474

Role of Matrix Metalloproteinases

in Human Periodontal Diseases*
Henning Birkedal-Hansen
Matrix metalloproteinases (MMP) are a family of proteolytic enzymes that mediate the degradation of extracellular matrix macromolecules, including interstitial and
basement membrane collagens, fibronectin, laminin, and proteoglycan core protein. The
enzymes are secreted or released in latent form and become activated in the pericellular
environment by disruption of a Zn++-cysteine bond which blocks the reactivity of the
active site. The major cell types in inflamed and healthy periodontal tissues (fibroblasts,
keratinocytes, endothelial cells, and macrophages) are capable of responding to growth
factors and cytokines, as well as to products released from the microbial flora by induction
of transcription of 1 or more MMP genes. Cytokines that are likely to regulate expression
of MMP genes in periodontal tissues include IL-1, TNF-a, and TGF-a. In addition,
triggered PMN leukocytes which express only 2 MMP (PMN-CL and Mr 92K GL) release
these enzymes from specific granule storage sites in response to a number of stimuli.
The evidence that MMP are involved in tissue destruction in human periodontal diseases
is still indirect and circumstantial. Cells isolated from normal and inflamed gingiva are
capable of expressing a wide complement of MMP in culture and several MMP can be
detected in cells of human gingiva in vivo. In addition, PMN-CL and Mr 92K GL are
readily detected in gingival crevicular fluid from gingivitis and Periodontitis patients.

Osteoclastic bone resorption does not appear to directly involve MMP, but a body of
evidence suggests that bone resorption is initiated by removal of the osteoid layer by
osteoblasts by means of a collagenase-dependent process. J Periodontol 1993; 64:474484.

Key Words: Enzymes, proteolytic; cytokines; growth factors; periodontal diseases/path-

ogenesis;

bone

resorption; leukocytes; proteinases.

METABOLIC DEGRADATION OF THE

EXTRACELLULAR MATRIX
Degradation of the extracellular matrix may involve as many
as 4 apparently distinct pathways (Fig. 1). A body of evidence suggests that matrix components may be dissolved
by extracellular matrix metalloproteinase (MMP)-dependent or plasmin (Pln)-dependent cleavage reactions, and
that larger fragments of matrix may be disposed of by a
phagocytic pathway by way of cleavage by lysosomal proteinases. Mineralized matrices appear to be degraded by a
complex extra/pericellular process mediated by osteoclasts
(osteoclastic pathway) which relies on degradation by lysosomal proteinases in a narrow pericellular compartment.
This review focuses specifically on matrix metalloproteinases and their role in the metabolic degradation of the extracellular matrix in human periodontal diseases. A comprehensive review of the matrix metalloproteinase field
including a complete bibliography of the literature through

*Department of Oral Biology and Research Center in Oral Biology, School

Dentistry, University of Alabama at Birmingham, Birmingham, AL.

of

Figure 1. Pathways for degradation of the extracellular matrix.

overlap are indicated by boxes.

Areas

of

1990 compiled by Woessner1 was recently published.2 For

shorter general discussions of the role of MMP in health
and disease the reader is referred to recent reviews.3,4 At
the outset it is important to recognize that much of the
evidence for the role of MMP in specific developmental or
pathological processes remains circumstantial and indirect.
That is certainly true for the involvement of MMP in human
periodontal diseases. It has proven difficult to specifically
identify the natural substrates for most MMP in part because
of their relatively broad and overlapping substrate specific-

Volume 64
Number 5

BIRKEDAL-HANSEN

monocytes/macrophages,14*15 osteoblasts,16*17 and chondrocytes.18 The highly glycosylated PMN enzyme has a considerably larger molecular mass than FIB-CL (Mr 75,000
vs. 57,000/52,000) but the 2 protein cores are of virtually
identical size. The 2 collagenases differ markedly in terms
of transcriptional regulation. PMN-type collagenase is rapidly released from specific granule storage sites of triggered

(Table 1). Moreover, the relationship of the MMPdependent pathway to other routes of matrix degradation
has not been adequately clarified. Yet the observation that
MMP transcripts or protein frequently are present in cells,
tissues, and interstitial fluids in vivo strengthens the notion
ities

that these enzymes are involved in the metabolic remodeling of the extracellular matrix.

PMNs whereas FIB-CL is synthesized on demand by initiating transcription of the corresponding gene. This process
causes a lag period of 6 to 12 hours before enzyme can be
detected in the extracellular environment.
The stromelysin group of MMP includes SL-1 and SL-2
which have virtually identical substrate specificities and cleave
a wide range of extracellular matrix proteins (proteoglycan
core protein, type IV and V collagen, fibronectin, and laminin.)19 SL-1 is expressed by stromal cells either constitutively or after induction by growth factors/cytokines [ILl,20 EGF,21 TNF-a,20 PDGF21] or phorbol esters.22*23 The
enzyme does not appear to be expressed by PMN leukocytes
and keratinocytes in the human, but a SL-1 (or SL-2) homologue is induced in murine skin epidermis by treatment with
the tumor promoter TPA (12-O-tetradecanoyl-phorbol acetate).23*24 SL-2 is expressed less abundantly than SL-1 and
does not appear to respond to growth factors (EGF and IL-

Matrix Metalloproteinases
The MMP gene family encodes 9 or more metal-dependent
endopeptidases which collectively are capable of degrading
most, if not all, extracellular matrix macromolecules (Table
1). The enzymes share extensive sequence homology but
differ somewhat in terms of substrate specificity and transcriptional regulation. All MMP may be regarded as derivatives of a 5-domain prototype structure, formed either by
addition or deletion of regulatory domains as shown in Figure 2. A short signal sequence is followed in order by a
propeptide which endows the virgin enzyme with catalytic
latency, a catalytic domain which contains the active site
and the catalytic machinery, a proline-rich hinge region,
and a pexin-like COOH-terminal domain which plays a role
in determining substrate specificity. In addition, the 2 gelatinases contain a gelatin-binding insert (fibronectin type
II-like repeats) in the catalytic domain.5*6 Each of the enzymes contains a tridentate Zn++-binding site believed to
constitute the active site.7 The pexin-like domain is absent
from the smallest MMP, PUMP-1.
The PMN-CL gene is only expressed by PMN leukocytes
whereas the highly homologous FIB-CL gene is expressed
by fibroblasts,8*9 keratinocytes,10,11 endothelial cells,12*13
Table 1: Matrix

1) or phorbol esters.

