Regulation of Chondrogenesis and Cartilage Maturation In Vitro: Role of TGF-beta1, Thyroid Hormone, and Wnt Signaling

摘要

Endochondral skeletal development involves the commitment and differentiation of mesenchymal cells into chondrocytes, and the subsequent, progressive proSiferation, hypertrophy, and mineralization of the growth cartilage that lead to invasive vascularization and the replacement of the cartilage anlage by newly formed bone. Regulation of the maturation program of cartilage is therefore essential for proper skeleta! formation. We have used two in vitro model systems to investigate the mechanisms regulating chondrocyte maturation. In the first system, chick embryonic Jimb bud mesenchymal cells are isolated at Hamburger-Hamilton Stage 23/24 prior to overt chondrogenesis, and plated as high cell density micromass cultures. Within 3-4 days, these cells differentiate into chondrocytes that, upon long-term culture up to 28 days, undergo progressive proliferation, hypertrophy, and mineralization. This maturation process is accompanied by cessation of cellular proliferation and the onset of programmed cell death (apoptosis), characterized by internucleosomal DNA fragmentation, appearance of apoptotic bodies, and TUNEL reactivity. Using this system, we have evaluated the effect of transforming growth factor-beta1 (TGF-IJ1) and the thyroid hormone, triiodothyronine (T3), on chondrocyte hypertrophy. T3 stimulates chondrocyte hypertrophy and apoptosis in a dose and culture time-dependent manner. Interestingly, in the presence of both factors, TGF-beta1 inhibits the hypertrophy/apoptosis-stimulating effect of T3. These observations strongly suggest that T3 and TGF-IJ1, which are both found in the growth cartilage in vivo, act to regulate cartilage maturation by modulating chondrocyte apoptosis. Using this system, we have recently examined whether members of the Wnt family of signaling molecules that are expressed in the embryonic limb and regulate mesenchymal chondrogenesis also influence cartilage maturation. Cultures are transfected prior to plating with replication-competent retroviral expression constructs of chicken Wnt-5a and Wnt-7a, as well as Chfz-S and Chfz-7, two putative Wnt receptors of the Frizzled gene family. Mis-expression of Wnt-7alpha results in severe inhibition of chondrogenesis, resulting in the formation of a fibroblastic cell mass, completely devoid of cartilage phenotype, by culture Day 28. Wnt-5a mis-expression elicits a slight retardation of maturation during the "pre-hypertrophic stage", but the cultures appear to recover in their maturation program by Day 28. Interestingly, mis-expression of Chfz-7 also inhibits chondrogenesis and retards chondrocyte maturation and hypertrophy. These observations strongly suggest that Wnt functions in cartilage involve both mesenchymal chondrogenesis and chondrocyte maturation and hypertrophy. In the second experimental system, chondrocytes isolated from the upper and lower sternum of the chick embryo, which differ in their ability to mature and undergo hypertrophy, are examined and compared in terms of the expression profiles of genes specific for the hypertrophic and apoptotic programs, to analyze the nature of the cross-talk between these two pathways in the regulation of cartilage maturation.