Retinoic acid (RA) is known to play a role in various aspects of skeletal development in vivo, including morphogenesis, growth plate maturation, and apoptosis. In cell culture, RA treatment of chondrocytes suppresses the differentiated phenotype characterized by production of type II collagen and aggrecan. In an effort to discover molecules involved in regulation of the chondrocyte phenotype or related to developmental processes such as chondrogenesis, mRNAs from bovine chondrocytes cultured with and without RA were amplified by reverse transcription-polymerase chain reaction (PCR) and compared by differential display. PCR products whose expression was inhibited by RA treatment were cloned. One cDNA encodes a molecule we call cartilage-derived retinoic acid-sensitive protein (CD-RAP), and its properties are described here. The full-length bovine CD-RAP mRNA was cloned after amplification by the rapid amplification of cDNA ends procedure, and a part of the rat CD-RAP mRNA was amplified by reverse transcription-PCR using sequence-specific primers. The bovine CD-RAP mRNA contains an open reading frame of 130 amino acids. CD-RAP mRNA expression, as determined by Northern blot analysis and in situ hybridization, was present only in cartilage primordia and cartilage. The inhibition of CD-RAP mRNA expression by RA in vitro was time-and dose-dependent and was tested over concentrations from 10 ؊8 to 10 ؊6 M. Southern blot analysis of genomic DNA indicated that CD-RAP was encoded by a single copy gene and that no other genes were closely related. What appears to be the human homologue of CD-RAP was recently isolated and cloned from a melanoma cell line and shown to function as a growth inhibitory protein (Blesch, A., Boberhoff, A.-K., Apfel, R., Behl, C., Hessdoerfer, B., Schmitt, A., Jachimcza, P., Lottspeich, F., Buettner, R., and Bogdahn, U. (1994) Cancer Res. 54, 5695-5701). Neither CD-RAP nor this protein showed any homology to known proteins. We speculate that, in vivo, CD-RAP functions during cartilage development and maintenance.

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Here we report on the structure, expression, and function of a novel cartilage-specific gene coding for a 17-kDa small, highly charged, and secreted protein that we termed Ucma (unique cartilage matrix-associated protein). The protein is processed by a furin-like protease into an N-terminal peptide of 37 amino acids and a C-terminal fragment (Ucma-C) of 74 amino acids. Ucma is highly conserved between mouse, rat, human, dog, clawed frog, and zebrafish, but has no homology to other known proteins. Remarkable are 1-2 tyrosine sulfate residues/molecule and dense clusters of acidic and basic residues in the C-terminal part. In the developing mouse skeleton Ucma mRNA is expressed in resting chondrocytes in the distal and peripheral zones of epiphyseal and vertebral cartilage. Ucma is secreted into the extracellular matrix as an uncleaved precursor and shows the same restricted distribution pattern in cartilage as Ucma mRNA. In contrast, antibodies prepared against the processed C-terminal fragment located Ucma-C in the entire cartilage matrix, indicating that it either diffuses or is retained until chondrocytes reach hypertrophy. During differentiation of an MC615 chondrocyte subclone in vitro, Ucma expression parallels largely the expression of collagen II and decreases with maturation toward hypertrophic cells. Recombinant Ucma-C does not affect expression of chondrocyte-specific genes or proliferation of chondrocytes, but interferes with osteogenic differentiation of primary osteoblasts, mesenchymal stem cells, and MC3T3-E1 pre-osteoblasts. These findings suggest that Ucma may be involved in the negative control of osteogenic differentiation of osteochondrogenic precursor cells in peripheral zones of fetal cartilage and at the cartilage-bone interface.Elucidation of molecular mechanisms underlying chondrocyte differentiation is not only important for our understanding of skeletal development, but also of particular interest for our knowledge on the behavior of chondrocytes following articular cartilage damage during cartilage repair and treatment of degenerative cartilage diseases. Initial steps of chondrogenesis, i.e. the formation of a cartilage blastema from limb bud mesenchymal cells, include cell condensation and onset of chondrocyte differentiation marked by the expression of cartilage-specific matrix proteins such as aggrecan, collagen II, IX, and XI and others (1, 2). These events are regulated by the orchestrated action of several growth factors including BMPs, Wnt factors, FGFs, and the transcription factors Sox5,6, and 9 (3,4). Further steps of chondrocyte growth, maturation, and replacement by bone in the growth plate of long bones, ribs, and vertebrae during endochondral ossification can be defined by the stepwise onset or decline of differentially expressed genes: collagen II and Sox9 for resting and proliferating, FGFR3 for proliferating and prehypertrophic, Ihh and PTHrP receptor for prehypertrophic, collagen X for hypertrophic, and Runx2, osteocalcin, and MMP13 for late hypertrophic chondrocytes (...

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