The Mr 72K GL is perhaps the most widely distributed

of all MMP and has been identified in fibroblasts,25 keratinocytes,26 endothelial cells,27 monocytes/macrophages,28
osteoblasts,29 and chondrocytes.30 Mr 72K GL does not
appear, however, to be expressed by PMN leukocytes but
is present in a circulating form in plasma.31 The Mr 92K

Metalloproteinase Family

Enzyme
Interstitial Collagenases
Fibroblast-type
Collagenase
PMN-type
Collagenase
Stromelysins
Stromelysin-1
Stomelysin-2

Abbreviation

Extracellular Matrix
Substrates

Mr

MMP#

FIB-CL

MMP-1

57,000/52,000

PMN-CL

MMP-8

75,000

Gelatin
Same as FIB-CL

SL-1

MMP-3

60,000/55,000

PG Core

SL-2

MMP-10

60,000/55,000

Same

MMP-2

72,000

MMP-9

92,000

Gelatin, Collagen IV, V, VII, X,

Elastin, Fibronectin
Gelatin, Collagen IV, V, Elastin

MMP-11
MMP-7

n.d.

n.d.

28,000

Fibronectin, Laminin,
Collagen IV, Gelatin, proCL,

Collagen I, II, III, VII, VIII, X,

Protein, Fibronectin,
Laminin, Collagen IV, V,
IX, X, Elastin
as

SL-1

Gelatinases
Mr 72K Gelatinase/
Mr 72K GL
Type IV Collagenase
Mr 92K Gelatinase/
Mr 92K GL
Type IV Collagenase

Other

Stromelysin-3

PUMP-1

Macrophage

Metallo-

SL-3
PUMP-1
MME

elastase
MMP

numbering according to Nagase et al.13

55,000

475

PG Core Protein
Elastin

J Periodontol

476

MATRIX METALLOPROTEINASES
HINGE
PROPEPTIDE CATALYTIC DOMAIN
PEXIN-LIKE DOMAIN
,

May

Table 2. Transcriptional Effect of Growth Factors and

Matrix Metalloproteinases

Reference

Induction
PROTOTYPE
FIBRONECTIN-LIKE
INSERT

PROLINE-HICH
HINGE

Mr 72 K GL,
Mr 92 K GL

SL-1,-2,-3
PMN-CL, FIB-CL

IL-1 a,
TNF-a
TGF-a
EGF
PDGF
bFGF
NGF

20, 45
20, 46, 47

TGF-

26, 41

49
21

Repression
TGF-
IFN-7

(Supplement)
Cytokines on
Reference

50, 52
53, 54

21, 48
44, 50
51

Propeptido

PUMP-1

Figure 2.

1993

Domain structure

of matrix metalloproteinases.

GL is produced by PMN leukocytes,32'33 keratinocytes,26'34

and monocytes/macrophages,35 and occasionally by fibroblasts. It is interesting to note that PMN which express a
unique and distinct CL gene utilize the same Mr 92K GL
gene as other cells although in a manner which yields a
storable rather than a secreted enzyme. Mr 72K GL is expressed constitutively by most cells in culture and responds
only moderately to growth factors which induce the Mr 92K
GL. The Mr 72K and Mr 92K GL cleave a number of
peptide bonds in denatured collagen chains to yield small
peptides.25'36 Besides gelatin, gelatinases also cleave types
IV, V, VII, and X collagens37"39 and elastin.40
PUMP-1 cleaves a wide range of substrates including
fibronectin, laminin, and gelatin. The enzyme is expressed
in gingival fibroblasts41 and has been isolated from the involuting rat uterus and from rectal carcinoma cells.42,43 SL-3
transcripts are expressed by mesenchymal cells of human
mammary carcinomas, often adjacent to invading epithelial
tumor cells. SL-3 expression is also induced in embryonic
fibroblasts by several growth factors. It is unknown whether
SL-3 is produced by cells of human periodontal tissues.
The amino acid sequence deduced from cDNA suggests that
SL-3 encodes a functional metalloproteinase, but the enzyme protein has not yet been isolated or characterized.
SL-3 has the same principal domain structure as CLs and
SL-1 and SL-2, but differs by an insert of 10 residues at
the autolytic activation site.44

Regulation of MMP Activity

The activity of MMP against extracellular matrix substrates
is regulated at 4 "gates": 1) by transcriptional regulation
of MMP genes; 2) by precursor activation; 3) by differences
in substrate specificity; and 4) by MMP inhibitors.
Transcriptional regulation. The transcription of most
MMP genes is regulated by endogenous growth factors and
cytokines (Table 2). Stimulation or repression of growth
factor- and cytokine-responsive MMP genes results in up
to a 50-fold change in mRNA and protein levels. Transcription of the CL and SL-1 genes (and in some cells the
SL-3 and Mr 92K GL genes as well) is induced by IL-

LATENT

Figure 3. Activation of MMP precursors

cysteine switch.

