Molecular cloning and cartilage gene expression of equine stromelysin 1 (matrix metalloproteinase 3).

The research article focuses on the cloning and molecular structure determination of equine (horse) stromelysin 1, a type of matrix metalloproteinase (enzyme), and its expression in cartilage tissue. The findings show increased expression of stromelysin in arthritic cartilage and susceptibility to regulation by Interleukin 1.

Molecular Cloning and Structure Determination

• RNA was extracted from horse arthritic cartilage samples and reverse transcription was applied to develop complementary DNA (cDNA) clones. Altogether, four overlapping clones provided a full-length sequence of equine stromelysin.
• The obtained cDNA sequences were ligated into plasmid constructs and subcloned into bacterial expression vectors.
• The sequence was determined by automated dye terminator sequencing.
• The coding region of 1,431 base pairs was well conserved between species, having 86%, 83%, and 78% sequence homology with human, rabbit, and mouse stromelysin, respectively.
• The translated portion coded for the prepropeptide of stromelysin, including parts of untranslated 5′ and 3′ regions. Predicted amino-acid sequence data indicated highly conserved features.
• When compared, the horse amino acid sequence revealed 89%, 88%, and 84% homology to the human, rabbit, and mouse stromelysin respectively.

Stromelysin Expression in Cartilage

• The expression of stromelysin mRNA in normal and arthritic cartilage and synovium was verified with northern blotting.
• The level of stromelysin mRNA was found to be minimal in normal cartilage and synovium tissue, but evidenced a considerable increase in arthritic cartilage.

Regulation by Interleukin 1

• The study also explored the impact of Interleukin 1 (IL-1) on stromelysin’s transcriptional activity in articular chondrocytes cultured with varying concentrations of IL-1.
• A marked dose-dependent up-regulation in stromelysin’s transcriptional activity was observed in chondrocytes exposed to 20 and 50 ng/ml of IL-1.

Conclusion

• The findings suggest that stromelysin’s DNA sequence in horses bears similitude to human and rodent sequences.
• While the level of stromelysin message in the normal cartilage and synovium is low, it increases significantly in arthritic cartilage and under exposure to IL-1.