MT1-MMP M NEUTROPHILS: POTENTIAL MECHANISM FOR COLLAGENASE ACTNATION

Jim Yuan Lai

A thesis submined in conformity with the requirements for the degree of Master of Science (Periodontology)

Graduate Department of Dentistry University of Toronto

O Copyright by Jim Yuan Lai (2000)

National Library 191 of Canada Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibliographie Services services bibliographiques

395 Wellington Street 395, rue Wellington Oîtawa ON K i A O N 4 Ottawa ON KI A ON4 Canada Canada

The author has granted a non- exclusive licence dowing the National Library of Canada to reproduce, loan, distribute or sel1 copies of this thesis in rnicroforrn, paper or eleclronic formats.

The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts fiom it may be printed or otherwise reproduced without the author's permission.

L'auteur a accordé une licence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/film, de reproduction sur papier ou sur format électronique.

L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.

MTl-MMP in Neutrophils: Potential Mechanism for Collagenase Activation Jim Yuan Lai Faculty of Dentistry, University of Toronto M.%. 2000

ABSTRACT

Matrix metalloproteinases (MMPs) are important enzymes in the destruction of

extracellulz natriccs in perioilontai diseases. One of these enzymes, MMP-8, is derived

largely from neutrophils. The active f o m of MMP-8 is found in the gingival crevicular

fluid of progressive periodontitis lesions but the rnechanism by which the latent enzyme

is converted to active forms in vivo is poorly understood. As activation of MMP-2 cm be

mediated by a membrane-bound MMP, membrane-type-1 MMP (MT1-MW), 1 tested

the hypothesis that MT-MMPs are expressed in neutrophils and can activate latent MMP-

8. My objectives were: 1) to assess the presence and location of MT-MMP in peripheral

blood neutrophils; 2) to detemine if MT-MMPs c m activate MMP-8. By RT-PCR, 1

found that human peripheral blood neutrophils expressed MTI-MMP mRNA.* The

plasma membrane and specific granule fractions of neutrophils were isolated by

discontinuous Percoll gradients and from these fractions 1 found MT1-MMP in the

plasma membrane fraction. Soluble biotinylated collagen assays showed some

collagenase activity (-28% collagen digestion) in the membrane fraction. The specific

granule fraction contained latent MMP-8 that could be activated by APMA. The

collagenase activity of a plasma membrane fraction combined with specific granules of

neutrophils (54% digestion) was higher than the collagenase activity of the individual

fractions (28% for plasma membrane, 1% for specific granules). These results are

consistent with the notion that MTI-MMP may contribute to the activation of MMPd on

the ce11 surface of peripherai blood neutrophils .

ACKNOWLEDGEMENTS

1 would like to express my gratitude and appreciation for the tremendous support and

assistance offered by the members of CIHR group in Periodontal Physiology.

My deepest thanks and admiration goes to Chns McCulIoch. He was not only a

supervisor, but a mentor who offered vaiuable advice and guidance on many things. His

expenence, patience and support guided me to the completion of rny thesis.

I would also like to acknowledge the help 1 received in the lab fiom Maki MacGillivray,

Jiaxu Wang, Pam Arora, Wilson Lee, Cheung Lo, and Kevin Ko. I especially would like

to thank Carol Laschinger. Her thoroughness, patience and guidance were extremely

valuable and much appreciated.

Finaily, I thank my parents for their unconditional love and support. Their trust and

encouragement have helped me achieve my goals and find happiness in life.

TABLE OF CONTENTS

. . Abstract ............................................................................................... 11 ... Ac knowledgements ................................................................................ .HL

Table of Contents ................................................................................... iv List of Figures ....................................................................................... vi .. Abbreviations ..................................................................................... ..vil

Review of the Literature .......................................................................... 1

Periodontd Diseases ........................................................................ 1 A . Classification, Naturai History and Clinical Course ............................... 1

7 B . Infiammation and Destruction of Extracellular Matrices ...................... ..., C . Role of Neutrophils in Lnflarnmation and Connective Tissue Destruction ...... 4

II . Destruction of Extracellular Matrices .................................................... 6 . A General Overview of the MMP family .............................................. 7

B . Domain Structure and Function of Collagenase .................................... 8 .................................................. . C Substmte Specificity of Collagenase 9

D . Activation Mechanisms of Collagenase ............................................ I O

III . Membrane-type Matrix Metalloproteinases ............................................ 12 . A Domain Structure and Function ...................................................... 12 . B Regulation & Activation of MT-MMP ............................................. 15 . C Substrate Specificity of MT-MMP .................................................. 17

D . Relationship to TIMP-2 .............................................................. 18 E . Membrane Fixation .................................................................. -20

[V . Introduction and Statement of the Problem ............................................. 21

Materials and Methods ......................................................................... - 2 2 33 Reagents ......................................................................................

Isolation of Polymorphonuclear Neutrophils 33 by Human Plasma/Percoll Blood Ce11 Separation ..............................

Isolation of PMN Plasma Membrane (y-fraction) and Specific Granules (p-fraction) By Discontinuous Percoll Gradients ........................... - 2 3

....................... Identification of MT 1 -MMP mRNA expression by RTPCR 24 hmuniocaiization of MT 1 MMP ..................................................... -25 Western BIot Analysis ................................................................... 2 6 Soluble Biotinylated Collagen Assay (SBA) ........................................ - 2 6 Preparation of samples for SBA ...................................................... 2 7

ResuIts ............................................................................................ -29 ............................................ A . MT1-MMP mRNA expression in PMN -29

8 . Immunolocalization of MT 1 -MW in HGF and PMN ............................ 29 ........................... C . Identification of MT1-MMP by Western Blot Andysis 33

..... D . Verification of Specific Granule @-fraction) as a source of latent MMP-8 33 .............. E . Collagenase Activity in NeutrophiI Plasma Membrane (yfraction) 35

F . Increased Collagenase Activity of Specific Granules @-fraction) Latent MMP-8 by Neutrophil Plasma Membrane .......................... 38

........................................................................................ Discussion -39 ..................... A . Expression of MT 1 - M W in Peripheral B lood Neutrophils -39

.................................................................. B . Foms of MT I MMP -40 ................................................... C . MMP-8 activation by MT I .MMP -42

D . Summary and Suggestions for Future S ~ d i e s ..................................... -45

...................................................................................... Conclusions -47

........................................................................................ References A8

LIST OF FIGURES

Figure 1 : MT1 -MMP mRNA Expression in HGF and PMN ............................... 30

Figure 2: Immunolocdization of MT1 -MMP in KGF ...................................... 31

Figure 3: immunolocalization of MT1 -MMP in PMN ...................................... 32

Figure 4: Identification of MT1 -MMP ........................................................ 34

Figure 5: Identification of MMP-8

Figure 6: Collagenase Activity in Plasma Membrane (y-Fraction) And Specific Granules (p.fraction) ................................................ 37

MMP:

MT-MMP:

TIMP:

Pm:

LPS:

ECM:

SBA:

RT-PCR:

PMA:

Dl-r:

APMA:

DAPI:

NHS-LC-Biotin:

matrix metalloproteinase

membrane-type matnx metalloproteinase

tissue inhibitor of matrix metalloproteinase

polynorphonuclear leukocyte

Iipopolysacc haride

extracellular matrices

soluble biotinylated collagen assay

reverse transcription-polymerase chah reaction

phorbol myristate acetate

dithiothreitol

P-aminophenylmercuric acetate

4,6-Diamidino 2-p henylindole

N-hydroxysul fosuccinimide- long chain- B iotin

vii

REVIEW OF THE LITERATURE

1. Periodontat Diseases

A. Classification, Natural History and Clinical Course

There are two major categories of inflammatory periodontai diseases, gingivitis and

periodontitis, the division of which is based on destruction of alveolar bone. Gingivitis is

a reversible inflamrnatory disease of only the gingiva while periodontitis involves the

destniction of the marginal supporting structures of the tooth including the alveolar bone.

The hailmark of gingivitis is gingival inflammation which is caused by adherent

subgingival bacterial plaques (Loe et al., 1965). Based on a sequence of retrospectively

analyzed histopathologic events, idammatory processes of the periodontium have been

separated into

and Schroeder

three hypothetical stages: the initial, earl y and established lesions

The initial lesion is an acute inHammatory reaction that occurs 2 to

4 days after the onset of subgingival plaque formation. This lesion is characterized by the

loss of perivascular collagen and increased migration of neutrophils through high

endothelial venules, across the junctiond epithelium and into the gingival sulcus. From 4

to 7 days, the initial lesion progresses to the early lesion in which 60-70% of the collagen

in the marginal gingival comective tissue may be destroyed and the inflammatory ce11

infiltrate is dominated by lymphocytes and macrophages. Afier 2 to 3 weeks, the

established lesion is formed which is synonymous with chronic aduit gingivitis. The key

histological feature of the established lesion is the presence of plasma cells in the

idammatory infiltrate. This lesion cm persist for months or years without progression to

periodontitis.

In a small proportion of individuals with gingivitis, some of the affected sites progress to

periodontitis (Loe et al. 1986). Several classifications of periodontitis have been

suggested but a recent review by Armitage (1 999) suggests that there are four major types

of periodontitis: i) chronic periodontitis; ii) aggressive periodontitis; iii) periodontitis as a

manifestation of systemic diseases; and iv) necrotizing ulcerative periodontitis. It has

been suggested that each disease type has a different etiology, a different rate of disease

progression and response to treatment, but a comrnon pathway of extracellular matrix

(ECM) destruction. Indeed the degradation of ECM rnacromolecules including collagen

fibers, elastic fibers, proteoglycans and glycoproteins is the structural basis for the

destruction of the periodontal ligament, the disruption of its attachent to cementum, and

ultimately the resorption of alveolar bone (Page and Schroeder 1976, Page et al. 1997).

B. Inflammation and Destruction of ECM

In periodontitis, there are several different mechanisms that rnay lead to degradation of

ECM rnacromolecules (Birkedal-Hansen 1993). One proposed mechanism is that

proteolytic enzymes from putative pathogenic bactena can directly degrade the ECM. For

example, Porphyrornonas gingivalis and dctinobacilhs actinomycetemcomitans produce

hydrolytic enzymes that cleave native type I and III collagen fibrils at physiologicai pH

and temperature (Birkedal-Hansen et al. 1988; Robertson et al. 1984). However. the

relative abundance of these bacterial enzymes in the lamina propria of the gingiva and the

periodontal ligament may not be sufficient to account for the arnount of collagen

degradation seen in periodontitis.

A second complementary mechanism is the release of bacterial virulence factors that

may act directly on host cells to perturb homeostasis. For example, in response to the

vinilence factor lipopolysaccharide (LPS) that is present in the envelope of several gram

negative anaembic pathogens, macrophages undergo major shifts in their expressed - gene

repertoire which can result in the induction, for exarnple, of matrix metailoproteinases

(Wahl et ai. 1974; Welgus et al. 1990). Other virulence factors such as proteinases from

the pathogen P. gingivalis have been demonstrated to induce the expression and mediate

the activation of MMPs from mucosal keratinocytes and fibroblasts (Birkedal-Hansen et.

a1 1984).

The third and most studied mechanism is a cytokine-dependent, host immune.

inflammatory response to the antigenic challenge of subgingival pathogens such as

Porphyromonus gingivalis, Bacteroides forsyrhus, and ndctinobacilltrs

actinomycetemcornitans. These pathogens colonize teeth and grow to form a biofilm

(Darveau et al. 1997) which eventudly extends subgingivally if left undisturbed

(Haffajee and Socransky 1994). The biofilm and its metabolites disrupt the anachment of

the junctional epitheliurn to the 100th. Subsequently, in a susceptible host, the presence of

viruience factors from perîodontal pathogens can tngger the synthesis and release of

proinflammatory cytokines such as IL4 P, TNF-a and IFN-y. These cytokines, in turn.

induce and enhance host ce11 pathways for the degradation of the ECM (Page 1998).

C. Role of Neutrophiis in Infiammation and Connective Tissue Destruction

One of the key inflammatory cells involved in the degradation of the ECM is the

neutrophil. Neutrophils comprise 4040% of the circulating leukocytes in the peripheral

blood and are a first line of defense against microbial attack. Neutrophils are produced in

the bone marrow and after maturation, are released into the bloodstream with a

circulatory half Iife of 6 to 7 hours (Gordon 1994).