ACTIVE

is initiated

by opening of the

I,20,45 TNF-a,20'46'47 PDGF,21,48 TGF-a,49 EGF,21

bFGF,20,50 NGF,51 and with few exceptions26 abrogated by
TGF-50,52 and IFN-7.53,54 TGF- which ablates transcription of CL and SL-150,52 upregulates Mr 72K GL by 2- to
4-fold and Mr 92K by up to 8-fold.26,41 These findings are
of considerable interest because TGF- in general appears
to down-regulate rather than stimulate MMP expression.
Transcription of MMP genes may also be regulated by other
endogenous pathways. FIB-CL is induced or stimulated in
osteoblasts by PTH and 1,25 di(OH)D3,17,55'56 and FIB-CL
and SL-1 are down-regulated by glucocorticoids and by
retinoids.57 59 The proximity of a viable microbial plaque,
moreover, creates an unequaled opportunity for direct transcriptional effects by microbial metabolites on human gingival and periodontal cells. A potentially important inter-

action that falls in this category is the induction of FIB-CL

in macrophages by LPS.60'61 In addition, microbial
proteinases62,63 and lectins64 may also play a role in the
transcriptional regulation of MMP expression.
Activation of precursors. The latency of MMP precursors appears to be maintained, at least in part, by a coordinate bond which links an unpaired Cys residue in the
propeptide to the active site Zn++65,66 (Fig. 3). Disruption
of the Cys-Zn++ bond is a prerequisite to activation and
may be achieved in a number of different ways: 1) by
interaction or modification of the Cys residue by organomercurials, metal ions, thiol reagents, and oxidants; 2) by
conformational change of the Polypeptide backbone induced by certain chaotropic agents (KI, NaSCN) and detergents (SDS); and 3) by excision of a portion of the propepeptide by proteolytic enzymes (trypsin, plasmin,
chymotrypsin, neutrophil elastate, cathepsin B, and plasma
kallikrein). The enzyme subsequently catalyzes 1 or more69
autolytic cleavages to generate the fully-processed form.66

Volume 64
Number 5

BIRKEDAL-HANSEN

Although MMP can readily be activated in the test tube by

any or all of these mechanisms, the biologic activation
mechanisms are still poorly understood. Several studies have
suggested that plasmin may play a role in activation of FIB-

CL and SL-1, at least in cell culture systems, and that SL1 is perhaps involved in the processing of the FIB-CL precursor to a particularly highly-active form.70-72 There is still
insufficient evidence to determine whether these reactions
also take place in vivo. It is of note, however, that the
degradation of gelatin and collagen by PMN leukocytes73'74
involves activation of CL and Mr 92K GL by oxidative
pathways, presumably by oxidation of the unpaired propeptide Cys residue by HOC1.
Substrate Specificity. A certain level of regulation of
MMP activity is encoded at the level of the substrate. Although the enzymes have somewhat overlapping substrate
specificities, there are also notable differences, particularly
with respect to the cleavage of collagens.2 Virtually all of
the enzymes cleave gelatin and fibronectin at some rate,
and most cleave type IV and V collagens at sufficiently
high temperatures. The characteristic ability of FIB-CL and
PMN-type collagenases to dissolve interstitial collagen fibrils by cleavage of the component molecules in the proteinase-resistant triple-helical domain, however, is not shared
by other members of the family.
Inhibition

a-Macroglobulins. Active

MMP

captured by a-macroglobulins by unique venus-fly-trap mechanism activated by cleavage of a bond in the "bait region."75,76 This
cleavage leads to hydrolysis of a labile internal thiol-ester
bond and covalent cross-linking of a nascent glytamyl residue to lysyl side chains exposed on the surface of the
attacking proteinase.77 The rapid capture rates, particularly
with CL, suggest that a-macroglobulins, particularly alM, play an important role in the regulation of MMP activity.
are

Tissue inhibitors of metalloproteinases (TIMPs). TIMPS

form classical non-covalent bimolecular complexes with the
active forms of MMPs and, in some instances, with latent
MMP precursors as well. TIMPs appear to regulate matrix
degradation both by proteinase elimination and by blockage
of autolytic MMP activation.78,79 Two members of the TIMP
family, TIMP-1 and TIMP-2, have been identified but more
may exist.13,80"83 TIMPs are widely distributed in tissues
and fluids84,85 and are expressed by many cell types in-

cluding fibroblasts,86 keratinocytes,87 monocytes/macrophages,15 and endothelial cells.13 TIMP-1 is a Mr 28K glycosylated protein with a Mr 20K protein core.80,86 TIMP-1
forms complexes with active collagenase, but not procollagenase, in a 1:1 stoichiometry with rCjS of 10~9M to
10" 10M.88,89 Complex formation with active MMP does not
appear to depend on cleavage of the inhibitor and fully

functional inhibitor can be recovered from the complex.90

TIMP-1 also forms a complex with the zymogen form of
Mr 92K GL.5,79,91 TIMP-2, a Mr 22,000 unglycosylated
protein, is expressed by fibroblasts and endothelial cells and

477

perhaps by other cells as well.13,92,93 The inhibitor forms a

complex with the zymogen form of Mr 72K GL.5,89 TIMP2 is 2- to 10-fold more effective than TIMP-1 against the
2 GL whereas TIMP-1 appears to more effectively inhibit
CL.91 The 2 TIMP genes are differently regulated. TIMP1 expression is stimulated by growth factors (EGF, TNFa, IL-1, TGF-), phorbol esters, retinoids, and glucocorticoids, whereas TIMP-2 expression is down-regulated by
TGF- and fails to respond to phorbol esters.41,94 98
ROLE OF MMP IN HUMAN PERIODONTAL
DISEASES

of MMP by Different Cell Types of Human

Periodontal Tissues
Each of the major cell types of human periodontal tissues
is capable of expressing a unique complement of MMP
when properly stimulated as summarized in Table 3. PMN
leukocytes share with many other cell types the ability to
express the common 92K GL gene but, for reasons that are
unknown, utilize a distinct gene for interstitial collagenase
(PMN-CL). Both enzymes are stored in specific granules
and rapidly released when the cells are triggered.99 Fibroblasts express a somewhat larger complement of MMP,

Expression

including an interstitial, fibroblast-type collagenase (FIBCL), SL-1, SL-3, Mr 72K GL, and PUMP-1, but not Mr
92K GL. Keratinocytes express FIB-CL, Mr 72K GL, and
Mr 92 K GL, but not SL-1. Macrophages and endothelial
cells each express FIB-CL,
the Mr 92K GL.12,13,20,39

