Molecular cloning and cartilage gene expression of equine stromelysin 1 (matrix metalloproteinase 3).
OBJECTIVE
To clone and determine molecular structure of equine stromelysin 1 (matrix metalloproteinase 3) and examine stromelysin expression in articular cartilage. SAMPLES AND PROCEDURE: Total RNA was harvested from equine arthritic cartilage specimens and was used for reverse transcription and polymerase chain reaction amplification to develop overlapping complementary DNA (cDNA) clones. Four cDNA sequences were ligated into plasmid (pGEM3Z) constructs and subcloned into bacterial expression vectors, and sequence was determined by automated dye terminator sequencing. Stromelysin mRNA expression was assessed in normal and arthritic cartilage and synovium by northern blotting. Interleukin 1 (IL-1) regulation of stromelysin transcriptional activity in articular chondrocytes cultured in the presence of 0, 20, and 50 ng of IL-1 alpha/ml was assessed by northern blotting of total RNA isolated from the cell layer and probed with 32P-labeled stromelysin cDNA.


RESULTS
4 overlapping clones provided the full-length cDNA sequence of equine stromelysin, including portions of untranslated 5' and 3' regions, and the entire translated portion coding for the stromelysin prepropeptide. The coding region of 1,431 base pairs was well conserved between species, with 86, 83, and 78% sequence homology to that of human, rabbit, and mouse stromelysin, respectively. Predicted amino-acid (AA) sequence data indicated highly conserved features. Comparison of the equine AA sequence revealed 89, 88, and 84% homology to the AA structure in human, rabbit, and mouse stromelysin, respectively. Minimal stromelysin mRNA expression was evident in normal cartilage and synovium, and increased expression was evident in arthritic cartilage. Marked dose-dependent up-regulation of stromelysin transcriptional activity was evident in chondrocyte cultures exposed to 20 and 50 ng of IL-1/ml.


CONCLUSIONS
Stromelysin DNA sequence in horses is similar to that in people and rodents. Constitutive stromelysin message amounts in normal cartilage and synovium are low, but considerably increased in arthritic cartilage and in chondrocytes exposed to IL-1.
Balkman
C E
CE
Comparative Orthopaedics Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Nixon
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000201:enzymology / Q000473:pathology / Q000662:veterinary; D001483:Base Sequence; D002358:Cartilage, Articular / Q000166:cytology / Q000201:enzymology* / Q000473:pathology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006734:Horse Diseases; D006736:Horses; D006801:Humans; D007794:Lameness, Animal; D019278:Matrix Metalloproteinase 3 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D011994:Recombinant Proteins; D019278:Matrix Metalloproteinase 3