Objectives: Activation of proMMP-2 by membrane type matrix metalloproteinase-1 (MT1-MMP) requires formation of an activation complex involving the hemopexin-like domain of proMMP-2 (PEX), the carboxyl-terminal domain of TIMP-2 (C-TIMP-2) and MT1-MMP. The goal of these experiments was to demonstrate that excess of extraneous recombinant (r) PEX or rC-TIMP-2 may interrupt the activation complex and therefore inhibit MMP-2 activation.
Methods: rC-TIMP-2 and rPEX were expressed in E. coli and purified to homogeneity by Ni3+ affinity chromatography as determined by SDS-PAGE. Human fibrosarcoma (HT1080) and rat osteosarcoma (ROS) cell lines were co-cultured with a concentration range of either purified rC-TIMP-2 or rPEX for 24 hours. The level of activation of proMMP-2 secreted into the culture medium was analyzed by gelatin zymography for relative amounts of latent 66 kDa and activated 59-kDa forms of MMP-2. Gel images were digitized and intensities of the two forms of MMP-2 were quantified.
Results: With concanavlin-A induction, the ratio of active:latent MMP-2 was 78.6 in HT1080 and 2.5 in ROS culture media. When treated with 7 μM rC-TIMP-2, these ratios decreased to 2.8 in HT1080 and 0.5 in ROS conditioned media, reflecting 96% and 80% inhibition of proMMP-2 activation for HT1080 and ROS cells, respectively. Addition of 2.2 μM rPEX decreased the ratio to 1.5 and 0.22 for HT1080 and ROS, respectively, corresponding to 98% and 91% inhibition of proMMP-2 activation. The inhibition of proMMP-2 activation by rC-TIMP-2 and rPEX was concentration dependent.
Conclusion: These results showed that rC-TIMP-2 and rPEX can inhibit activation of proMMP-2 in cancer cells by a mechanism that most likely involves competitive interruption of the interactions between PEX and TIMP-2. This approach constitutes a potential future strategy for MMP-2 inhibition in periodontal disease and oral cancer. (Supported by DE 017139 (COSTAR), DE 018135 and DE 017139).
S. BABER, B, S., Z, C., M, M., Dr. Sanjay Pal, and Xu, X., “Competitive Inhibition of proMMP-2 Activation”, J Dent Res , vol. 87, no. A, p. 1012, 2008.