SL-1, and both the Mr 72K and

Cell Type-Specific Differences in Regulation of MMP

The synthesis and packaging of MMP in specific granules
are essentially complete when the PMN enters the blood
stream. The triggered release of MMP from specific granules is clearly not a transcriptional event and it is still uncertain to what extent it is regulated by growth factors and
cytokines. The response pattern of the PMN leukocyte is
clearly unique and different from that of any other cell type
not only in the complement of MMP expressed but also in
the utilization of the enzymes. The PMN leukocyte has
evolved to respond rapidly and in full force to external
stimuli. No other cell type is capable of "dumping" such
large quantities of destructive enzymes in a matter of minutes. The response is sustained, when needed, by continuous recruitment of new cells and not by prolonged release
from cells already triggered. Fibroblasts, keratinocytes,
macrophages, and endothelial cells share an essentially
common response to catabolic growth factors such as IL1, TNF-a, TGF-a, EGF, bFGF, PDGF, namely induction
or stimulation of transcription of MMP genes. However,
there are important cell-specific and cytokine-specific differences among these responses. In fibroblasts, IL-l induces primarily SL-1, TNF-a primarily CL,20 IFN-7 represses SL-1 but moderately upregulates CL,53 TGF-
represses CL and SL-1, but upregulates 72K GL. In kera-

478

MATRIX METALLOPROTEINASES
Table 3.
Tissues

May
Expression

of Matrix

Metalloproteinases by Five Major Cell Types

Type
Enzymes
expressed

Leukocyte
PMN-CL,

Mr 92K GL

Fibroblast

FIB-CL, SL-1,
Mr 7K GL,
PUMP-1,

activation

FIB-CL,

Macrophage

Endothelial
Cell

FIB-CL, SL-1,
Mr 72K GL,

FIB-CL, SL-1,

IL-1, TNF-a,
TNF-a, EGF,
EGF, TGF-a, TGF-a,
PDGF, TPA
TGF-,

TPA

TPA

Transcriptional

Transcriptional

SL-3

Transcriptional

Keratinocyte

(Supplement)

of Human Periodontal

PMNCell

J Periodontol
1993

Mr 72 K GL,
Mr 92K GL,
SL-2

Mr 92K GL

Mr 72K GL,
Mr 92K GL

TPA

Mobilization
of enzymes

Granule
Release

Transcriptional

Transcriptional

Response

Seconds

6 to 12 hours

6 to 12 hours

Response

Minutes

Days

activation

activation

time

duration

tinocytes, TGF- upregulates both the Mr 72 K and the Mr

92K GL;26 on the other hand, IL-l, which is the most
potent known inducer of CL- and SL-l-expression by fibroblasts, has no effect on keratinocytes, at least not in the
human.11 Recent studies in our own laboratory have shown
that growth factors and cytokines which induce expression
of SL-1 in stromal cells induce exclusively SL-2 in keratinocytes (unpublished data). The findings summarized above
show: 1) that different cell types express different complements of MMP; 2) different cytokines elicit different transcriptional effects in the same cell type; and 3) that different
cell types do not necessarily respond in the same fashion
to a given cytokine. In the light of these findings, and in
the light of the many potential synergistic and antagonistic
interactions that exist between 2 or more cytokines, it is
apparent that different inflammatory infiltrates may vary
considerably in destructive potential. This is perhaps the
key to understanding why inflammation of the human gingiva may or may not give rise to tissue destruction and

attachment loss.
Recent studies have started to shed light into the mechanisms that enable growth factors and cytokines to elicit
overlapping, yet occasionally distinct, transcriptional effects. The AP-1 binding site is a necessary, but not sufficient, requirement for transcriptional activation of MMP
expression by many growth factors and cytokines. For instance, the Mr 92K GL gene which contains 3 copies of
the AP-1 site is inducible by catabolic growth factors whereas
the Mr 72K GL gene which contains no such elements is
not.100-101 The transcriptional activity of the AP-1 site depends on binding of c-fos/c-jun dimers to the TGAGTCA
sequence. The role of fos, however, is quite complex and
even among transcriptional pathways that operate through
the AP-1 site, some but not all, are c-fos-dependent. For
instance, induction of SL-1 by PDGF is fos-dependent
whereas that of EGF is not.21 Moreover, abrogation of FIBCL and SL-1 transcription by TGF- is also dependent on
c-fos although the action of this growth factor is indepen-

activation
6 to 12 hours

6 to 12 hours

Days

dent of the AP-1 site and instead

bp TGF--responsive

activation

depends

on a

unique

10

element.50,52 Recent studies have

identified promoter and enhancer elements required for

maximal transcriptional activation that are specific for each
MMP. Such is the case for the PEA-3 site in the FIB-CL
promoter and the NIP protein binding site in the SL-1 pro-

moter.102"104

The second messenger-signaling pathways which mediate growth factor and cytokine transcriptional effects on
MMP gene expression are still incompletely understood. A
body of evidence suggests that protein kinase C (PKC), a
cellular receptor for TPA, acts as a messenger in the transcriptional regulation of growth factor-responsive MMP genes
in some but not all cases.53,105 cAMP may be involved as
a second messenger in some responses but apparently through
activation of a different (Jun B-dependent) transcriptional
machinery than either TPA or growth factors/cytokines.58,106 cAMP stimulates MMP expression in some cell
types; in others leads to repression. For instance, expression
of CL and SL-1 by guinea pig peritoneal macrophages, rat
UMR-106 osteosarcoma cells, and rat fibroblasts is stimulated by cAMP,24,56,107 whereas the constitutive expression of CL by human GM637 fibroblasts,104 and the IL-1,
EGF- and oncogene-induced transcription of FIB-CL and
SL-1 genes by rat and human fibroblasts is repressed.52,108
What Are the Important Cytokines and Growth
Factors?
The complexity of the cytokine networks and the many
different cellular responses among resident and immigrant
cells of inflamed periodontal tissues give rise to an almost
unlimited number of putative pathogenic processes. Clearly
not all of these are equally important, but it is not yet
possible to identify those cellular interactions that actually
drive attachment loss. It is likely that IL-l plays an important role in regulating MMP expression in inflamed human gingiva. IL-la and - are present in concentrations of
10"9-10_8M109,110 which are clearly sufficient to induce

