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Membrane proteases: roles in tissue remodeling and tumour invasion
Membrane proteases: roles in tissue remodeling and tumour Wen-Tien Georgetown
University
School
invasion
Chen
of Medicine,
Washington,
District
of Columbia,
USA
The
coordinated control of extracellular matrix degradation on the cell surface involves three crucial elements: secreted proteases and their inhibitors, surface protease receptors and integral membrane proteases. The roles that each of these elements play in cell surface proteolysis are described. The localization of proteases to the cell surface, protease activation, and regulation of cell surface proteolysis by protease inhibitors are key issues for elucidating the role of membrane proteases in tissue remodeling and tumour invasion.
Current
Opinion
in Cell
Biology
Introduction
localization
802
MMP-matrix activator; @
Current
Abbreviations metalloprotease; uPA-urokinase
Biology
of active
extracellular
proteases
Invading cells may interact with the ECM through distinct yet coordinated biochemical mechanisms of adhesion and degradation (Fig. 1). In addition to focal adhesion assemblies, whose molecular components are being increasingly identified [7], cell surface extensions called invadopodia have been characterized, which invade the ECM by means of extracellular proteolysis. In areas of the ECM where extensive degradation occurs, the invadopodia can be readily identified by immunofluorescence, phase contrast microscopy and electron microscopy ([8-l; T Kelly et al., unpublished data). The invadopodia may provide a structural entity to bind or activate proteases and allow us to test the hypothesis that membrane-associated proteases become concentrated at the invadopodia where they degrade a wide variety of ECM molecules, thereby pem&ing tumour cell invasion and metastasis. Analogous cell surface specializations also appear in a wide variety of tissue cells of monocytic origin such as macrophages and osteoclasts. ‘Ruffled border’ membranes of osteoclasts appear similar in structure and function to invadopodia, while ‘podosomes’, as described in some cases, present most of the proteins commonly found in focal adhesions and may simply re-
Studies of biochemically defined membrane fractions of normal or tumour tissue suggest that the active form of proteases is membrane-associated. Membrane-associated, ECM-degrading, secreted enzymes (Table 1) include plasminogen activators, the tumour associated trypsin-like proteases, cathepsins B and L and matrix metalloproteases (Mh4Ps). Cells must have receptors that bind secreted proteases to the cell surface. ECM-degrading proteases integral to the plasma membrane are less well known. Thus, there are three crucial elements that control turnover of the ECM on the cell surface: pro-
matrix; plasminogen
4BO2-809
teases and protease inhibitors clustering at focal invasion and adhesion sites; cell receptors recruiting intracellular and secreted proteases to the cell surface; and integral membrane proteases activating a protease cascade. Here, I will review how proteases are localized to the ceU surface, activated and regulated during tissue remodeling and tumour invasion,
For nearly thirty years since the discovery of collagenases (reviewed in [lo*] >, biologists have inferred that tissue remodeling and tumour invasion require controlled degradation of extraceUular matrix (ECM) macromolecules following their ceU surface deposition. Several reviews have appeared during the past year that document the role of proteases in the ECM degradation by tissue cells during biological processes such as morphogenesis, cartilage and bone repair, wound healing, angiogenesis and neutrophil migration, and the increased proteolysis exhibited by cells in disease states such as tumour cells during invasion and metastasis 12451. In o&o studies strongly suggest that the ECM degradative processes are con fined to localized extracellular sites that are delineated by the interactions of cells with the ECM. Proteolysis of the ECM may be due to the expression of integral membrane proteases and cysteine proteases on the cell surface (Fig. la), or the binding and activation of secreted latent serine and metalloproteases on the cell surface (Fig. lb). Most of the secreted proteases that have been described are abundant, although only a small percentage of the total secreted proteases are actually in the active form.
ECM-extracellular tPA-tissue-type
1992,
TIMP-tissue plasminogen
Ltd
ISSN
0955674
inhibitor activator;
of metalloprotease; uPAR-uPA
receptor.
Membrane
proteases
Chen
(a) Adhesion Cd) lnvadopodia
n
Extracellular
B Adhesion
matrix receptor
ligand
Lx
Secreted
(integrin)
D
Inhibitor
protease
0
Protease
0
integral
receptor membrane
protease
Fig. 1. Schematic representation of the cell surface localization and activation of four classes of extracellular matrix degrading proteases: integral membrane proteases, cathepsins, matrix metalloproteases and serine proteases. (a) Adhesion of cell to extracellular matrix. (b) A possible route of cell surface expression for integral membrane proteases that may bind and activate secreted proteases kathepsins, serineand metalloproteases) at invadopodia. (c) A possible mechanism whereby surface expression of protease receptors bind to and activate secreted proteases kathepsins, serineand metalloproteases) at invadopodia. (d) Invading cells utilize either protease receptors or integral membrane proteases to bind and activate secreted proteases.
fer to adhesive contacts (SC Mueller, Y Yeh, W-T Chen, unpublished data). Regulation of proteolytic activity of invadopodia may occur by changing adhesive properties at focal adhesion. For example, in transformed cells invading fibronectin-coated matrix beads, pl integrins are localized to invadopodia and their surrounding focal adhesions [8-l. Plasma membrane proteins associated with the ,invadopodium-rich ECM contact fraction of transformed cells have 30.fold increases in tyrosine phosphorylation compared with that observed in focal adhesionrich ECM contact fraction of normal cells [9]. Integrinmediated contact at focal adhesions of the cellular periphety may result in an extremely tight cellular adherence to the matrix bead, which prevents the influx of
protease inhibitors into the central area containing the invadopodia [10-l. On the other hand, integrin-mediated adhesion of invadopodia in the central cellular region may stabilize invadopodia and also mediate endocytic clearance of degraded libronectin matrix material
[@I.
Matrix
metalloproteases
MMPs, including the extensively studied 72 and 92 kD MMPs, are secreted in zymogen form, and activation is required to achieve ECM degradation. Both active and latent forms of MMPs are complexed with and regu-
803
804
Cell-to-cell
contact
and extracellular
matrix
. rable
1. Major
proteases
on
the
cell
surface.
Molecular Subunits
Matrix
weight
Pro-enzyme
Mode
fkD)
of surface
metalloproteases
Collagenase
52
52
42
Secreted,
?
Celatinase
fMMP2)
72
72
62, 59
Secreted,
7
Celatinase
fMMP9)
92
92
83,
75
Secreted,
7
57
57
45,
28
Secreted,
I
50, 33
Stromelysin Serine
association
Active
(MMPI)
fMMP3)
proteases
Urokinase
plasminogen
55
55
Secreted,
receptor
bound
70
70
Secreted,
receptor
bound
90
90
Secreted,
receptor
bound
Elastase
Secreted,
I
Thrombin
Secreted,
?
