Andrew J. P. Docherty

Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor.

Exposure of quiescent MRC‐5 human fibroblasts to growth factors such as epidermal growth factor, basic fibroblast growth factor or embryonal carcinoma‐derived growth factor resulted in the induction of mRNA transcripts encoding the metalloproteinases collagenase and stromelysin and the specific metalloproteinase inhibitor TIMP, whilst expression of collagen and fibronectin was relatively unaffected. Exposure of quiescent cells to growth factors in the presence of transforming growth factor beta (TGF‐beta) resulted in inhibition of collagenase induction and a synergistic increase in TIMP expression. TGF‐beta alone did not significantly induce metalloproteinase or TIMP expression. These effects on mRNA transcripts were reflected in increased secretion of TIMP protein and collagenase activity. Nuclear run‐off analysis of growth factor‐induced transcription revealed that the TGF‐beta modulation of TIMP and collagenase expression was due to transcriptional mechanisms. The observations suggest that TGF‐beta exerts a selective effect on extracellular matrix deposition by modulating the action of other growth factors on metalloproteinase and TIMP expression.

TNF‐α converting enzyme (TACE) is inhibited by TIMP‐3

TNF‐α converting enzyme (TACE; ADAM‐17) is a membrane‐bound disintegrin metalloproteinase that processes the membrane‐associated cytokine proTNF‐α to a soluble form. Because of its putative involvement in inflammatory diseases, TACE represents a significant target for the design of specific synthetic inhibitors as therapeutic agents. In order to study its inhibition by tissue inhibitors of metalloproteinases (TIMPs) and synthetic inhibitors of metalloproteinases, the catalytic domain of mouse TACE (rTACE) was overexpressed as a soluble Ig fusion protein from NS0 cells. rTACE was found to be well inhibited by peptide hydroxamate inhibitors as well as by TIMP‐3 but not by TIMP‐1, ‐2 and ‐4. These results suggest that TIMP‐3, unlike the other TIMPs, may be important in the modulation of pathological events in which TNF‐α secretion is involved.

The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3

A recombinant soluble form of the catalytic domain of human ADAM‐10 was expressed as an Fc fusion protein from myeloma cells. The ADAM‐10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described ‘metallosheddase’ cleavage sites of tumour necrosis factor α, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM‐10. The natural MMP inhibitors, TIMP‐2 and TIMP‐4 were unable to inhibit ADAM‐10, but TIMP‐1 and TIMP‐3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM‐10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP‐1) and 0.9 nM (TIMP‐3). The TIMP‐1 inhibition of ADAM‐10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP‐3, in cell based assays.

Regulation of Matrix Metalloproteinase Activitya

Matrix metalloproteinases (MMPs) are thought to initiate the degradation of the extracellular matrix during the remodeling of connective tissues. A clear understanding of the mechanisms governing the regulation of their activity during normal physiological processes should give further insights into the uncontrolled remodeling occurring in degradative pathologies. Regulation of the MMPs occurs at the level of gene expression, with precise spatial and temporal compartmentalization of both synthesis and secretion by resident cells as well as by those cells invading the tissue. Extracellularly, MMPs are further regulated by the extent of processing of the proform to an active enzyme and by the relative production of the specific inhibitors of MMPs, the tissue inhibitors of metalloproteinases (TIMPs). Furthermore, the potential for association of MMPs with the cell surface or extracellular matrix components further constrains their relationship with substrates, activators and inhibitors, acting as a further regulator of MMP activity. We initiated a program of study of the MMPs and TIMPs to ascertain the relation between their structure and their function, with particular emphasis on the mechanisms of biological regulation. Recombinant wild-type proteins and specific mutants, including deletion and site mutations, have been prepared using a mammalian expression system.’ Aspects of our findings, which illustrate the fundamental importance of the domain structure of MMPs and TIMPs in their biology, are presented here.

The matrix metalloproteinases and their natural inhibitors: prospects for treating degenerative tissue diseases

Uncontrolled matrix metalloproteinase activity is thought to be a cause of the tissue damage observed in many disease processes. None of the drugs currently in use can prevent tissue destruction, and strategies for the development of synthetic inhibitors have been hampered by a poor understanding of the biochemistry of matrix metalloproteinases. Recent cDNA cloning efforts and characterization of recombinant human matrix metalloproteinases have permitted structure-function analysis of the enzymes and their inhibitors. Progress in this area should help indicate a route to rational strategies for designing lead therapeutic compounds.

Tissue inhibitor of metalloproteinases-2 inhibits the activation of 72 kDa progelatinase by fibroblast membranes

We report that monolayers of human fibroblasts stimulated with concanavalin A were able to activate 72 kDa progelatinase but not 95 kDa progelatinase. The activating capacity of fibroblasts appeared approx. 6 h after concanavalin A stimulation and was blocked by cycloheximide. The activation of 72 kDa progelatinase was readily inhibited by TIMP-2 but only poorly by TIMP-1. Plasma membranes isolated from the fibroblasts were capable of activating 72 kDa progelatinase. The cleavage products of the plasma membrane-mediated activation of 72 kDa progelatinase corresponded to those of organomercurial-induced self-cleavage. Only inhibitors of metalloproteinase self-cleavage inhibited the activating capacity of plasma membrane preparations, although the activating capacity was destroyed by trypsin and heat. As with the fibroblast monolayers, TIMP-2 was a potent inhibitor of the membrane-mediated activation whereas TIMP-1 was less so.