Volume 64
Number 5
Table 4. Tissue Destructive

BIRKEDAL-HANSEN

Enzymes

and Inhibitors in

Inflammatory Exudates, Saliva,

479

and Plasma

Concentration

M-g/ml

Reference

GCF

1-10

115, Estimate

Saliva

0.340

127

Plasma
GCF

0.5-0.6
0.1-0.5

31
Estimate

Fluid

Enzyme/Inhibitor
Collagenase

(PMN)

Mr 92K
Mr 72K

gelatinase
gelatinase

Plasminogen

GCF

40-200

Plasma

TIMP

Synovial

fluid

a2-Macroglobulin

Synovial

fluid

Plasma
GCF

GCF
Plasma

Estimate
140

200
0.3-4.6
1.0-1.5
0.2-1.0

128
Estimate

700-1100

143

200-2,400
2,250

143

expression of SL-1 and CL in cultured fibroblasts. (10~91010 M). IL-1 is also a potent inducer of bone resorption,

but it is not known whether this response is related to the

inductive effect of IL-1 on MMP. TNF-a is also present in
inducing concentrations in the range 0.1-13 nM.111 It is
reasonable to assume that TGF-a may also be involved,
although the concentration of TGF-a in GCF has not yet
been determined. TGF-a is a potent inducer of CL and Mr
92K GL in keratinocytes, and recent studies have shown
that the EGF-receptor (which also serves as a receptor for
TGF-a) is expressed abundantly on epithelial cells of the
oral mucosa.112
MMP Are Expressed by Gingival/Periodontal Cells In
Vivo
Woolley and Davis113 investigated the expression of MMP
in normal or inflamed human gingiva by immunolocalization methods. FIB-CL was associated with cells in the gingival connective tissue often in close proximity of the junctional and pocket epithelia but not in any epithelial cells.
Enzyme was also associated with collagen fibrils and oc-

casionally with inflammatory infiltrates. Our own studies

have shown that only low levels of MMP are expressed in
any one section. Cells scattered throughout the connective
tissue (fibroblasts and/or macrophages) show staining of the
perinuclear region with antibodies to FIB-CL. Mr 72K GL
is not infrequently associated with blood vessels and capillaries. Even heavily-inflamed human gingiva shows only
sporadic expression of MMP with no discernible pattern of
distribution (unpublished data). In the aggregate, immunolocalization studies have proven fairly inconclusive and
have given few, if any clues, to the level of involvement
of MMP in periodontal diseases.
MMP in GCF and Saliva
GCF and saliva contain at least 2 MMP, CL, and Mr 92K
GL.114"116 CL appears to be exclusively or predominantly
of the PMN-type.117,118 The apparent absence of SL-1 and
Mr 72K GL under conditions that readily permit detection

141, 142

120, 121, 122

of Mr 92K GL leads us to believe that the Mr 92 K GL,

too, is of PMN origin. These observations suggest that PMN
leukocytes are the dominant, and perhaps the only significant, contributors of MMP in GCF. If so, the question
arises whether the enzymes are indeed released from PMNs
in the tissue or the crevicular fluid, or perhaps still inside
the cells at the time of sampling and only secondarily released as a sampling artifact. "GCF enzymes" produced
by PMN leukocytes and perhaps carried to the GCF still
inside the cells include CL, Mr 92K GL, elastase, and
myeloperoxidase. Measurement of these activities in oral
fluid samples appears to be essentially an enzyme-based
PMN count, a concern previously voiced by Cimasoni.119
Metabolites in GCF may originate from 3 different compartments: from plasma, from resident cells/stroma, and
from PMN leukocytes (Table 4). Serum components including a-2M, albumin, and Igs typically are present at
high concentrations, varying between 20% and 100% of
plasma values. a2-M concentrations, for instance, vary between 0.2-2.4 mg/ml or 10 to 1000 times higher than any
of the MMP.120123 Because of the high concentration of
a2-M, it is unlikely that any MMP exists in GCF in an
active form. This notion apparently conflicts with the find(versus latent) MMP in Periodontitis
ing of more active
126
as previously alluded to it is unbut
patients/sites,123
certain whether "active" MMP in GCF exist as such or are
generated by solubilization or lysis of PMNs.
Plasminogen is by far the most abundant proteinase in
GCF. Although the concentration of Plasminogen has not
been accurately determined, we predict (based on the concentration of other plasma proteins) that it is as high as 40
to 200 |xg/ml. By comparison, we have previously estimated that the concentration of CL may reach 10 |xg/ml,
but it is probably much lower. Since Mr 72K GL is present
in plasma at a concentration of approximately 0.5 (xg/ml,31
we also predict that this enzyme, although undetectable by
zymographic analysis, exists in GCF at a concentration of
0.1 to 0.5 |xg/ml even in the absence of any local contribution from the tissues. Mr 92K GL, presumably of PMN

480

J Periodontol

MATRIX METALLOPROTEINASES

is present in saliva in relatively high levels (340 ng/

ml).127 It is also readily detectable in GCF112 but the concentration is not known. TIMP-1 (or TIMP-2) levels in GCF
are unknown but based on the plasma concentration (1.01.5 u,g/ml)128 we estimate that TIMP concentrations of GCF
are at least 0.2 to 1.0 Lig/ml.
Metabolites that originate from the resident cell/stroma
presumably are washed away by the outward fluid flow
provided they escape local capture and elimination. It is
likely the PGE2, IL-1, and TNF-a are true metabolites released from cells indigenous to the area or perhaps newlyrecruited macrophages. Recent studies have shown, however, that triggered PMN leukocytes activate transcription
of the IL-1 gene.129 It therefore cannot be excluded that
PMN leukocytes are a major source of IL-1 in GCF.

origin,

Association of GCF MMP With Disease Severity

A number of studies have aimed at correlating MMP activities with disease severity. Several experiments have shown
that when group means are compared, CL activity increases
with disease severity (Periodontitis > gingivitis > health)
and that treatment tends to return GCF values to control
levels.115'124-126 It has not been possible to convincingly
demonstrate similar correlations for individual sites, presumably because of the high site-to-site, person-to-person,
and day-to-day variation. Neither the "level" nor the "pattern" of activity at the site seems to correlate well with
"bone loss" over a 6-month period.130,131 When monitoring a particular site over an extended period of time, the
high degree of week-to-week variation suggests that a single
measurement taken at a particular point in time has little,
if any, predictive value. Sporadic "high" values never remain high for long and do not necessarily herald impending
bone loss.130,131

May
REFERENCES
1. Woessner JF, Jr. Literature

2.