In the blood flow through capillaries, there are continuous reversible rolling contacts

between neutrophils and uninilamed endotheliurn. These transient, adhesive rolling

contacts are mediated by L-selectin, a ce11 surface protein that is constitutively expressed

on the surface of the neutrophil (Smith et al. 199 1). However, in the presence of

proinfiammatory cytokines such as Il- 1 P and TNF-a. endothelial cells rapidly translocate

P-selectins from Weibel-Palade bodies (McEver et al. 1989) to the ce11 surface and

induce biosynthesis of E-selectins (Bevilacqua et al. 1987). The carbohydrate-bearing

moieties on the neutrophil surface, gpl50-Lewis x and siaiyl Lewis-X, will respectively

bind to P- and E-selectins (Fukuda et al. 1984: Symington et of. 1985). This

consequently strengthens the adhesive interactions between the neutrophils and

endothelial cells and increases the number of neutrophils rolling and attaching to

inflamed postcapillary high endothelial vendes. Subsequently, interleukin-8 (a product of

endothelial cells) induces neutrophils to shed L-selectin and express p2-integins (LFA-1'

Mac-1, CR4) that are stored in the specific granules (Miyasaki 1996). LFA- 1 and Mac-1

bind tightiy to the endotheliai adhesion molecules (ICAM-I and -2) and this binding

initiates the tramendothelid migration of the neutrophil through the endothelid ce11

junctions into the extravascular cornpartment, a process known as diapedesis.

In periodontal tissues, neutrophils infiltrate the perivascdar connective tissue, migrate

dong a gradient of chemoattractants generated by subgingival bactena and pass through

the junctional epithelium to fom a defensive barrier between the subgingival plaque and

the gingival tissues (Theilade et al. 1985). Upon encountenng bacteria the neutrophil

utilizes various bactenal killing mechanisms which can be broadly classified into

oxidative and nonoxidative system. Oxidative killing involves the production of toxic

superoxide anions (023, hyarogen peroxide (H202), and hydroxyl radicals (OH).

However, no studies have conclusively s h o w that these metabolites alone are capable of

producing toxic effects under pathophysiologicai conditions. Nonoxidative mechanisms

involve secretion of antimicrobial components stored in granules. For exarnple, the

primary or anirophilic granules contain myeloperoxidase. defensins, and lysozyme while

secondary or specific granules contain lactofemn and lysozyme. (Van Dyke 1994).

In the presence of bactenal factors or complement factors (e.g. C k ) , the granule contents

are released extraceilulariy either by secretion or when the neutrophil undergoes

apoptosis or cytolysis (Miyasaki 1991). Although the primary function of the neutrophil

is host protection, the extracellular release of the granule contents may lead to excessive

tissue breakdown because proteolytic enzymes such as elastase. collagenase and

gelatinase are also found within these granules and are able to attack key components of

the extracellular matrix (Weiss 1 989). Notabl y, the neutrop hil collagenase (MW-8)

found in the specific granules appears to have a direct role in the tissue destruction of

periodontitis. In periodontitis, much of the collagenase activity is denved from

neutrophils and not from bacteria or other host cells, as based on the pattern of collagen

substrate degradation (Gangbar et al. 1990, Lee et al. 199 1 & 1995, O v e d l et al. 1991).

A longitudinal cohort study that examined gingival crevicular fluid in patients

demonstrated that active collagenase activity was 5 - 6 fold higher in groups with active

periodontitis than groups with gingivitis (Lee et cil. 1995). Large increases of active

collagenase were detected concurent with connective tissue anachment loss. In contrat,

latent collagenase activity was 2-fold higher in patients with inflammation but no

destruction.

II. Destruction of Extracellular Matrices

Birkedal-Hansen (1993) identified five distinct pathways that lead to degradation of the

extracellular matrices of the penodontium. The first pathway involves the conversion of

plasminogen into plasmin, a senne protease that cleaves fibrin and fibronectin (Dano et

al. 1985). The second pathway is the neutrophil-serine proteinase pathway. When

released, the neutrophil serine proteinases, elastase and cathepsin G, cleave a variety of

ECM molecules such as type N collagen, laminin, fibronectin and proteoglycans (Weiss

1989). The third pathway involves intracellular degradation by fibroblasts and

macrophages that phagocytize collagen fibrils and degrade them within phagolysosomes.

This intracellular pathway is particularly important in sites of rapid collagen turnover

such as the healthy gingiva or periodontai Ligament (Melcher and Chan 1981). The

fourth, osteoclastic bone resorption pathway is unique in that removal of minerai (i.e.

hydroxyapatite) is required before degradation of matrix proteins cm be initiated (Vaes

1988). The f i f i pathway is the matrix rnetalloproteinase pathway. This pathway will be

discussed in detail in the following section as it is centrai to the objectives of this thesis.

A. General Ovewiew of the MMP family

The MMP family is a group of metal-dependent endopeptidases which are capable of

degrading most extracellular matrix macromolecules. MMPs are involved in comective

tissue rernodeling and degradation, embryonic growth and development, and diseases

such as rheumatoid arthntis, periodontitis, tumour growth and metastasis (Birkedai-

Hansen et al. 1993). The MMPs share sequence homology but differ in terms of substrate

specificity and transcriptional regulation.

Based on substrate specificity, the MMPs are classified into four broad categones: i)

collagenases ii) gelatinases iii) stromelysins including matrilysin and metalloelastase and

iv) membrane-type MMPs (Polette et al. 1998). The collagenases (MMP-1, MMP-8.

MMP-13) cleave type I, II, III, VI1 and X coliagen. Neutrophil collagenase (MMP-8) is

produced largely by neutrophils whereas interstitial collagenase (MMP-1) is produced by

many ce11 types such as fibroblasts, keratinocytes, endothelid cells, macrophages.

chondrocytes and osteoblasts (Birkedaf-Hansen rf al. 1993). A recent paper (Hanernaaijer

et al. 1997) has s h o w that other cells can produce MMP-8 but the pathophysiologicd

significance of this is unclear. MMP-13 is produced by both epithelial and mesenchymd

cells in inflarned and remodelling connecting tissues. Its ability to cleave type II collagen,

type X collagen and cartilage aggrecan suggests that MMP-13 plays a significant role in

cartilage collagen degradation (Freije et al. L994, Mitchell et al. 1996, Knauper et al.

1976). MMP- 13 ha also been identified in gingival crevicular Buid (Mancini et al. 1999)

but the degradative impact of MMP-13 on periodontal connective tissues has not been

assessed. Gelatinases (MMP-2, MMP-9) are also synthesized by a wide variety of cells.

In addition to gelatin, gelatinases also cleave type IV collagen found in basement

membranes. The expression of gelatinases is often associated with invasive and

metastatic tumours (Liotta 1980). Stromelysins degrade a wide variety of collagenous

and noncollagenous ECM substrates which include collagen. gelatin, laminin and

proteoglycans. The final group, membrane-type MMPs, often contain a transmembrane

domain. This hydrophobic stretch of amino acids anchors the protein to the plasma

membrane and leaves the catalytic domain exposed extracellularly. In contrast, the other

MMPs are released extracellularly as soluble proteinases.

B. Domain Structure and Function of Collagenase

Collagenases have a five-domain modular structure that is shared by the other MMPs

(Fig. A) (Birkedal-Hansen ef al. 1993). The hydrophobic signal sequence of about 1 7-29

residues is followed by a 77-87 residue propeptide domain that constitutes, for example,

the NH2-terminal domain of the secreted MMP-8 precursor. This propeptide contains a

highly conserved sequence (PRCGVPD) which maintains the latency of the enzyme

extracellularly until it is removed in the process of enzyme activation (Springman et al.

1990). The caialytic domain of about 160 residues contains a highly conserved 2nZ'

binding active site (Lovejoy er al. 1994). A 5-50 residue proline-nch hinge region

c o ~ e c t s the catalytic domain with a 200 residue pexin-like COOH domain which plays a

role in substrate specificity.

Figure A: Domain structure of Collagenase a) hydrophobic signal sequence; b) NH2-terminal propeptide; c) catalytic domain with zinc molecule; d) proline-rich hinge region; e) hemopexin-like COOH terminal domain

While the MMPs share certain structural motifs, there are sorne notable differences

between closely related enzymes such as the neutrophil collagenase (MMP-8) and the

fibroblast collagenase (MMP-1). MMP-8 has a higher molecular mass than MMP-1

(80kDd75kDa vs. 57 kDa/SZkDa) because of the greater glycosylation of MMP-8. It is

speculated that die carbohydrate moieties of MMP-8 encode targeting signals that direct

the enzyme to specific granule storage sites (Birkedal-Hansen et al. 1993). Another

significant difference between the two enzymes is the mode of transcriptional regulation.

MMP-8 is synthesized during neutrophil maturation and is rapidly released fiom the

specific granules after neutrophil activation whereas MMP-1 is not stored in granules but

instead is synthesized in response to a wide variety of stimuli including IL-1 and TNF-a.

C. Substrate Specificity of Collagenase

Collagenases can cleave the native triple helix of type I1 II, and III collagens at a single

site in each polypeptide chah at physiological temperature and pH. In type 1 collagen, the

cleavage occurs at the glycine775-isoleucine776 bond in the alpha 1 chah and at the

glycine775-leucine bond776 in the alpha 2 c h a h This single cut produces a N-terminal K

and a C-terminal % collagen fragment. Subsequent degradation of the collagen may be

mediated by the gelatinases. Wu et al. (1990) demonstrated by site-directed mutagenesis

that the primary structure of the collagenase-sensitive site in collagen is an important

factor in determining the susceptibility to collagenase. However, the susceptibility of this

site cannot be accounted entirely by the amino acid sequence of collagen alone. The

collagenase-sensitive region rnay also be a locus that at 37OC more readily unfolds and

relaxes its triple helicai structure than other regions (Birkedal-Hansen 1987). In

cornparison to the fibroblast collagenase, the neutrophil collagenase exhibits 10 to 30 fold

higher catalytic efficiency on al1 substrates except for type III collagen (Netzell-Arnett et

al. 1991).

D. Activation Mechanisms of Collagenase

The activation of the latent collagenase is a critical regdatory step especially with MMP-

8. As mentioned above. latent forms of MMP-8 are stored in specific granules of

neutrophils and upon neutrophil activation, the granule contents are released

extracellularly (Weiss et al. 1985, Desrochers et ul. 1992). However, collagen

degradation cannot occur until MMP-8 is activated. The latency of the collagenase is

rnaintained by the formation of a cysteine9n" bond that links the unpaired propeptide

cysteine residue to the active site ~ n " (Van Wart el al. 1990). In enzyrne activation.

dissociation of the bond between the cysteine thiolate moiety and zinc atom is assumed to

be a crucial step. This process is described by Van Wart (1990) as the cysteine switch

activation mechanism. In vitro studies have demonstrated that there are different ways in

how t h i s activation c m be achieved.

Organrnercurials, metal ions, thiol reagents and oxidants can directly interact with the

cysteine residue to disrupt the cpteine-~n" bond in vitro. This interaction results in a

confirmation shift that triggen a senes of autolytic cleavages to occur. The first cleavage

leads to a reduction in molecular mass but not activation. The second cleavage. which

occurs at ~ s ~ ~ - ~ e t ~ ' , results in 40% increase of the mêuimum enzymatic activity. The

final cleavage occurs at ~ h e ' ~ - ~ e t ' ~ or ~ e t ' * - ~ e u ~ ' after prolonged incubation with the

activator (Knauper el al. 1 990, Blaser et al. 199 1).

Another activation system is based on Ni vitro studies using chaotropic agents (KI,

NaSCN) or detergents (SDS) to disnipt the cysteine-~n" bond, a procedure which

induced conformational changes in the polypeptide backbone. This conformational shift

also leads to several autolytic cleavages in which a fully processed active form of the

enzyme is generated (Nagase 1997).

A third activation process involves proteolytic enzymes (trypsin, plasmin, chymotrypsin,

neutrophii elastase, cathepsin B, plasma kallikrein). These enzymes excise a portion of

the propeptide which causes the cysteine switch to open. Activation requires the cleavage

of peptide bonds probably between residues 70 and 82. specifically, ~ h e ' ~ . et" and

eu" (Nagase 1990).

However, despite these advances based on MMP-8 activation systems studied in vitro,

the biologicai mechanism by which MMP-8 activation actually occurs in vivo is poorly

understood. The pathway involvhg proteolytic enzymes may be the closest surrogate to

the in vivo situation. Activation in vivo is frequentiy associated with a decrease in

molecular mass due to the removal of the propeptide. For example. latent MMP-8 in

gingival crevicular fluid is 78 kDa while the active form is 60 kDa (RomaneIli et al.

