changes in the osteogenic capability while downregulating Dnmt3a with lentivirus in DOP mice in vivo. Results The proliferative ability of DOP-ASCs was lower than that of control ASCs. DOP-ASCs showed a decrease in osteogenic differentiation capacity, lower Wnt/β-catenin signaling pathway activity, and a higher level of Dnmt3a than control ASCs. When Dnmt3a was downregulated by siRNA and shRNA, osteogenic-related factors Runt-related transcription factor 2 and osteopontin, and activity of Wnt/β-catenin signaling pathway were increased, which rescued the poor osteogenic potential of DOP-ASCs. When Dnmt3a was upregulated by LVRNA-Dnmt3a, the osteogenic ability was inhibited. The same results were obtained in vivo. Conclusions Dnmt3a silencing rescues the negative effects of DOP on ASCs and provides a possible approach for bone tissue regeneration in patients with diabetic osteoporosis.

Cell signaling and transcriptional regulation of osteoblast lineage commitment, differentiation, bone formation, and homeostasis

“…A recent study found that Dnmt3a may inhibit osteoblast differentiation via regulating Wnt/β-catenin signaling and Runx2, Ocn activity (Fig. 5d ; Table 3 ) 422 . Demethylation of the promoter’s CG region is linked to the ten-eleven translocases (TET) protein, which may alter chromatin status and encourage osteoblast differentiation 422 .…”

“… 408 DNA methylases Dnmt3a Inhibits osteoblast differentiation via regulating Wnt/β-catenin signaling and Runx2, Ocn activity. 422 DNA demethylases TET1/2 Converts 5mCpG to 5hmCpG. 423 – 425 Chromatin remodeling Baf45a/ Baf45d Promote the activation of genes crucial for osteoblast maturation and mineralization 415 , 419 , 420 …”

Resistin targets TAZ to promote osteogenic differentiation through PI3K/AKT/mTOR pathway

“…Firstly, although we identified that resistin targeted TAZ to induce OD through PI3K/AKT/mTOR pathway, the crosstalk with other signaling pathways cannot be ruled out, and the interaction between resistin and the OD-related genes remains to be further revealed. We next sought to explore other possible pathways, including Wnt/β-catenin pathway 42 , 43 and MAPK pathway, 44 , 45 and carry out relevant clinical studies. Secondly, bone defect remodeling is a dynamic process involving osteogenesis (bone formation) and osteoclastogenesis (bone resorption).…”

The role of circadian rhythm in osteoporosis; a review

“…Osteoporosis is a complex pathogenic metabolic disease ( Loffler et al, 2020 ; Zhang et al, 2022 ) caused by imbalanced bone formation and resorption ( Johnston & Slemenda, 1995 ; Gu et al, 2020 ). By 2050, the number of hip fracture patients in Asia will more than double, accounting for 50% of the total hip fracture in the world ( Wu et al, 2021 ).…”

“…We found that the number of articles in the past two or 3 years has increased significantly, as shown in the figure below ( Figure 1 ). The Wnt signaling pathway is believed to play an important role in bone homeostasis and bone related diseases, hence new osteoporosis therapies targeting Wnt signaling are emerging ( Manolagas, 2014 ; Zhang et al, 2022 ). Similarly, the protein kinase B (Akt) signaling pathway is closely associated with osteoporosis treatments ( Liu et al, 2018 ; Jiang et al, 2022 ).…”