Canine TIMP-2: purification, characterization and molecular detection

Abstract

Matrix metalloproteinases (MMPs), which degrade tissues in health and disease are under the control of the tissue inhibitors of MMPs, the TIMPs. TIMP-2 is particularly important for control of MMP-2 and both have been implicated in many pathological processes from arthritis to tumour invasion. This study characterized and detected TIMP-2 from canine cells; including synovial fibroblasts and three tumour-derived canine cell lines, K1, K6 and DH82. Gelatin zymography demonstrated that pro-MMP-2 is produced by synovial fibroblasts and the three cells lines. Reverse zymograms showed that all the cell sources tested secrete both TIMP-1 and TIMP-2. The 22 kDa band was purified and n-terminal amino acid sequencing showed it to be highly homologous to equine and human TIMP-2. Analysis of purified canine MMP-2 and MMP-9 showed that TIMP-2 is associated, and co-purifies with MMP-2. Polymerase chain reaction, using consensus primers, was used to detect TIMP-2 mRNA from the cell sources and proved positive in all cases. This work highlights the importance of TIMP-2 as the main inhibitor for MMP-2 and, therefore, opens the possibilities of targeting TIMP-2 for therapeutic intervention against connective amino acid tissue degradation in a range of diseases.