Loss of Dnmt3b in Chondrocytes Leads to Delayed Endochondral Ossification and Fracture Repair

Abstract

Despite advanced understanding of signaling mediated by local and systemic factors, the role of epigenetic factors in the regulation of bone regeneration remains vague. The DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b have tissue specific expression patterns and create unique methylation signatures to regulate gene expression. Using a stabilized murine tibia fracture model we find that Dnmt3b is induced early in fracture healing, peaks at 10 days post fracture (dpf), and declines to nearly undetectable levels by 28 dpf. Dnmt3b expression was cell-specific and stage-specific. High levels were observed in chondrogenic lineage cells within the fracture callus. To determine the role of Dnmt3b in fracture healing, Agc1CreERT2;Dnmt3bf/f (Dnmt3bAgc1ER) mice were generated to delete Dnmt3b in chondrogenic cells. Dnmt3bAgc1ER fracture displayed chondrogenesis and chondrocyte maturation defect, and a delay in the later events of angiogenesis, ossification, and bone remodeling. Biomechanical studies demonstrated markedly reduced strength in Dnmt3bAgc1ER fractures and confirmed the delay in repair. The angiogenic response was reduced in both vessel number and volume at 10 and 14 dpf in Dnmt3bAgc1ER mice. Immunohistochemistry showed decreased CD31 expression, consistent with the reduced angiogenesis. Finally, in vitro angiogenesis assays with human umbilical vein endothelial cells (HUVECs) revealed that loss of Dnmt3b in chondrocytes significantly reduced tube formation and endothelial migration. To identify specific angiogenic factors involved in the decreased callus vascularization, a protein array was performed using conditioned media isolated from control and Dnmt3b loss-of-function chondrocytes. Several angiogenic factors, including CXCL12 and osteopontin (OPN) were reduced in chondrocytes following loss of Dnmt3b. DNA methylation analysis further identified hypomethylation in Cxcl12 promoter region. Importantly, the defects in tube formation and cell migration could be rescued by administration of CXCL12 and/or OPN. Altogether, our findings establish that Dnmt3b positively regulates chondrocyte maturation process, and its genetic ablation leads to delayed angiogenesis and fracture repair.

Dnmt3b ablation impairs fracture repair through upregulation of Notch pathway

“…Recent studies defined essential roles of Dnmt3a and Dnmt3b in adult stem cell and hematopoietic stem cell differentiation (17,18) and in osteoclastogenesis (19). Moreover, our recent findings provided evidence that Dnmt3b is highly expressed in chondrocytes and progenitor cells of fracture calluses and showed that Dnmt3b ablation in chondrocytes results in a chondrogenic differentiation defect and delayed fracture repair (20). This suggests that Dnmt3b has an essential role in regulation of progenitor cell differentiation.…”

Phenotypic instability of chondrocytes in osteoarthritis: on a path to hypertrophy

“…Changes in DNA methylation are also associated with gene activation during chondrogenesis . TET1, which catalyzes the conversion of 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC), regulates chondrocyte‐specific gene expression, and the chondrocyte‐specific loss of Dnmt3b , which can methylate previously un‐methylated DNA, leads to defects in chondrogenesis and chondrocyte maturation, thus delaying endochondral ossification . In addition, histone acetyltransferases or demethylases are known to interact with and enable SOX9‐driven transactivation of chondrocyte‐specific genes.…”

“…57 TET1, which catalyzes the conversion of 5methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), regulates chondrocyte-specific gene expression, 58 and the chondrocyte-specific loss of Dnmt3b, which can methylate previously un-methylated DNA, leads to defects in chondrogenesis and chondrocyte maturation, thus delaying endochondral ossification. 59 In addition, histone acetyltransferases 11 or demethylases 60 are known to interact with and enable SOX9-driven transactivation of chondrocyte-specific genes. The activities of the ESET and EZH2 histone methyltransferases, which catalyze methylation of histone 3 at lysine 9 (H3K9) and trimethylation of histone 3 lysine 27 (H3K27me3), respectively, are required for optimal chondrocyte hypertrophy and skeletal development.…”

Epigenetics as a New Frontier in Orthopedic Regenerative Medicine and Oncology

“…Many developmental programs are reactivated in response to injury. Wang et al showed that DMNT3B is expressed in early stages of fracture repair and declines at later stages of repair. DMNT3B expression in chondrocytes is necessary for appropriate callus formation and coupling to angiogenesis and ossification of the stabilized fracture due to hypomethylation and suppression of CXCL12 and osteopontin.…”