ADAM10 is a principal 'sheddase' of the low-affinity immunoglobulin E receptor CD23

CD23, the low-affinity immunoglobulin E receptor, is an important modulator of the allergic response and of diseases such as rheumatoid arthritis. The proteolytic release of CD23 from cells is considered a key event in the allergic response. Here we used loss-of-function and gain-of-function experiments with cells lacking or overexpressing candidate CD23-releasing enzymes (ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM33), ADAM-knockout mice and a selective inhibitor to identify ADAM10 as the main CD23-releasing enzyme in vivo. Our findings provide a likely target for the treatment of allergic reactions and set the stage for further studies of the involvement of ADAM10 in CD23-dependent pathologies.

Functional heterogeneity of osteoclasts: matrix metalloproteinases participate in osteoclastic resorption of calvarial bone but not in resorption of long bone

Data in the literature suggest that site‐specific differences exist in the skeleton with respect to digestion of bone by osteoclasts. Therefore, we investigated whether bone resorption by calvarial osteoclasts (intramembranous bone) differs from resorption by long bone osteoclasts (endochondral bone). The involvement of two major classes of proteolytic enzymes, the cysteine proteinases (CPs) and matrix metalloproteinases (MMPs), was studied by analyzing the effects of selective low molecular weight inhibitors of these enzymes on bone resorption. Mouse tissue explants (calvariae and long bones) as well as rabbit osteoclasts, which had been isolated from both skeletal sites and subsequently seeded on bone slices, were cultured in the presence of inhibitors and resorption was analyzed. The activity of the CP cathepsins B and K and of MMPs was determined biochemically (CPs and MMPs) and enzyme histochemically (CPs) in explants and isolated osteoclasts. We show that osteoclastic resorption of calvarial bone depends on activity of both CPs and MMPs, whereas long bone resorption depends on CPs, but not on the activity of MMPs. Furthermore, significantly higher levels of cathepsin B and cathepsin K activities were expressed by long bone osteoclasts than by calvarial osteoclasts. Resorption of slices of bovine skull or cortical bone by osteoclasts isolated from long bones was not affected by MMP inhibitors, whereas resorption by calvarial osteoclasts was inhibited. Inhibition of CP activity affected the resorption by the two populations of osteoclasts in a similar way. We conclude that this is the first report to show that significant differences exist between osteoclasts of calvariae and long bones with respect to their bone resorbing activities. Resorption by calvarial osteoclasts depends on the activity of CPs and MMPs, whereas resorption by long bone osteoclasts depends primarily on the activity of CPs. We hypothesize that functionally different subpopulations of osteoclasts, such as those described here, originate from different sets of progenitors.—Everts, V., Korper, W., Jansen, D. C., Steinfort, J., Lammerse, I., Heera, S., Docherty, A. J. P., Beertsen, W. Functional heterogeneity of osteoclasts: matrix metalloproteinases participate in osteoclastic resorption of calvarial bone but not in resorption of long bone. FASEB J. 13, 1219–1230 (1999)

The enzymatic activity of ADAM8 and ADAM9 is not regulated by TIMPs

The ADAM family of proteases are type I transmembrane proteins with both metalloproteinase and disintegrin containing extracellular domains. ADAMs are implicated in the proteolytic processing of membrane‐bound precursors and involved in modulating cell–cell and cell–matrix interactions. ADAM8 (MS2, CD156) has been identified in myeloid and B cells. In this report we demonstrate that soluble ADAM8 is an active metalloprotease in vitro and is able to hydrolyse myelin basic protein and a variety of peptide substrates based on the cleavage sites of membrane‐bound cytokines, growth factors and receptors which are known to be processed by metalloproteinases. Interestingly, although ADAM8 was inhibited by a number of peptide analogue hydroxamate inhibitors, it was not inhibited by the tissue inhibitors of metalloproteinases (TIMPs). We also demonstrate that the activity of recombinant soluble ADAM9 (meltrin‐γ, MDC9) lacks inhibition by the TIMPs, but can be inhibited by hydroxamate inhibitors. The lack of TIMP inhibition of ADAM8 and 9 contrasts with other membrane‐associated metalloproteinases characterised to date in this respect (ADAM10, 12, 17, and the membrane‐type metalloproteinases) which have been implicated in protein processing at the cell surface.

Gelatinase A (MMP-2) and cysteine proteinases are essential for the degradation of collagen in soft connective tissue

The degradation of soft connective tissue collagen is considered to depend on the activity of various proteolytic enzymes, particularly those belonging to the group of matrix metalloproteinases and cysteine proteinases. In the present study, we investigated the contribution of these enzymes to this process. Using a general inhibitor of MMPs (SC44463), collagen degradation was strongly inhibited, by about 40% after 24 h and up to 80% after 72 h of culturing. Blockage of cysteine proteinase activity (with leupeptin or E-64) reduced breakdown at these time intervals by 50% and 20%, respectively. Given the abundant presence of gelatinases--in particular gelatinase A (MMP-2)--in the tissue, the effect of an inhibitor selective for gelatinases (CT1166) was studied. Gelatinase inhibition resulted in a dose-dependent decrease of collagen breakdown up to 90% after 48 h. The ability of gelatinase A to degrade collagens was demonstrated by the induction of breakdown in devitalized explants by addition of activated gelatinase A, or by activation of endogenous enzyme with 4-aminophenylmercuric acetate. This latter effect was not found with plasmin, an activator of MMPs other than gelatinase A. Finally, the relevance of gelatinase A to the in vivo degradation of soft connective tissue collagen was implicated by the significant correlation found between its activity and the collagen turnover rates of four soft connective tissues (tooth pulp, periodontal ligament, molar gingiva and skin). We conclude that collagen degradation in soft connective tissue is mediated by MMPs and to a lesser extent by cysteine proteinases. Our data are the first to attach a key role to gelatinase A in this process.