Type I collagen is one of a few physiological substances that caninduce the activation of MMP-2, a tissue degrading enzyme involved invarious physiological and pathological phenomena. In the present study,specificity for the types and structures of collagen capable of thisinduction has been studied. MMP-2 activation was monitored by zymography.Treatment of the cell cultures by addition of collagen in solution, insteadof growing cells oil collagen gels, was developed to avoid unknown effectsof trapped serum proteins, and for ease in manipulating collagenconcentrations. Addition of recombinant, purified, or partially purifiedcollagens into the culture of normal human skin fibroblasts demonstratedthe specificity of fibrillar-type collagen for induction of MMP-2activation. Reduction of MMP-2 fibril formation by treatment of thecultures with dry monomeric or alkali-treated collagen, or afibril-inhibitory collagen peptide, abrogated the MMP-2 activation.Moreover the periodate-sensitive features of collagen, such as thecarbohydrate molecules, were required for the appropriate cellular responseto collagen, whereas the presence or absence of telopeptide made littledifference to induction of MMP-2 activation. Both mRNA and protein levelsof the prominent MMP-2 activator, MT1-MMP, were consistently correlatedwith the amount of MMP-2 activation. The importance of MT1-MMP wassupported by the relative lack of MMP-2 activation after collagenstimulation of MM1-MMP% mouse fibroblasts. The role of collagen in this process was further assessed inascorbate-treated fibroblast cultures, which secrete and deposit endogenouscollagen, and better represent ~iin vivo~i conditions. Endogenous collageninduced a similar profile of MMP-2 activation and MT1-MMP expression asexogenous collagen. Temporal resolution of transcriptional andnon-transcriptional regulation of MT1-MMP by collagen was also observed inthis culture system. The essential role of collagen for MMP-2 activation inthis system was confirmed with prolyl-4-hydroxylase inhibitors, and withfibroblasts derived from the Mov-13 mouse, in which the collagen (+,a)1 (I)gene is not expressed. Inhibition of the endogenous collagen cross-linkingby BAPN had no effect on the activation of MMP-2. Surprisingly,pC-procollagen, produced by Mov13-5 CM mouse fibroblasts transfected with adefective (+,a)(,1)(I) chain, retained the ability to induce MMP-2activation, despite their inability to form fibrils. This result suggestedthat a non-fibrillar collagen structure can induce MMP-2 activation.Furthermore, ascorbate-treated cultures of MT1-MMP -/- mouse fibroblastsshowed considerable MMP-2 activation, which appears to be due to anintracellular activation. Collectively this study confirms an importantrole of type I collagen in both MT1-MMP- dependent and - independent activation ofMMP-2, and the ability of non-fibrillar collagen structures to induce MMP-2activation in ~iin vivo~i-simulated cultures of fibroblasts.