Susan P. Hawkes

Murine tissue inhibitor of metalloproteinases-4 (Timp-4): cDNA isolation and expression in adult mouse tissues

We have isolated cDNA clones corresponding to a new member of the murine tissue inhibitor of metalloproteinase (TIMP) family, designated Timp‐4. The nucleotide sequence predicts a protein of 22 609 Da that contains the characteristic 12 cysteine TIMP signature. TIMP‐4 is more closely related to TIMP‐2 and TIMP‐3 than to TIMP‐1 (48%, 45% and 38% identity, respectively). Analysis of Timp‐4 mRNA expression in adult mouse tissues indicated a 1.2 kb transcript in brain, heart, ovary and skeletal muscle. This pattern of expression distinguishes Timp‐4 from other Timps, suggesting that the TIMP‐4 protein may be an important tissue‐specific regulator of extracellular matrix remodelling.

Sorsby's fundus dystrophy tissue inhibitor of metalloproteinases-3 (TIMP-3) mutants have unimpaired matrix metalloproteinase inhibitory activities, but affect cell adhesion to the extracellular matrix

The TIMP family of matrix metalloproteinase inhibitors consists of four members, of which TIMP-1, -2 and -4 are secreted, freely diffusible proteins, whereas TIMP-3 is ECM-associated. Mutations in the TIMP3 gene have been linked to Sorsbys fundus dystrophy (SFD), an autosomal dominant inherited retinal degenerative disease that leads to blindness. The SFD mutations characterized result in introduction of an unpaired cysteine residue in the C-terminal domain of TIMP-3. We have expressed four SFD mutant TIMP-3 proteins in baby hamster kidney (BHK) cells and evaluated their characteristics alongside wild-type TIMP-3. Analysis of the mutant proteins (Ser156Cys, Gly167Cys, Tyr168Cys and Ser181Cys) by SDS-PAGE and reverse zymography revealed that each of the mutants retained gelatinase A and gelatinase B inhibitory activity, and were localized to the ECM. Association rate constants for Ser156Cys TIMP-3 with gelatinase-A, gelatinase-B, stromelysin-1 and collagenase-3 were only moderately reduced compared to wild-type TIMP-3. However, all of the mutants displayed aberrant protein-protein interactions, resulting in the presence of additional proteins or complexes in ECM preparations. Two of the mutants (Ser156Cys and Ser181Cys) showed a marked propensity to form multiple higher molecular-weight complexes that retained TIMP activity on reverse zymography. Expression of the SFD mutant TIMP-3 (and to a lesser extent, wild-type TIMP-3) proteins in BHK cells conferred increased cell adhesiveness to the ECM. Our findings indicate that the pathogenesis of Sorsbys fundus dystrophy cannot be attributed to a failure to localize SFD TIMP-3 proteins to the ECM or defects in MMP inhibition, but may involve the formation of aberrant TIMP-3-containing protein complexes and altered cell adhesion.

Identification and characterization of human tissue inhibitor of metalloproteinase-3 and detection of three additional metalloproteinase inhibitor activities in extracellular matrix

We have identified and characterized a novel human tissue inhibitor of metalloproteinase (TIMP). It is found exclusively in the extracellular matrix of a large number of cultured human cells, including: primary embryonal kidney (293), neuroblastoma (SK-N-SH), normal whole embryo (FHs 173We), cervical carcinoma (HeLa S3), colon adenocarcinoma (Caco-2), ileocecal adenocarcinoma (HCT-8), fibrosarcomas (SW 684 and Hs 913T) and normal gingival fibroblasts (GF11 and 1292). It was not detected in the conditioned media from any of these cell lines. Its apparent molecular mass of 24-25 kDa, as determined by its migration on protease-substrate gels, is intermediate between TIMP-1 (28.5 kDa) and TIMP-2 (21 kDa). Like the latter two proteins, human TIMP-3 contains intrachain disulfide bonds and displays altered electrophoretic mobility in the presence of beta-mercaptoethanol. The N-terminal, amino acid sequence of the protein is identical to that of chicken TIMP-3 (ChIMP-3), and its amino acid composition is similar. The protein is not N-glycosylated, as determined by treatment with N-glycosidase-F. Finally, it is recognized by antisera raised against pure ChIMP-3 but not by anti-human TIMP-1 or anti-human TIMP-2 antibodies. Based on these properties, we propose that this protein is TIMP-3 and is the human counterpart of ChIMP-3 (Pavloff et al., J. Biol. Chem. 267: 17321-17326, 1992). Two additional inhibitors detected in the matrix of human cell lines, designated inhibitor of metalloproteinase (IMP)-a and IMP-b, migrate with apparent masses of 29 kDa and 30 kDa. Both are N-glycosylated. A fourth inhibitor activity, which is smaller in mass than TIMP-3 and is also pecifically located in the matrix, is detectable in some cell lines.

Cloning, expression, and characterization of chicken tissue inhibitor of metalloproteinase-2 (TIMP-2) in normal and transformed chicken embryo fibroblasts

Rous sarcoma virus‐transformed chicken embryo fibroblasts (RSVCEF), when compared to normal CEF, produce elevated levels of matrix metalloproteinase‐2 (MMP‐2) that exists in a form free of complexed tissue inhibitor of metalloproteinase‐2 (TIMP‐2). In order to ascertain whether the increased levels of TIMP‐free MMP‐2 in RSVCEF cultures are due to diminished expression of TIMP‐2 or alterations in TIMP‐2 that diminish its MMP‐2 binding ability, it was necessary to clone, characterize, and express chicken TIMP‐2 cDNA. The TIMP‐2 cDNA was cloned from a chick embryo λgt11 library by RT‐PCR using primers based on amino‐acid sequences determined from isolated TIMP‐2. The deduced amino acid sequence for chicken TIMP‐2 is 81% identical to human TIMP‐2; most of the sequence differences lie in the carboxyl terminal portion of chicken TIMP‐2. Northern analysis of mRNA levels in CEF and RSVCEF demonstrates that TIMP‐2 mRNA levels are increased in RSVCEF. However, TIMP‐2 protein levels, relative to proMMP‐2 levels, appear to decrease upon transformation and suggest additional control of TIMP‐2 at the post‐transcriptional level. Addition of recombinantly expressed TIMP‐2 to RSVCEF cultures causes a disappearance of TIMP‐free (TF) proMMP‐2 with a corresponding increase in the TIMP‐complexed (TC) proMMP‐2 levels, demonstrating that TF proMMP‐2 is capable of converting to TC pro‐MMP‐2 when free TIMP‐2 is available. Surprisingly, RSVCEF cultures manifest a TIMP‐2 population that is not complexed to MMP‐2, despite the coexistence of TIMP‐free proMMP‐2. Gel‐filtration analysis indicates that this uncomplexed TIMP‐2 exhibits an apparent molecular weight of 50 kDa, indicating it is not free TIMP‐2 and that it exists in transformed cultures in a noncovalent complex with an undefined molecule. Thus transformed cells can alter the TIMP‐2/MMP‐2 balance by transcriptional and post‐translational modifications, yielding a population of inhibitor‐free, proteolytically active MMP2. J. Cell. Physiol. 174:342–352, 1998.