(A,B) TGF-β stimulates Smurf2 and inhibits ColX expression in chondrocytes from growth plates (A) and sterna (B). (C) PTHrP inhibits ColX but not Smurf2 expression in growth plate chondrocytes. Chondrocytes were cultured in the presence or absence of PTHrP for 2 days, and the mRNA levels of Smurf2 and ColX were determined by real time RT-PCR. Error bars represent the standard deviation of the mean of the results (n = 4).

(A,B) Relative expression levels of Smurf2 and ColX in growth plate (GP) chondrocytes (A) and sternal chondrocytes (B). Growth plate chondrocytes and sternal chondrocytes were cultured at 37°C for 2, 4, 6, and 8 days. Total RNA was extracted from the cells and the mRNA levels of Smurf2 and ColX were determined by real time RT-PCR. Error bars represent the standard deviation of the mean of the results (n = 3).

(A,B) Morphology of ulna growth plate at HH stage 36–37 (A) and stage 43 (B) stained with hematoxylin and eosin (H&E). a′, b′, and c′ are the enlarged regions of a, b, and c in (A). e′, f′, and g′ are the enlarged regions of e, f, and g in (B). (C,D) Relative expression levels of Smurf2 and ColX in growth plate (C) and sterna (D). Total RNA was extracted from the growth plate and sternum at HH stages 37, 40, and 43. The mRNA levels were determined by real time RT-PCR. Error bars represent the standard deviation of the mean of the results (n = 4). *p < 0.05, **p < 0.01.

(A) Smurf2 is overexpressed in chondrocytes infected with Smurf2-RCAS. Arrows indicate the accumulation of flag-tagged Smurf2 protein in the nucleus of the chondrocytes stained with a rhodamine-coupled antiflag antibody (left). Arrowheads indicate the background in the chondrocytes infected with Smurf2-RCAS (left), or empty RCAS (right). (B) Overexpression of Smurf2 in chondrocytes inhibits TGF-β signaling. Chondrocytes infected with Smurf2-RCAS (Smurf2-RCAS), RCAS (RCAS), and vehicle without any of the virus (Mock) were transfected with p3TP-Lux. Luciferase activity was determined after 40 h. (C,D) Injection of chick wing buds with Smurf2-RCAS accelerated cartilage maturation and endochondral ossification. Smurf2-RCAS (C) or RCAS(D) was injected into the right wing bud at HH stage 20–23, and the left wing buds were uninfected controls. Wing buds were harvested at HH stage 30–34, and stained with Alcian blue and Alizarin red. Red arrow indicates an expanded ossification domain in the humerus infected with Smurf2-RCAS; black arrows indicate small patches of ossification in the control humerus. Double arrowhead lines indicate the distance between the hypertrophic domain and the articular surface. An arrowhead indicates the hypertrophic zone. H, humerus; R, radius; U, ulna. (E) Quantitative analysis of the distance between the hypertrophic (hy) zone and the ends of long bones. Error bars represent the standard deviation of the mean of the results (n = 4). *p < 0.05.