Tissue-type
activator
plasminogen
Plasminogen
activator
Cathepsins Cathepsins
8, H, L
30
30
Cathepsins
C, J, K
> 250
> 250
Integral CD10
membrane
100
or CALLA
90, 110 membrane
Secreted,
altered
processing
Secreted,
altered
processing
proteases
Meprins 170 kD
25, 5
protease
?
lated by tissue inhibitors of metalloproteases (TIMPs) [ 1 l-161, and the molecular interactions between MMPs and T’IMPs are an important regulatory point for ECM proteolysis. Other points of regulation, including binding of MMPs to the cell surface and physiological activation of pro-enzymes, are less well characterized. Previously, several morphological and biochemical observations have suggested that activation takes place after the binding of pro-enzymes or their complexes with TIMPs to the plasma membrane [17-201. There is more recent evidence for this hypothesis. The 72 kD MMP can be activated by incubation with isolated fibroblast membranes, and the resulting activity is blocked by TIMP2 [ 211. The carboxyl-terminal domain of collagenase and stromelysin is essential for membrane activation and modulates interactions with TIMPs, but is not required for catalysis [22,23*]. Our recent studies using antiinvadopodia monoclonal antibody CPl, have shown that both biotinyfated latent recombinant 72 kD MMP and the MMP-inhibitor complex bind to the plasma membrane at invadopodia (W-T Chen et al, unpublished data). However, only the antibody that recognized the active form of the 72kD MMP labeled the invadopodia, suggesting active MMPs are at these sites. Consistent with this, a novel cell fractionation technique isolating cell membranes in contact with the ECM from transformed cells ( [8.1; T Kelly et al, unpublished data) and a rapid procedure of isolating membrane-bound proteases from tumour cells of different invasive potential (WL Monsky et al, unpublished data) have shown that the 72 kD MMP associated with the cell membrane appears to be the active form. Expression of different MMPs has been correlated with many biological processes. These include early mouse development [24], development of the mammary gland [ 251, development of chick neural retina [ 261, ovulation [ 271, bone resorption by osteoclasts [ 281, cornea remod-
100 400
tetramer ?
Integral,
altered
processing
tetramer
Integral,
altered
processing
170
Integral,
altered
processing
eling [ 291, neovascularization [30], blastocyst outgrowths [31], mononuclear phagocyte differentiation [ 32,331 and proteolytic opening of the blood-brain barrier [34]. In diseases, MMPs are expressed in high levels in highly metastatic tumour cells [35,36], Rous sarcoma virustransformed cells [37] and cells involved in rheumatoid arthritis [38]. Inhibition studies using antibodies, TIMPs and other protease inhibitors have shown the role of MMPs in invasion of human cytotrophoblasts [39] and tumour cells [4(X43]. Such inhibitory effects may be mediated by interactions with the highly conserved peptide sequence from the MMP pro-segment, perhaps due to its potential membrane-binding activity [44-l. Localization studies of protein and mRNA show an increased expression of the 72 kD MMP in human colonic adenocarcinoma [45] and of the 72 and 92 kD MMPs in human skin cancers [46], suggesting that enhanced expression of the MMPs may be a marker of human tumour invasiveness. Conversely, levels of TIMPs are found to be decreased in human colon cancer [47], a situation that favors proteolysis by inhibitor-free MMPs. The 72 and 92 kD MMPs have a fibronectin-like collagen-binding domain [48*], and stromelysin appears to have a collagenbinding site at the carboxyl-terminal domain [49-l. The presence of collagen-binding domains in MMPs is significant, as it allows the localization and concentration of latent forms of MMPs to the ECM.
Serine
proteases
It has been suggested that serine proteases are probably not directly responsible for matrix degradation but are useful as activators for the metalloproteases that serve as the workhorses [2]. The serine proteases implicated in
Membrane
this process so far include the well characterized urokinase plasminogen activator-receptor ( UPA-uPAR) system, leukocyte elastase and the tumor-associated trypsin. The detailed domain structure of uPA has been determined [50*], and conformational similarities between one-chain and two-chain tissue-type plasminogen activator (tPA) have been resolved [ 511. Both uPA (50 kD) and tPA (70 kD) can activate the abundant serum protein plasminogen (900) to the broad-specificity protease plasmin by cleavage of a single bond. Plasmin is active in librinolysis, tissue remodeling and tumor invasion. The uPA-catalyzed plasminogen activation is strongly enhanced when both uPA/pro-uPA and plasminogen are bound to the ceU surface [52**,53**]. The cell surface receptor for uPA (uPAR) is a single-chain 5560 kD glycoprotein. The physiological activator of uPA has not been identified, but other membrane-associated enzymes appear to be involved. Evidence that supports this hypothesis comes from the demonstration that the singlechain pro-uPA is converted into the active two-chain form and exhibits its enzymatic activity upon binding to the cell surface of transformed cells [54*], monocytes [ 55*] and liver cells [ 56.1. Cytofluorometty showed that the uPA ligand bound preferentially to the ceU surface, and the bound uPA was involved in the breakdown of fibrin [57*]. Surface-bound uPA became phospholylated [58], and enzymatic properties of the phosphotylated uPA were altered [59]. Interestingly, a 19 amino acid synthetic peptide derived from the E8 fragment of the laminin A chain (Cys-Ser-Arg-Ala-Arg-Lys-Gln-AlaAla-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp-Arg-NH2) was identified, which promotes metastasis and stimulates MMP activity in the culture medium of BIG melanoma cells 1601. This peptide is also a potent stimulator of tPA-catalyzed plasminogen activation, resulting in a 22-fold increase in the activation reaction [ 6I*]. Expression of different serine proteases and their cell surface receptors has also been correlated with many biological processes. Tumor infiltrates contain many more uPA-positive cells than normal tissues, but the staining is confined to iibroblast-like cells and endothelial cells in the tumor stroma [62], whereas uPAR is detected in the malignant epithelial cells (631. These iindings may indicate that colon cancer cells recruit stromal cells to produce uPA involved in degradation of the ECM during invasive growth. This in zdvo paracrine interaction between uPA and its receptor may indeed play an im portant role in tumor ceU invasion as shown in experimental metastases [52**,53**]. Consistent with this finding, desulfatohirudin, a highly specific thrombin inhibitor, can block experimental melanoma metastasis [64]. Inhibitors of the serine proteases, free or complexed with proteases, interact with the ceU surface and lead to protease internalization. For example, intemalization of the urokinase-plasminogen activator-inhibitor type-l (PAIl, 45 kD) complex is mediated by the uPAR [65*]. The anti-carcinogenic Bowman-Birk protease inhibitor complexed with its target protease is internalized and inhibits the growth-regulated activity displayed by the protease [66*]. In addition, proteases and their inhibitors bind to the ceU surface at different sites. For
proteases
Chen
example, PAIl is localized in the focal adhesions but not in invadopodia (W-T Chen, unpublished data). This is interesting because ECM substrates are degraded by transformed cells and tumor cells at sites of ECM contact with invadopodial membranes but not at focal adhesions. Here, the uPA system may function to release cells from the substratum during migration, rather than mediating the active ECM degradation that occurs at the invadopodia of transformed cells.
