Role of tissue inhibitor of metalloproteinases-2 (TIMP-2) in regulation of pro-gelatinase A activation catalyzed by membrane-type matrix metalloproteinase-1 (MT1-MRP) in human cancer cells

To clarify the regulatory mechanism of pro-gelatinase A (proGelA) activation at a cellular level, expression of gelatinase A (GelA), three MT-RIMPs, and TIMP-2 was examined with 11 human cancer cell lines cultured in the presence and absence of stimulants. MT1-MMP mRNA was expressed in 8 cell lines, while MT2-MMP and MT3-MMP mRNAs were expressed in fewer cell lines, The cells with high proGelA activation strongly expressed MTI-MMP mRNA but not MTS-MMP and MT3-MMP mRNAs, suggesting that MT1-MMP was responsible for the proGelA activation in the cancer cells. Treatments with concanavalin A (Con A) and a phorbor ester (TPA) enhanced the MTI-MMP expression, but only Con A stimulated the proGelA activation in many cell lines. In HT1080 fibrosarcoma cells, however, TPA also stimulated the activation. The level of TIMP-8 secreted into culture medium inversely correlated with proGelA activation, For example, 2 squamous cell carcinoma lines (HSC-3 and HSC-4) and 3 HT1080 clones, which efficiently activated proGelA, secreted little TIMP-8 into medium, whereas other cell lines and other HT1080 clones, which hardly activated proGelA, secreted TIMP-8 at high levels, When HSC-3 cells were incubated with TIMP-8 protein or transfected with TIMP-8 cDNA, the proGelA activation was strongly inhibited, These results indicated that. extracellular TIMP-8 was an important negative regulator of proGelA activation. However, the level of extracellular TIMP-8 was not consistent with that of TIMP-2 mRNA in some cell lines. Other experimental results suggested that TIMP-2 might be rapidly metabolized after binding to MT1-MMP, and Con A treatment might stabilize the complex of TIMP-8 and MT1-MMP on cell membranes.