3.
4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

Role of MMP in Bone Resorption

A number of studies have suggested that osteoblasts express
FIB-CL when stimulated by bone-resorbing agents.16,56 These
observations have led to the hypothesis that osteoclastic
bone resorption is initiated by an Osteoblast response to
resorptive signals such as PTH, which includes expression
of FIB-CL and perhaps other MMP, and result in dissolution of the unmineralized collagenous osteoid layer.132
Osteoblasts later vacate the surface as newly-recruited osteoclasts move in. Osteoclasts do not appear to express
MMP, but utilize a distinct acidic cathepsin-dependent
mechanism for dissolution of mineralized matrices.133'134 It
is possible, although not yet proven, that expression of
MMP is an early key event in bone resorption, a finding
that may help explain why bone resorption is so highly
sensitive to IL-1 and TNF-a.135138

Acknowledgments
This study was supported by
DE06028.

14.

15.

16.

17.

18.

19.

NIH grants DE08228 and

20.

1993

(Supplement)

on vertebrate matrix metalloproteinases

and their inhibitors. In: Birkedal-Hansen H, Werb Z, Welgus HG,
Van Hart HE, eds. Matrix Metalloproteinases and Inhibitors. Stuttgart: Gustav Fischer Verlag; 1992:425-501.
Birkedal-Hansen H, Werb Z, Welgus HG, Van Wart HE, eds. Matrix
Metalloproteinases and Inhibitors. Stuttgart: Gustav Fischer Verlag;
1992.
Woessner JF Jr. Matrix metalloproteinases and their inhibitors in
connective tissue remodeling. FASEB J 1991; 5:2145-2154.
Birkedal-Hansen H. Matrix Metalloproteinases. A Review. In: Alvarez OA, ed. Critical Reviews in Oral Biology and Medicine. Boca
Raton, FL: CRC Press; 1993; 4:197-250.
Goldberg Gl, Marmer BL, Grant GA, Eisen AZ, Wilhelm S, He C.
Human 72-kilodalton type IV collagenase forms a complex with a
tissue inhibitor of metalloproteases designated TIMP-2. Proc Natl
Acad Sei (USA) 1989; 86:8207-8211.
Collier IE, Krasnov PA, Strongin YA, Birkedal-Hansen H, Goldberg
Gl. Alanine scanning mutagenesis and functional analysis of the
fibronectin-like collagen binding domain from human 92kDa type
IV collagenase. / Biol Chem 1992; 267:6776-6781.
Vallee BL, Auld DS. Active zinc binding sites of zinc metalloenzymes. In: Birkedal-Hansen H, Werb Z, Welgus HG, Van Wart HE,
eds. Matrix Metalloproteinases and Inhibitors. Stuttgart: Gustav
Fischer Verlag; 1992:5-19.
Goldberg Gl, Wilhelm SM, Kronberger A, Bauer EA, Grant GE,
Eisen AZ. Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein. JBiol Chem 1986; 261:6600-6605.
Stricklin GP, Bauer EA, Jeffrey JJ, Eisen AZ. Human skin collagenase:
Isolation of precursor and active forms from both fibroblast and
organ cultures. Biochemistry 1977; 16:1607-1615.
Lin HY, Wells BR, Taylor RE, Birkedal-Hansen H. Degradation of
type I collagen by rat mucosal keratinocytes. / Biol Chem 1987;
262:6823-6831.
Petersen MJ, Woodley DT, Stricklin GP, O'Keefe EJ. Production
of procollagenase by cultured human keratinocytes. / Biol Chem
1987; 262:835-840.
Moscatelli D, Jaffe E, Rifkin DB. Tetradecanoyl phorbol acetate
stimulates latent collagenase production by cultured human endothelial cells. Cell 1980; 20:343-351.
Herron GS, Banda MJ, Clark EJ, Gavrilovic J, and Werb Z. Secretion of metalloproteinases by stimulated capillary endothelial cells.
II. Expression of collagenase and stromelysin activities is regulated
by endogenous inhibitors. J Biol Chem 1986; 261:2814-2818.
Welgus HG, Campbell EJ, Bar-Shavit Z, Senior RM, Teitelbaum
SL. Human alveolar macrophages produce a fibroblast-like collagenase
and collagenase inhibitor. J Clin Invest 1985; 76:219-224.
Campbell EJ, Cury JD, Lazarus CJ, Welgus HG. Monocyte procollagenase and tissue inhibitor of metalloproteinases. / Biol Chem
1987; 262:15862-15868.
Otsuka K, Sodek J, Limeback H. Synthesis of collagenase and
collagenase inhibitors by osteoblast-like cells in culture. EurJ Biochem
1984; 145:123-129.
Quinn CO, Scott DK, Brinckerhoff CE, Matrisian LM, Jeffrey JJ,
Partridge NC. Rat collagenase. Cloning, amino acid sequence comparison, and parathyroid hormone regulation in osteoblastic cells. /
Biol Chem 1990; 265:22342-22347.
Lefebvre V, Peeters-Joris C, Vaes G. Modulation by interleukin-1
and tumor necrosis factor a of production of collagenase, tissue
inhibitor of metalloproteinases and collagen types in differentiated
and dedifferentiated articular chondrocytes. Biochim Biophys Acta
1990; 1052:366-378.
Nicholson R, Murphy G, Breathnach R. Human and rat malignanttumor-associated mRNAs encode stromelysin-like metalloproteinases. Biochemistry 1989; 28:5195-5203.
MacNaul KL, Chartrain N, Lark M, Tocci MJ, Hutchinson NI.

