CLONING OF A RETINOIC ACID-SENSITIVE MESSENGER-RNA EXPRESSED IN CARTILAGE AND DURING CHONDROGENESIS

Retinoic acid (RA) is known to play a role in various aspects of skele tal development in vivo, including morphogenesis, growth plate maturat ion, and apoptosis, In cell culture, RA treatment of chondrocytes supp resses the differentiated phenotype characterized by production of typ e II collagen and aggrecan, In an effort to discover molecules involve d in regulation of the chondrocyte phenotype or related to development al processes such as chondrogenesis, mRNAs from bovine chondrocytes cu ltured with and without RA were amplified by reverse transcription-pol ymerase 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-se nsitive protein (CD-RAP), and its properties are described here. The f ull-length bovine CD-BAP mRNA was cloned after amplification by the ra pid amplification of cDNA ends procedure, and a part of the rat CD-RAP mRNA was amplified by reverse transcription-PCR using sequence-specif ic primers, The bovine CD-RAP mRNA contains an open reading frame of 1 30 amino acids, CD-RAP mRNA expression, as determined by Northern blot analysis and in situ hybridization, was present only in cartilage pri mordia and cartilage, The inhibition of CD-RAP mRNA expression by RA i n vitro was time- and dose-dependent and was tested over concentration s from 10(-8) to 10(-6) M, Southern blot analysis of genomic DNA indic ated that CD-RAP was encoded by a single copy gene and that no other g enes were closely related, What appears to be the human homologue of C D-RAP was recently isolated and cloned from a melanoma cell line and s hown 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 c artilage development and maintenance.