Homeobox Protein Msx2 Acts as a Molecular Defense Mechanism for Preventing Ossification in Ligament Fibroblasts

Abstract

Ligaments and tendons are comprised of tough yet flexible connective tissue. Little is known, however, about the precise characteristics of the cells in ligaments and tendons due to the absence of specific markers and cell lines. We recently reported a periodontal ligament cell line, PDL-L2, with suppressed Runx2/Osf2 transcriptional activity and an inability to form mineralized nodules. The present study demonstrates that the homeobox protein Msx2 is a key factor in suppressing those two functions. Msx2 colocalizes with Runx2/Osf2 and suppresses its activity cooperatively, acting with another corepressor, TLE1, as a complex to recruit histone deacetylase 1 activity. Reverse transcription-PCR and in situ hybridization demonstrated that Msx2 expression is higher in periodontal ligament and tendon cells than in osteoblasts. Stable reduction of Msx2 expression in PDL-L2 cells induces osteoblastic differentiation, thereby causing matrix mineralization. Conversely, stable, forced Msx2 expression in MC3T3-E1 cells prevented osteoblast differentiation and matrix mineralization. Msx2-induced suppression of osteoblast differentiation was repressed by bone morphogenetic protein 2. In addition, Msx2 was downregulated in a symptom- and calcification-dependent manner at the affected region in patients with ossification of the posterior longitudinal ligament. Our findings indicate that Msx2 plays a central role in preventing ligaments and tendons from mineralizing.

We are grateful to Paul Denny and Gerard Karsenty for generous gifts of the antibodies for Msx2 and Runx2/Osf2, respectively, and to Masato Kobori of Yamanouchi Pharmaceutical Co., Ltd., for invaluable technical advice and discussion.

This work was supported in part by the Novartis Foundation (Japan) for the Promotion of Science (T.Y.), by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (nos. 10470388, 12470389, and 14370591 to H.K.), a grant from Ground Research for Space Utilization promoted by the National Space Development Agency and Japan Space Forum, and by grants from Yamanouchi Pharmaceutical Co., Ltd., and Novartis Pharma Japan.