1999). Since activation of collagenase is a cntical regulatory step, there have been

continuing searches for an activation mechanism that truly reflects the in vivo situation.

The recently discovered membrane-type MMP was identified as the first physiological

activator of gelatinase A (Sato 1994) but little is known about the role of membrane-type

MMPs in the activation of M W - 8 .

III. Membrane-Wpe Matrix Metallo~roteinase

A. Domain structure and Function of MT-MMP

The majority of the MMPs are in a soluble form. However, by the use of RT-PCR and

screening a human placenta cDNA library, Sato (1 994) discovered a protein of 582 amino

acids that had the cornmon MMP five domain structure with three unique insertions.

First, there is an insertion of I l amino acids between the propeptide and the catalytic

domain. This insertion contains a stromeiysin-3-like RXKR furin cleavage motif. The

second insertion is an 8 arnino acid residue within the cataiytic domain whose h c t i o n

remains undehed while the third insertion of 24 hydrophobic arnino acids in the C-

terminus represents the trammembrane domain. The trammembrane domain allows the

pmtein to be anchored to the plasma membrane with its catalytic domain exposed to the

extracellular space. Hence, this protein is cailed membrane-type MMP. Currently, the

family of MT-MMP has been expanded to include MTI-, MT& MT3-, MT4-, MT5-,

MT6-MMPs (Sato 1994, Will 1995, Takino 1995. Puente 1996, Pei 1999 & 1999a).

Many tissues and cells express MTI-MMP. For example, during mouse embryogenesis,

MTl-MMP is expressed at hi& levels in developing blood vessels, kidney,

osteocartilaginous and musculotendinous tissues (Kinoh et al. 1 996, Apte et ai. 1997,

Sato & Seiki 1996). MTI-MMP is also found in lung tissue, kidneys, microglia in the

brain, ocular tissues, enamel and the pulp organ of teeth (Takino et al. 1995a & b, Sato et

al. 1994, Caron et al. 1998). At sites of vascular injury during wound healing, demal

fibroblasts express MT1 -W. Other cells that express MT I -MMP include smooth

muscle cells, endothelid cells, ameloblasts, odontoblasts, chondrocytes. and osteoclasts

(ha i et al. 1997, Sato & Seiki 1996, Sato ef al. 1997)

In various tumours of lung, stomach, colon, breast, ovary, cervix. urethra, bladder and

pancreas, stroma1 and cancer cells have elevated MTI-MMP expression (Sato & Seiki

1996). Notably, MTl-MMP expression levels correlate with gelatinase A activation and

with the malignant potential and invasiveness of the turnour. In an experimental

metastasis assay, MTI-MMP expression has been shown to enhance metastatic activity

(Tsunezuka et al. 1996). In the context of cancer biology, gelatinase A plays a cntical

role in the invasion of tumour cells through the basement membrane (Stetler-Stevenson et

aL 1993). Because progelaûnase A is produced constitutively in high concentration by

many ce11 types, its activity is regulated mainly at the level of proenzyme activation

whereas the expression of other MMPs such as fibroblast collagenase, strornelysin and

gelatinase B is enhanced by various growth factors (Birkedal-Hansen et al. 1993). By

gelatin zymography, MTI-MMP has been demonstrated to induce progelatinase A

activation (Sato et al. 1994). This rnechanism will be discussed below.

There is growing evidence that MT-MMP rnay be a key regulator of ECM turnover.

MTI-MMP deficient mice exhibit inadequate collagen turnover which leads to dwarfism,

osteopenia, arthritis, and connective tissue disease (Holmbeck er al. 1999). MTI-MMP

deficiency greatly affects the skeleton, seen in reduced longitudinal growth, cranial

dysmorphism, osteoclasia and osteopenia. MT1 -MMP is also essential for growth plate

function and secondary ossification. As bone formation requires remodeling of

unmineralized comective tissue, when MTI-MMP is deficient, the remodeling of the

periskeletal soft tissues is impaired. The block of remodelling leads to increased bone

resorption and osteoclastic activity. Collectively. these finding demonstrate the

importance of MTI-MMP since this enzyme cannot be adequately compensated by other

MMPs or by other collagen degrading mechanisms (Holmbeck et al. 1999). In

corroboration of these fmdings, Zhou et al. (2000) have also demonstrated that MT1-

MMP deficient mice have impaired endochondral ossification in skeletal development

and impaired angiogenesis. However. neither of these studies examined perturbations of

inflammation, so the relative importance of MTI-MMP in the idammatory response is

unknown*

B. Replation & Activation of MT-MMP

The processes that regulate the synthesis and activation of MT1-MMP are not clearly

understood. In some papers (Lehti et al. 1998, Stanton et al. 1988, Hernandez-Barrantes

et al. 2000), the latent form of MT1-MMP is reported to be 60 kDa and the active form is

57 kDa However, the active MTl-MMP can be M e r processed to a functionaily

inactive form (44 kDa) that lacks the entire catalytic domain but maintains the

hemopexin-like domain and hinge region (Hemandez-Barrantes et al. 2000). MTI-MMP

expression is regulated in part by cytoskeleton-ECM interactions. It is induced in

fibroblasts by culturing in three dimensional collagen matrices, by mechanical stretching

and by treatment of cells with the cytoskeleton disrupting agent cytochalasin D (Gilles et

al. 1997, Tyagi et al., 1998, Ailenberge Br Silveman 1996).

MTl-MMP is unlike most other MMP genes. The majority of MMP genes are either not

expressed in unstimulated cells or are expressed at low levels; their gene expression is

increased in the presence of proinflammatory cytokines. On the other hand, in vitro,

MTl-MMP is constitutively expressed by different ce11 types including fibroblasts,

endothelid cells and smooth muscle cells. Its expression is not dfected by TGF-P but is

decreased by dexamethasone and only modestly enhanced by PMA, TNF-a, and the

lectin concanavalin A (Lohi et al. 1996). Thus. other aspects of MT-MMP regulation

need to be considered such as its activation mechanism.

Like the other W s , MTI-MMP contains a propeptide domain that maintains latency.

As mentioned before, an 11 amino acid sequence that contains a stromelysin-3-like

RXKR furin cleavage

facilitate constitutive

motif precedes the catalytic domain. This motif has been shown to

intracellular processing and activation of stomelysin-3 (Pei and

Weiss 1995) and MTI-MMP itself. The sequence is recognized by furin, a proprotein

convertase present in the Golgi apparatus that is able to activate recombinant MTl-MMP

(Sato et al. 1996). When fin specifically cleaves MTI-MMP in vitro between Arg"'-

T ~ T " ~ , MTl-MMP is activated, which in tum enables MTI-MMP to activate

progelatinase A. This contention is supported by Pei and Weiss (1996) who reported

activation of MTI-MMP using a mutant protein lacking the trammembrane domain

purified From MDCK cells. However, Cao et al. (1 996) demonstrated that hirin-induced

activation of MTI-MMP is not a prerequisite for progelatinase activation. Activation of

gelatinase continued despite the presence of aiPIpirr, a M n inhibitor, and within COS4

cells cotransfected with furin and mutant forms of MT1 -MMP in the RRKR"' site. The

discrepancy between these studies is explained by Yana & Weiss (2000). First, they

reported that alPIpm h i n inhibitor is unable to efficiently inhibit the proprotein

convertase-dependent pathways. Second, it was discovered that the MTl-MMP contains

a secondary M n cleavage site of mg9, which was only used when RRKR"' was

mutated. Thus in Cao's study, îürin converted mutant MTl-MMP to an active form

through cleavage at the secondary site. The activated MTI-MMP was then able to active

progelatinase A.

Another possible activation mechanism suggests that plasmin can activate the pro-MT1 -

MMP by cleavage of Arglo8 and ~ r ~ " ' (Okumura 1997). These authors suggested that

the pro-MT1 -MMP is activated by plasmin extraceIIularIy.

C. Substrate Specifieity of MT-MMP

MT1 -MMPY s substrates include a variety of ECM proteins such a s type 1, II and III

collagens, gelatin, fibronectin, vitronectin, tenascin, entactin and laminin-1 (Ochuchi et

al. 1997, d'Orth0 et al. 1997). In cornparison to MMP- 1, MT1-MMP is 5-7.1 fold less

efficient at cleaving type 1 collagen while its gelatinolytic activity is 8-fold higher. MTI-

MMP is also an efficient fibrinolytic proteinase: overexpression of MT1 -MMP enables

non-fibrinolytic cells to invade fibrin gels (Hiraoka er al. 1998). However, more

significantly, other MMPs are substrates for MT-MMPs. MTI-MMP has been implicated

in the activation of pro-MMP2 and pro MMP 13 (Sato et al. 1994, Murphy et al. 1999.

Knauper et a[. 1996a).

nie fint MMP substrate discovered for a MT-MMP was progelatinase A. Sato et al.

( 1994) transfected MT I -MMP plasmid into human fibrosarcoma UT 1080 cells which

constitutively secrete pro-gelatainase A into the culture medium. MT1 -MMP generated

two new gelatinolytic bands which represented the intermediate and active forrns of

gelatinase A. MT1 -MW recognizes the ~ s n ~ ~ - ~ e u bond (Kinoshita et al. 1996). After

cleavage, the progelatinase A is subsequently processed to an intennediate form. Then. an

autoproteolytic reaction occurs to produce the fully active fom which is dependent on the

presence of gelatinase A at the ce11 surface (Sato et al. 1996a). This enzymatic activity is

inhibited by TIMP-2 and TIMP3, but not by n M P l (Will et al. 1996). TIMP-2 has a

complex and critical regulatory role in the activity of MTI-MW. In vitro experiments

revealed that both MT1-MMP and TIMP-2 are required for the binding of gelatinase A to

the MTI-MMP at the cell surface and for subsequent membrane activation of gelatinase

A (Strongin et al. 1995, Atkinson et al. 1995).

D. Relationship to TIMP-2

TIMP-2, a 21 kDa non-glycosylated protein. belongs to a family of endogenous

inhibitors, the tissue inhibitors of metalloproteinases. These proteins fonn non-covalent

stoichiometric complexes with both latent and active MMPs. TIMPs regulate rnatrix

degradation mainly by bloc kage of autolytic MMP activation (DeClerck et al. 1 99 1 ).

Previous studies have s hown that at low concentrations, TIMP -2 stimulates progelatinase

A activation whereas at high concentrations. it inhibits activation (Strongin et al. 1995.

Kinoshita et al. 1998). Strongin (1 995) also demonstrated in cross-linking experiments

that TIMP-2 binds to MT1-MMP and to the hemopexin-like domain of progelatinase A.

Based on these observations. a mode1 for activation of progelatinase A was proposed in

which the catalytic domain of MTLMMP binds to the N-terminus of TMP-2 while the

C-terminus of TIMP- binds the C-terminus of progelatinase A (Strongin et al. 1995,

Butler et al. 1998). This complex enables progelatinase A to cluster at the ce11 surface

near a second active MTI-MMP molecule (Fig. B). If not bound by TIMP-2, this second

MTI-MMP wi11 then cleave the propeptide of progelatinase A to initiate activation of

geiatinaseA.

Figure 8: Activation of MMP-2 (progelatinase A) by MT1 -MMP

Figure C: Excess of TIMP-2, no activation of MMP-2

Because TIMP-2 is a potent and specific inhibitor of MTI-MMP and gelatinase A, the

optimal concentration of TIMP-2 to enable gelatinase A activation is quite low and is

within a narrow range. This mechanism permits active MTI-MMP to activate the

progelatinase A bound ont0 the complex. An excess of TIMP-2 will lead to the inhibition

of the activation reaction (Fig. C).

In addition to regulation of progelatinase A activation, TIMP- has an unique interaction

with MTI-MMP in tems of the activity of the enzyme on the ceIl surface. With

recombinant vaccinia viruses encoding Full length MT1-MMP or TIMP-2. Hernandez-

Barrantes (2000) was able to control various expression levels of TIMP-2 in mammalian

cells. The resdts demonstrated that TIMP-2 directly and positively regulated the

concentration of active M T 1 - W . In the absence of TIMP-2, there is a significant

decrease in the amount of active MT1-MMP on the cell surface due to the generation of

membrane-bound inactive 44 kDa species. However, in cells that CO-express MT 1 -MMP

and TIMP-2, cleavage at the ~ l ~ - ~ l # ~ ~ and the subsequent generation of the inactive

f o m is significantly inhibited by TIMP-2. The authors of this study concluded that

because TIMP-2 and not TIMP-1 inhibits the generation of 44 kDa species, the MTI-

MMP processing is an autocatalytic event. Thus, by binding and inhibiting a fraction of

active MTI-MMP, TIMP-2 reduces the extent of autocatalytic processing of free active

57 kDa species and therefore promotes its accumulation on the ce11 surface. This in tum

enhances progelatirme A activation.