Cathepsins Lysosomal cysteine protease cathepsins are typically associated with intracellular compartments; however, digestion within the lysosomes can also contribute to the process of ECM degradation. Cathepsins B, H and L exist in single-chain forms (3OkD) and two-chain forms (25 and 5 kD), while cathepsins C, J and K are oligomeric in structure and contain extremely long propeptides [67]. Cathepsins B and L, and a cathepsin L-like 70 kD protease from bone tissue can degrade coUagens and appear necessary for bone resorption [68]. Bone tissue cells, like osteoclasts, secret cathepsins to a compartmentalized extraceUular space delimited by podosome focal adhesions where an acidic micro-environment optimal for cathepsin activity may be maintained. Malignantly transformed cells have apparent increases in ceU surface association of cathepsins B [69*,70] and L [71*]. Cathepsin B associated with the ceU surface can degrade ECM components at both acidic and neutral pH [69]. Proteolytic processing and glycosylation of cathepsin B have been suggested to achieve ceU surface expression and pH dependence of cathepsin B-catalyzed hydrolysis during ECM invasion of tumor cells [72*-74-l.
Integral
membrane
proteases
Very little attention has been paid to membrane proteases integral to the ceU surface that degrade the ECM, primarily because of their low abundance and the difficulty of identifying invasive ceU lines that highly express these membrane proteases. A few plasma membrane proteases, however, have been identified from biochemically defined ‘membrane’ fractions derived from tissue homogenates. CeU fractionation and immunological assays have localized CD10 or CALL4 (the 1OOkD common acute lymphoblastic leukemia antigen) to the surface of a variety of tissue and tumor cells [75,76]. CDlO, a type II transmembrane glycoprotein, was identified as neutral endopeptidase 24.11, an integral membrane zinc metallo-endoproteinase. Meprins are ceU surface oligomeric glycoproteins from kidney tubule brush borders that have metallo-endopeptidase activity [n]. Recent cloning studies show that meprins are part of a family of metallo-endopeptidases, the ‘astacin family [78]. Finally, hepsin has been recently characterized as a plasma membrane protease [79]. These proteases prob-
805
806
Cell-to-cell
contact
and extracellular
matrix
.
ably have functions that are related to the function of microvillar membranes of epithelia in intestine and kidney, although it is not yet clear what significance these proteases have for matrix remodeling and the invasion process. Since the identification of cell contact-related protease activities from Rous sarcoma virus-transformed cells [&IO], one of the main aims of our laboratory has been to find integral membrane protease molecules of invasive cells that are involved in the detachment of cells from the ECM leading to cell invasion. Using fibronectin-coated crosslinked gelatin films as immobilized substrates to identify the invasiveness of tumor cells, we have identified three groups of neutral membrane proteases: serine, metallo- [19], and sulfhydryl proteases [Sl] from transformed and tumor cells that degrade the ECM. Recently, we have identitied a novel sulfhydtyl membrane protease with high molecular weight (170 kD), that is tightly associated with the plasma membrane of invadopodia of transformed cells in contact with the ECM (T Kelly et al, unpublished data). The 170kD membrane protease activity of embtyonic cells, similar to the 170kD membrane protease of human malignant melanoma ceU line LOX cells ([81]; WL Monsky et al, unpublished data), is maximal at neutral pH, and has a peculiar cysteine protease inhibitor profile: its activity is enhanced by EDTA and dithiothreitol, but inhibited partially (80%) by the cysteine-protease inhibitors, N-ethyl maleimide and phenylrnethylsulfonyl fluoride. Consistent with the possible cysteine active site, these membrane proteases can bind to an organomercurial adsorbent (T Kelly et al. unpublished data), but not to the serine protease inhibitor paminobenzamidine. We suggest that expression of this group of related membrane proteases in invadopodia contributes to the localized degradation of the ECM occurring in invading cells.
their functions. Attempts to exploit these opportunities may weU result in the development of new reagents for the early detection and treatment of various diseases.
Acknowledgements I am indebted to Thomas Kelly, Chen-Yong Susette C Mueller and Yunyun Yeh for helpful was supported by USPHS grant number R01 33711.
tin, Wayne Monsky, discussions. This work CA-39077 and ROl HL-
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HOWARD EW. BANDA MJ: Biding of Tissue Inhibitor of MetaUoproteinases 2 to Two Distinct Sites on Human 72-kDa Gelatinase. Identification of a Stabilization Site. J Eiol Uw?n 1991, 26617972-17977.
21.
28.
MJ: Regulation of the Progelatinase by Tis/ Biol Cbem 1991,
16.
18.
J
29.
EW,
Tissue teinase 15.
BANDA
Human 72-kD Metalloproteinases-2.
of
of 72- and 92.kDa Metalloproteinases-2. 14.
EC,
Inhibitor
Droteases
43.
44. .
DECLERCK YA, PEW TAYLOR SM: Inhibition
with an Inhibitor 52:701-708.
REDWOOD
SM,
Abrogation by using
of the Protease
LIP BC-S.
A, ALBINI
hibition of Tumor Peptlde Sequence zyme Prosegment.
L~NGL!~
KE:
N, SHI~IADA H. BOONE TC, LANCLFY KE, of Invasion and Metastasis in CeUs
Transfected Res 1992,
MELCHIOIU
H.
Muscle CeU Layers Inhibitor. Cancer
of MetalIoproteinases.
WEIRS RE, HODGE
DE,
Cancer DROLLER
MJ:
Invasion of Human Bladder Tumor CeUs Inhibitor(s). Cancer 1992, 691212-1219. A, RAN JM, STETLER-STEVENSON WG: InCeU Invasion by a Highly Conserved from the Matrix MetaUoproteinase EnCancer Res 1992, 52:23532356.
807
808
Cell-to-cell
&tact
and
The highly conserved peptide not only inhibits MMF’ activity
extracellular sequence from but also blocks
matrix the MMP pro-segment tumor cell invasion in
ulm
45.
