The process of endochondral ossification consists of multiple stages. The first stage is the condensation of mesenchymal cells, which subsequently differentiate into chondrocytes and produce cartilage matrix proteins including type 2 collagen (Col2a1) and Aggrecan, and form cartilage rudiments. In the second stage, chondrocytes undergo proliferation and maturation into hypertrophy, and express hypertrophic chondrocyte specific marker, type X collagen (ColX). Finally, calcification of matrix and invasion of blood vessels takes place, hypertrophic chondrocytes undergo apoptosis, and cartilage is removed and replaced by bone.
During the long bone lengthening through endochondral ossification, chondrocyte proliferation and maturation are tightly regulated by several growth factors such as Indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), and members of the TGF-β superfamily. TGF-β exists in three isoforms, TGF-β1, -β2, and -β3, and has diverse functions in the regulation of chondrocyte differentiation and maturation. TGF-β induces chondrogenesis and Col2a1
expression in limb bud cartilage and mesenchymal stem cell culture.1–3
On the other hand, TGF-β is a potent inhibitor of chondrocyte maturation, evidenced in part by accelerated maturational progression when TGF-β signaling is reduced.4,5
Ihh is expressed by prehypertrophic chondrocytes and coordinates with PTHrP expressed by periarticular chondrocytes to form a negative feedback loop regulating the growth and differentiation of chondrocytes.6
TGF-β2, expressed in the perichondrium, mediates the effect of Ihh on the expression or acts upstream of PTHrP in this feedback loop.7
In addition, PTHrP may also act downstream of TGF-β, mediating its inhibitory effect on chondrocyte hypertrophic differentiation.8
TGF-β signaling is regulated at various levels. The expression level of TGF-β in the chondrocytes of the growth plate does not always correlate with the levels of its biologically active polypeptide in the matrix environment due to post-transcriptional mechanisms including regulation and activation of TGF-β latent forms by vitamin D3 and matrix metalloproteinases (MMPs).9–11
In addition, TGF-β intracellular signaling is modulated by the expression levels of TGF-β receptors (type II and type I)and TGF-β receptor regulated Smads (Smad2 and Smad3). Furthermore, the TGF-β signaling cascade within the cells is regulated by intracellular signals such as inhibitory Smads and Smurfs. For example, Smad7 exerts an inhibitory function through binding to the TGF-β type I receptor and prevention of recruitment and phosphorylation of Smad3.12
Smad7 also acts as an adaptor to promote Smurf2-mediated ubiquitination and degradation of the TGF-β type I receptor.13
Although Smurf2 ubiquitinates both BMP and TGF-β activated Smads for degradation in mammalian cells,14,15
we found that Smurf2 specifically targets TGF-β receptor activated Smads for ubiquitination and degradation in primary articular chondrocytes, and leads to advanced cell maturation due to loss of TGF-β signaling.16
However, how Smurf2 affects chondrocyte maturation during embryonic development is totally unknown. In this study, we monitored the expression levels of endogenous Smurf2 in chondrocytes during embryonic development and overexpressed Smurf2 in a developing chick wing bud, and found: (1) the expression levels of Smurf2 are associated with chondrocyte stage during embryonic development, and (2) ectopic overexpression of Smurf2 in chick wing buds accelerated cartilagematuration and endochondral ossification.