E. Membrane Fixation

The complex of the MTI-MhPTIMP-2 with progelatinase A illustrates the concept of

pericellular proteolysis (Werb 199'7). As mentioned before, MMPs are the major enzymes

that degrade the ECM. The majority of these enzymes are soluble and are released

extracellularly. However, previous studies have shown that ECM degradation in vivo is

confined to the immediate pericellular environment of the ce11 (Andreasen et al. 1997.

Nakahara et al. 1997). This Ieads to the question of how the proteinases that degrade the

ECM can operate in a spatially confined manner. One possible mechanism is the

involvement of membrane-bound enzymes such as MT-MMP. Not only is MTI-MMP

capable of degrading ECM molecules, it is also capable of localizing and activating other

MMPs at the cell surface such as gelatinase A. This activation and Iocalization

mechanism may be applied to other MMPs and other disease processes but this has not

yet been investigated, most notably for MMP-8.

IV. Introduction and Statement of the riroblem

Periodontal disrases are a group of chronic infiammatory disorders which involve the

destruction of extracellular matrices (ECM). One of the destructive enzymes that

degrades collagen in the ECM is the neutrophil collagenase or matrix metalloproteinase-8

(MW-8). MMP-8 is released as an inactive zyrnogen which is subsequently activated

extracellularly by cleavage of the propeptide. Activation of MMP-2 (gelatinase) by a

membrane bound MMP, membrane-type- 1 MMP (MT 1 -MMP) has been demonstrated in

fibroblasts, chondrocytes, and various tumour tissue ce11 lines (Sato et ai. 1994). There is

growing evidence that MT-MMPs may be key regulators of ECM turnover. For exarnple,

MTI-MMP deficient mice exhibits inadequate collagen turnover which leads to

dwarfism, osteopenia, arthntis, and connective tissue disease (Holrnbeck et al. 1999).

However, there is limited information on activation of MMP-8 by MT-MMPs in

neutrophils or indeed whether MT-MMPs are expressed on the neutrophil plasma

membrane at ail. My hypothesis is that MT-MMPs are expressed in neutrophils and can

activate latent MMP-8. To address this hypothesis. rny objectives were to:

1) Determine if MTI-MMP mRNA is expressed in peripheral blood neutrophils by RT-

PCR

2) Determine if MTl-MMP protein is found in the plasma membrane or in granules of

penpheral blood neutrophils

3) Compare by SBA the amount of MMP-8 activation by neutrophil membrane Fractions

that may contain MT-MMPs.

MATERIALS AND METHODS

A. Reagents

h u n o P u r e @ NHS-LC-Biotin for collagen Iabeling was purchased korn Pierce

(Rockford, Illinois). Anti-MT1-MMP rabbit polyclonal antibody used for

irnmunofluorescence and Westem blots was from Sigma (St. Louis, Missouri). Anti-

nebulin monoclonai antibody used for immunofluorescence was from Sigma. Rhodamine

(TEüTC)-conjugated affinipure F(ab')? fragment donkey anti-rabbit IgG (WL) used for

irnmunofluorescence was fiom Jackson Irnmunoresearch Lab, Inc. (West Grove,

Pennsylvania). Anti-MMP-8 mouse monoclonal antibody (IgG) used for Westem blots

was from Calbiochem (San Diego, California). ECL reagents were fiom Amersham

International (Buckinghamshire, UK). Anti-mouse IgGi HRP conjugates (Caltag

Laboratories, Burlingame, CA) and anti-rabbit IgG HRP conjugates used for Western

blots were from Arnersharn Life Science (Buckinghamshire, UK.) Puified MMP-8 was

from Calbiochem (San Diego, California). P-aminophenylmercuric acetate (APMA) was

fiom Sigma. Pefabloc was from Boehringer Mannheim (Laval, Quebec, Canada). MTI-

MMP PCR primers were kindly donated by Dr. C. Overall (UBC, Vancouver, BC).

B. Isolation of PoIymorphonuclear Neutrophils by Human Plasma/Percoll Blood

Ce11 Separation

Blood (80 ml) was drawn from male donors with a 19 gauge butterfly neede and

collected into 3.8% sodium citrate. The blood was centrifuged at room temperature for 20

minutes at 175 x g (1080 rpm, Beckman GPR centrifuge) to separate the platelet nch

plasma (PRP) layer from red ce11 fraction. The PRP layer was centrifuged at 1000 x g

(3400 rpm) at room temperature for 15 minutes to obtain the platelet poor plasma (PPP)

layer. Dextran sedimentation was performed on the red ce11 fraction for 30 minutes to

facilitate sedimentation of erythrocytes. and the leukocyte-rich supematant was aspirated.

Percoll in PPP (2 ml of 42%; v/v) was layered under the leukocyte-rich supematant with

a baked pasteur pipette and then, 2 ml of 51% (vlv) Percoll in PPP was layered under the

42% layer to form a plasma/Percoll gradient. Centrifugation was camed out at 180 x g

(1 l8Orpm) at room temperature for 10 minutes to separate the rnononuclear, neutrophil

and erythrocyte layers. The neutrophil layer was collected and resuspended in Kreb's

Ringer Phosphate with Dextrose (KRPD) buffer at a concentration of 8x10~ cellsfml.

Under light microscopy, the number and purity of neutrophils were determined. The yield

was approximately 1 . 5 ~ 1 o8 cells containhg 90-95% neutrophils, 1 2 % erythrocytes, 3-

5% eosinophils and < 0.5% rnononuclear cells.

C. Isolation of PMN Plasma Membranes (y-fraction) and Specific Granules (B-

fraction) by Discontinuous Percoll Gradients

The protocol used to isolate the plasma membrane and specific granules was based on

Borregaard (1 983) with some modifications. Isolated neutrophils were resuspended in 1X

PIPES with Pefabloc at 5x10' cellslml. Nitrogen cavitation was performed at 4°C for 8

minutes at 700 psi to disrupt the cells. The cavitate was collected in IOX EGTA and

centnfuged at 4°C for 10 minutes at 1600 rpm to remove the nuclei and whole cells. For

discontinuous Percoll gradients, 4.5 ml of Percoll with a density of 1.120 g/ml? was

Iayered under 4.5 ml of Percoll (density of 1.030 g/ml). The sample (1 52 .0 ml) was

applied on top and centrifugation was carried out at 12,000rpm at 4OC for 45 minutes.

The density of the gradient was estimated fiom the migration of caiibration beads of

known density (Pharmacia Fine Chemicals) in gradients run in parallel. The mean density

of the a, p, and y fractions were 1.135 glml. 1 .O84 g M , and 1 .O26 g/ml respectively. The

three fractions were removed and ultracentrifuged at 4OC for 90 minutes at 35.000 T m to

remove the Percoll.

D. Identification of MT1-MMP mRNA Expression by RT-PCR

Total RNA was isolated fiom cells by the QIAGEN RNAeasy Total RNA kit according

to the manufacturer's instructions and quantified by spectrophotometry (Ultrospec 3000;

Pharmacia Biotech; Montreal, Quebec). cDNA was produced fIom 5 pg of total RNA per

sarnple, using Superscript II reverse transcriptase (GIBCO) primed with random hexamer

prirners. RNA (5 pg) was incubated with random hexamers (5.0 pl of 100 @pl in

DEPC-water) to a final volume of 27 pl at 70°C for 10 minutes. The buffer contained 6

pl of 0.1 M DTT, 8 pi of 5 m M mixed dNTP, 12 pl of 5X fint strand buffer and 3 pl of

RNA guard and the reaction was initiated at 25OC for 5 minutes. Superscript II (3 pl) was

added and incubated at 2S°C for an additional I O minutes and then heated to 42°C for 1

hour. The terminai reaction was conducted at 70°C for 15 minutes. As a negative control.

a separate reaction was performed without reverse transcriptase to ensure no genomic

DNA were present PCR primers were as follows:

MT1 -MMP: 5'-GGGCCCAACATCTGTGAC-3' and 5'-CCCATCCAGTCC-3'

GADPH: 5 '-GGCATGGACTGTGGTCATGA-3 'and5'-TCACCACCATGGAGAAGGC-3'

PCR reactions were started at 94°C for 1 minutes, continued for 35 cycles at 94OC for 30

seconds, 50°C for 30 seconds, and 7 2 T for 30 seconds followed by a 10 minute

extension at 72OC. PCR reactions were carried out in separate reaction vessels in a

volume of 50 @ with 0.5 pbf primes. 1.5 mM MCJCI~: 3 111 cDNA, 200 p.!! ~NTPs a d

2.5 U Taq DNA polymerase. Products were run on 1.5% agarose gels at 120 V for 1 hour

and visuaiized under UV light.

E. Imrnunolocalization of MT1-MMP

In 8-chamber well glass slides (LaboTeka), neutrophils (5 x 10' per well) were incubated

at 37°C for 5 minutes to allow neutrophil adherence to the slides. The cells were fixed

with ice-cold methanol for 6 minutes at -ZO°C, washed twice with PBS and non-specific

binding sites were blocked with 0.2% BSA at room temperature for 10 minutes. After

washing two times with PBS, the cells were incubated with anti-MT1-MMP antibody (10

pg/ml) in a humidor at 4°C overnight. The cells were washed twice with PBS and

blocked with 0.2% BSA at room temperature for 10 minutes. TRITC goat anti-rabbit

F(ab')2 fragment (1.5 mg/ml at 1: 100) was added for 2 hours at 4°C. Cells were washed

twice with PBS, stained with DAPI (1 pg/ml in 0.01% Nonidet and PBS) for 2 minutes

and washed again with PBS. The cells were covered with irnmunofloure (ICN

Biomedicais. Aurom, OH) rnounted and examined irnmediately by epifluorescence

microscopy. Fluorescence micrognphs were recorded on Kodak High-Speed TMX

P3200 film. The positive controls included human gingival fibroblasts that were grown to

subconfluence in wells coated with fibronectin (10 pg/ml). Negative controls were

neutrophils treated without primary antibody or neutrophils treated with anti-nebulin

antibody (76 pg/ml) as the primary antibody.

F. Western Blot Analysis

Human gingival fibmblast lysates, neutrophil lysates and neutrophil cellular fractions (a,

p, y) were incubated in electrophoresis sample buffer (0.05 M Tris-HCI pH 6.8, 8 M urea,

2% (w/v) SDS, 8% (vh) Bromphenol blue) and 0.15 M of dithiothreitol ( D m

(SBDTT), boiled for 5 minutes and separated by SDS-PAGE on 10% cross-linked

minigels at 120 V for 1 % hours and transferred for 2 hours (64 &gel) onto

nitrocellulose membranes. M e r blocking with 5% ( w h ) Carnation milk, membranes

were incubated with anti-MT1-MMP rabbit antibody (2 pg/ml) or with anti-MMP-8

mouse antibody (5 pg/rnl) for 3 hours at room temperature, waçhed and incubated with

either anti-rabbit IgG HRP (1:3000 dilution) or anti-mouse IgGi HRP (1 2000 dilution)

for I h o u at room temperature. The bands were visualized with ECL reagents and

molecular weights of the fragments were detemined by cornparison with prestained

molecular weight standards. Treatment of blots with secondary antibody alone sho wed no

reactivity when the secondary antibody was incubated in 5% (wh) Carnation milk in

TBS-Tween.

G. Soluble Biotinylated Collagen Assay (SBA)

Aliquots of samples were incubated with biotinylated collagen at ratios of 10 ng

biotinylated collagedpl sample, for 21 hours at room temperature in the presence of

collagenase assay buf5er (CAB - 0.05M Tris, 0.2 M NaCl containing 5 mM CaCl?, 0.5

pVml BRU 35 and 0.2 @ml NaN3). Biotinylated collagen was prepared as described in

Mancini et al. (1999). Reactions were terminated by the addition of SBiDTT and boiled

for 5 minutes. Collagen fragments were separated on 7.5% cross-linked SDS-PAGE gels

and transferred to a nitrocellulose membrane using the PHAST system (Pharmacia). After

blocking with 5% (wh) Carnation milk in TBS-Tween, the membranes were rinsed with

TBS (0.02 M Tris-HC1. 0.14 M NaCl pH 7.6 containing 0.1% Tween 20) and incubated

with HRP-labeled streptavidin (Amersham) diluted Ill 500 in Tris-HCI b a e r pH 7.6.

