Parathyroid Hormone–related Peptide (pthrp)-dependent and -independent Effects of Transforming Growth Factor ␤ (tgf-␤ ) on Endochondral Bone Formation

Previously, we showed that expression of a dominant-negative form of the transforming growth factor ␤ (TGF-␤) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-␤ in limiting terminal differentiation in vivo. Parathyroid hormone–related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hyper-trophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-␤ and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embry-onic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-␤ inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-␤ 1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-␤ in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-␤ acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-␤ has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation. Key words: chondrocyte differentiation • skeletal development • perichondrium • organ culture • transforming growth factor ␤ receptors T HE rate of cartilage differentiation must be carefully regulated so that bones attain the proper shape and length. Early in skeletal development, mesenchy-mal cells condense and differentiate into chondroblasts, which form the initial shape of the bone rudiment. Chon-droblasts then undergo a complex program of proliferation , maturation, and hypertrophy. Hypertrophic cartilage is then replaced with bone. Endochondral bone formation is complex and requires the coordination of signals from several factors and multiple cell types (reviewed in Can-cedda et al., 1995; Erlebacher et al., 1995). Chondrocyte differentiation is regulated by factors synthesized by both chondrocytes and cells in the perichondrium, the layer of mesenchyme that surrounds the cartilage rudiment. Parathyroid hormone–related peptide (PTHrP) 1 was first identified as a factor associated with humoral hyper-calcemia of malignancy (Broadus and Stewart, 1994). PTHrP is expressed in a wide variety of adult and embry-onic cell types, including …