Localization of the Death Domain of Tissue Inhibitor of Metalloproteinase-3 to the N Terminus

doi:10.1074/jbc.M007929200

Localization of the Death Domain of Tissue Inhibitor of Metalloproteinase-3 to the N Terminus

METALLOPROTEINASE INHIBITION IS ASSOCIATED WITH PROAPOPTOTIC ACTIVITY*

From the ^‡Bristol Heart Institute, Level 7, Bristol Royal Infirmary, University of Bristol, Bristol BS2 8HW, United Kingdom, the ^¶School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom, the ^‖Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge CB2 2QQ, United Kingdom, and the ^**Department of Medicine and Therapeutics, University of Glasgow, Glasgow G11 6NT, United Kingdom

Abstract

The tissue inhibitors of metalloproteinases (TIMPs) are a family of four secreted inhibitors of matrix metalloproteinases (MMPs). Recently, additional functions have been attributed to the TIMPs, including cell growth and inhibition of angiogenesis. In particular, we demonstrated that TIMP-3 overexpression using gene transfer induces apoptosis in a variety of cell types and can inhibit vascular neointima formation in vivo. However, little is know about the mechanisms underlying TIMP-3-mediated apoptosis. Here, using both purified recombinant proteins and novel adenoviral vectors we demonstrate that the prodeath domain of TIMP-3 is located within the N-terminal three loops of TIMP-3. Although both wild type and N-terminal TIMP-3 proteins promoted apoptosis, a T-2/T-3 chimera, in which the N-terminal three loops of TIMP-3 are replaced by those of TIMP-2, failed to induce cell death. Furthermore, a point mutation at residue 1 of TIMP-3 totally abolished MMP-inhibitory activity of TIMP-3 and also failed to promote apoptosis. This study demonstrates, using multiple apoptosis assays, that the prodeath function of TIMP-3 is located within the N-terminal three loops and the presence of functional metalloproteinase-inhibitory activity is associated with the induction of apoptosis.

Footnotes

↵* This work was funded by the British Heart Foundation, the Arthritis Research Campaign, and the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed: Tel.: 44-0-117-928-3587; Fax: 44-0-117-928-3581; E-mail: Mark.bond@bris.ac.uk.
Published, JBC Papers in Press, September 27, 2000, DOI 10.1074/jbc.M007929200
Abbreviations:

TIMP

tissue inhibitor of metalloproteinase

TIMP-3_Cys1-Ser

cysteine 1 to serine mutant of tissue inhibitor of metalloproteinase-3

wtTIMP-3

wild type tissue inhibitor of metalloproteinase-3

ISEL

in situ end labeling

MMP

matrix metalloproteinase

RAd

recombinant adenovirus

SFD

Sorsby's fundus dystrophy

SMC

smooth muscle cell

TNF-α

tumor necrosis factor α

TACE

TNF-α-converting enzyme

N-TIMP-3

N-terminal three loops of TIMP-3

C-TIMP-3

C-terminal three loops of TIMP-3

PCR

polymerase chain reaction

pfu

plaque-forming units

PBS

phosphate-buffered saline
- Received August 30, 2000.
- Revision received September 19, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.