Afier a second wash, ECL reagents were used for detection of biotinylated collagen

fragments by cherniluminescence. Autoradiographs were scanned and full Iength and %

a-chahs were quantified by cornputer analysis using the IP Lab Gel Scientific Image

Processing prograrn (Signal Analytics- V i e ~ a , Virginia USA). The estimation of

collagenase activity was based on the densitometric data in terms of percentage

biotinylated collagen degradation into % a-chains.

% collagen degraded = densitv of Yt a1 (1) chahs X IO0 densities of % al (1) chains + a l (1) chains

H. Preparation of samples for SBA

The total protein yield of y-fractions and p-fractions fiom the discontinuous Percoll

gradients was dependent on the donor and the initial amount of neutrophils isolated.

From 1.5~10' cells, the total protein obtained was 96 pg and 287 pg for y-fractions and P-

fractions respectively. In preparation for SBA andysis, various combination of these

fractions were rnixed and incubated for 4 hours rotating at 37OC. The samptes included

150 p1 of y, 150 pl y + 50 pl P, 150 pl of P with a total protein concentration of O. 130

pg/pI for the y-fraction and 0.379 pg/$ for the p-fraction. An aliquot (100 pl) of the P

fraction was activated with the addition of 50 p1 1 m M APMA and incubated at room

temperature for 45 minutes. Then, 50 pl from samples were removed and cornbined with

6 pl 10X CAB and 2 pl biotinylated collagen for SBA analysis.

A. MT1-MMP mRNA expression in PMN

Expression of MTI-MMP mRNA was exarnined in whole ce11 lysates of peripheral blood

neutrophils and human gingival fibroblasts by RT-PCR. PCR primers for a 236 bp

GAPDH product were used as a positive control to ensure the presence of cellular mRNA

(Fig. 1). The MT1-MMP primes identified a 580 bp MTI-MMP product in both the

neutrophil and HGF samples. No gene pmduct was identified in a separate reaction

without reverse transcriptase to ensure the absence of genomic DNA.

B. Immunolocalization of MT1-MMP in HGF and PMN

Immunolocalization studies were done to detemine the presence of MTl-MMP. As a

positive control, human gingival fibroblasts (HGF) exhibited strong staining for MT1 - MMP (Fig. 2A) and within the same field, the DAPl stained nuclei were evident (Fig.

2B). No significant immunoreactivity was observed in HGF preparations stained with an

irrelevant antibody, anti-nebulin, (Fig. 2C) or with no primary antibody (Fig. 2E).

Peripheral blood neutrophils stained with ah-MT1-MMP also showed bright staining

(Fig. 3A). The neutrophils were identified based on morphology under light microscopy

and DAPI staining demonstrated the multilobuiar appearance of the neutrophil nuclei.

Neutrophil preparations stained with an irrelevant antibody, anti-nebulin (Fig. 3C) or with

no primary antibody (Fig. 3E) showed no significant immunoreactivity. 1 used a nebulin

antibody as a control because nebulin is a hi& molecular weight protein that is

specifically localized in skeletal muscle rnyofibrils. This immunolocdization study was

HGF PMN

236 bp GAPDH

Figure 1: MTI-MMP mRNA expression in HGF and PMN. MTI-MMP mRNA expression was tested by RT-PCR on samples of whole ce11 lysates of human gingival fibroblasts (HGF) and peripheral blood neutrophils (PMN). Rimers 142 and 90 identified a 580 bp o f MTI-MMP in PMN and HGF. The primer for GAPDH positive control also appears in both samples as 236 bp. -RT lanes were reactions with no reverse transcriptase produced no gene products.

Figure 2: Immunolocaihation of MT1-MMP. A) Preparation of human gingival fibroblasts (HGF) stained wi th anti-MT 1 -MMP primary antibody and TRITC-conjugated secondary antibody. B) same field shown in panel A stained with DAPI. C) PMN stained with ad-nebulin primary antibody and TNTC-conjugated secondary antibody. D) same field shown in panel C stained with DAPI. E) PMN s h e d with no phary antibody and TRITC- conjugated secondary antibody. F) same field shown in panel E s h e d with

I DAPI,

- --

Figure 3: Immunolocaluation of MT1-MMP. A) Reparation of peripheral blood neutrophils (PMN) stained with anti-MT 1 -MMP primary antibody and TRITC-conjugated secondary antibody. B) same field shown in panel A stained with DAPI. C) PMN stained with anti-nebulin primary antibody and TMTC-conjugated secondary antibody. D) same field shown in panel C stained with DAPI. E) PMN stained with no primary antibody and TRITC-conjugated secondary antibody. F) same field shown in panel E stained with DAPI.

completed on neutrophil samples from three different donors with similar results. In ail

samples, the neutrophils incubated with anti-MT1-MMP antibody as the primary

antibody exhibited strong fluorescence with minimal background signais.

C. Identification of MTl-MMP by Western Blot Analysis

Sarnples of human gingival fibroblasts, penpheral blood neutrophils and fractions thereof

were separated by SDS-PAGE and transferred onto nitrocellulose membranes. M e r

incubation with anti-MT1-MMP, three bands were identified at 86 kDa, 80 ma, and 62

kDa for the human gingival fibroblast sample (HGF) (Fig. 4). The same banding pattern

appeared for the plasma membrane fraction (y) fkom neutrophils. For the whole ce11

neutrophil lysate sample (PMN), the 80 kDa and 62 kDa bands were present with

additional bands at 69 kDa and 66 kDa. No bands were detected in the a and P fractions

(Fig. 4). This Western Blot anaiysis was repeated with neutrophil samples from three

other human donon and each blot produced the same banding pattern. The 62 kDa band

is the latent forrn of MTI-MMP and was present in the human gingival fibroblast (HGF),

whole ce11 neutrophil lysates (PMN) and the plasma membrane fraction (y). MT1-MMP

was not present in the specific (p-fraction) or in azurophilic (a-fraction) granules.

D. Verification of specific granules (p-fraction) as a source of latent MMP-8

Samples of purified human MMP-8 (Calbiochem). neutrophil plasma membrane (y-

fraction) and specific granules @-fraction) were separated by SDS-PAGE and transferred

onIo nitrocellulose membrane. M e r incubation with anti-MMP-8, the purified human

MMP-8 sample exhibited two bands at 65 kDa and 55 kDa which represented the latent

PMN HGF a B Y PMN HGF

+ + no primary Ab anti-MT1-MMP primary Ab

Figure 4: Identification of MT1-MMP. Samples of whole ce11 lysates o f peripheral blood neutrophils (PMN) and postnuclear cavitate fractions of y (plasma membrane), f3 (specific granules), a (azurophil granules) were tested by Western blot for immunoreactive MTI-MMP. The proform of MTI-MMP of 62 kDa is predominant in PMN and y (plasma membrane) fhctions. The positive control of human gingival fibroblast (HGF) also demonstrates the presence of MTI-MMP. The samples incubated with no primary antibody demonstrate minimal cross-reactivity.

and active form of MMP-8 respectively. The specific granules sample @-fraction)

exhibited o d y the 65 kDA latent form of MMP-8 whereas in the neutrophil plasma

membrane sample (y-fraction), MMP-8 was not detected (Fig. 5).

Collagenase activity of the specific granules sample Ip-fraction) was tested hy SBA (Fip.

6). Analysis of an diquot (56.9 pg total protein) representing 6.5% of the p-fraction

reveded negligible (1%) digestion of the biotinylated collagen. An equivalent aliquot

(6.5% or 58.1 pg total protein) of specific granules from another donor digested 3% of

the collagen. However, when an aliquot representing 4.3% or 37.3 pg total protein of P-

fraction was treated with APMA, the collagenase activity of the specific granules sample

generated 86% collagen digestion. Thus, the specific granules (P-hction) contain latent

MMP-8 that could be activated. Both specific granule sarnples (P-Fraction) that were

obtained fiom two difTerent human donors produced similar results from the anti-MMP-8

Westem Blots analysis and SBA.

E. Collagenase activity in neutrophil plasma membrane (y-fraction)

The neutrophil plasma membrane sample (y-fraction) contained MT1-MMP as

demonstrated by the ad-MU-MMP immunofluorescence (Fig. 3A) and Westem blot

analysis (Fig. J), but the sample did not contain any forms of MMP-8 as demonstrated by

the anti-blMP-8 Westem Blot anal ysis (Fig. 5).

However, the neutrophil plasma membrane sample (y-fraction) did exhibit some

collagenase activity. Analysis by SBA showed that an aliquot (6.7% or 19.5 pg total

Figure 5: Identification of MMP-8 Western blot analysis with anti- MMP-8 monoclonal antibody demonstrates the presence of latent 65kDa MMP-8 in the P fractions and absence of MMP-8 in the y hctions. Rire human MMP-8 serves as a positive control dernonstrating the presence of both the latent and active form of MMP-8.

APMA

1 % % Digestion

Figure 6: Collagenase activity in plasma membrane (y-fraction) and opecific granules (p-fraction). y, P, APMA activated p, and

samples were tested by SBA for collagenase activity. y alone had some intrinsic collagenase activity while P alone had no activity. B can be activated by the addition of APMA. y + P demonstrated an increase in digestion when combined.

protein) of plasma membrane fraction generated 28% collagen digestion (Fig. 6) . Another

equivalent aliquot (6.7% or 20.4 pg total protein) of plasma membrane fraction from

another donor digested 24% of the collagen.

F. Increased collagenase activity of specific granules @-fraction) latent MMP-8 by

neutrophil plasma membrane

The incubation of an aliquot (19 pg total protein) representing 5% of neutrophil plasma

membrane fraction with an diquot (1 -6% or 19 pg total protein) of specific granules

sample @-fraction) digested 54% of the collagen (Fig. 6). Aliquot mixtures of similar

proportions fiom another donor generated 40% collagen digestion.

DISCUSSION

The main findings of this study are that MTI-MMP is expressed in peripheral blood

neutrophils and is e ~ c h e d in the plasma membrane fraction of these celis. In vitro

experiments showed that cornponents of the plasma membrane fraction can activate the

latent collagenase From the specific granules of neutrophils.

A. Expression of MTI-MMP in Penp heral BIood Neutrophils

RT-PCR demonstrated that peripheral blood neutrophils expressed MTI-MMP mRNA

while immunofluorescence and Western blots showed the presence of the MTI-MMP

protein. I used human gingival fibroblasts as positive controls for MTI-MMP and, as

expected, these cells showed constitutive expression of MTI-MW mRNA and protein as

previously described (Atkinson et al. 1995, Gilles et al. 1997). Many other ce11 types

such as smooth muscle cells, endothelid cells, arneloblasts, odontoblasts, chondrocytes.

and osteoclasts express MTI-MMP (Imai et al. 1997, Sato & Seiki 1996, Sato et al.

1997) however to the best of my knowledge, this is the first report of MTl-MMP

expression by neutrophils.

In experiments using nitrogen-cavitated cells separated by density centrifugation in

Percoll gradients, f localized MTl-MMP to the plasma membrane fractions as

demonstrated by Western blots of the various neutrophil fractions. This finding is

consistent with previous studies showing that MT1-MMP is found in the plasma

membrane preparation of transfected COS4 cells, human skin fibroblasts and osteoclasts

(Sato et al. 1994, Atkinson et al. 1995, Sato et al. 1997). In view of the structure of MT1-

h4MP which contains a 24 amino acid hydrophobie h?uis-membrane domain, it would be

expected that MTI-MMP wouid be tethered to the neutrophil membrane by the tram-

membrane region.

B. Forms of MTI-MMP

By Westem blotting 1 only found the 62 kDa (latent) form of MTI-MMP in neutrophils.

MTl-MMP exists in three forms. The latent fom that contains a propeptide domain is 60

D a form. Cleavage of this propeptide domain results in a 57 kDa form which c m

activate progelatinase A. However, in the absence of TIMP-2, this 57 kDa fom

undergoes autocatalytic conversion to a functionally inactive form (44 kDa) that lacks the

entire cataiytic domain but maintains the hemopexin-like domain and hinge region

(Hemandez-Barrantes et al. 2000). The absence of the active and the functionally

inactive MTl-MMP foms in the Westem blots may be amibuted in part to the neutrophil

preparation method since isolation procedures can exert profound effects on the

activation systems that regulate the proteolytic machinery of these cells (Pabst 1994).

