Study design: Gene expression and protein localization of osteopontin (OPN) in spinal hyperostosis of the twy mouse by means of in situ hybridization, immunohistochemistry, and Northern blot analysis.
Objective: To verify the involvement of OPN in spinal hyperostosis in the twy mouse and elucidate its ossification pattern at molecular levels.
Summary of background data: OPN is a molecule that consistently colocalizes with ectopic calcification in human pathologic conditions. The twy mouse, which shows ectopic calcification of the spinal ligament resulting in hind limb paralysis, is considered to be a model for human ossification of the posterior longitudinal ligament of the spine.
Methods: Twenty-eight each of age-matched twy, heterozygote, and wild-type mice were killed at 2, 4, 8, 12, and 16 weeks old and subject to histologic and/or molecular analyses. Sections were hybridized with RNA probes for OPN and also stained with anti-OPN antibodies. Total cellular RNA was extracted from the cervicothoracic spine of each genotype at 2- and 16-week-old, and gene expression for OPN and COL10A1 was quantified by Northern blot analysis.
Results: Enhanced expression of OPN mRNA was observed in spinal hyperostotic lesions of the twy mouse, specifically in cells of the spinal ligament and chondrogenic cells in the outer layer of the anulus fibrosus. These trends were also confirmed by immunohistochemical analyses. Northern blot analysis showed that a considerable amount of OPN transcripts was detected in all genotypes at 2 weeks old, but the robust expression of OPN mRNA was maintained only in twy mice at 16 weeks old. COL10A1 transcripts were hardly detected regardless of the genotype at 16 weeks old.
Conclusion: OPN was overexpressed in the hyperostotic spinal lesions of twy mice, and the hyperostosis was induced mainly by ectopic ossification of the spinal ligament. Because OPN is considered to be an inhibitor of calcification, further studies will be necessary to verify whether OPN overexpressed in the twy mouse is functional.