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Hypermethylation in Calca Promoter Inhibited ASC Osteogenic Differentiation in Rats with Type 2 Diabetic Mellitus
Lei Wang1, Feng Ding1, Shaojie Shi1
1State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Engineering Research Center for Dental Materials and Advanced Manufacture, Department of Implantology, School of Stomatology, The Fourth Military Medical University, Xi'an, China.
Abstract:
The abnormal environment of type 2 diabetes mellitus (T2DM) leads to a substantial decrease in osteogenic function of stem cells. However, the gene sequence does not vary before and after disease for the patient. This phenomenon may be related to changes in osteogenesis-related gene expression caused by DNA methylation. In this study, we established T2DM models to extract adipose-derived stem cells (ASCs) for different gene identifications through DNA methylation sequencing. Specific fragments of methylation changes in the target gene (Calca) were identified by IGV analysis. CGRP was applied to compare the effects on ASCs-T2DM morphology via phalloidin staining, proliferation through CCK-8 assay, and osteogenic differentiation with osteogenic staining, qPCR, and repair of calvarial defect. Furthermore, 5-azacytidine (5-az) was used to intervene ASCs-T2DM to verify the relationship between the methylation level of the target fragment and expression of Calca. We found that the DNA methylation level of target fragment of Calca in ASCs-T2DM was higher than that in ASCs-C. CGRP intervention showed that it did not change the morphology of ASCs-T2DM but could improve proliferation within a certain range. Meanwhile, it could significantly enhance the formation of ALP and calcium nodules in ASCs-T2DM, increase the expression of osteogenesis-related genes in vitro, and promote the healing of calvarial defects of T2DM rat in a concentration-dependent manner. 5-az intervention indicated that the reduction of the methylation level in Calca target fragment of ASCs-T2DM indeed escalated the gene expression, which may be related to DNMT1. Taken together, the environment of T2DM could upregulate the methylation level in the promoter region of Calca and then decrease the Calca expression. The coding product of Calca revealed a promoting role for osteogenic differentiation of ASCs-T2DM. This result provides an implication for us to understand the mechanism of the decreased osteogenic ability of ASCs-T2DM and improve its osteogenic capacity.
Insights
Type 2 diabetes mellitus (T2DM) impairs stem cell bone formation via increased DNA methylation of the Calca gene. CGRP treatment and 5-azacytidine intervention restored osteogenic function in T2DM stem cells.
Area of Science:
- Stem cell biology
- Epigenetics
- Endocrinology
Background:
- Type 2 diabetes mellitus (T2DM) environment significantly reduces osteogenic differentiation of adipose-derived stem cells (ASCs).
- Gene sequence remains unchanged, suggesting epigenetic modifications like DNA methylation may underlie impaired osteogenesis.
Purpose of the Study:
- Investigate DNA methylation changes in ASCs from T2DM models.
- Identify the role of the Calca gene in T2DM-induced stem cell dysfunction.
- Evaluate therapeutic potential of CGRP and 5-azacytidine for restoring osteogenic capacity.
Main Methods:
- Establishment of T2DM rat models and isolation of ASCs.
- DNA methylation sequencing and IGV analysis to identify methylation changes in the Calca gene.
- Assessment of ASC morphology, proliferation, and osteogenic differentiation following CGRP and 5-azacytidine treatment.
- In vivo evaluation of calvarial defect repair in T2DM rats.
Main Results:
- Increased DNA methylation of a specific Calca gene fragment was observed in ASCs from T2DM models.
- CGRP treatment improved proliferation and significantly enhanced osteogenic markers (ALP, calcium nodules) and gene expression in ASCs-T2DM.
- CGRP promoted calvarial defect healing in T2DM rats in a dose-dependent manner.
- 5-azacytidine intervention reduced Calca methylation, increasing gene expression, suggesting a role for DNMT1.
Conclusions:
- T2DM upregulates DNA methylation in the Calca promoter region, decreasing its expression and impairing osteogenesis.
- Calca gene product promotes osteogenic differentiation of ASCs-T2DM.
- Targeting Calca methylation offers a potential strategy to improve osteogenic capacity in T2DM patients.