Cognimt of the tendency of neutrophils to be readily activated by inappropriate isolation

procedures, special precautions were taken to prevent ce11 activation following

venipuncture in healthy adult human volunteers. For example, as neutrophils can be

activated by trace amounts of lipopolysaccharides (LPS), 1 used LPS-fiee solutions.

disposable plastics and glassware baked at 180°C for several hours. It is thought that if

these special precautions are taken, neutrophils isolated From circulating biood are largly

in a "resting state" (Haslett et al. 1985). Thus in resting neutrophils, the annamentarium

used for bacterial defence is inactive. Notably, the components of the NADPH oxidase

system are stored in granules and therefore in resting neutrophils the granule contents are

not assembled and are not prepared for destruction of phagocytosed organisms (Weiss

1989). Similarly, the neutrophil collagenase (MMP-8) is stored in the latent, pro-enzyme

form in the specific granules pnor to release and extracellular activation (Doherty et al.

1994). By analogy 1 suggest that in resting neutrophils the MT1 -MMP is in a latent fnm

because it has not been activated to engage in proteolysis. Thus the 57 D a active form

and the 44 kDa functionally inactive form of MTl-MMP were not detected in the

Western blot analysis. Currently, it is not known what mechanism rnay activate MTI-

MMP on the surface of neutrophils. However, 1 speculate that the 57 kDa active form is

present in activated neutrophils. It is likely that this activation mechanism would exert an

important effect on the abiiity of MTI-MMP to activate MMP-8 and thereby initiate

degradation of rnatrix proteins. Thus, if MT1 -MMP cm cleave the N-terminal propeptide

of MW-8, it may be able to regulate the collagenolytic activity of MMP-8.

In vitro, a number of activation mechanisms have been studied which produce different

forms of MMP-8 and with varying levels of collagenolytic activity. For example,

stromelysin-2 processes MMP-8 by a single-step activation mechanism by cleavage of

the ~ l ~ ~ ~ - ~ h e ~ ~ peptide bond in the N-terminal propeptide domain. This active W - 8

displays very high specific collagenolytic activity (Knauper ef al. 1996b). On the other

hand. trypsin requires a two-step activation mechanism in which the fmt cleavage occurs

at Ar&~he' '~ to generate an intermediate latent forrn and then a second cleavage at

~ r ~ ' ~ - C ~ s ' ' to produce an active MMP-8 (Knauper et al. 1990). MMP-8 activation by

HgClz follows a three-step mechanism where the first cleavage is at ~ s n ' ~ - ~ a l ~ ~ . Then,

autoproteolytic cleavage of ~ s ~ ~ ~ - ~ e t ~ ~ produces an intermediate form ( ~ e t ~ j N-

terminus) which displays only about 40% of the maximum collagenolytic activity. Final

activation occurs d e r autoproteolytic cleavage of either P he7'-~et80 or ~ e t ' ' - ~ e u ~ ' (Blaser et al. 199 1).

C. MMP-8 activation by MT1-MMP

Currently, the system(s) by which MMP-8 is activated in vivo is not known although, as

discussed above, a large nurnber of in vitro studies have implicated stromelysin (Knauper

et al. 1996b), oxidants (Nagase 1997), as well as other proteases such as trypsin and

chymotrp ysin (Knauper et al. 1 990).

Initiai pilot experiments to determine if MTI-MMP can activate MMP-8 involved the

incubation of recombinant MTI-MMP with latent rat MMP-8. Based on the SBA, this

combination resulted in complete digestion of the biotinylated collagen. However, the

latent rat MMP-8 which was supposed to be latent produced > 50% digestion.

Unfortunately, this experiment was not reproduced due to the lack of more recombinant

MTl-MMP. Analysis of MTl-MMP mediated activation of MW-8 was also

cornplicated by the dificulty in obtaining a source of pure latent MW-8. The latent rat

MMP-8 most likely was zctivated during shipment or during storage, so I decided to use

the specific grandes from neutrophils as a source of latent MMP-8. As dernonstrated by

the Western biots (Fig. 5) and SBA (Fig. 6), the specific granules were a viable source of

latent MMP-8 than can be activated by APMA.

Since only a limited amount of recombinant MTI-MMP was available, 1 tried to generate

recombinant MTl-MMP by transfections of CHO cells with MT1-MMP plasmids. While

the transfection was successful, the predominant form of MTI-MMP generated was the

inactive form that lacked the catalytic domain. Consequently, the neutrophil plasma

membrane was used as a source of MTI-MMP, a source that required large number of

cells and arduous procedures.

As I was able to show the presence of MTl-MMP in association with the ce11 surface of

neutrophils, 1 conducted expenments in which I combined neutrophil plasma membrane

fractions with the contents of specific granules that contained latent MMP-8. In these

expenments, the membrane preparation exhibited abundant MT I -MMP and moderate

collagenase activity while the specific granules contained abundant latent MMP-8 but

minimal collagenase activity. Afier combuiing the two fractions, collagenase activity was

doubled. This finding indicates that components of the neutrophil membrane including

MTI-MMP have the potential to activate latent MMP-8. While MTI-MMP exhibits

collagenase activity, it is 5-7.1 fold less eficient than MMP-8 (Ohuchi el al. 1997).

However, if MTI-MMP can process the latent form of MMP-8 to an active form, then

this would provide an efficient and spatially segregated mechanisrn for activating MMP-8

on the surface of neutrophils. In this context, previous studies have demonstrated that

MTI-MMP activates other MMPs such as latent gelatinase A and collagenase-3 (Sato et

al. 1994, Knauper et al. 1996). Sato (1 994) transfected MTI-MMP plasmid into hurnan

fibrosarcoma HT1080. These ce11 lines also secreted progelatinase A (66 kDa) into the

culture supernatant. Plasma membrane fiactions of the transfected HT1080 (20 pg

protein) were incubated with the conditioned medium fiom HT1080 that contained

progelatinase A for two hours at 37°C. Gelatin zymography demonstrated that the plasma

membrane generated the processed 64 kDa and 62 kDa gelatinase A. Similarly, Knauper

(1996) incubated fibroblast-derived plasma membranes that contained MT1 -MMP (1 5p1;

Img/ml protein) with 50 ng of procollagenase-3. The plasma membranes processed the

procollagenase-3 to a 48 kDa active enzyme as demonstrated by Western blot analysis.

Taken together, these studies indicate that MT1-MMP activation of MMP-8 is feasible.

However, that MTI-MMP c m directly activate MMP-8 still needs to be established.

As mentioned above, in gingivitis, MMP-8 is predominantly latent, whereas, in

periodontitis, it is conceivable that the combination of a susceptible host and the presence

of periodontal pathogens provides a situation in which an activation cascade can lead to

active MMP-8. 1 suggest that vinilence factors and inflammatory cytokines may trigger

specific enzymes which lead to activation of enzymes such as funn or plasmin. These

enzymes in turn can activate latent MT1 -MMP. The active MT1 -MMP will then activate

MMP-8 which leads to destruction of the periodontium. As many cells types express

MTI-MMP, it is possible that within the penodontiurn or gingival crevice. other ce11

types (e.g. fibrblasts) that express MT1 -MW may be responsible for activation of MMP-

8. However, because neutrophils are so abundant in acute inflammation and are the

primary producers of MMP-8, it seems unlikely that other cells such as fibroblasts play a

significant role in M W - 8 activation. That extensive but revenible destruction of

gingival tissues occur in gingivitis suggests that there may be fundamental differences in

the rnechanism of tissue degradation between periodontitis and gingivitis.

D. Summary and Suggestions for Future Studies

In summary, my results demonstrate that neutrophils express MTl-MMP and the protein

is located on the plasma membrane. The combination of plasma membrane fractions with

latent MMP-8 indicates that components of the neutrophil membrane may be able to

activate MMP-8. I suggest that one of these activating molecules may be M T l - W .

However, the activation of MMP-8 by MT1-MMP still needs to be confmed by more

definitive experiments. For example, a potential future study could involve the use of an

antibody that blocks the enzymatic activity of MT1-MMP or an assay that

immunodepletes MTI-MMP From the plasma membrane fraction prior to incubation with

latent MMP-8. Under these conditions, selective removal of the MTl-MMP by antibody

should reduce the activation of M W - 8 and lead to lower collagenase activity as detected

by the SBA assay.

In considenng activation systems, 1 should also point out the involvement of TIMP-2 in

the hypotheticd activation of MMP-8 by MTl-MMP as there is increasing evidence that

TIMP-2 plays an important role in regulating the activity of MTI-MMP. Strongin et al.

(1995) suggested that TIMP-2 is required for progelatinase A (MMP-2) activation and

Hemandez-Barrantes (2000) demonstrated that TIMP-2 regulated the effective cell-

surface "concentration" of active MTl-MMP by adjusting the autocatalysis of the

enzyme and consequently its availability for interacting with gelatinase A. Notably, in the

absence of TIMP-2, there is uncontrolled autocatalysis of MTl-MMP that leads to

production of the inactive 44 kDa form. However, when there is an excess of TIMP-2, the

enzymatic action of MTI-MMP is also inhibited. In effect, a fiiture mode1 of MTI-MMP

activation of MMP-8 may involve controlling the level of TIMP-2 at the ce11 surface for

appropriate regulation of MTI-MMP and its subsequent activation of MMP-8.

The ability to regulate the activity of MMP-8 at the ce11 surface confines collagenase

activity close to the cell. This illustrates the concept of pericellular proteolysis (Werb

1997). Even though MMP-8 is released extracellularly, ECM degradation in vivo is

confined to the irnmediate pencellular environment of the ce11 (Ancireasen el al. 1997,

Nakahara et al. 1997). Because MT1-MMP is bound on the neutrophil plasma membrane

surface, it provides the oppominity to concentrate the various components involved with

ECM degradation close to the neutrophil. Thus, if MTI-MMP can activate MMP-8, then

MTl-MMP may provide a mechanisrn for controlled localized degradation of the

collagen matrix in periodontitis and other diseases in which there is a marked neutrophil

inflammatory infiltration.

CONCLUSIONS

MT I -MMP is expressed in periphed blood neutrophils.

MTl-MMP is found in the plasma membrane and not in the specific and azurophilic granules.

Specific granules fiom the P-fraction of neutrophils are a source of latent MMP-8 t h t

can be activated by APMA.

The plasma membrane hction has collagenase activity.

The collagenase activity of a plasma membrane fraction cornbined with specific granules of neutrophils is higher than the collagenase activity of the individual fractions.

REFERENCES

Ailenberg, M., Silverman, M. (1996). Ce1lda.r activation of mesangial gelatinase A by cytochalasin D is accompanied by enhanced mRNA expression of both gelatinase A and its membrane-associated gelatinase A activator (MT-MW). Biochemica! Journal 3 13, 879-884.

Ancireasen, P.A., Kjoller, L., Christensen, L, Dum, M.J. (1997). The urokinase-type plasminogen activator system in cancer metastasis: a review. Inrernntional Jozlrna! of Cancer 72, 1-22.

Apte, S.S., Fukai, N., Beier, D.R., Olsen, B.R. (1997). The matrix metalloproteinase-14 (MMP-14) gene is stmcturally distinct from other MMP genes and is CO-expressed with the TIMP-2 gene during mouse embryogenesis. JO urnal of Biological Chemistry 272, 2551 1-25517.

Armitage, G.C. (1999). Development of a Classification System for Periodontai Diseases and Conditions. Annals of Periodontology 4, 1-6

Atkinson, S.J., Crabbe, T., Cowell, S., Ward, R.V., Butler, M.J., Sato, H. Seiki, M., Reynolds, J.J., Murphy, G. (1995) Intermolecular autolpic cleavage can contribute to the activation of progelatinase A by ce11 membranes. Journal of'diological Chemistry 2 70, 30479-30285.

Bevilacqua, M.P., Pober, J.S., Mendrick, D.L., Cotran, R.S., Gimbrone, M.A. (1 987). Identification of an inducible endothelial-leukocyte adhesion molecuie, ELAM-1. Proceedkgs of Nafional Academy of Science, USA 84,9238-9242.

Birkedai-Hansen, H., Wells, B.R., Lin, H-Y., Caufield, P.W., Taylor, R.E. (1984). Activation of keratinocyte-mediated collagen (type 1) breakdown by suspected hurnan periodontopathogen: Evidence of a novel mechanism of connective tissue breakdown. Journal of Periodontal Research 19,645-650.

Birkedal-Hansen, H. (1 987). catabolism and turnover of collagens: collagenases. ltlethods in Enzymology 144: 140- 1 7 1

Birkedal-Hansen, H., Taylo, R.E., Zarnbon, J.J., Barwa, P.K., Neiden, M. (1988). Characterization of colIagenoIytic activity from strains of Bacteroides gingivalis. JO urnd of P eriodonial Research 23,258-264.

