No abstract

The mRNA level of basic helix-loop-helix transcription factor DEC1 (BHLHB2)/Stra13/Sharp2 was up-regulated during chondrocyte differentiation in cultures of ATDC5 cells and growth plate chondrocytes, and in growth plate cartilage in vivo. Forced expression of DEC1 in ATDC5 cells induced chondrogenic differentiation, and insulin increased this effect of DEC1 overexpression. Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) suppressed DEC1 expression and the differentiation of ATDC5 cells, but DEC1 overexpression antagonized this inhibitory action of PTH/PTHrP. Transforming growth factor-␤ or bone morphogenetic protein-2, as well as insulin, induced DEC1 expression in ATDC5 cultures where it induced chondrogenic differentiation. In pellet cultures of bone marrow mesenchymal stem cells exposed to transforming growth factor-␤ and insulin, DEC1 was induced at the earliest stage of chondrocyte differentiation and also at the hypertrophic stage. Overexpression of DEC1 in the mesenchymal cells induced the mRNA expressions of type II collagen, Indian hedgehog, and Runx2, as well as cartilage matrix accumulation; overexpression of DEC1 in growth plate chondrocytes at the prehypertrophic stage increased the mRNA levels of Indian hedgehog, Runx2, and type X collagen, and also increased alkaline phosphatase activity and mineralization. To our knowledge, DEC1 is the first transcription factor that can promote both chondrogenic differentiation and terminal differentiation.The development of the vertebrate long bones occurs through the process of endochondral ossification, which is initiated in the embryo with the condensation of mesenchymal cells and then progresses with their commitment and differentiation into chondrogenic cells. By the late embryonic stage, the epiphyseal growth plate has developed with distinguishable, well organized and spatially distinct zones of resting, proliferating, and post-proliferative hypertrophic chondrocytes. The hypertrophic cartilage calcifies and is invaded by capillaries, and is subsequently replaced by new bone (1). Recent studies have identified several transcription factors involved in endochondral ossification. Among these, Sox9 is required for the condensation of prechondrogenic mesenchymal cells, and Sox5 and Sox6, as well as Sox9, are required for the activation of type II collagen expression during chondrogenesis (2). In addition, different sets of Smads are involved in stimulation or inhibition of chondrocyte hypertrophy by transforming growth factor-␤ (TGF-␤) superfamily members (3, 4). Runx2/Cbfa1/AML3/PEBP2␣-A is essential for intramembranous ossification, and mutations in this gene are responsible for cleidocranial dysplasia, a syndrome characterized by open fontanelles and hypoplastic clavicles (5-8). Furthermore, Runx2 plays a crucial role in endochondral ossification. In Runx2-deficient mice, chondrocyte hypertrophy, mineralization, and vascular invasion are suppressed in most parts of the skeleton (9 -13). On the other hand, little is known about the role of the bas...

The experiments described herein tested the effects of CaCl2 and ZnCl2, added at various concentrations in the culture medium, upon the synthesis of collagen and proteoglycan by adult and fetal (articular, epiphyseal and hypertrophic) bovine chondrocytes maintained in high density multilayer cultures. CaCl2 concentrations below 0.5 mM or the addition of 1-50 microM ZnCl2 to the medium selectively promoted the production of collagen by all four populations of chondrocytes but had no effect on fibroblasts. Further, these changes had no statistically significant effect on the incorporation of 35S-sulfate into macromolecules or on the synthesis of gelatinase A, measured by gelatin zymography. The addition of CaCl2 and ZnCl2 at these concentrations did not result in a change in the relative proportion of non-crosslinked 3H-collagen molecules (synthesized in the presence of beta-aminopropionitrile) partitioning in the cell layer and medium compartments, and did not appreciably alter the pattern of collagens synthesized by any of the cell populations. The hypertrophic cells synthesized high levels of collagen type X in the presence as well as absence of exogenously added cations. However, CaCl2 at 10 mM caused a marked upregulation of collagen type X synthesis by a preparation of chondrocytes derived from the entire growth plate, consistent with the view that calcium at that concentration stimulated the differentiation of some of the cells into hypertrophic chondrocytes.

No abstract