46
LEVY AT, CKKE V, SOBEL MF.. GAREZI~A S, GRIGIONI WF, L~OTTA IA, STEIUR-STEVENSON WG: Increased Expression of the Mr 72000 Type IV Collagenase in Human Colonic Adenocarcinema Cancer Res 1991, 51:439-444. PYKE C, RWKLWI E, HLIHTA P, HLII~KAINEN T, DANO K, TRYGGVAsON & Localization of Messenger RNA for M, 72000 and 92000 Type IV Collagenases in Human Skin Cancers by in sih~ Hyixidization. Cancer Res 1992, 52:133&341.
47.
L.IJ X, UVY M, WEINsTEtN 5, SANTEUA RM: lmtnurtological Quantiation of Levels of Tiiue Inhibitor of MetaUoproteinase-1 in Human Colon Cancer. Cancer Res 1991, 51:62316235.
48. .
COUIER IE, KIWNOV PA, STRONG~N AY, BIRKEDAL-HANSEN H, G~IDBERG Gl: Alanine Scanning Mutagenesis and Functional Analysis of the Fibronectin-like Collagen-binding Domain &om Human 92-kDa m N Collagenase. J Biol cbem 1992, 267:6ntL6?81.
Alanine scanning mutagenesis was used to probe structural elements in the hbronectin-like collagen-binding domain of human 92 kD MMP. Changes in this domain are su&ient to alter the gelatin-binding ability of the fusion proteins. 49. .
AUAN JA, HEMBRY RM, ANGAL S, REYNOIDS JJ, MUR~HY G: Binding of Latent and High M, Active Forms of Stromelysin to Collagen is Mediated by the C-terminal Domain. J Cell Sci 1991, 99:7a9-795. Strome$xm has its collagen-binding site at the carboxyl-terminal domain. N~~~~ATNY V, MEDVED I, MA?AR A, MARCOITE P, HENX!N J. INGHMl K: Domain Structure and Interactions of Recombit Urokinase-type Plasminogen Activator. J Biol Ghem 1992, 267:387t%3885. Describes the detailed domain structure of uPA
The authors showed that secreted single-chain pro-uPA in Rous sarcoma virus-transformed cells was activated by an endogenous, plasminindependent mechanism upon binding of the pro-uPA to the cell surface of transformed cells. MANCHANDA N, SCHWARTZ BS: Single Chain Urokinase. Aug mentation of Enzymatic Activity Upon winding to Monocytes. J Biol &em 1991, 266145%14584. This paper examin es the ceU surface actimtion of singlexhain uPA in detail.
55. .
56. .
KUIPER J, RrJle~ DC, DE MUNK GAW, VAN BERKEL rJC: In Viuo and in Vitro Interaction of High and Low Molecular Weight Single-chain Urokinase-type Plasminogen Activator with Rat Liver Cells. J Biol Cbem 1992, 267:15891595. The plasma clearance and the interaction of high and low molecular weight single-chain UPA with rat liver cells was determined. A new UPA recognition site on liver ceUs was identified and this site was involved in the turnover of uPA
TAKAHA~H~ K, GOJOBORI T, TANIFUJI M: Two-color Cytofluorometry and Cellular Properties of the Urokinase Receptor Associated with a Human Metastatic Carcinomatous CeU Line. I&p Cell Res 1991, 192:405-413. Cytotluorometry showed that uPA bound preferentially to cells that had been exposed to acidic pH. The data obtained by zymography of the cellular proteins suggested that the uPA was bound to the receptor. Bound uPA was found to degrade fibrin. 57. .
58.
TAKAHA%~~ K, Kwm HC, IKEO K, KOH E: Phosphorylation of a Surface Receptor Bound Urokinase-type Plasminogen Activator in a Human Metastatic Carcinomatous CeU Line. Biocbem Biopbys Res Commun 1992, 18231461472.
59.
TAKAH+SHI K, KWAAN HC, KOH E, TANABE M: Enzymatic Properties of the Phosphotylated Urokinase-type Plasminogen Activator Isolated from a Human Carcinomatous CeU Line. Bic&em Bicpbys Re.s Commun 1992, 182:1473-1481.
60.
KANEMOTO T. REICH R, ROYCE L, GRF.ATOREX D. ADLER SH, SHIRU~HI N, MARTIN GR, YAMADA Y. KLEINMAN HK: ldentilication of an Amino Acid Sequence from the Laminin A Chain that Stimulates Metastasis and Collagenase N Production. Proc Nat1 Acad Sci USA 1990, 8732279-2283.
61. .
STACK S, GRAY RD, PIZZO SV: Modulation of Plasminogen vation and Type N Collagenase Activity by a Synthetic tide Derived from the hninin A-chain. Biochemists
50. .
51.
NIENABER VI, YOUNG SI, BIIXTOFT JJ. HIGGINS DL, BERIINER LJ: Conformational Similarities Between One-chain and Twochain Tissue Plasminogen Activator (t-PA) implications to the Activation Mechanism on One-chain t-PA Bicdemistty 1992, 31:3852-3861.
ActiPep1331,
30:20732on.
52.
Osso’JGKI 1 CLUNIE G, M.YJCCI M-T, B~ASI F: In Viva .. Paracttne Interaction Between Urokinase and its ReceptoT: Effect on Tumor Cell Invasion. J Cell Biol 1991, 115:1107-1112. An in vi00 model of invasion, consisting of the chorio-allantoic membrane of chicken embryos, was used in this study. Only cells that concurrently expressed both uPA and uPAFt, and in which the receptor was saturated with uPA, were &cient in invasion. Transfection experiments have determined that uPA produced by one ceil can, in a patacrine fashion, a&t the invasive capacity of a receptor-expressing cell.
The 19 amino acid synthetic peptide derived from the E8 fragment of the laminin A-chain is a potent stimulator of &‘A-catalyzed plasminogen activation. The activity of purified type I and type IV collagenase was. however, inhibited by this peptide. This study supports the idea that this peptide stimulates plasminogen activation, subsequently generating coUagense activity. 62.
GRANDAHL-HANSEN LLINO LR, DANO K: gen Activator in Colon in Humans.
QUAX PH, VAN MU~AN GN, WEENING-VERHOEFF EJ, LIJNO LR, DANO K, RLIITER DJ, VERHEIJEN JH: hjetastatic Behavior of Human M oma Cell Lines in Nude Mice Correlates with Uro iFi!Ese -type Plasminogen Activator, its w-1 Inhibitor, and Urokinase-mediated Degradation J Cell Biol 1991, 115:191-199. Plasmin-dependent degradation of a smooth muscle cell ECM by some human melanoma cell lines was mediated by tPA and others by uPA The UPA and PAll producing cell lines showed the highest frequency to form spontaneous lung metastases after subcutaneous inoculation. The authors conclude that uPA-mediated matrix degradation in uitro and production of uPA and PAIl by human melanoma cell lines correlated with their ability to form spontaneous lung metastasis in nude mice.
