cDNA cloning and expression of a metalloproteinase inhibitor related to tissue inhibitor of metalloproteinases.

Abstract

The purification and characterization of a metalloproteinase inhibitor (MI) from bovine aortic endothelial cells, and the demonstration that it is related to, but distinct from, tissue inhibitor of metalloproteinases (TIMP) 2). These zinc-dependent endopeptidases include interstitial collagenase (MMP-1), 72-kDa gelatinase (type IV collagenase, MMP-2), 92-kDa gelatinase, and stromelysin (proteoglycanase, transin, MMP-3). Virtually all extracellular matrix constituents can be degraded by this family of enzymes, and while there is some specificity in substrate recognition, there is also considerable overlap (2).The MMPs are synthesized and secreted as inactive zymogens, and their activity in the extracellular milieu is regulated by activators and inhibitors (2). Changes in the balance between active enzymes and their inhibitors may be involved in a number of human diseases, including arthritis, fibrosis, and tumor invasion (see review, ref. 3).Several inhibitors of metalloproteinases have been described and characterized. These include small cationic proteins extracted from cartilage and large blood vessels (4); a2-macroglobulin, a major serum inhibitor with Mr -725,000 (5); and tissue inhibitor of metalloproteinases (TIMP), a glycoprotein of Mr 'z30,000 (6, 7) for which the human cDNA has been cloned and sequenced (8,9). TIMP is ubiquitous among tissues and species and is considered a major regulatory inhibitor in the extracellular milieu (see review, ref. 2).We recently described the purification and characterization of TIMP and a novel metalloproteinase inhibitor (designated MI), both secreted by bovine aortic endothelial cells (10). The MI was shown to have an apparent Mr of 27,500, to be unglycosylated, and to have 51% identity to the bovine TIMP in the first 45 (8,9). Deoxyinosine (I) was used at codon nucleotide positions of fourfold degeneracy (11)

Stromelysin-1 (MMP-3) is a target and a regulator of Wnt1-induced epithelial-mesenchymal transition (EMT)

“…To evaluate the sustained expression of Wnt1 in our transfected clones, RT-PCR were performed using the following primers: 5'-CAT CGA GTC CTG CAC CTG-3'; 5'-TGG GCG ATT TCT CGA AGT AG-3'. 47 In some conditions, cells were cultured in the presence of the natural MMP inhibitor rTIMP-2 (10 µg/ ml), 48 the synthetic broad spectrum MMP inhibitors AG3340 (10 µg/ml) (Prinomastat, Agouron/Pfizer, Inc., San Diego, CA), or N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid (NNGH, 50 µM) 49,50 (Enzo Life Sciences, Inc., Plymouth Meeting, PA). Immortalized mouse fibroblast L cells and Wnt3a-expressing L cells (ATCC, Manassas, VA) were maintained in DMEM containing 10% FCS.…”

Tissue inhibitor of metalloproteinases-3 (TIMP-3) expression is increased during serum deprivation-induced neuronal apoptosis in vitro and in the G93A mouse model of amyotrophic lateral sclerosis: A potential modulator of Fas-mediated apoptosis

“…The MMPs play dynamic roles in developmental morphogenesis and in wound healing and repair during progression of tissue injury and pathologic diseases such as arthritis, cancer, and diabetes (Woessner, 1998;Visse and Nagase, 2003;Nagase et al, 2006). The activity of MMPs is regulated by four tissue inhibitors of matrix metalloproteinases (TIMPs), endogenous inhibitors of MMPs (Docherty et al, 1985;Boone et al, 1990;Apte et al, 1994;Greene et al, 1996).…”

Gene encoding a novel murine tissue inhibitor of metalloproteinases (TIMP), TIMP‐3, is expressed in developing mouse epithelia, cartilage, and muscle, and is located on mouse chromosome 10

“…TIMP-1 is a 184 amino acid glycoprotein of M, = 28.5 x lo3 (Gasson et al, 1985;Docherty et al, 19851, while TIMP-2 is a non-glycosylated, 194 amino acid residue long protein of M, = 21-23 x lo3 (Stetler-Stevenson et al, 1989;Boone et al, 1990). TIMP-1 and TIMP-2 share structural similarity as well as a n overlap in the range of MMP inhibitory activities (Ward et al, 1991).…”