Biochimica et Biophysica Acta, 1071 (1991) 149-158 © 1991 Elsevier Science Publishers B.V. 0304-4157/91/$03.50 ADONIS 0304415791000578

!49

BBAREV85379

Review

Cellular functions of metallo-endoproteinases W i l l i a m J. Lennarz 1 a n d Warren J. Strittmatter ~ t Department of Biochemistry and Cell Biology, State Univer.~ity of New York at Stony Brook, Stony Brook, N Y (U.S.A.) and 2 Departments of NeuroloD,, Biochemistry and Neuroscience, Baylor College of Medicine, Houston, TX (US.A) (Received 13 August 1990) (Revised manuscript received 19 November 1990)

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Mechanism of proteinase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B Cellular localization of metallo-endoproteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1,~9 149 150

I1.

Regulation of metallo-endoproteinase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Transcriptional regulation of metalio-endoproteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B Secretion and activation of proproteinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C Natural inhibitors of metallo-endoproteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D Synthetic inhibitors of metallo-endoproteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

150 151 151 151 152

I11.

Intracellular metallo-endoprotein~ses in post-translational protein processing . . . . . . . . . . . . . . .

152

IV.

Extracellular metallo-endoproteinases in protein and neuropeptide hydrolysis . . . . . . . . . . . . . . .

153

V.

Metallo-endoproteinases in exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Inhibition of exocytosis by proteinase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B Mechanisms of action . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .

154 154 155

VI.

Metallo-endoproteinases in cell-cell fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Sperm-egg fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B Myoblast fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C Primary mesenehyme cell fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

155 ! 55 155 156

VII.

Secreted metalio-endoproteinases and the extracellular matrix . . . . . . . . . . . . . . . . . . . . . . . . . . A Secreted metallo-endoproteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B Metallo-endoproteinases in cellular metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C Metallo-endoproteinases in synovial m e m b r a n e destruction . . . . . . . . . . . . . . . . . . . . . . . . . .

156 157 157 157

VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. Introduction

I-A. Mechanism of proteinase activity Metallo-endoproteinases hydrolyze peptide bonds in proteins at neutral pH, and require metal ion at the

Correspondence: W.J. Strittmatter, Dept. Neurology, Baylor College of Medicine, Houston, TX 77030, U.S.A.

157 157 157

active site for catalytic activity. Zinc is the metal ion required for three well characterized metallo-endoproteinases (collagenase, proteinase 24.11 and meprin), although other divalent metals, such as cobalt, can reconstitute activity in the inactive apoenzyme [1]. The mechanism of peptide bond hydrolysis by mammalian metallo-endoproteinases is not known. However, the bacterial metallo-endoproteinase, thermolysin, has been extensively studied by X-ray crystallography, employing transition state analogues [2-6]. The following

150 \1/ A.

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+HIS-

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\1/ B.

-GLU c"0 "OH

Zr~* .....÷HIS0 I

._o.C.~_. R i

\1/ C.

-GLU c"0 "OH

Zr~+ 0

H_O-~

HIS-

H'NsH

k, Fig. 1. Proposed mechanism of peptide bond hydrolysis (modified from Ref, 6). See Section I-A for more details.

mechanism, shown in Fig. 1, has been proposed [2,6]: The carbonyl oxygen of the peptide bond being hydroiyzed coordinates with the zinc ion of the metaUo-endoproteinase, forcing the zinc-liganded water toward the carboxylate group of the glutamic acid residue (Glu 143) in the proteinase active site (Fig. 1A). The water molecule, activated by both the carboxylate and zinc ion, attacks the carbonyl carbon to form a tetrahedral intermediate (Fig. 1B). The histidine (His TM) stabilizes the transition state through hydrogen bonding and donates a proton to the leaving group nitrogen, leading to bond cleavage (Fig. 1C). Although the mammalian neutral metallo-endoproteinase (NEP, EC 3.2.24.11) has little overall sequence homology with thermolysin, conservation of sequences in their active sites is observed, including the two histidine residues which coordinate zinc (His 583 and His 587 in NEP and His t42 and His 146 in thermolysin) and two essential amino acids involved in catalysis (Glu 5a7 and His 637 in NEP and Glu 143 and His 231 in thermolysin) [7-9]. Site directed mutagenesis of residue Glu 584 in NEP to either valine or aspartic acid completely abolishes enzymatic activity without changing its affinity for a substrate related inhibitor. Substitution of His 5s3 or His 587 with phenylalanine also abolishes enzymatic activity [8,9]. The consensus sequence Val-lle-Gly-His-Glu-lle-Thr-His-Gly-Phe-Asp is found in neutral metallo-endoproteinases [10]. These studies on enzyme mechanism are important not only for their insight into the molecular mechanism, but because they have helped in the development of active site inhibitors which are invaluable in studying the biological roles of metaUo-endoproteinases, and for use therapeutically. The peptide bonds hydrolyzed by metallo-endoproteinases vary depending on the individual metallo-endoproteinase. Typically, however, metallo-endoproteinases hydrolyze the peptide bond on the amino side of the P~ amino acid if it is an uncharged aromatic amino acid or

a large aliphatic amino acid. The amino acid on the carboxy side of the hydrolyzed peptide bond (the Pt amino acid) also influences hydrolysis, with serine and threonine increasing both the K m and Vmax of hydrolysis, as compared with glycine [11]. In vitro, metallo-endoproteinases may hydrolyze proteins (e.g., insulin) as well as small peptides (e.g., enkephalin). The well-defined substrate specificity of metallo-endoproteinases has permitted the development of small di- and tripeptide inhibitors which block catalytic activity of a large number of these enzymes. Synthetic peptide inhibitors and substrates have been particularly useful in studies examining metallo-endoproteinase function in intact cells because of their relatively low toxicity.