63.
HOLLX? W, BAISI F. Bovo D: Role of the Urokinase Receptor in Facilitating Exvacellular Matrix Invasion by Cultured Colon Cancer. Cancer Res 1991, 51:369&3695.
64.
ESUMI N, FAN D, FIDLER IJ: Inhibition of Murine Melanoma Experimental Metastasis by Recombinant Desuffitohirudin, a Highly SpeciJic Thrombin Inhibitor. Cancer Res 1991. 51:4549-4556.
53. ..
54. .
BERKENPAS MB, QIJ~G~EY JP: Transformation-dependent Activation of Urokinase-type Plasminogen Activator by a Plasmindependent Mechanism: Involvement of Cell Surface Membranes. Proc Nat1 Acad Sci USA 1991, 88:7768-7772.
J, RALF~(IAER E, KIRKEBY LT, KIUSIFNSEN P, Localization of Urokinase-type PlasminoStromal Cells in Adenocarinomas of tbe Am J Patbol 1991, 138:111-117.
65. .
OISON D. P&I&EN J, HOYER-HANSEN G, RONNE E, SAKAGUCH~ K, WUN T-C, APPEUA E, DANO K, BIA?I F: Internalization of the Urokinase-plasminogen Activator Inhibitor Type-l Complex is Mediated by the Urokinase Receptor. J Biol Ckm 1992, 267:912$9133. This paper documents the internalization of the PAIl and UPA-PAJ complexes mediated by the uPAR 66. .
BILLINGS PC, HARBREs JM: A Growth-regulated tivity that is Inhibited by the Anticarcinogenic
Protease AcBowman-
Membrane Birk Rotease 89:312&3124. This paper shows hibitor complexed the growth-regulated 67.
ISHIIXIH
68
Pnx
N&l
A&
.Sci USA
that the anti-carcinogenic Bowman-Birk with its target protease is internalized activity displayed by the protease.
1992,
protease inand inhibits
D. SATO N, KO~IIN~U E: Molecular Cloning for Rat Cathepsin C. Cathepsin C, a Cysteine with an Extremely Long Propeptide. J Biol Cbem
K. MUNO
of cDNA Protein% 1991,
Inhibitor.
75.
LEDENT P, VAES G: Collagenolytic Cysteine Proteinases of Bone Tissue. Cathepsin B, @ro)Cathepsin L and a Cathepsin L-like 70kDa Proteinase. Biocbem J 1991,
.
dation of Extracellular-matrix sin B from Normal and
DAY N&
Tumour
HONN
282:273-278. This paper shows that cathepsin B associated with tumor cells can degrade ECM components at both pH. Thus, an active cathepsin B on the cell surface
CathepJ 1992,
the cell surface of acidic and neutral is implicated.
Apfor the Deby Breast-
CUUEN
BM. HALLIDAY IM, KAY G, NELSON J, WALKER B: The
plication tection tumour
of a Novel Biotinylated Affinity Label of a Cathepsin B-like Precursor Produced CeUs in Culture. Biochem J 1992, 283:461465.
71.
KANE SE, G~ITESMAN
.
nant
72.
MACH L, SrrIw~
.
Processing and Glycosylation the Primary Structure of the
MM: The Role of Cathepsin L in MaligSemis Cancer Biol 1990, 1:127-136. The paper presents a comprehensive oveniew of the role of cathepsin L in malignant transformation.
Transformation.
Carbohydrate
Moiety Enzyme. Biodwm
of the
77.
J 1992,
78.
.
79.
see 74. .
4 MORT JS: Characterization of Recombinant Rat Cathepsin B and Nonglycosylated Mutants Expressed in Yeast. New Insights into the pH Dependence of Cathepsin B-catalyzed Hydrolyses. J Biol Uwm
1~92, [7r*].
J 1991,
Sc07-r
CS, TURNER
AJ,
KENNY
AJ:
A
278:417421.
SHI~P MA, STEFANO GB,
SXVITZER SN, GRIFFIN JD,
MZ, WOIZ RL, GORBM -B. CeU Surface Endopeptidases
KOUNNAS
JUNG
1991, W,
REINHERZ EL:
CM, BOND JS: Meprin-A of the Mouse Kidney.
and J Biol
266:1735&17357.
G~RBEA
CM,
F~ANNERY AV.
BEYNON
RJ, GM
GA
TSLJI A, Tow-ROSADO A, ARAI T, LE BEAU MM, LEMONS RS. CHOU S-H, KLJRACHI K: Hepsin, a CeU Membrane-associated
Rotease.
Characterization,
Localization. 80.
CHEN
sion grade
W-T,
of
81.
Tissue
J Biol
C&em
OIDEN
K,
1991,
Distribution,
at
CeU
and
Gene
2(X%16948-16953.
BERNARD
Transformation-associated
Fibronectin 98:154&1555.
282:577-582.
HASNAIN S, HIR~ZA T. Ttil
H,
BOND JS: The a Subunit of Meprin A: Molecular Cloning and Sequencing, DifTerential Expression in lnbred Mouse Strains, and Evidence for Divergent Evolution of the a and p Subunits. J Biol Uwm 1992, 267: in press.
see 174.1. 73.
S, MURRAY
&WI
K. HAGEN A, BALL%UN C, GLOSSL J: Proteolytic
of Cathepsin B. The role of Latent Precursor and of the for CeU-type-specilic Molecular Forms
in Sublines of a Human Colon Cancer Metastatic Potentials. / Biol Gem 1992,
CD10 (CALLA)/Neuual Endopeptidase 24.11 Modulates Inflammatory Peptide-induced Changes in Neutrophil Morphology, Migration, and Adhesion Proteins and is itself Reg ulated by Neutrophil Activation. BIG& 1991, 78:1834-1841.
KV, SIXXNE BF: Degra-
Proteins by Human Tissues. Biochn
HOWEU
B&hem 76.
BLICK MR. KARIJSTIS DG,
809
Highly Sensitive e.1.i.s.a. for Endopeptidase-24.11, the Common Acute-lymphoblastic-leukaemia Antigen (CALLA, CD10). Applicable to Material of Porcine and Human Origin.
DEW.SS~ J-M,
69.
Chen
267:5700-5711. Three excellent papers [72*-74.1 deal with the proteolytic processing and glycosyiation of cathepsin B. These properties are thought to be involved in ceU surface expression of cathepsin B, and pH dependence of cathepsin B-catalyzed hydrolysis during ECM invasion of tumor cells.
26616312-16317.
279:167-174.
70.
brane Glycoproteins Exhibiting Distinct
Droteases
BA,
CHU
F-F:
Protease Contact
Sites.
