Abstract
We investigated the effects of bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta superfamily, on the regulation of the chondrocyte phenotype, and we identified signaling molecules involved in this regulation. BMP-2 triggers three concomitant responses in mouse primary chondrocytes and chondrocytic MC615 cells. First, BMP-2 stimulates expression or synthesis of type II collagen. Second, BMP-2 induces expression of molecular markers characteristic of pre- and hypertrophic chondrocytes, such as Indian hedgehog, parathyroid hormone/parathyroid hormone-related peptide receptor, type X collagen, and alkaline phosphatase. Third, BMP-2 induces osteocalcin expression, a specific trait of osteoblasts. Constitutively active forms of transforming growth factor-beta family type I receptors and Smad proteins were overexpressed to address their role in this process. Activin receptor-like kinase (ALK)-1, ALK-2, ALK-3, and ALK-6 were able to reproduce the hypertrophic maturation of chondrocytes induced by BMP-2. In addition, ALK-2 mimicked further the osteoblastic differentiation of chondrocytes induced by BMP-2. In the presence of BMP-2, Smad1, Smad5, and Smad8 potentiated the hypertrophic maturation of chondrocytes, but failed to induce osteocalcin expression. Smad6 and Smad7 impaired chondrocytic expression and osteoblastic differentiation induced by BMP-2. Thus, our results indicate that Smad-mediated pathways are essential for the regulation of the different steps of chondrocyte and osteoblast differentiation and suggest that additional Smad-independent pathways might be activated by ALK-2.
Highlights
The development of long bones involves first the formation of cartilage primordia, which prefigure the future skeletal elements, and the replacement of the cartilage by bone
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis showed that the basal level of aggrecan and Sox9 expression was very low in MC615 cells grown in 1% FBS and was stimulated in the presence of Bone morphogenetic proteins (BMPs)-2 (Fig. 1A)
Our RT-PCR analysis indicated that chondrogenic expression was stronger in Primary embryonic chondrocytes (PEC) than in MC615 cells, as attested by the expression of Col2a1 restricted to the IIB form and by the higher levels of aggrecan and Sox9 expression in PEC
Summary
Cell Culture—The MC615 cell line has been characterized previously [17]. For the studies with freshly isolated chondrocytes, embryonic mouse chondrocytes were prepared from the ventral parts of the rib cages of 17.5-day post coitum mice, as described [23]. The MC615 cells and rib chondrocytes were maintained in 1:1 high glucose DMEM/ Ham’s F-12 containing 10% FBS and supplemented with 2 mM Lglutamine, 100 units/ml penicillin, 100 g/ml streptomycin (all products from Invitrogen) on Corning dishes, as previously described [22]. MC615 cells were seeded in six-well tissue culture plates with 1.75ϫ105 cells/well and were incubated for 2 days with 1:1 high glucose DMEM/Ham’s F-12 supplemented with 1% FBS Ϯ BMP-2. After washing twice with PBS, cells were incubated for 20 min with a mixture of 0.1 mg/ml naphthol AS-MX phosphate (Sigma), 0.5% N,N-dimethylformamide, 2 mM MgCl2, and 0.6 mg/ml fast blue BB salt (Sigma) in 0.1 M Tris-HCl, pH 8.5, at 37 °C, followed by observation using phase-contrast microscopy.
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