I-B. Cellular localization of metallo-endoproteinases Metallo-endoproteinases are found in several cellular compartments. The brush border in the gut and kidney, for example, have plasma membrane proteinases, discussed later, which are integral membrane proteins with their catalytic site on the outside of the cell. These proteinases may be involved in hydrolyzing protein in the gut and in the kidney lumen as a prerequisite step in uptake and utilization of the component amino acids. Other cells, such as the inflammatory macrophages, embryonic cells, and metastatic cells secrete metallo-endoproteinases into the extracellular space where they may degrade basement membrane protein as a necessary step in a diversity of processes, including metastasis, synovium destruction in arthritis and tissue modeling during embryogenesis. As discussed later, these secreted metallo-endoproteinases are exquisitely regulated by a variety of mechanisms. Metallo-endoproteinases are also found in the cytoplasm of exocytotic cells, such as neurons, adrenal chromaffin cells, and mast cells, where they have been implicated in exocytosis. Finally, metallo-endoproteinases are found in the rough endoplasmic reticulum and mitochondria, where they function to remove the signal sequence of proteins during their synthesis and insertion or passage through these membranes. The roles of metallo-endoproteinases in specific cellular locales are now being extensively studied. In some instances, for example basement membrane hydrolysis, the role of metallo-endoproteinases has been well established by extensive biochemical, pharmacologic and molecular studies. With respect to other potential roles, such as membrane fusion, the evidence supporting the direct involvement of metallo-endoproteinases is less compelling, and is primarily pharmacologic. Clearly, this is an avenue for further investigation. 11. Regulation of metallo-endoproteinase activity In view of the fact that metallo-endoproteinases play important roles in a variety of cellular functions it is not

151 surprizing that several mechanisms operate in controlling the activities of these enzymes. Thus far, studies on the regulation of metallo-endoproteinases have focused on the secreted matrix proteinases, collagenase and stromelysin.

II-A. Transcriptional teinases

regulation of metallo-endopro-

The mRNA encoding secreted matrix metalio-endoproteinases are induced by the exposure of "~lls to growth factors, oncogenes and tumor promotors [13-15]. The oncogenes Ha-ras and v-mos [16], the tumor promoter phorbol esters [16], and platelet derived growth factor [17] increase the levels of mRNAs encoding both collagenase and stromelysin. These agents appear to stimulate metallo-endoproteinase mRNAs synthesis via Fos protein since their effects on metallo-endoproteinase induction is blocked by antisense c-fosRNA [16,17]. The proto-oncogene c-Jun also appears to increase metailo-endoproteinases mRNA [18]. Tumor necrosis factor-a causes prolonged activation of both c-Jun and collagenase gene expression, and the c-Jun product forms a heterodimeric complex with specific DNA binding domains which serve as a promotor for both strome!ysin and collagenase [18]. Induction of metallo-endoproteinase gene expression therefore utilizes pathways involving at least c-Fos and c-Jun. Heat shock stress also induces the elevation of mRNAs for both collagenase and stromelysin, which also have putative heat shock consensus sequences flanking the 5' region of the genes [19]. Metallo-endoproteinase expression is also negatively regulated. Glucocorticoids and tumor necrosis factor-fl block expression of metallo-endoproteinases at the level of mRNA transcription [13].

II-B. Secretion and activation of proproteinase Another site of regulation of metallo-endoproteinases is at the level of conversion of the inactive polypeptide to an active form. For example, the secreted form of collagenase is a catalytically inactive proproteinase which must be proteolytically processed outside the cell for activation. Human fibroblast collagenase contains two sites for proteolytic cleavage, cleavage at one site results in the activation of the proteinase, whereas cleavage at the other site results in inactivation (Fig. 2). The full length, inactive proproteinase is 52 kDa. In in 0

r

82

251

m

450

i

~ Zn binding A COOH domain I / Activation Degradation site site Fig. 2. Human fibroblast collagenase showing sites for proteolytic activation, inactivation and zinc binding. (Modified from Ref. 20.) NH 2

vitro studies, the latent collagenase can be activated by a two step proteolysis. First, in vitro, trypsin hydrolyzes the Arga6-Asn37 bond to generate an inactive 46 kDa form. Subsequent hydrolysis between Gin 8° and Leu 83 yields a catalytically active 42 kDa proteinase. Autohydrolysis of the Pro25°-lle TM bond inactivates the proteinase (Fig. 2) [20]. The inactive procollagenase can be activated in vitro not only by extracellular proteolytic processing by trypsin or plasmin, but also by sulfhydryl alkylating agents, oxidants or disulfide compounds. The model of in vitro activation based on these observations is that Cys 73 in the inactive proproteinase is complexed with the catalytic zinc atom. All modes of activation appear to disrupt this bond, forming a functional active site. This activation mechanism for human collagenase may also apply to other metallo-endoproteinases [20].