Birkedd-Hansen, H. (1993). Role of cytokines and inflammatory mediators in tissue destruction. Journal of Periodonfal Research 28,500-5 10.

Birkedal-Hansen, H., Moore, W.G.I., Bodden, M.K., Windsor, L.J., Birkedal-Hansen, B., Decarlo, A., Engler, LA. (1993). Matrix metailoproteinases: a review. Criticai Revie~vs in Oral Bioiogy and Medicine 4, 197-250.

Blaser, J., Knauper, V., Osthues, A., Reinke, H., Tschesche, H. (1991). Mercurial activation of human polymorphonuclear leucocyte procollagenase. European Journal of Biochemistry 202, 12234230.

Borregaard, N., Heiple, J.M., Skons, E.R., Clark, R.A. (1 983). Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. Journal of Cell Biology 97,5206 1.

Butler, G.S., Butler, M.J., Atkinson, S.J., Will, H., Tamura, T., van Westrum, S.S., Crabbe, T., Clements, J., d'Orth, M.P., Murphy, G. (1998). Journal of Biological Chemistry 273,871-880.

Cao, J., Rehmtulla, A., Bahou, W., Zucker, S. (1996) Membrane type matrix metalloproteinase 1 activates pro-gelatinase A without f i n cleavage of the N-temiinal domain. Journal ofBiological Chemistry 27 1,30 174-30 1 80.

Cao, J., Drew, M., Lee, H.M., Corner, C., Bahou, W.F., Zucker, S. (1998). The propeptide domain of membrane type 1 matrix metalloproteinase is required for binding of tissue inhibitor of metalloproteinases and for activation of pro-geiatinase A. Journal of Biological Chemistry 273,34745-34752.

Caron, C. Xue, J., Bartlett, J.D. (1998) Expression and localization of'membrane type 1 matrix metalloproteinase in tooth tissues. Matrtr Biology 17,jO 1 -5 1 1

Dano, K., Andreasen, P.A., Grondahl-Hansen, J., Knstensen, P., Nielsen, L.S., Skriver, L. (1985) Plasrninogen Activators, Tissue Degradation and Cancer. ildvances in Cancer Research 44, 1 3 9-266.

Danteau, R.P., Tanner, A.. Page, R.C. (1997). The microbial challenge in periodontitis. Periodontology 2000 14, 1 2-3 2.

DeClerck, Y.A., Yean, T.D., Lu, H.S., Ting, J. Langley, K.E. (1991). Inhibition of autoproteolytic activation of interstitial procollagenase by recombinant metalloproteinase inhibitor MVT'MP-2. Journal of Biological Chemistïy. 266,3893-3839

Desroches, P.E., Mookhtiar, K., Van Wart, H.E., Hasty, KA., Weiss, S. J. (1992) Proteolytic inactivation of alpha 1-proteinase inhibitor and alpha 1 -antichymorrypsin by oxidatively activated human neutrophil metalloproteinases. Jotirnal of Biological Chemistry 267,5005-50 12.

Docherty, N.S., Janusz M.J. (1994). 4. Neutrophil protease: their physiological and pathologicd roles. Immunopharrnacology of Neutmphils. eds. Hellewell, P.G, Williams. T. J. Academic Press Limited.

d'ortho, M.P., Will, H., Atkinson, S., Butler, G.' Messent, A., Gavrilovic, J., Smith, B., Timpl, R., Zardi, L., Murphy, G. (1997). Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spechum proteolytic capacities comparable to many matr;x metalloproteinases. European Journal of Biochemistry 250, 75 1-757.

Freije, J.M., Diez-Itza, I., Balbin, M., Sanchez, L.M., Blasco, R., Tolivia, J., Lopez-Otin, C., (1 994) Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. Journal of Biologicol Chernistry 269, 16766- 16773-

Fukuda, M., Spooncer, E.S., Oates, LE., Dell, A., Klock, J.C. (1984). Stmcuture of sialyated fucosyl lactoaminoglycan isolated from human granulocytes. Journal of Biologieal Chemistry 259, 10925- 10935.

Gangbar, S., Overall, C.M., McCdloch, C.A., Sodek, J. (1990). Identification of polymorphonuclear collagenase and gelatinase activities in mouthrinse samples: correlation with periodontal disease activity in adult and juvenile periodontitis. Journal of Periodontal Research 25,257-267.

Gilles, C., Polette, M., Seiki, M., Birembaut, P., Thompson, E.W. (1997). Implication of collagen type 1-induced membrane-type 1 -matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma. Laboratory Imestigution 76,65 1-660.

Gordon, M.Y. (1994). 2. Origin and Development of Neutrophils. Irnrnunopharmacology of Neutrophils. eds. Hellewell, P.G, Williams, T. I. Acadernic Press Limited

HafTajee. A.D., Socransky, S.S. (1994) Microbial etiological agents of destructive periodontal diseases. Periodontology 2000 5. 78-1 1 1.

Hanemaaijer, R. Sorsa, T, Konttinen, Y.T.? Ding, Y., Sutinen, M., Visser, H., van Hinsbergh, V.W.M., Helaakoski, T., Kainulainen, T., Ronka. H., Tschesche, H., Salo, T. (1997). Matrix metalloproteinase-8 is expressed in rheurnatoid synovial fibroblasts and endothelial cells. Journal of BioZogical Chemisny 272,3 1504-3 1509.

Haslett, C., Guthrie, L.A., Kapaniak, M.M., Johnston, R.B., Henson, P.M. (1985). Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bactenal lipopolysaccharide. American Jozirnal of Pathology 1 19, 1 O 1 - 109.

Hemandez-Barrantes, S., Toth, M., Bemardo, MM., Yurkova, M.. Gervasi, D.C., Raz, Y. Sang, Q.A, Fndman, R. (2000). Binding of active (57kDa) membrane type 1-matrix metalloproteinase (MT I - M W ) to tissue inhibitor of metailoproteinase (TIMP-2) regdates MTI-MMP processing and pro-MMP-2 activation. Journal of Biological Chernistry 275,12080- 12089.

Hiraoka, N., Allen, E., Apel, I.J., Gyetko, M.R., Weiss, S.J., (1998). Matrix metalloproteinases regulate neovascularization by acting as pericellular fibrinolysins. Cell 95,365-377.

Holmbeck, K., Bianco, P., Caterina, J., Yamada, S., Kromer, M., Kuznetsov, S.A., Mankani, M., Robey, P.G., Poole, A. R., Pidow, I., Ward, J.M. Birkedd-Hansen, H. (1 999) MT1-MW-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell99, 8 1-92.

Imai, K., Ohta, S., Matsumoto, T., Fujimoto, N., Sato, H., Motoham, S., Okada, Y. (1997) Expression of membrane-type 1 matrix metalloproteinase and activation of progelatinase A in human osteoarthntic cartilage. American Journal of Pathology 151 , 245-256

Kinoh, H., Sato, H., Tsunemka, Y., Takino, T., Kawashima, A., Okada, Y., Seiki, M. (1996). MT-MW, the cell surface activator of proMMP-2 (pro-gelatinase A), is expressed with its substrate in mouse tissue during embryogenesis. Journal of Cell Science 109,953-959.

Kinoshita, T., Sato, H., Takino, T., Itoh, M., Akizawa, T., Seiki, M. (1996). Processing of a precursor of 72-kilodalton type IV collgenase/gelatinase A by a recombinant membrane-type 1 matrix metalloproteinase. Cancer Research 56,2535-2538.

Kinoshita, T., Sato, H., Okada, A., Ohuchi. E., Imai, K., Okada, Y., Seiki, M. (1998). TIMP-2 promotes activation of progelatinase A by membrane-type 1 matrix metalloproteinase immobilized on agarose beads. Journal of Biological Chemistry 273, 16098-16103.

Kleiner, D.E., Stetler-Stevenson, W.G. (1999) Matrix metailoproteinases and metastasis. Cancer Chemotherapy and Phurmacology 43(suppl), S42-S5 1 .

Knauper, V., Krarner, S., Reinke, H., Tschesche (1990). Characterization and activation of procollagenase from human ppolymorphonuclear leucocytes. N-terminai sequence determination of the proenzyme and various proteolytically activated forms. Ewopean Journal of Biochemistry 1 89,295-300.

Knauper, V., Will, H., Lopez-Otin, C. Smith, B., Atkinson, S.J., Stanton, H.. Hembry, R.M., Murphy, G. (1 996a) Cellular rnechanisms for human procollagenase-3 (MMP- 13) activation. Evidence that MTI-MMP (MME'-14) and gelatinase a (MMP-2) are able to generate active enzyme. Journal of Biological Chemistry 27 1 . 1 7 1 24- 17 13 1.

Knauper, V., Mwphy, G., Tschesche, H. (1996b). Activation of human neutrophil procollagenase by strornelysin 2. . European Journal of Biochemistry 23 5 , 1 87- 19 1.

Knauper, V., Lopez-Otin, C., Smith, B., Knight, G., characterization of human collagenase-3. Journal of 1550.

Murphy, G. (1996). Biochemical Biological Chemistry 27 1 , 1 544-

Lamster, I.B., Oshrain, R.L., Harper, D.S., Celenti, R.S., Hodiaras, C.A., Gordon, J.M., Enxyrne activity in crevicular fluid for dtection and prediction of clinical attachment loss in patients with chronic adult periodontitis; six month results. Journal of Periodontology, 59,5 16-523.

Lee, W., Aitken, S., Kulkarni, Ci., Birek, P., Overall, CM., Sodek, J., McCulloch, C.A. (1 99 1). Collagenase activity in recment periodontitis: relationship to disease progression and doxycylcine therapy. Journal of Periodontal Research 26,479-485

Lee, W., Aitken, S., Sodek, J., McCulloch, C.A. (1 995). Evidence of a direct relationship between neutrophil collagenase activity and penodontal tissue destruction in vivo: role of active enzyme in in human periodontitis. Journal of Periodontal Research 30,23-33.

Lehti, K., Lohi, J., Valtanen, H., Keski-Oja, J. (1998) Proteolytic processing of membrane-type4 matrix metalloproteinase is associated with gelatinase A activation at the ce11 surface. Biochemical Journal 334,345-353.

Liotta, L.A., Tryggvason, K., Garbisa, S., Hart, I., Foltz, CM., Shafie, S. (i980). Metastatic potential correlates with enzymic degradation of basiment membrane collagen. Nature 284,67-68.

Lohi, J., Lehti, K., Westemarck, J., Kahari, V-M., Keski-Oja, J. (1996). Regulation of membrane-type matrix metalloproteinase-1 (MT-MMP-1) expression by growth factors and the phorbol ester PMN. European Journal of Biochernistry 239,239-247.

Loe, H., Theilade E., and Jensen, S.B. (1965). Experirnental gingivitis in man. Journal of Periodontology 36, 1 77- 1 87.

Loe, H., Anerud, A.? Boysen, H.. and Momson, E. (1986). Natural history of periodontal disease in man. Rapid, moderate and no loss of attachent in S n Lankan laborers 14 to 46 years of age. Journal of Clinical Periodontology 1 3,43 1 -445.

Lovejoy, B. Cleasby, A. Hassell, A.M., Longley, K., Luther, MA., Wegl, D., McGeehan, G., McElroy. A.B., Drewry, D., Lambert, M.H., Jordan, J.R. (1994). Structure of the catalytic domain of fibroblast collagenase complexed with an inhibitor. Science 263. 375

Massova, I., Kotra, L.P., Fridrnan? R.? Mobashery, S. (1998). Matnx metalloproteinases: structures, evohtion, and diversification. FASEB Journal 12, 1075- 1 095.

McEver, R.P., Beckstead, J.H.. Moore, K.L.. Marshall-Carlson, L., Bainton, D.F. (1 989). GMP-140, a platelet alpha-granule membrane protein is also synthesised by vascular

endothelid cells and is locdised in the Weibel-Palade bodies. Journal of Clinical Investigation 84,92-99.

Melcher, A.H., Chan, J. (1981). Phagocytosis and digestion of collagen by gingival fibroblasts in vivo: a study of senai sections. Journal of Ultrastructural Research 77, 1- 36.

Mitchell, P.G., Magna, H.A., Reeves, LM., Lopresti-Morrow, L.L., Yocum, S.A., Rosner, P.J., Geoghegan, K.F., Hambor, J.E. (1996). Cloning, expression. and type 11 collagenoiytic activiw of ma& metailoproteinase- 1 3 fiom hurnan osteoarthntic cartilage. Journal of Clinical Investigation 97,76 1-768.

