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Targeting Runx2 expression in hypertrophic chondrocytes impairs endochondral ossification during early skeletal development.

ABSTRACT: Runx2 is a known master transcription factor for osteoblast differentiation, as well as an essential regulator for chondrocyte maturation. Recently, more and more data has shown that Runx2 regulates hypertrophic chondrocyte-specific type X collagen gene (Col10a1) expression in different species. However, how Runx2 regulation of Col10a1 expression impacts chondrocyte maturation, an essential step of endochondral bone formation, remains unknown. We have recently generated transgenic mice in which Flag-tagged Runx2 was driven by a cell-specific Col10a1 control element. Significantly increased level of Runx2 and Col10a1 mRNA transcripts were detected in transgenic mouse limbs at both E17.5 (embryonic day 17.5) and P1 (post-natal day1) stages, suggesting an in vivo correlation of Runx2 and Col10a1 expression. Surprisingly, skeletal staining suggested delayed ossification in both the axial and the appendicular skeleton of transgenic mice from E14.5 until P6. Histological analysis showed elongated hypertrophic zones in transgenic mice, with less von Kossa and TUNEL staining in long bone sections at both E17.5 and P1 stages, suggesting defective mineralization due to delayed chondrocyte maturation or apoptosis. Indeed, we detected increased level of anti-apoptotic genes B-cell leukemia/lymphoma 2, Osteopontin, and Sox9 in transgenic mice by real-time RT-PCR. Moreover, immunohistochemistry and Western blotting analysis also suggested increased Sox9 expression in hypertrophic chondrocytes of transgenic mice. Together, our data suggest that targeting Runx2 in hypertrophic chondrocytes upregulates expression of Col10a1 and other marker genes (such as Sox9). This will change the local matrix environment, delay chondrocyte maturation, reduce apoptosis and matrix mineralization, and eventually, lead to impaired endochondral ossification.

SUBMITTER: Ding M

PROVIDER: S-EPMC3359397 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Targeting Runx2 expression in hypertrophic chondrocytes impairs endochondral ossification during early skeletal development.

Ding Ming M Lu Yaojuan Y Abbassi Sam S Li Feifei F Li Xin X Song Yu Y Geoffroy Valérie V Im Hee-Jeong HJ Zheng Qiping Q

Journal of cellular physiology 20121001 10

Runx2 is a known master transcription factor for osteoblast differentiation, as well as an essential regulator for chondrocyte maturation. Recently, more and more data has shown that Runx2 regulates hypertrophic chondrocyte-specific type X collagen gene (Col10a1) expression in different species. However, how Runx2 regulation of Col10a1 expression impacts chondrocyte maturation, an essential step of endochondral bone formation, remains unknown. We have recently generated transgenic mice in which ...[more]

PMID: 22223437

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Project description: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. © 2017 American Society for Bone and Mineral Research.

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Targeting Runx2 expression in hypertrophic chondrocytes impairs endochondral ossification during early skeletal development.

Publications

Targeting Runx2 expression in hypertrophic chondrocytes impairs endochondral ossification during early skeletal development.

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