J Cell
Expresthat DeBiol
1984,
AOYAMA 4 CHEN W-T:
A 170.kDa Membrane-bound Rotease is Associated with the Expression of Invasiveness by Human Malignant Melanoma Cells. Proc N&l Ad Sci USA 1990,
87:8296-8300.
267:4713-i721.
SAITOH
cosylation
0, WANG
W-C,
LAXAN R, FLIKLJIIA
and CeU Surface
Expression
M: Differential
of Lysosomal
Gly-
Mem-
W-T Chen. Department of Anatomy and Cell Biology, Georgetown versity School of Medicine, 3900 Reservoir Road N.W., Washington, trict of Columbia 20007. USA.
UniDis-
University
School
invasion
Chen
of Medicine,
Washington,
District
of Columbia,
USA
The
coordinated control of extracellular matrix degradation on the cell surface involves three crucial elements: secreted proteases and their inhibitors, surface protease receptors and integral membrane proteases. The roles that each of these elements play in cell surface proteolysis are described. The localization of proteases to the cell surface, protease activation, and regulation of cell surface proteolysis by protease inhibitors are key issues for elucidating the role of membrane proteases in tissue remodeling and tumour invasion.
Current
Opinion
in Cell
Biology
Introduction
localization
802
MMP-matrix activator; @
Current
Abbreviations metalloprotease; uPA-urokinase
Biology
of active
extracellular
proteases
Invading cells may interact with the ECM through distinct yet coordinated biochemical mechanisms of adhesion and degradation (Fig. 1). In addition to focal adhesion assemblies, whose molecular components are being increasingly identified [7], cell surface extensions called invadopodia have been characterized, which invade the ECM by means of extracellular proteolysis. In areas of the ECM where extensive degradation occurs, the invadopodia can be readily identified by immunofluorescence, phase contrast microscopy and electron microscopy ([8-l; T Kelly et al., unpublished data). The invadopodia may provide a structural entity to bind or activate proteases and allow us to test the hypothesis that membrane-associated proteases become concentrated at the invadopodia where they degrade a wide variety of ECM molecules, thereby pem&ing tumour cell invasion and metastasis. Analogous cell surface specializations also appear in a wide variety of tissue cells of monocytic origin such as macrophages and osteoclasts. ‘Ruffled border’ membranes of osteoclasts appear similar in structure and function to invadopodia, while ‘podosomes’, as described in some cases, present most of the proteins commonly found in focal adhesions and may simply re-
Studies of biochemically defined membrane fractions of normal or tumour tissue suggest that the active form of proteases is membrane-associated. Membrane-associated, ECM-degrading, secreted enzymes (Table 1) include plasminogen activators, the tumour associated trypsin-like proteases, cathepsins B and L and matrix metalloproteases (Mh4Ps). Cells must have receptors that bind secreted proteases to the cell surface. ECM-degrading proteases integral to the plasma membrane are less well known. Thus, there are three crucial elements that control turnover of the ECM on the cell surface: pro-
matrix; plasminogen
4BO2-809
teases and protease inhibitors clustering at focal invasion and adhesion sites; cell receptors recruiting intracellular and secreted proteases to the cell surface; and integral membrane proteases activating a protease cascade. Here, I will review how proteases are localized to the ceU surface, activated and regulated during tissue remodeling and tumour invasion,
For nearly thirty years since the discovery of collagenases (reviewed in [lo*] >, biologists have inferred that tissue remodeling and tumour invasion require controlled degradation of extraceUular matrix (ECM) macromolecules following their ceU surface deposition. Several reviews have appeared during the past year that document the role of proteases in the ECM degradation by tissue cells during biological processes such as morphogenesis, cartilage and bone repair, wound healing, angiogenesis and neutrophil migration, and the increased proteolysis exhibited by cells in disease states such as tumour cells during invasion and metastasis 12451. In o&o studies strongly suggest that the ECM degradative processes are con fined to localized extracellular sites that are delineated by the interactions of cells with the ECM. Proteolysis of the ECM may be due to the expression of integral membrane proteases and cysteine proteases on the cell surface (Fig. la), or the binding and activation of secreted latent serine and metalloproteases on the cell surface (Fig. lb). Most of the secreted proteases that have been described are abundant, although only a small percentage of the total secreted proteases are actually in the active form.
ECM-extracellular tPA-tissue-type
1992,
TIMP-tissue plasminogen
Ltd
ISSN
0955674
inhibitor activator;
of metalloprotease; uPAR-uPA
receptor.
Membrane
proteases
Chen
(a) Adhesion Cd) lnvadopodia
n
Extracellular
B Adhesion
matrix receptor
ligand
Lx
Secreted
(integrin)
D
Inhibitor
protease
0
Protease
0
integral
receptor membrane
protease
Fig. 1. Schematic representation of the cell surface localization and activation of four classes of extracellular matrix degrading proteases: integral membrane proteases, cathepsins, matrix metalloproteases and serine proteases. (a) Adhesion of cell to extracellular matrix. (b) A possible route of cell surface expression for integral membrane proteases that may bind and activate secreted proteases kathepsins, serineand metalloproteases) at invadopodia. (c) A possible mechanism whereby surface expression of protease receptors bind to and activate secreted proteases kathepsins, serineand metalloproteases) at invadopodia. (d) Invading cells utilize either protease receptors or integral membrane proteases to bind and activate secreted proteases.
fer to adhesive contacts (SC Mueller, Y Yeh, W-T Chen, unpublished data). Regulation of proteolytic activity of invadopodia may occur by changing adhesive properties at focal adhesion. For example, in transformed cells invading fibronectin-coated matrix beads, pl integrins are localized to invadopodia and their surrounding focal adhesions [8-l. Plasma membrane proteins associated with the ,invadopodium-rich ECM contact fraction of transformed cells have 30.fold increases in tyrosine phosphorylation compared with that observed in focal adhesionrich ECM contact fraction of normal cells [9]. Integrinmediated contact at focal adhesions of the cellular periphety may result in an extremely tight cellular adherence to the matrix bead, which prevents the influx of
protease inhibitors into the central area containing the invadopodia [10-l. On the other hand, integrin-mediated adhesion of invadopodia in the central cellular region may stabilize invadopodia and also mediate endocytic clearance of degraded libronectin matrix material
[@I.
Matrix
metalloproteases
MMPs, including the extensively studied 72 and 92 kD MMPs, are secreted in zymogen form, and activation is required to achieve ECM degradation. Both active and latent forms of MMPs are complexed with and regu-
803
804
Cell-to-cell
contact
and extracellular
matrix
. rable
1. Major
proteases
on
the
cell
surface.