II-C. Natural inhibitors of me.allo-endoproteinases The activity of secreted metallo-endoproteinases is also regulated by protein inhibitors released by various tissue explants and cells in culture [21]. The most specific of these protein metallo-endoproteinase inhibitors belong to a growing list of Tissue _Inhibitors of Metalloendo_Proteinases (TIMP) [21]. The first TIMP characterized, TIMP-1, is a glycoprotein of 28.5 kDa, which forms a 1" 1 complex with collagenase Type I, IV, V, stromelysin and other matrix metallo-endoproteinases. The secreted form of TIMP-1 deduced from its cDNA contains 184 amino acids [22]. TIMP-1 is found in many body fluids including synovial fluid, amniotic fluid and in explants of bone, synovium, cartilage and aorta [21]. Cells which synthesize and release matrix metallo-endoproteinases, such as capillary endothelial cells and fibroblasts, also synthesize and release TIMP-1 [23]. Synthesis and secretion of matrix metallo-endoprot¢inase and TIMP appear to be independently modulated in these cells: glucocorticoids and tumor necrosis factor-fl decrease collagenase production whereas they increase TIMP-1 production [24]. TIMP-2 is a 24 kDa protein that forms a stable, noncovalent complex with human type IV collagenase [25]. Other less specific endogenous proteinase inhibitors also complex and inhibit metallo-endoproteinases. a2-Macroglobulin, a glycoprotein of 725 kDa inhibits many metallo-endoproteinases [21]. A third group of inhibitors, of lower molecular weight, (10-20 kDa) are found in extracts of bone, cartilage and aorta, but thus far have been only poorly characterized [21]. The regulation of matrix metallo-endoproteinase activity in the extracellular spare appears important in normal physiologic function and also in disease mechanisms, such as cartilage and synovium destruction in rheumatoid arthritis and in cellular migration in tumor metastasis. It is not unreasonable to speculate that

152 naturally occurring metallo-endoproteinase inhibitors may play a role in these disease processes, and therefore they may find applications as therapeutic agents. Metallo-endoproteinase inhibitors isolated from Actinomycetes have proven useful in elucidating the catalytic mechanism of metallo-endoproteinases, as well as serving as prototypes for the development of other high affinity, high specificity synthetic inhibitors [6]. Phosphoramidon (( N-a-L-rhamnopyranosyl-hydroxyphosphinyl)-L-leucyi L-tryptophan) inhibits some mammalian metallo-endoproteinases [6] such as the neutral endopeptidase E.C. 3.4.24.11 with nanomolar affinity, whereas it has no effect on some others, such as the soluble metallo-endoproteinase E.C. 3.4.24.15.

H-D. Synthetic inhibitors of metailo-endoproteinases Synthetic inhibitors of metallo-endoproteinases have been used for two major purposes: (1) to explore the cellular roles of metallo-endoproteinases by inhibiting these proteinases and determining whether or not a specific cellular function is altered and (2) to alter disease processes promoted by abnormal metallo-endoproteinase activity, i.e., as drugs. Of course, these inhibitors are also useful in classifying proteinases. A vast literature describing the synthetic metallo-endoproteinase inhibitors cannot be extensively reviewed here [26]. Since all metallo-endoproteinases require metal for catalytic activity, chelating compounds such as EDTA, EGTA and 1,10 phenanthroline inhibit these enzymes. The ability of a metal chelator to inhibit enzymatic activity does not by necessity mean however that metal is required for the catalytic mechanism, since many proteins bind metal to stabilize tertiary structure. Synthetic metallo-endoproteinase inhibitors contain a moiety which interacts with the active site zinc atom and a peptidyl moiety recognized and bound by the active site. Rational inhibitor design therefore requires knowledge of the substrate specificity of the particular proteinase. A series of di- and tri-peptides have been examined for their effects on the activity of the metallo-endoproteinase, with the goal of optimizing both the affinity of interaction with the proteinase and the specificity for a particular proteinase. These peptides have been synthesized with different reactive groups, such as N-caxboxyalkyl, thiol or hydroxamate, that interact with the catalytic zinc. Such rational design of metallo-endoproteinase inhibitors by detailed examination of substrate-active site interactions has produced compounds of high specificity and affinity. One such naturally designed inhibitor, captopril, inhibits the metallodipeptidase angiotensin-l-converting enzyme and is widely used clinically for treating hypertension [27]. Of course, compounds which inhibit metalloendoproteinases may also inhibit other enzymes, or may inhibit cellular processes by less specific mechanisms.

Therefore, functions ascribed to specific enzymes solely by observing the effects of such inhibitors must always be tentative.

