Molecular characterization and methylation study of matrix gla protein in articular cartilage from pig with osteochondrosis

Abstract

Osteochondrosis (OC) or leg weakness is an economically important disease of young fast growing pigs and is a concern of animal welfare. The etiology and pathogenesis of osteochondrosis is not fully understood yet, but any abnormalities in the formation of hypertrophic chondrocytes and disrupted blood supply to the growth cartilage are very important predisposing factors. Matrix gla protein (MGP) as a potential calcification inhibitor of extracellular matrix might contribute to the development of OC. Molecular characterization, polymorphisms analysis, methylation at promoter region and expression of MGP gene and protein were performed in both healthy and OC cartilage collected from a Duroc × Pietrain resource population. The porcine MGP gene consists of 4 exons and 3 introns. The full-length MGP cDNA isolated from articular cartilage consists of 606 bp with a 69-bp 5′ UTR, a 312-bp open reading frame with a start codon, a 225-bp 3′ UTR. Three single-nucleotide polymorphisms (SNP) were detected in the intron 1 (A-115G, C-1073T and C-1135A) and one in the 3′UTR (C-3767T). The relative abundance of MGP mRNA was lower ( P < 0.05) in OC compared with healthy cartilage. Moreover, the intensity of MGP band was lower ( P < 0.05) in OC group when quantified by western blot. Furthermore, one CpG region was identified in MGP promoter and DNA methylation of three CG sites were higher in OC compared with normal cartilage. This suggested that the high DNA methylation at specific CG sites in the MGP promoter might be involved in the down regulation of MGP in OC. Immunofluorescence of normal cartilage collected from pigs of different ages revealed that MGP signals were higher in younger pigs and decreased in the older pigs. The MGP protein was expressed more near to the cartilage canals. These results suggest that the MGP gene might be a potential candidate gene for the development of OC in pigs.