Molecular Subunits
Matrix
weight
Pro-enzyme
Mode
fkD)
of surface
metalloproteases
Collagenase
52
52
42
Secreted,
?
Celatinase
fMMP2)
72
72
62, 59
Secreted,
7
Celatinase
fMMP9)
92
92
83,
75
Secreted,
7
57
57
45,
28
Secreted,
I
50, 33
Stromelysin Serine
association
Active
(MMPI)
fMMP3)
proteases
Urokinase
plasminogen
55
55
Secreted,
receptor
bound
70
70
Secreted,
receptor
bound
90
90
Secreted,
receptor
bound
Elastase
Secreted,
I
Thrombin
Secreted,
?
Tissue-type
activator
plasminogen
Plasminogen
activator
Cathepsins Cathepsins
8, H, L
30
30
Cathepsins
C, J, K
> 250
> 250
Integral CD10
membrane
100
or CALLA
90, 110 membrane
Secreted,
altered
processing
Secreted,
altered
processing
proteases
Meprins 170 kD
25, 5
protease
?
lated by tissue inhibitors of metalloproteases (TIMPs) [ 1 l-161, and the molecular interactions between MMPs and T’IMPs are an important regulatory point for ECM proteolysis. Other points of regulation, including binding of MMPs to the cell surface and physiological activation of pro-enzymes, are less well characterized. Previously, several morphological and biochemical observations have suggested that activation takes place after the binding of pro-enzymes or their complexes with TIMPs to the plasma membrane [17-201. There is more recent evidence for this hypothesis. The 72 kD MMP can be activated by incubation with isolated fibroblast membranes, and the resulting activity is blocked by TIMP2 [ 211. The carboxyl-terminal domain of collagenase and stromelysin is essential for membrane activation and modulates interactions with TIMPs, but is not required for catalysis [22,23*]. Our recent studies using antiinvadopodia monoclonal antibody CPl, have shown that both biotinyfated latent recombinant 72 kD MMP and the MMP-inhibitor complex bind to the plasma membrane at invadopodia (W-T Chen et al, unpublished data). However, only the antibody that recognized the active form of the 72kD MMP labeled the invadopodia, suggesting active MMPs are at these sites. Consistent with this, a novel cell fractionation technique isolating cell membranes in contact with the ECM from transformed cells ( [8.1; T Kelly et al, unpublished data) and a rapid procedure of isolating membrane-bound proteases from tumour cells of different invasive potential (WL Monsky et al, unpublished data) have shown that the 72 kD MMP associated with the cell membrane appears to be the active form. Expression of different MMPs has been correlated with many biological processes. These include early mouse development [24], development of the mammary gland [ 251, development of chick neural retina [ 261, ovulation [ 271, bone resorption by osteoclasts [ 281, cornea remod-
100 400
tetramer ?
Integral,
altered
processing
tetramer
Integral,
altered
processing
170
Integral,
altered
processing
eling [ 291, neovascularization [30], blastocyst outgrowths [31], mononuclear phagocyte differentiation [ 32,331 and proteolytic opening of the blood-brain barrier [34]. In diseases, MMPs are expressed in high levels in highly metastatic tumour cells [35,36], Rous sarcoma virustransformed cells [37] and cells involved in rheumatoid arthritis [38]. Inhibition studies using antibodies, TIMPs and other protease inhibitors have shown the role of MMPs in invasion of human cytotrophoblasts [39] and tumour cells [4(X43]. Such inhibitory effects may be mediated by interactions with the highly conserved peptide sequence from the MMP pro-segment, perhaps due to its potential membrane-binding activity [44-l. Localization studies of protein and mRNA show an increased expression of the 72 kD MMP in human colonic adenocarcinoma [45] and of the 72 and 92 kD MMPs in human skin cancers [46], suggesting that enhanced expression of the MMPs may be a marker of human tumour invasiveness. Conversely, levels of TIMPs are found to be decreased in human colon cancer [47], a situation that favors proteolysis by inhibitor-free MMPs. The 72 and 92 kD MMPs have a fibronectin-like collagen-binding domain [48*], and stromelysin appears to have a collagenbinding site at the carboxyl-terminal domain [49-l. The presence of collagen-binding domains in MMPs is significant, as it allows the localization and concentration of latent forms of MMPs to the ECM.
Serine
proteases
It has been suggested that serine proteases are probably not directly responsible for matrix degradation but are useful as activators for the metalloproteases that serve as the workhorses [2]. The serine proteases implicated in
Membrane
this process so far include the well characterized urokinase plasminogen activator-receptor ( UPA-uPAR) system, leukocyte elastase and the tumor-associated trypsin. The detailed domain structure of uPA has been determined [50*], and conformational similarities between one-chain and two-chain tissue-type plasminogen activator (tPA) have been resolved [ 511. Both uPA (50 kD) and tPA (70 kD) can activate the abundant serum protein plasminogen (900) to the broad-specificity protease plasmin by cleavage of a single bond. Plasmin is active in librinolysis, tissue remodeling and tumor invasion. The uPA-catalyzed plasminogen activation is strongly enhanced when both uPA/pro-uPA and plasminogen are bound to the ceU surface [52**,53**]. The cell surface receptor for uPA (uPAR) is a single-chain 5560 kD glycoprotein. The physiological activator of uPA has not been identified, but other membrane-associated enzymes appear to be involved. Evidence that supports this hypothesis comes from the demonstration that the singlechain pro-uPA is converted into the active two-chain form and exhibits its enzymatic activity upon binding to the cell surface of transformed cells [54*], monocytes [ 55*] and liver cells [ 56.1. Cytofluorometty showed that the uPA ligand bound preferentially to the ceU surface, and the bound uPA was involved in the breakdown of fibrin [57*]. Surface-bound uPA became phospholylated [58], and enzymatic properties of the phosphotylated uPA were altered [59]. Interestingly, a 19 amino acid synthetic peptide derived from the E8 fragment of the laminin A chain (Cys-Ser-Arg-Ala-Arg-Lys-Gln-AlaAla-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp-Arg-NH2) was identified, which promotes metastasis and stimulates MMP activity in the culture medium of BIG melanoma cells 1601. This peptide is also a potent stimulator of tPA-catalyzed plasminogen activation, resulting in a 22-fold increase in the activation reaction [ 6I*]. Expression of different serine proteases and their cell surface receptors has also been correlated with many biological processes. Tumor infiltrates contain many more uPA-positive cells than normal tissues, but the staining is confined to iibroblast-like cells and endothelial cells in the tumor stroma [62], whereas uPAR is detected in the malignant epithelial cells (631. These iindings may indicate that colon cancer cells recruit stromal cells to produce uPA involved in degradation of the ECM during invasive growth. This in zdvo paracrine interaction between uPA and its receptor may indeed play an im portant role in tumor ceU invasion as shown in experimental metastases [52**,53**]. Consistent with this finding, desulfatohirudin, a highly specific thrombin inhibitor, can block experimental melanoma metastasis [64]. Inhibitors of the serine proteases, free or complexed with proteases, interact with the ceU surface and lead to protease internalization. For example, intemalization of the urokinase-plasminogen activator-inhibitor type-l (PAIl, 45 kD) complex is mediated by the uPAR [65*]. The anti-carcinogenic Bowman-Birk protease inhibitor complexed with its target protease is internalized and inhibits the growth-regulated activity displayed by the protease [66*]. In addition, proteases and their inhibitors bind to the ceU surface at different sites. For
proteases
Chen
example, PAIl is localized in the focal adhesions but not in invadopodia (W-T Chen, unpublished data). This is interesting because ECM substrates are degraded by transformed cells and tumor cells at sites of ECM contact with invadopodial membranes but not at focal adhesions. Here, the uPA system may function to release cells from the substratum during migration, rather than mediating the active ECM degradation that occurs at the invadopodia of transformed cells.