Iii. lntracellular metailo-endoproteinases in post-translational protein processing Proteins located within intraceilular vesicles, such as the mitochondria and secretory vesicles, are translated as larger proteins which are then proteolytically cleaved, typically at the amino terminus, to yield a smaller product. Often secreted l~roteins undergo several hydrolytic processing steps before the peptides are secreted by the cell in their biologically active form. Several metallo-endoproteinases have been identified in such post-translational protein processing events. The best characterized processing metallo-endoproteinase is in the mitochondrial matrix [28-30]. Many of the polypeptides found within mitochondria (either as integral proteins within the mitochondrial membrane or as soluble proteins in the matrix) are initially translated by ribosomes in the cytoplasm as soluble proteins. These proteins contain an amino-terminal presequence that enables the precursor to enter the mitochondrial matrix where it is then proteolytically removed. Proteolyric cleavage of this sequence is not necessary for import of these mitochondrial proteins but is necessary for assembly into larger functional complexes with other proteins. This processing proteinase is dependent on divalent metals (Zn 2+ and Co 2+) and is inhibited by metal chelators, (1,10 phenanthroline and EDTA). However, inhibition of catalytic activity by metal chelators does not however necessarily indicate the presence of a metallo-endoproteinase, since metal-stabilized proteinases would also be inhibited. One such processing proteinase, isolated from Neurospora, is a soluble matrix protein with a molecular mass of 57 kDa [31]. The purified proteinase has processing activity which is !ow, but activity can be fully restored by recombination with another protein, the processing enhancing protein, (PEP; molecular mass 52 kDa), which itself appears to have no proteinase activity. The PEP, unlike the soluble processing protease, is associated with the mitochondrial membrane. The mechanism of interaction between the catalytic, processing proteinase and the noncatalytic processing enhancer protein is not known. Two mutants of Saccharomyces, mas-1 and mas-2, are temperature-sensitive for the import of proteins into the mitochondria. Both mutants accumulate uncleaved protein precursors outside the mitochondrial inner membrane and fail to import protein at normal rates. The proteinase cleaving the amino-terminus of these matrix proteins is a metallo-endoproteinase consisting of two non-identical subunits of 48 and 51 kDa [32]. Separation of these two subunits causes loss of proteolytic activity. The small subunit is the product of the

153 MAS1 gene and the larger subunit the product of the MAS2 gene [33]. The observation that both temperature-sensitive mutants not only fail to proteolytically process the amino-terminus of the mitochondrial proteins, but also fail to import them, raises the possibility that this two protein complex plays an additional role in protein targeting or protein import. Proteins translated on the rough endoplasmie reticulum commonly have signal peptides responsible for initial targeting of the protein to the endoplasmic reticulum. Such signal peptide sequences on the amino terminus are usually hydrolyzed at arg-arg, lys-lys, or arg-lys bonds. A less common site of processing of the proprotein occurs at arg-pro. The active peptide adrenorphin, for example, is an opiate agonist in brain and adrenal, which is processed intracellulady by a metallo-endoproteinase which cleaves the arg-pro bond in the inactive precursor [34]. Intracellular, soluble metallo-endoproteinases within mitochondria and secretory vesicles therefore appear required for selective processing of protein precursors to produce biologically active peptides. As described in the next section, metallo-endoproteinases outside the cell play important roles in terminating the effects of biologically active peptides by hydrolyzing them to smaller, biologically inactive fragments.

IV. Extracellular metallo-endoproteinases in protein and neuropeptide hydrolysis Microvilli in the kidney and in the gut contain zincdependent endoproteinases [35]. One such proteinase, E.C. 3.4.24.11, resembles bacterial metallo-endoproteinases in its mechanism of hydrolysis and in its inhibition by the bacterial proteinase inhibitor phosphoramidon [36]. This plasma membrane metallo-endoproteinase, which is also found in brain [37], spermatozoa and pancreas [38], is an integral membrane protein with both the catalytic and zinc binding sites facing the outside of the cell [38]. The primary amino acid sequence of the endoproteinase in rabbit kidney deduced from eDNA, contains 750 amino acids, with the 27 amino acid amino te,.m~_/nus in the cytoplasmic compartment, a 23 amino acid single membrane spanning segment and the remainder in the extracellular space [39]. In vitro, this proteinase hydrolyzes a number of peptides including angiotensin, bradykinin, substance P and oxytocin. Since this plasma membrane metallo-endoproteinase has its catalytic domain outside of the cell, it hydrolyzes peptides filtered in the glomerulus of the kidney. Mouse kidney brush border contains another plasma membrane metallo-endoproteinase, meprin. Unlike the neutral endoproteinase E.C. 3.4.24.11, meprin does not hydrolyze peptides containing less than six amino acid

