Book of abstract 2008

Dimerization of endogenous MT1-MMP is a regulatory step in
the activation of the 72 kDa gelatinase, MMP-2, on fibroblasts and
fibrosarcoma cells
Signe Ingvarsen1, Daniel H. Madsen1, Thore Hillig1, Leif R. Lund1, Kenn Holmbeck2,
Niels Behrendt1, Lars H. Engelholm1
1The Finsen Laboratory, Rigshospitalet, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark; 2Oral &
Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of
Health, 30 Convent Drive, Bethesda, MD 20892, USA
Matrix metalloproteases (MMPs) are centrally engaged in the processes of extracellular
matrix turnover that occur during cancer invasion. An important MMP cascade reaction
is initiated by the membrane-anchored matrix metalloprotease, MT1-MMP, which
serves to activate the proenzyme form of the secreted gelatinase, matrix metalloprotease-2
(MMP-2). This reaction occurs in an interplay with the matrix metalloprotease inhibitor,
TIMP-2, and the proposed mechanism involves two molecules of MT1-MMP in complex
with one TIMP-2 molecule. To study this, as well as other roles of MT1-MMP, we have
now raised a panel of monoclonal antibodies against the protein. These antibodies have
been raised in MT1-MMP knock-out mice and react against conserved epitopes in murine
and human MT1-MMP. Using one of these antibodies we provide positive evidence
that proMMP-2 activation is governed by dimerization of MT1-MMP on the surface of
fibroblasts and fibrosarcoma cells. The antibody in question binds specifically to MT1-
MMP on the cell surface, as shown by immunofluorescence experiments. It is directed
against the hemopexin domain of MT1-MMP and has no effect on the catalytic activity of
the protease domain. The antibody induces dimerization of the endogenous MT1-MMP
on the cell surface. Through this reaction, it markedly stimulates the formation of the
62 kDa active MMP-2 and the processing into a 59 kDa product that retains gelatinolytic
activity.
p18
This effect is indeed a consequence of MT1-MMP dimerization because it requires
the divalent monoclonal antibody with no effect being obtained with monovalent Fab
fragments. Since only a negligible level of proMMP-2 activation is obtained with MT1-
MMP expressing cells in the absence of dimerization, our results identify the dimerization
event as a critical level of proteolytic cascade regulation.
100