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Carminerin contributes to chondrocyte calcification during endochondral ossification

Abstract

Endochondral ossification is an essential process not only for physiological skeletal development and growth, but also for pathological disorders. We recently identified a novel cartilage-specific molecule, carminerin (also known as cystatin 10 and encoded by Cst10), which is upregulated in synchrony with cartilage maturation and stimulates the later differentiation of cultured chondrocytes1. Although carminerin-deficient (Cst10−/−) mice developed and grew normally, they had a microscopic decrease in the calcification of hypertrophic chondrocytes at the growth plate. When we created experimental models of pathological endochondral ossification, we observed suppression of chondrocyte calcification during formation of osteoarthritic osteophytes, age-related ectopic ossification and healing of bone fractures in Cst10−/− mice. Cultured Cst10−/− chondrocytes showed a reduction in calcification with activation of an SRY site in the promoter of the gene encoding nucleotide pyrophosphatase phosphodiesterase 1 (NPP1, encoded by Enpp1). Functional NPP1 is required for carminerin deficiency to suppress the pathological endochondral ossifications listed above. Carminerin is the first cartilage-specific protein that contributes to chondrocyte calcification during endochondral ossification under physiological and pathological conditions through the transcriptional inhibition of NPP1.

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Figure 1: Targeted disruption of mouse gene encoding carminerin (Cst10).
Figure 2: Radiological and histological findings of the long bones in wild-type (Cst10+/+) and Cst10−/− littermates at 8 weeks of age under physiological conditions.
Figure 3: Histological and radiological findings of osteoarthritic joints in wild-type (Cst10+/+) and Cst10−/− littermates.
Figure 4: Radiological and histological findings of bone fracture healing in wild-type (Cst10+/+) and Cst10−/− littermates.

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Acknowledgements

This study was supported by a Grant-in-aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (#14370454), and by the Investigation Committee on the Ossification of Spinal Ligaments, Japanese Ministry of Public Health and Welfare.

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Correspondence to Hiroshi Kawaguchi.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Radiological and histological findings of tibias and vertebrae under physiological conditions. (PDF 587 kb)

Supplementary Fig. 2

Radiological and histological findings of age-related ectopic ossification. (PDF 469 kb)

Supplementary Fig. 3

Ex vivo cultures of growth plate chondrocytes and calvarial osteoblasts. (PDF 989 kb)

Supplementary Fig. 4

Effects of putative cytokines on the carminerin signaling to inhibit NPP1. (PDF 190 kb)

Supplementary Fig. 5

Osteoarthritic osteophyte formation, age-related ectopic ossification, and high phosphate-induced auricular ossification in mice of four genotypes. (PDF 567 kb)

Supplementary Table 1

Dissociation constants of complexes between cystatins and cysteine proteinases. (PDF 133 kb)

Supplementary Methods (PDF 120 kb)

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Yamada, T., Kawano, H., Koshizuka, Y. et al. Carminerin contributes to chondrocyte calcification during endochondral ossification. Nat Med 12, 665–670 (2006). https://doi.org/10.1038/nm1409

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