residues [40,41]. Also, in contrast to neutral endoproteinase, meprin is not inhibited by phosphoramidon. Proteinases with similar characterizatics have been isolated from rat kidney microvilli, termed rat endopeptidase-2 [42] and in human intestinal brush border, termed 'PABA-peptide hydrolase' [43]. The neutral metallo-endoproteinase (EC 3.4.24.11) in brain was originally termed 'enkephalinase' since the proteinase was originally identified by its ability to hydrolyze, and therefore inactive, enkephalin. Since this metallo-endoproteinase also hydrolyses and inactivates other neuropeptides ineluding substance P, atrial natriuretic factor, neurotensin and cholecystokinin and fl-endorphin, it may regulate synaptic function by inactivation of these peptide transmitters. Immunohistoehemical study of E.C. 3.4.24.11 shows highest levels of this proteinase in brain regions containing the highest levels of the neuropeptides substance P and Leu-enkephalin and also demonstrates that the proteinase is found primarily in neural and not glial, plasma membranes [45]. Brain contains other metallo-endoproteinases. One of these is a soluble 67 kDa metallo-endoproteinase (E.C. 3.4.24.15) that is clearly distinguished from the plasma membrane proteinase (E.C. 3.4.24.11), since the former is not inhibited by phosphoramidon, does not hydrolyze leu- or met-enkephalin, but does hydrolyze and inactivate, L H R H , substance P, neurotensin and bradykinin [46]. This proteinase rapidly converts several enkephalin containing peptides, such as a-neoendorphin, fl-neoendorphin, into the biologically active enkephalins. A membrane-bound metallo-endoproteinase with substrate specificity and inhibitor profile similar to the soluble E.C. 24.11 has been identified [47]. Four other metallo-endoproteinases have been identified in rat brain with molecular masses of 112, 92, 70 and 65 kDa [48]. These proteinases are inhibited by 1,10 phenanthroline and not by serine, eysteine, or aspartic proteinase inhibitors. Their cellular localizations and functions are yet not known. Lymphoid cells, including most acute lymphoblastic leukemias and normal fl lymphoid progenitors, possess an integral membrane glyeoprotein of 749 amino acids named the common acute lymphoblastie leukemia antigen (CALLA), or CD10. Sequencing of this protein from human cells revealed 94% identity with metalloendoproteinase 3.4.24.11 from rat and rabbit. Transfeetion of CALLA eDNA into cell lines resulted in expression of a protein with metallo-endoproteinase activity [49]. The role of CALLA in lymphoid development and in lymphoblastic leukemias is not known. Since the chemostatic peptide f-Met-Leu-Phe and peptide hormones including enkephalin, oxytocin, substance P and angiotensin I and II are inactived by CALLA, functional or developmental roles of the proteinase are likely.

154

V. Metallo-endoproteinases in exocytosis The regulated secretion process results in the release of neurotransmitters, enzymes and hormones from cells by precisely controlled fusion of secretory vesicles with the plasma membrane. Regulated exocytosis is initiated by ligand binding to a cell surface receptor, which then results in depolarization of the cell by transient opening of potassium channels. Depolarization then caus,~s an increase in intracellular free calcium, with calcium entering the cell cytoplasm via the plasma membrane or the endoplasmic reticulum membrane. The elevation of intracellular free calcium appears to be a necessary signal that triggers fusion of the secretory vesicles with the plasma membrane, permitting the release of the vesicle contents into the extracellular space. The biochemical mechanism by which calcium ions trigger exocytotic fusion is extensively studied and has been reviewed [50,51 ].

V.A. Inhibition of exocytosis by proteinase inhibitors A role for metallo-endoproteinases in exocytosis has been proposed because a number of synthetic metalloendoproteinase inhibitors block regulated exocytosis in several cell types and these cells are known to contain metallo-endoproteinases. If a metallo-endoproteinase is required for exocytosis, substrates oi" known metallo-endoproteinases should compete with the natural protein substrate and thus prevent exocytosis. Most metallo-endoproteinases will hydrolyze synthetic CBZ-dipeptide amides only with large aliphatic amino acids, such as leucine, or noncharged aromatic amino acids, such as phenylalanine, on the amino terminus of the hydrolyzed peptide bond. Therefore, CBZ-Ser-Leu-amide and CBZ-Tyr-Phe-amide have high affinities for metallo-endoproteinases and CBZ-Gly-Leu-amide and CBZ-Gly-Phe-amide have intermediate affinities, whereas CBZ-Gly-Gly-amide is not a substrate [11]. The affinity of a dipeptide substrate varies according to the particular metallo-endoproteinase, but generally is in the millimolar range. Many studies investigating the mechanism of exocytosis have employed primary cultures of dissociated bovine adrenal chromaffin cells. Exocytosis in these cells is initiated by eho~nergic agonists such as carbachol, binding to nicotine receptors and ultimately results in the exocytotic release of catecholamines [52]. Dipeptides that are meta!lo-endoproteinase substrates inhibit exocytotie release of catecholamine. The metallo-endoproteinase inhibitor 1,10-phenanthroline also prevents carbachol-induced exocytosis [53]. Addition of calcium, equimolar to added 1,10-phenanthroline, does not reverse this inhibition, suggesting that 1,10-phenanthroline inhibits exocytosis by chelating a metal other than calcium. The fluorogenic metallo-endoproteinase substrate 2-amino-

benzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide, used to characterize the adrenal metallo-endoproteinase, also inhibits receptor-mediated exocytosis in a dose-dependent fashion [53]. These observations demonstrate that exocytosis in adrenal chromaffin cells is blocked by compounds that interact with metallo-endoproteinases. Catecholamine secretion in adrenal chromaffin cells can be initiated directly by depolarization of the plasma membrane using high concentrations of potassium, which opens voltage-dependent calcium channels. Both 1,10-phenanthroline and the dipeptide substrates, CBZGly-Leu-NH 2 and CBZ-GIy-Phe-NH 2 block catecholamine secretion induced by potassium, while CBZ-GIyGly-NH 2 does not [53]. This observation indicates that these compounds are not inhibiting exocytosis by interacting at the ACh receptor itself, but rather are acting at some point distal to the receptor in inhibiting exocytosis. Furthermore, these same dipeptide metallo-endoproteinase inhibitors, as well as chelators and substrates, also prevent exocytosis in other cells, including mast cells [53] and cholinergic neurons in the peripheral nervous system [54] and neurons in the retina [55]. These cells also contain metallo-endoproteinases which hydrolyze synthetic peptide substrates. Metallo-endoproteinase activity in adrenal chromaffin cells can be detected using the fluorogenic metalloendoproteinase substrates 2-amino benzoyl-Ala-GlyLeu-Ala-4-nitrobenzylamide (AAGLAN) and succinylAla-Ala-Phe-4-aminomethylcoumarin. Metallo-endoproteinase activity is found in both the particulate fraction and the soluble fraction of homogenized cells. The pellet enzyme is inhibited by very low concentrations of phosphoramidon, whereas the soluble enzyme is not; these findings indicate the soluble enzyme is distinct from that found in the particulate fraction [57]. The acrosome reaction is a specialized case of exocytosis that occurs in sperm prior to egg fertilization. Studies in sea urchin sperm suggest that this exocytotic event also appears to be mediated by a metallo-endoproteinase [57]. In fact, the presence of a soluble metallo-endoproteinase was demonstrated and characterized in sperm homogenates using the fluorogenic proteinase substrate succinyl-alanine-alanine-phenylalanine-4-aminomethylcoumarin. The proteinase is inhibited by the metal chelators EDTA and 1,10phenanthroline and activity of the inactive apoenzyme could be reconstituted with Zn 2÷. The metallo-endoproteinase substrate and inhibitors block the acrosome reaction induced either by egg jelly coat or by ionophore, but have no effect on the influx of Ca 2+ [57]. These observations suggest that inhibition occurs at a step independent of Ca 2+ entry. Overall, there is strong, albeit indirect evidence that the acrosome reaction requires the action of metallo-endoproteinase. Additional evidence supporting this premise, but not excluding other possible indirect effects, has been reported in a

155 study on the acrosome reaction in human sperm [58]. Metallo-endoproteinase inhibitors also block the intracellular vesicular transport of proteins from endoplasmic reticulum to golgi, and also block endocytosis in hepatoma cells [59].

V-B. Mechanism of action Although the mechanism by which 1,10 phenanthroline and synthetic peptide metallo-endoproteinase inhibitors block fusion appears to be due to their ability to interact with metallo-endoproteinases it is possible that these compounds have other cellular effects. Consequently, since like all inhibitors, these compounds can have both specific and nonspecific actions any conclusions based on studies employing just inhibitors must be tentative. Indeed, two studies suggest that the effects of synthetic metallo-endoproteinase inhibitors on blocking exocytosis may be due to their effects on intracellular calcium homeostasis [60,61], while a third suggests a membrane perterbant effect [62]. The mechanism of action of these compounds and the possible role of metallo-endoproteinase in exocytosis needs to be further explored with both more specific inhibitors, including antibodies that inhibit the catalytic activity of the metallo-endoproteinase and the development of a cell free reconstituted exocytosis fusion model. Vi. Metallo-endoproteinases in cell-cell fusion Three examples of cell fusion will be considered with respect to the involvement of metallo-endoproteinase: fertilization, involving fusion of the sperm and egg plasma membranes; myoblast cell fusion, which results in multinucleated myotubes that differentiate into mature myofibrils; and primary mesenchyme cell fusion, a prerequisite to skeleton formation in the sea urchin embryo.

VI-A. Sperm-egg fusion Two membrane fusion events are involved in fertilization of the sea urchin gamete, fusion of the plasma membrane of the gametes and subsequent exocytosis [63] of the contents of the egg cortical granules. Substrates and inhibitors of metallo-endoproteinases prevent the membrane fusion event and sperm-egg fusion, but have no effect on egg cortical granule exocytosis [63]. Several observations indicate that the inhibitors and substrates specifically inhibit gamete fusion as a result of interaction with metallo-endoproteinase(s). First, the ability of peptides to inhibit fusion correlates well with their ability to interact with these proteinases. Succ-AIa-AIa-Phe-AMC and CBZ-Gly-Phe-NH2 interact with metallo-endoproteinases and block the fusion event in fertilization, whereas peptides that do not

interact with proteinase (e.g., Succ-Ala-Ala-AMC and CBZ-GIy-GIy-NH2) have no effect on fusion. Second, the metal chelators 1,10-phenanthroline and EDTA inhibit fertilization. Inhibition of this processes is reversed by Zn 2+, which reconstitutes the process of fertilization, suggesting that the effect is not merely due to an irreversible perturbation of the egg membrane by the chelators. Third, preincubation of the egg with 1,10phenanthroline followed by removal by washing has no effect on fertilization. The observations that metallo-endoproteinase inhibitors and substrates block the calcium-dependent exocytosis in the acrosome reaction in sperm discussed earlier, but have no effect on the calcium-dependent exocytosis of the cortical granule reaction in the egg, suggests different requirements for fusion in these two cell types. The lack of effect of the inhibitors and substrates on the ionophore-induced, cortical granule reaction in the egg is not due to failure to enter the egg because they also have no effect on the calcium-induced reaction in cell-free preparations of the isolated egg cortex.

