Cytokine-like 1 Knock-out Mice (Cytl1−/−) Show Normal Cartilage and Bone Development but Exhibit Augmented Osteoarthritic Cartilage Destruction

Jimin Jeon,Hwanhee Oh,Gyusuk Lee,Je-Hwang Ryu,Jinseol Rhee,Jin-Hong Kim,Kyung-Hwun Chung,Woo-Keun Song,Churl-Hong Chun,Jang-Soo Chun

doi:10.1074/jbc.m111.218065

Abstract

We have shown that cytokine-like 1 (Cytl1) is a novel autocrine regulatory factor that regulates chondrogenesis of mouse mesenchymal cells (Kim, J. S., Ryoo, Z. Y., and Chun, J. S. (2007) J. Biol. Chem. 282, 29359-29367). In this previous work, we found that Cytl1 expression was very low in mesenchymal cells, increased dramatically during chondrogenesis, and decreased during hypertrophic maturation, both in vivo and in vitro. Moreover, exogenous addition or ectopic expression of Cytl1 caused chondrogenic differentiation of mouse limb bud mesenchymal cells. In the current study, we generated a Cytl1 knock-out (Cytl1(-/-)) mouse to investigate the in vivo role of Cytl1. Deletion of the Cytl1 gene did not affect chondrogenesis or cartilage development. Cytl1(-/-) mice also showed normal endochondral ossification and long bone development. Additionally, ultrastructural features of articular cartilage, such as matrix organization and chondrocyte morphology, were similar in wild-type and Cytl1(-/-) mice. However, Cytl1(-/-) mice were more sensitive to osteoarthritic (OA) cartilage destruction. Compared with wild-type littermates, Cytl1(-/-) mice showed more severe OA cartilage destruction upon destabilization of the medial meniscus of mouse knee joints. In addition, expression levels of Cytl1 were markedly decreased in OA cartilage of humans and experimental mice. Taken together, our results suggest that, rather than regulating cartilage and bone development, Cytl1 is required for the maintenance of cartilage homeostasis, and loss of Cytl1 function is associated with experimental OA cartilage destruction in mice.

Highlights

R&D project (A101793). 1 To whom correspondence may be addressed: Department of Orthopedic teoglycan, to form cartilage tissue
We have previously shown that cytokine-like 1 (Cytl1) is a novel autocrine regulatory factor involved in chondrogenesis of mouse mesenchymal cells in vitro [8]
We demonstrated in this study that deletion of the Cytl1 gene in mice does not affect cartilage development, endochondral ossification, or long bone development, despite the in vitro chondrogenic effects of Cytl1

Summary

EXPERIMENTAL PROCEDURES

Animals—Male mice (C57BL/6, Cytl1Ϫ/Ϫ, STR/ort, and CBA/CaCrl) were used for cartilage and bone development and experimental OA studies. Short arm and long arm fragments were amplified by polymerase chain reaction (PCR) from genomic DNA obtained from 129/ SvJ mouse J1 embryonic stem cells. The 2.7-kb NheI-XhoI fragment (5Ј short arm) was amplified using forward (5Ј-gctagcaaaggagccacagaaatat-3Ј) and reverse (5Ј-ctcgagtgggacactgaattcttt-3Ј) primers containing NheI and XhoI sites, respectively (underlined). The 7.3-kb BamHI-NotI fragment (3Ј long arm) was amplified using forward (5Ј-ggatccagctcagtctctggccctct-3Ј) and reverse (5Ј-gcggccgccaccagtgaaaagaattaa-3Ј) primers containing BamHI and NotI sites, respectively (underlined). For Southern blot analysis, mouse genomic DNA obtained from 129/SvJ mouse J1 embryonic stem cells was digested with BamHI and hybridized with a 314-bp probe amplified from genomic DNA by PCR using the primers 5Јcctctacactctccacattcc-3Ј (forward) and 5Ј-aagggcatcgaaggagtcctt-3Ј (reverse). Mesenchymal cells obtained from E11.5 embryos of WT and Cytl1Ϫ/Ϫ mice were digested with 1% trypsin and 0.2% type II collagenase (381 units/mg solid; Sigma) for 10 min.

Primer sequences

RESULTS

DISCUSSION