Nell-1 Modulates Postnatal Cartilage Homeostasis and Endochondral Ossification

Objectives: Nel-like molecule-1 (Nell-1) has been shown to promote cartilage regeneration. More recently, the first case of interstitial 11p14.1-p15.3 deletion involving Nell-1 in a short statured girl has been reported. Here, we reveal the role of Nell-1 on postnatal cartilage and endochondral ossification with conditional Nell-1 knockout in Col2α1-expressed cells for the first time due to the neonatal lethality of global Nell-1 deficient mice in previous studies.
Methods: Nell-1^flox/flox; Col2α1-Cre (Nell-1 cKO) and Nell-1^flox/flox (WT) mice were used. In-vivo Micro-CT was scanned to dynamically observe secondary ossification center (SOC) formation at P7, P14, P21 and P28. Bone parameter analysis (length, BMD, BV, cortical/trabecular thickness), morphological evaluations and dynamic histomorphometry of femur and tibia at different time points were performed. Expression and distribution of Col-2, Col-10, Ihh and PTHrP were examined by Immunohistochemistry (IHC). Primary sternal chondrocytes were cultured in chondrogenic medium for Alcian blue staining and gene expression analyses (Col-2, Col-10, Aggrecan, Ihh, Patched-1, Gli-1, PTHrP) by qPCR, with/without adding Nell-1. Proliferation was observed by BrdU incorporation and MTT assay.
Results: cKO of Nell-1 did not affect the longevity of mice, but resulted in a smaller size compared to WT littermates in young adulthood. In Nell-1 cKO mice, retarded SOC formation, slower bone growth rate, shorter femur/tibia, smaller cortical and trabecular bone volume were observed. Histological analyses showed thinner and disorganized growth plates, reduced chondrocytes proliferation, and lower level of Col-2, Col-10, Ihh and PTHrP expression in cKO mice. Decreased osteoblast and increased osteoclast were also observed in metaphyseal trabecular bones. Gene expression of all mentioned markers went down significantly in Nell-1 cKO chondrocytes. Addition of Nell-1 rescued these deficiencies to certain extent.
Conclusions: Nell-1 modulates postnatal cartilage homeostasis and endochondral ossification by upregulating Ihh/PTHrP signaling pathway. Chondrocyte-specific Nell-1 deficiency may result in postnatal dwarfism and premature osteoporosis.