VI-B. Myoblast fusion During muscle development, mononucleated myoblasts fuse to form multinucleated myotubes. Whereas myoblast fusion requires many months in vivo, in tissue culture it occurs in a few days. Myoblast fusion has been studied in vitro using primary cultures of embryonic or newborn muscle and with muscle cell lines. The myoblasts proliferate for several days and then withdraw from the cell cycle. Subsequently, they fuse to form large multinucleated myotubes, which then accumulate muscle-specific proteins and develop fibers and contractile ability. In order to study myoblast fusion, the fusion process itself must be distinguished from the process by which myoblasts become fusion competent. By switching cells to low calcium (270/tM) about 24 h after plating, the cells proliferate and withdraw from the cell cycle. If kept in low calcium medium, the cells synthesize muscle-specific proteins, indicating that they continue to differentiate, even though they cannot fuse. When 1.4 mM calcium is added to these cultures, they fuse rapidly over 3-24 h. This fusion does not require further cell division, protein, or RNA synthesis [64]. Metalloproteinase inhibitors prevent fusion when added to the medium of calcium-deprived myoblasts just before fusion is triggered by the addition of calcium [65]. The metal chelator 1,10-phenanthroline prevents myoblast fusion. Normal fusion occurs if 1,10-phenanthroline is chelated with an equimolar concentration of ZnCI 2 before being added to the cultures. Increasing the calcium concentration does not alter inhibition by 1,10-phenanthroline.

156 Other metalloproteinase inhibitors also prevent myoblast fusion [53]. CBZ-L-phenylalanine, a competitive inhibitor of metallo-endoproteinases prevents myoblast fusion with half-maximal inhibition of fusion at approx. 1.5 raM. The inhibition by CBZ-L-phenylalanine is stereospecific. The ability of dipeptide derivatives to inhibit myoblast fusion correlates with their ability to bind to characterized neutral metallo-endoproteinases, with the potency series being CBZ-TyrLeu-Amide -- CBZ-Ser-Leu-amide > CBZ-Gly-Phe-amide --- CBZ-Gly-Leu-amide >> CBZ-Gly-Gly-amide. The dipeptide substrate CBZ-Gly-Phe-amide inhibits fusion and interacts with the metallo-endoproteinases only if both the carbobenzoxy or amide blocking groups are present. The close correlation between the potency of these dipeptides in inhibiting myoblast fusion and their ability to interact with metallo-endoproteinases makes it likely that they inhibit fusion by interacting with the metallo-endoproteinase. Nonspecific effects are unlikely, since inhibition is reversible and stereo-specific. Myoblasts contain both soluble and particulate metallooendoproteinases [66]. The particulate myoblast metaUo-endoproteinase resembles the Class E.C. 3.4.24.11 proteinases: it is particulate (i.e., possibly membrane-bound) and is inhibited by both phosphorarnidon, a hydroxamic acid derivative that does not block fusion and by homo-Phe-[N]-L-Phe-fl-Ala. This enzyme is not inhibited by CBZ-Gly-Leu-amide or the irreversible inhibitor C1CH2CO(N-OH)-Phe-Ala-AlaNH 2. In contrast, the soluble metallo-endoproteinase is blocked by 1,10-phenanthroline, CBZ-Gly-Leu-amide, and the irreversible inhibitor CICH2CO(N-OH)-PheAIa-AIa-NH2, all of which also block myoblast fusion. The soluble metallo-endoproteinase may therefore be involved in myoblast fusion, whereas it is unlikely that the particulate proteinase is. 1,10-Phenanthroline, a hydrophobic molecule which diffuses through the plasma membrane, inhibits myoblast fusion, while bathophenanthroline, a polar molecule which does not readily enter this cell, does not inhibit myoblast fusion, suggesting that an intracellular metallo-endoproteinase may be involved [67]. Two soluble intracellular metaUoendoproteinases have been identified in myoblasts [66].

to inhibit fusion events in other systems discussed earlier were added to cultures of embryos or to cultures of primary mesenchyme cells isolated from the embryos. 1,10-Phenanthroline blocked the formation of spicules by primary mesenchyme cells both in the isolated cell and the embryos. Similarly, spiculogenesis was inhibited by the metallo-endoproteinase dipeptide substrate, CBZ-GIy-Phe-NH2, both in cultures of primary mesenchyme cells and embryos. A control dipeptide, CBZ-Gly-GIy-NH2, that is not a substrate for metalloendoproteinases had no effect on the cells. Thus, the terminal differentiation of primary mesenchyme cells, the formation of CaCO3-containing spicules within a syncytial cavity, may require the activity of a metalloendoproteinase. In support of this idea, the activity of such a metallo-endoproteinase was directly established in homogenates of primary mesenchyme cells [68]. Clearly, further experiments are necessary to more precisely define the locus of action of the enzyme. In this context it should be noted that roles of proteinases and their inhibitors in development have been recently reviewed elsewhere [69].