Miyasaki, K.T. (1 99 1). The Neutrophil: Mechanism of controlling periodontal bacteria. Journal of Periodontology 62,76 1-774.

Miyasaki, K.T. (1996). Cha ter 10: Altered Leukocyte Function and Periodontal Disease. R Clinical Periodontology 8' Ed Carraiua F.A., Newman, M.G. eds.. W.B. Saunders Company, Toronto.

Murphy, G., Stanton, H., Cowel, S., Butler, G., Knauper, V., Atkinson, S., Gavrilovic, J. (1 999) Mechanisms for pro ma& metailoproteinase activation. Acta pathologica et micro biologica Scandinavica 1 07,3 8-44.

Nagase, H., Enghld, J.J., Suzuki, K., Salvesen, G. (1990) Stepwise activation mechanisms of precursor of matrix metalloproteinase 3 (stomelysin) by proteinases and (4-arnino-phenyl) mercurîc acetate. Biochemishy 29,57834789.

Nagase, H. (1997). Activation mechanisms of matnc rnetalloproteinases. Biological Chemistry 378, 15 1-1 60.

Nakahara, H. Howard, L, Thompson, E.W., Sato, H., Seiki, M., Yeh, Y., Chen, W-T. (1 997) Transmembrane/cytoplasmic domain-mediated membrane type 1 -matrix metailoprotease docking to invadopodia is required for ce11 invasion. Proceedings of National Academy of Science USA 94,7959-7964.

Netzel-Arnett, S., Fields, G., Birkedal-Hansen, H., Van Wart, H.E. (1 99 1). Sequence specificities of human fibroblast and neutrophil collagenases. Journal of Biological Chemistry 266,6747-6755.

Ochuchi, E., Imai, K.? Fujii, Y., Sato, H., Seiki, M., Okada Y. (1 997JMembrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. Journal of Biological Chemistry 272,2446-245 1 .

Okumura, Y., Sato, H.. Seiki, M., Edo, H. (1997). Proteoiytic activation of the precursor of membrane type 1 ma& metailoproteinase by huma. plasmin. A possible ce11 surface activator. Federaiion of European Biochemical Socieiies Le [fers 402, 1 8 1 - 1 84.

Overall, C.M., Sodek, I. McCulloch, C.A., Birek, P. (1991). Evidence for polymorphonuclear leukocyte collagenase and 92-kilodalton gelatinase in gingival crevicular fluid. infection & Immunity 59,4687-4892.

Pabst, M.J. (1 994). 9. Priming of Neutrophils. Immunopharmacology of Neutrophils. rds. HeIlewelI, P.G, Williams, T. J. Academic Press Limited.

Page, R.C., and Schroeder, H.E. (1976). Pathogenesis of inflammatory periodontai disease. A summary of curent work. Lnboratory Investigation 34,235-249.

Page, R.C., Offenbacher, S., Schroeder, H.E., Seymour, G.J., Komman, K.S. (1997). Advances in the pathogenesis of penodontitis: summary of developrnents. clinical implications and future directions. P eriodontology 2000 14,2 16-248.

Page, R.C., (1998). The pathobiology of periodontal diseases may dfiect systemic diseases: inversion of a paradigm. Annals of Periodontology. 3(1), 108- 120

Pei., D ., Weiss, S. (1 99 5). Furin-dependent intracellular activation of the human stromelysin-3 zyrnogen. Nature 375,244-247.

Pei, D., Weiss. S. (1 996). Transmembrane-deletion mutants of the membrane-type matrix rnetalloproteinase-l process progelatinase A and express intrinsic matnx-degrading activity. Journal ofBiologicu1 Chemistry 27 1,9 135-9 140.

Pei, D. (1999). Identification and characterization of the fifth membrane-type matrix metalloproteinase MTS-MMP. Journal of Biologicaf Chemistry 274,8925-8932.

Pei, D. (1999a). Leukolysin/MMP25/MT6-W: a novel matrix rnetalloproteinase specificail y expressed in the leukocyte lineage. Cell Research. 9,29 1-303.

Polette, M. Birembaut, P. (1998) Membrane-type metalloproteinases in Nmor invasion. The International Journal Of Biochemistry & Cell Biology 20, 1 195-1202.

Puente, X.S., Penda, A.M., Liano, E., Vaiasco, G., Lopez-Otin, C. (1996). Molecular cloning of a novel membrane-type matrix metailoproteinase from a hurnan breast carcinoma. Cancer Research 56,944-949.

Roberston, P.B., Lantz, M., Manrcha, P.T., Kornman, K.S., Tnrmmel, CL., Holt. S.C. (1982). Collagenolytic activity associated with bacteroides species and Actinobacillus actinomycetemcomitans. Journal of Periodontu[ Research 17,275-283.

RomaneIli. R., Mancini, S., Laschinger, C., Overall, CM., Sodek, J., McCulloch, C.A.G. (1999). Activation of neutrophil collagenase in penodontitis. Infection and lmmunity 67. 23 19-2326.

Sato, H., Takino, T., Okada, Y. Cao, J., Shinagawa, A., Yamaoto, E., Seiki. M. (1 994). A matrix metalloproteinase expressed on the surface of invasive tumour celis. Nature 370, 61-65.

Sato, H., Seiki, M. (1996). Membrane-type rnatrix metalloproteinase ( M T - W s ) in tumour metastasis. Journal of Biochemistry 1 19,209-2 15.

Sato, H., Kinoshita, T., Takino, T., Nakayama, K., Seiki, M. (1996). Activation of a recombinant membrane-type lrnatrix rnetalloproteinase (MTl-MMP) by funn and its interaction with tissue inhibitor of metalloproteinase (TIMP-2). FEBS Leu. 393, 10 1 - 104.

Sato, H., Takino, T., Kinoshita. K., Imai, K., Okada, Y., Stetler-Stevenson. W.G., Seiki, M. (1996). Ce11 surface binding and activation of gelatinase A induced by expression of membrane-type- 1 -matrix metalloproteinase (MT1 -MMP). FEBS Lett. 3 85.23 8-240.

Sato, T., del Carmen Ovejero, M., Hou, P., Heegaard, A., Kumegawa, M., Foged, N.T.. Delaisse, J. Identification of the membrane-type matrix metalloproteinase MT1 -MMP in osteoclasts. Journal of Ce11 Science 1 10, 589-596.

Smith, C.W., Kishimoto, T.K., Abbass, O., Hughes, R.J., Rothiein, R.. Mclntyre, L.V., Butcher, E. Anderson, D.C. (1991). Chernotactic factors reguiate lectin adhesion molecule- 1 (LECAM- 1 )-dependent neutrophil adhesion to cytokine-stimuiated endotheliai cells in vitro. Journal of Clinical Investigation 87,609-6 18.

Springman, E.B., Angleton, E.L., Birkedal-Hansen, H., Van Wart, H.E. (1990). Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a cysteine switch mechanism for activation. Proceedings ofNuiiona1 Acaderny of Science USA 87, 364.

Stanton, H., Gavrilovic, J. Atkinson, S. J., d70rtho, M.P., Yamada. K.M., Zardi, L. Murphy, G. (1 998) The activation of ProMMP-2 (gelatinase A) by HT 1 O80 fibrosarcoma cells is promoted by culture on a fibronectin substrate and is concomitant with an increase in processing of MTI-MMP (MMP-14) to a 45 kDa fom. Joztrnal of Cell Science 1 1 1,2789-2798

Stetler-Stevenson, W .G.. Amavoorian, S., Liotta, L. A. (1 993). Tumour cell interaction with the extracellular matrix during invasion and metastasis. Annztal Revieiv of Cell Biology 9,54 1 -5 73.

Strongin, A.Y., Collier, I., Bannikov, G.. Marner, B.L.. Grant. G.A.. Goldberg, G.I. (1995). Mechanism of ce11 surface activation of 72 kDa type IV collagenase. Isolation of the activated fo rm of the membrane metalloprotease. JO urnal of Biologicol C h e m i s v 270,533 1-5338.

Symington, F.W., Hedges, DL, Hakornori, S.I. (1985). Glycolipid antigens of human polymorphonuclear neutrophiis and the inducible HL-60 myeloid leukaemia line. Journal of Imrnunology 134,2498-2506.

Takino, T., Sato, H., Yamamoto, E.9 Seiki, M. (1995a). Cloning of a human gene potentially encoding a novel matnx metalloproteinase having a C-teminal transmembrane domain. Gene 155,293-298.

Takino, T. Sato, H., Shinagawa, A., Seiki, M. (1995b). Identification of the second membrane-rype matrix rnetalloproteinase (MT-MMP-2) gene from a human placenta cDNA library: MT-MMP form a unique membrane-type subclass in the MMP family. Journal of Biological Chemishy 270,23 0 1 3 -23020.

Theilade, J., Attstrom, R. (1985). Distribution and ultrastructure of subgingival plaque in beagle dogs with gingival inflammation. Journal of Periodontal Research 10. 13 1 - 145.

Tsunezuka, Y. Kinoh, H., Takino, T., Watanabe, Y., Okada, Y., Shinagawa, A., Sato, H., Seiki, M. (1 996) Expression of membrane-type matrix metalloproteinase 1 (MT 1 -MMP in tumour cells enhances pulmonary metastasis in an experirnental metastasis assay. Cancer Research 56,5678-5683.

Tyagi, S.C., Lewis, K., Pikes, D., Marcello, A., Mujumdar, V.S., Smilcy, LM., Moore, C.K. (1 998). Stretch-induced membrane type matrix metalloproteinase and tissue plasminogen activator in cardiac fibroblast cells. Journal of Ce12 Physiology 176, 374- 382.

Vaes, G. (1988). Cellular biology and biochemical mechanism of bone resorption Clin. Orrhop. Rel. Res. 23 1,239-27 1.

Van D yke, T.E., Vaikuntam, J. (1 994). Neutrophil function and dys function in peridontal disease. Ctirrent Opinion in Periodontology 19-27

Van Wart, H., Birkedd-Hansen, H. (1 990). The cysteine switch: a rpinciple of regulation of metalloproteinase activity with potentiai applicability to the entire matrix me tallo pro teinase gene famil y. Proceedings of l e iVational A cademy of Sciences USA 87, 5578-5582.

Wahl. L., Wahl? S.M., Mergenhagen, S.E., Martin, GR. (1974). Collagenase production by endotoxin-activated macrophages. Proceedings of the National Academy of Sciences USA 71,3598-3601.

Weiss, S.J., Peppin. G., Ortiz, X., Ragsdale, C., Test, S.T. (1985) Oxidative autoactivation of latent collagenase by human neutrophils. Science 227,747-749.

Weiss, S.J. (1989). Tissue destruction by neutrophils. New EngZund Joirrnal of Medicine 320,365-376.

Welgus, H.G., Campbell, E.J., Cury, J.D. (1990). Neutra1 metalloproteinases produced by human mononuclear phagocytes. Enzyme profile. regulation, and expression during cellular development. Journal of Clinical investigation 86, 1496- 1 502.

Werb, 2. (1997). ECM and ceil surface proteolysis: regulating cellular ecoiogy. Cell91, 43 9-442. Will, H., Hinzmann, B. (1995). cDNA sequence and mRNA tissue distribution of a novel hurnan matrix metalloproteinase with a potential trammembrane segment. European Journal ~~'Biochemistry 23 1,602-608.

Will, H., Atkinson, S.J., Butler, G.S., Smith, B., Murphy, G. (1996). The soluble catalytic domain of membrane type 1 matrix metalloproteinase cleave the propeptide of progelatinase A and initiates autoproteolytic activation. Journal of Biological Chemistry 271, 171 19-17123.

Wu. H., Byrne, M.H, Stacey, A., Goldring, M.B.. Birkhead, J.R., Jaenisch, R., Krane. S.M. (1990). Generation of collagenase-resistant collagen by site-directed mutagenesis of murine proal(I) collagen gene. Proceedings of the National Acodemy of Science USA 87. 5888-5892.

Yana, I., Weiss, S.J. (2000). Regdation o f membrane type4 matrix metalloprofeinase activation by proprotein convertases. Molecular Biology of the Cell 1 1. 2387-240 1.

Zhou, Z., Apte, S.S., Soininen, R., Cao, R., Baaklini. G.Y., Rauser. R.W.. Wang, J., Cao. Y., Tryggvason, K. (2000) lmpaired endochondnl ossification and angiogensis in mice deficient in membrane-type matrix rnetalloproteinase 1. Proceedings of National Acaderny of Science USA 97,4052-4057.