Tissue inhibitor of metalloproteinases-4 inhibits but does not support theactivation of gelatinase A via efficient inhibition of membrane type I-matrix metalloproteinase

The tissue inhibitors of metalloproteinases 1-4 (TIMPs) have discrete regul atory roles in the activation of matrix: metalloproteinase (MMP)-2 (gelatin ase A), an important basement membrane-degrading MMP pivotal to tumor metas tasis and angiogenesis. TIMP-2 binds to both the hemopexin C domain of prog elatinase A and the active site of membrane type-1 (MT1) MMP. This trimeric complex presents the cell surface-bound gelatinase A zymogen to a fret MT1 -MMP molecule for activation. To investigate the role of TIMP-4 in the acti vation process, we developed a new procedure for the expression and purific ation of recombinant human TIMP-4 from baby hamster kidney cells. The recom binant TIMP-4 was a potent inhibitor of gelatinase A {apparent K-i [K-i(app .)] less than or equal to 9 pM; k(on) (association rate constant), 4.57 +/- 0.13 x 10(6) M(-1)s(-1)} and was less dependent upon hemopexin C domain in teractions than TIMP-2 in its mode of binding and inhibition. Unlike TIMP-1 , TIMP-4 strongly inhibited MT1-MMP (K-i(app.) less than or equal to 100 pM ; k(on), 3.49 +/- 0.34 x 10(6) M(-1)s(-1)) and blocked the concanavalin A-i nduced cellular activation of progelatinase A. In concanavalin A-stimulated homozygous Timp2 -/- fibroblasts or unstimulated MT1-MMP-transfected Timp2 -/- cells, which cannot activate progelatinase A, activation was restored by the addition of 0.3-5 nM TIMP-2 but not by TIMP-4, unequivocally showing the TIMP-2. dependency of MT1-MMP-induced activation of gelatinase A and t he fact that TIMP-4 cannot support activation. The dominance of TIMP-2, in the activation process was further supported by the preferential binding of TIMP-2 compared with TIMP-4 to the hemopexin C domain of progelatinase A i n inhibitor mixtures and by the ability of TIMP-2 to displace TIMP-4 from t he hemopexin C domain. Hence, TIMP-4 regulates gelatinase A activity by eff icient inhibition of MT1-MMP-mediated activation and by inhibiting the acti vated enzyme and, thus, is a tumor resistance factor in the peritumor strom a.