VI.C. Primary mesenchyme cellfusion

92 kDA Type IV coilagenase (MMP-9) (3) Stromelysin (MMP-3)

92

Stromelysin-2 (MMP-10)

53

PUMP-1 (MMP-7)

28

As a prerequisite to skeleton formation in the sea urchin embryo, primary mesenchyme cells undergo fusion via filopodia to form syncytia. The spicule is formed as a result of deposition of a glycoprotein matrix and CaCO3 within the syncytial cable. To investigate the potential involvement of a metaUo-endoproteinase in spiculogenesis, the effect of inhibitors of this enzyme on skeleton formation was studied [68]. Two types of inhibitors of metallo-endoproteinases; 1,10-phenanthroline and dipeptide substrates of the proteinase, shown

Vll. Secreted metallo-endoproteinases and the extraceilular matrix The extracellular matrix is a complex structure of proteins and proteoglycans that provides structural support for tissues and organs, and serves as a physical barrier for cellular migration. The proteins comprising the extracellular matrix include a family of collagens, elastin, glycoproteins (laminin, fibronectin, entactin) and TABLE I Matrix.degrading metalloproteinases a

Name(s) (1) Interstitial collagenase (MMP-1) PMN coilagenase (MMP-8) (2) 72 kDA Type IV collagenase (MMP-2)

Size (kDa) 52

I, II, III collagen 75 72

57

Modified from Ref. 13. MMP, matrix metallo-endoproteinase.

a

Degrades

I, II, III collagen, fibronectin, gelatins IV, V collagen, gelatins Proteoglycans, laminin, fibronectin, Ill, IV, V collagen, gelatins III, IV, V collagen, fibronectin, gelatins Gelatins, fibronectin

157 proteoglycans (including heparin sulfate) [70]. Much attention is focusing on the dynamic restructuring of the extracellular matrix in both normal function and in disease and on the pivitol role of secreted metallo-endoproteinases in this process. VII-A. Secreted metallo-endoproteinases

The number of matrix metallo-endoproteinases that are secreted as inactive pro-proteinases and are inhibited by specific tissue inhibitors continues to grow. Many matrix hydrolyzing metallo-endoproteinases and their matrix substrates have been identified, as shown in Table I [13]. VII-B. Metallo-endoproteinases in cellular metastasis

The ability of cancer cells to metastasize is a complex, poorly understood sequence involving: (a) oncogene activation and proliferation of transformed cells; (b) local invasion and destruction of extracellular matrix; (c) migration through tissue; (d) penetration of the blood vessel wall and entry into the blood stream; (e) exit from the blood vessel; and (f) proliferation at the new site [71]. The role of metallo-endoproteinases in tumor metastasis is based on two primary lines of evidence. First, tumor cells secrete a variety of metalloendoproteinases capable of hydrolyzing various components of the extracellular matrix and the amount of proteinase correlates with the metastatic capacity of the tumor [72]. Second, inhibiting metallo-endoproteinases, with either synthetic metallo-endoproteinase inhibitors or with the tissue inhibitor of metallo-endoproteinase (TIMP), prevents cellular migration both in vitro and in vivo [73,74]. Since collagen is a major structural protein in tissue, the matrix metallo-endoproteinase collagenase has been a central focus of research in metastasis. Many tumors contain and secrete large amounts of collagenases that hydrolyze type I, II & III collagen and that appears to correlate with metastatic capacity. Epithelial cancers and adenocarcinomas secrete stromelysin, a matrix metallo-endoproteinase which hydrolyzes type IV collagen found in basement membrane, including the basement membrane in blood vessels [73]. Tumors secreting elevated levels of type IV collagenase have a higher capacity of metastasis [72]. Multiple cellular and biochemical mechanisms regulate the expression secretion and activation of matrix metallo-endoproteinases, as discussed in Section II. Each of these mechanisms could regulate the ability of the cell to metastasize and each could, in principle, be targeted toward develop,ne',t of therapeutic agents to prevent metastasis.

VII-C. Metallo-endoproteinases in synovial membrane destructio::

The synovium in rheumatoid arthritis in two animal models of arthritis is characterized by hypertrophy and hyperplasia of the normally thin synovium, due to the proliferation of fibroblast-like mesenchyme cells called synoviocytes. These cells predominate at sites of cartilage resorption and bone erosion in diseased joints, where they cause destruction of the joint. Cultures of synoviocytes, from both patients with rheumatoid disease and from animals with experimentally induced arthritis produce and secrete high levels of stromyelysin (identical to transin), a matrix metallo-endoproteinase which hydrolyzes collagens type Ill, IV and V and other matrix proteins [75]. Stromelysin is also secreted by chondrocytes [76] and osteoblasts [77,78] which are also found at sites of cartilage and bone re-absorption in both human rheumatoid arthritis, and animal models. These observations suggest that secretion and activation of matrix metallo-endoproteinase by synoviocytes, chondrocytes and osteoblasts play an important role in the extracellularly matrix destruction of rheumatoid arthritis and also suggest a potential role for selective matrix metallo-endoproteinase inhibitors as therapeutic agents.

Viii. Conclusion A number of major areas require greater understanding before a clear picture of the function of metallo-endoproteinases can emerge. This includes better knowledge of the regulation of their activity and the development of reagents to study the subcellular localization of both the inactive and active forms of these enzymes. However, in most systems, the most essential bit of information that is missing is the identification of the endogenous substrates. To understand how metallo-endoproteinases participate in metastatic processes, or in membrane fusion events it is essential to identify the substrate(s) and its subcellular site of localization. Knowledge of these two facts should pave the way to a new level of understanding the biological function of metallo-endoproteinases.

Acknowledgements The authors wish to acknowledge support from the National Institutes of Health (HD21483 and HD18590 to WJL and AG08664 and NS20596 to WJS) and the Fyfe Foundation and Cephalon Inc. to WJS.

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