Volume 64
Number 5

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.
34.

35.

36.

37.

38.

Discoordinate expression of stromelysin, collagenase, and tissue inhibitor of metalloproteinases-1 in rheumatoid human synovial fibroblasts. JBiol Chem 1990; 265:17238-17245.
Kerr LD, Holt JT, Matrisian LM. Growth factors regulate transin
gene expression by c-/os-dependent and o/os-independent pathways.
Science 1988; 242:1424-1427.
Chin JR, Murphy G, Werb Z. Stromelysin, a connective tissuedegrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. / Biol Chem 1985;
260:12367-12376.
Wilhelm SM, Collier IE, Kronberger A, et al. Human skin fibroblast
stromelysin: Structure, glycosylation substrate specificity, and differential expression in normal and tumorigenic cells. Proc NatlAcad
Sei (USA) 1987; 84:6725-6729.
Matrisian LM, Bowden GT, Krieg P, et al. The mRNA coding for
the secreted protease transin is expressed more abundantly in malignant than in benign tumors. Proc NatlAcad Sei (USA) 1986; 83:94139417.
Seltzer JL, Adams SA, Grant GA, Eisen AZ. Purification and properties of a gelatin-specific neutral protease from human skin. / Biol
Chem 1981; 256:4662-4668.
Salo T, Lyons JG, Rahemtulla F, Birkedal-Hansen H, Larjava H.
Transforming growth factor-l up-regulates type IV collagenase
expression in cultured human keratinocytes. / Biol Chem 1991;
266:11436-11441.
Kalebic T, Garbisa S, Glaser B, Liotta LA. Basement membrane
collagen: Degradation by migrating endothelial cells. Science 1983;
221:281-283.
Garbisa S, Ballin M, Daga-Gordini D, et al. Transient expression
of type IV collagenolytic metalloproteinase by human mononuclear
phagocytes. / Biol Chem 1986; 261:2369-2375.
Overall CM, Sodek J. Initial characterization of a neutral metalloproteinase, active on native 3/4-colIagen fragments, synthesized by
ROS 17/2.8 osteoblastic cells, periodontal fibroblasts, and identified
in gingival crevicular fluid. J Dent Res 1987; 66:1271-1282.
Lefebvre V, Peeters-Joris C, Vaes G. Production of gelatin-degrading matrix metalloproteinases ("type IV collagenases") and inhibitors by articular chondrocytes during their dedifferentiation by serial
subcultures and under stimulation by interleukin-1 and tumor necrosis factor a. Biochim Biophys Acta 1991; 1094:8-18.
Zucker S, Lysik RM, Gurfinkel MH, et al. Immunoassays of type
IV collagenase/gelatinase (MMP-2) in human plasma. ImmunolMeth
1992; 148:189-198.
Hibbs MS, Hasty KA, Seyer JM, Kang AH, Mainardi CM. Biochemical and immunological characterization of the secreted forms
of human neutrophil gelatinase. / Biol Chem 1985; 260:24932500.
Murphy G, Ward R, Hembry RM, Reynolds JJ, Kuhn K, Tryggvason K. Characterization of gelatinase from pig polymorphonuclear
leukocytes. Biochem J 1989; 258:463^172.
Wilhelm SM, Collier IE, Manner BL, Eisen AZ, Grant GA, Goldberg GI. SV40-transformed human lung fibroblasts secrete a 92kDa
type IV collagenase which is identical to that secreted by normal
human macrophages. / Biol Chem 1989; 264:17213-17221.
Mainardi CL, Hibbs MS, Hasty KA, Seyer JM. Purification of a
type IV collagen degrading metalloproteinase from rabbit alveolar
macrophages. Coll Relat Res 1984; 4:479-492.
Seltzer JL, Akers KT, Weingarten H, Grant GA, McCourt DW,
Eisen AZ. Cleavage specificity of human skin type IV collagenase
(gelatinase). JBiol Chem 1990; 265:20409-20413.
Seltzer JL, Eisen AZ, Bauer EA, Morris NP, Glanville RW, Burgeson RE. Cleavage of type VII collagen by interstitial collagenase
and type IV collagenase (gelatinase) derived from human skin. /
Biol Chem 1989; 264:3822-3826.
Gadher SJ, Schmid TM, Heck LW, Woolley DE. Cleavage of collagen type X by human synovial collagenase and neutrophil elastase.
Matrix 1989; 9:109-115.

BIRKEDAL-HANSEN
39.

481

Welgus HG, Fliszar a, Seltzer JL, Schmid TM, Jeffrey JJ. Differential susceptibility of type X collagen to cleavage by two mammalian interstitial collagenases and 72kDa type IV collagenase. /

Biol Chem 1990; 265:13521-13527.

