Molecular characterization and chromosomal assignment of equine cartilage derived retinoic acid sensitive protein (CD–RAP)/melanoma inhibitory activity (MIA)

Abstract

Cartilage-derived retinoic acid sensitive protein (CD–RAP) also known as melanoma inhibitory activity (MIA) has already been established as a marker for chondrocyte differentiation and a number of cancerous conditions in humans. Studies have also shown that CD–RAP/MIA is a potential marker of joint disease. The objective of this study was to characterize the equine CD–RAP/MIA gene and thus make it available as a marker in cartilage research and clinical studies. Gene analysis revealed that the equine gene (GenBank accession no. EF679787) consists of four exons and three introns, and the homology to the human gene is 90% for the translated region. The upstream sequence includes regulatory elements and putative transcription factor binding sites previously described in the human and murine promoter regions. The deduced amino acid sequence consists of 130 aa including a signal peptide of 23 aa, and has a 91% identity to the human protein. Using radiation hybrid mapping, the CD–RAP/MIA gene was localized to the p arm of equine chromosome 10 (ECA10p), which is in accordance with prediction based on the current human-equine comparative map. Gene expression studies showed expression of CD–RAP/MIA mRNA in articular cartilage and chondrocytes from horses with no signs of joint disease. The expression decreased as the cells dedifferentiated in monolayer culture. We also identified an equine CD–RAP/MIA splice variant similar to that reported in humans. The CD–RAP/MIA protein was detected in equine synovial fluid, serum and culture medium from chondrocyte cultures. In conclusion, CD–RAP/MIA is expressed in equine cartilage and chondrocytes, and the protein can be detected in equine serum, synovial fluid and in culture medium from chondrocyte cultures. The equine gene and resulting protein share great homology with the human gene, making future studies on CD–RAP/MIAs potential as a marker in joint disease possible using the equine joint as a model.