TIMP Independence of Matrix Metalloproteinase (MMP)-2 Activation by Membrane Type 2 (MT2)-MMP Is Determined by Contributions of Both the MT2-MMP Catalytic and Hemopexin C Domains

Charlotte J Morrison,Christopher M Overall

doi:10.1074/jbc.m603331200

Charlotte J Morrison, Christopher M Overall

Open Access

https://doi.org/10.1074/jbc.m603331200

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Abstract

The important and distinct contribution that membrane type 2 (MT2)-matrix metalloproteinase (MMP) makes to physiological and pathological processes is now being recognized. This contribution may be mediated in part through MMP-2 activation by MT2-MMP. Using Timp2-/- cells, we previously demonstrated that MT2-MMP activates MMP-2 to the fully active form in a pathway that is TIMP-2-independent but MMP-2 hemopexin carboxyl (C) domain-dependent. In this study cells expressing MT2-MMP as well as chimera proteins in which the C-terminal half of MT2-MMP and MT1-MMP were exchanged showed that the MT2-MMP catalytic domain has a higher propensity than that of MT1-MMP to initiate cleavage of the MMP-2 prodomain in the absence of TIMP-2. Although we demonstrate that MT2-MMP is a weak collagenase, this first activation cleavage was enhanced by growing the cells in type I collagen gels. The second activation cleavage to generate fully active MMP-2 was specifically enhanced by a soluble factor expressed by Timp2-/- cells and was MT2-MMP hemopexin C domain-dependent; however, the RGD sequence within this domain was not involved. Interestingly, in the presence of TIMP-2, a MT2-MMP.MMP-2 trimolecular complex formed, but activation was not enhanced. Similarly, TIMP-3 did not promote MT2-MMP-mediated MMP-2 activation but inhibited activation at higher concentrations. This study demonstrates the influence that both the catalytic and hemopexin C domains of MT2-MMP exert in determining TIMP independence in MMP-2 activation. In tissues or pathologies characterized by low TIMP-2 expression, this pathway may represent an alternative means of rapidly generating low levels of active MMP-2.

Highlights

Recruitment of secreted proteases to the cell surface increases the proteolytic repertoire of a cell and results in high local concentrations of the proteases
In this study we have shown that activation of proMMP-2 by MT2-matrix metalloproteinases (MMPs) occurs in a tissue inhibitors of metalloproteinases (TIMPs)-2-independent manner due in part to the greater propensity of the MT2-MMP versus MT1-MMP catalytic domain to make the initial first cleavage of the MMP-2 prodomain in the absence of TIMP-2 (Fig. 8C)
The second cleavage of the MMP-2 prodomain to generate fully active MMP-2 was enhanced by a secreted soluble protein (Fig. 5, A and B) and was MT2-MMP and MMP-2 hemopexin C domain-dependent (Fig. 1B)

Summary

EXPERIMENTAL PROCEDURES

Recombinant Protein Expression and Purification Hemopexin C Domains—The cDNA encoding the linker (L) and hemopexin C domain (CD) (Thr305-Cys559) of MT2-MMP (MT2-MMP LCD) was amplified by PCR and cloned into the bacterial expression vector pGYMX (51). Soluble MT-MMP (sMT-MMP)—PCR was used to introduce a FLAG sequence at the 3Ј end of the sMT1-MMP cDNA used previously in Bigg et al (42) This construct was cloned into the pPIC9 vector (Invitrogen) for expression in Pichia GS115 cells (Invitrogen). Timp2Ϫ/Ϫ cells expressing MT2-MMP or transfected with pGW1GH vector alone were grown on or in a native type I collagen matrix Vitrogen௡ (Cohesion Technologies, Inc.) (2.3 mg/ml). After 24 h of incubation at 37 °C, collagen gels with cells were washed extensively in PBS and incubated under serum-free conditions in the presence of 5 nM TIMP-2-free proMMP-2. After washing in PBS, 10-␮l aliquots of gelatin-Sepharose with bound proMMP-2 were incubated with 100 ␮l of TIMP-2 (200 ␮g/ml) or concentrated conditioned medium or PBS or DMEM for 2 h at 4 °C.

RESULTS

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DISCUSSION