Cathepsins Lysosomal cysteine protease cathepsins are typically associated with intracellular compartments; however, digestion within the lysosomes can also contribute to the process of ECM degradation. Cathepsins B, H and L exist in single-chain forms (3OkD) and two-chain forms (25 and 5 kD), while cathepsins C, J and K are oligomeric in structure and contain extremely long propeptides [67]. Cathepsins B and L, and a cathepsin L-like 70 kD protease from bone tissue can degrade coUagens and appear necessary for bone resorption [68]. Bone tissue cells, like osteoclasts, secret cathepsins to a compartmentalized extraceUular space delimited by podosome focal adhesions where an acidic micro-environment optimal for cathepsin activity may be maintained. Malignantly transformed cells have apparent increases in ceU surface association of cathepsins B [69*,70] and L [71*]. Cathepsin B associated with the ceU surface can degrade ECM components at both acidic and neutral pH [69]. Proteolytic processing and glycosylation of cathepsin B have been suggested to achieve ceU surface expression and pH dependence of cathepsin B-catalyzed hydrolysis during ECM invasion of tumor cells [72*-74-l.
Integral
membrane
proteases
Very little attention has been paid to membrane proteases integral to the ceU surface that degrade the ECM, primarily because of their low abundance and the difficulty of identifying invasive ceU lines that highly express these membrane proteases. A few plasma membrane proteases, however, have been identified from biochemically defined ‘membrane’ fractions derived from tissue homogenates. CeU fractionation and immunological assays have localized CD10 or CALL4 (the 1OOkD common acute lymphoblastic leukemia antigen) to the surface of a variety of tissue and tumor cells [75,76]. CDlO, a type II transmembrane glycoprotein, was identified as neutral endopeptidase 24.11, an integral membrane zinc metallo-endoproteinase. Meprins are ceU surface oligomeric glycoproteins from kidney tubule brush borders that have metallo-endopeptidase activity [n]. Recent cloning studies show that meprins are part of a family of metallo-endopeptidases, the ‘astacin family [78]. Finally, hepsin has been recently characterized as a plasma membrane protease [79]. These proteases prob-
805
806
Cell-to-cell
contact
and extracellular
matrix
.
ably have functions that are related to the function of microvillar membranes of epithelia in intestine and kidney, although it is not yet clear what significance these proteases have for matrix remodeling and the invasion process. Since the identification of cell contact-related protease activities from Rous sarcoma virus-transformed cells [&IO], one of the main aims of our laboratory has been to find integral membrane protease molecules of invasive cells that are involved in the detachment of cells from the ECM leading to cell invasion. Using fibronectin-coated crosslinked gelatin films as immobilized substrates to identify the invasiveness of tumor cells, we have identified three groups of neutral membrane proteases: serine, metallo- [19], and sulfhydryl proteases [Sl] from transformed and tumor cells that degrade the ECM. Recently, we have identitied a novel sulfhydtyl membrane protease with high molecular weight (170 kD), that is tightly associated with the plasma membrane of invadopodia of transformed cells in contact with the ECM (T Kelly et al, unpublished data). The 170kD membrane protease activity of embtyonic cells, similar to the 170kD membrane protease of human malignant melanoma ceU line LOX cells ([81]; WL Monsky et al, unpublished data), is maximal at neutral pH, and has a peculiar cysteine protease inhibitor profile: its activity is enhanced by EDTA and dithiothreitol, but inhibited partially (80%) by the cysteine-protease inhibitors, N-ethyl maleimide and phenylrnethylsulfonyl fluoride. Consistent with the possible cysteine active site, these membrane proteases can bind to an organomercurial adsorbent (T Kelly et al. unpublished data), but not to the serine protease inhibitor paminobenzamidine. We suggest that expression of this group of related membrane proteases in invadopodia contributes to the localized degradation of the ECM occurring in invading cells.
their functions. Attempts to exploit these opportunities may weU result in the development of new reagents for the early detection and treatment of various diseases.
Acknowledgements I am indebted to Thomas Kelly, Chen-Yong Susette C Mueller and Yunyun Yeh for helpful was supported by USPHS grant number R01 33711.
tin, Wayne Monsky, discussions. This work CA-39077 and ROl HL-
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CD10 (CALLA)/Neuual Endopeptidase 24.11 Modulates Inflammatory Peptide-induced Changes in Neutrophil Morphology, Migration, and Adhesion Proteins and is itself Reg ulated by Neutrophil Activation. BIG& 1991, 78:1834-1841.
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Highly Sensitive e.1.i.s.a. for Endopeptidase-24.11, the Common Acute-lymphoblastic-leukaemia Antigen (CALLA, CD10). Applicable to Material of Porcine and Human Origin.
DEW.SS~ J-M,
69.
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267:5700-5711. Three excellent papers [72*-74.1 deal with the proteolytic processing and glycosyiation of cathepsin B. These properties are thought to be involved in ceU surface expression of cathepsin B, and pH dependence of cathepsin B-catalyzed hydrolysis during ECM invasion of tumor cells.
26616312-16317.
279:167-174.
70.
brane Glycoproteins Exhibiting Distinct
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BA,
CHU
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SAITOH
cosylation
0, WANG
W-C,
LAXAN R, FLIKLJIIA
and CeU Surface
Expression
M: Differential
of Lysosomal
Gly-
Mem-
W-T Chen. Department of Anatomy and Cell Biology, Georgetown versity School of Medicine, 3900 Reservoir Road N.W., Washington, trict of Columbia 20007. USA.
UniDis-