40. Senior R, Griffin G, Fliszar CJ, Shapiro SD, Goldberg GI, Welgus
HG. Human 92- and 72-kilodalton type IV collagenases are elastases. JBiol Chem 1991; 266:7870-7875.
41. Overall CM, Wrana JL, Sodek J. Transcriptional and post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by transforming growth factor-l in human fibroblasts. J Biol Chem 1991;
266:14064-14071.
42. Woessner JF, Taplin CJ. Purification and properties of a small latent
matrix metalloproteinase of the rat uterus. / Biol Chem 1988;
263:16918-16925.
43. Miyazaki K, Hattori Y, Umenishi F, Yasumitsu H, Umeda M. Purification and characterization of extracellular matrix-degrading metalloproteinase, matrin (Pump-1) secreted from human rectal carcinoma cell line. Cancer Res 1990; 50:7758-7764.
44. Basset P, Bellocq JP, Wolf C, et al. A novel metalloproteinase gene
specifically expressed in stromal cells of breast carcinoma. Nature
1990; 348:699-704.
45. Wahl L, Wahl SM, Mergenhagen SE, Martin GR. Collagenase production by endotoxin-activated macrophages. Proc Natl Acad Sei
(USA) 1974; 71:3598-3601.
46. Uitto V-J, Larjava H, Heino J, Sorsa T. A protease from Bacteroides
gingivalis degrades cell surface and matrix glycoproteins of cultured
gingival fibroblasts and induces secretion of collagenase and Plasminogen activator. Infect Immun 1989; 57:213-218.
47. Birkedal-Hansen H, Wells BR, Lin H-Y, Caufield PW, Taylor RE.
Activation of keratinocyte-mediated collagen (type I) breakdown by
suspected human periodontopathogen: Evidence of a novel mechanism of connective tissue breakdown. J Periodont Res 1984; 19:645650.
48. Bauer EA, Cooper TW, Huang JS, Altman J, Deuel TF. Stimulation
of in vitro human skin collagenase expression by platelet-derived
growth factor. Proc NatlAcad Sei (USA) 1985; 82:4132-4136.
49. Lyons JG, Lin H-Y, Salo T, et al. Expression of collagen-cleaving
matrix metalloproteinases by keratinocytes. Effect of growth factors
and cytokines and of microbial mediators. In: Hamada S, Holt SC,
McGhee JR, eds. Periodontal Disease: Pathogens and Host Immune
Responses. Tokyo: Quintessence Publishing Co.; 1991:291-305.
50. Edwards DR, Murphy G, Reynolds JJ, et al. Transforming growth
factor beta modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J 1987; 6:1899-1904.
51. Machida CM, Scott JD, Ciment G. NGF-induction of the metalloproteinase transin/stromelysin in PC 12 cells: Involvement of multiple protein kinases. / Cell Biol 1991; 114:1037-1048.
52. Kerr LD, Miller DB, Matrisian LM. TFG-l Inhibition of transin/
stromelysin gene expression is mediated through a Fos binding sequence. Cell 1990; 61:267-278.
53. Unemori EN, Bair MJ, Bauer EA, Amento EP. Stromelysin expression regulates collagenase activation in human fibroblasts. Dissociable control of two metalloproteinases by interferon-7. J Biol Chem
1991; 266:23477-23482.
54. Andrews HJ, Bunning RAD, Plumpton TA, Clark IM, Russell RGG,
Cawston TE. Inhibition of interleukin-l-induced collagenase production in human articular chondrocytes in vitro by recombinant
human interferon-gamma. Arthritis Rheum 1990; 33:1733-1738.
55. Delaisse J-M, Eeckhout Y, Vaes G. Bone-resorbing agents affect
the production and distribution of procollagenase as well as the activity of collagenase in bone tissue. Endocrinology 1988; 123:264276.
56. Civitelli R, Hruska KA, Jeffrey JJ, Kahn AJ, Avioli LV, Partridge
NC. Second messenger signaling in the regulation of collagenase
production by osteogenic sarcoma cells Endocrinology 1989;
124:2928-2934.
57. Jonat C, Rahmsdorf HJ, Park KK, et al. Antitumor promotion and

482

MATRIX METALLOPROTEINASES

antiinflammation: Down-modulation of AP-1 (Fos/Jun) activity by

Cell 1990; 62:1189-1204.
58. Nicholson RC, Mader S, Nagpal S, Leid M, Rochette-Egly C,
Chambon P. Negative regulation of the rat stromelysin gene promoter by retinoic acid is mediated by an AP-1 binding site. EMBO
J 1990; 9:4443^1454.
59. Frisch SM, Ruley HE. Transcription from the stromelysin promoter
is induced by Interleukin-1 and repressed by dexamethasone. / Biol
Chem 1987; 262:16300-16304.
60. Wahl L, Wahl SM, Mergenhagen SE, Martin GE. Collagenase production by endotoxin-activated macrophages. Proc Natl Acad Sei
(USA) 1974; 71:3598-3601.
61. Cury J, Campbell EJ, Lazarus CJ, Albin RJ, Welgus HG. Selective
upregulation of human alveolar macrophage collagenase production
by lipopolysaccharide and comparison to collagenase production by
fibroblasts. / Immunol 1988; 141:4306-4312.
62. Dayer JM, Beutler B, Cerami A. Cachectin/tumor necrosis factor
stimulates collagenase and Prostaglandin E2 production by human
synovial cells and dermal fibroblasts. J Exp Med 1985; 162:21632168.
63. Brenner DA, O'Hara M, Angel P, Chojkier M, Karin M. Prolonged
activation of jun and collagenase genes by tumour necrosis factora. Nature 1989; 337:661-663.
64. Overall CM, Sodek J. Concanavalin A produces a matrix-degradative phenotype in human fibroblasts. J Biol Chem 1990; 265:2114121151.
65. Springman EB, Angleton EL, Birkedal-Hansen H, Van Wart HE.
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. Proc Natt Acad
Sei (USA) 1990; 87:364-368.
66. Van Wart HE, Birkedal-Hansen H. The cysteine switch: A principle
of regulation of metalloproteinase activity with potential applicability
to the entire matrix metalloproteinase gene family. Proc Natl Acad
Sei (USA) 1990; 87:5578-5582.
67. Grant GA, Eisen AZ, Marmer BL, Roswit WT, Goldberg GI. The
activation of human skin fibroblast procollagenase. J Biol Chem
1987; 262:5886-5889.
68. Nagase H, Enghild JJ, Suzuki K, Salvesen G. Stepwise activation
of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl)mercuric acetate. Biochemistry 1990;
29:5783-5789.
69. Suzuki K, Enghild JJ, Morodomi T, Salvesen G. The activation of
tissue procollagenase by matrix metalloproteinase 3 (stromelysin).
Biochemistry 1990; 29:10261-10270.
70. Mignatti P, Robbins E, Rifkin DB. Tumor invasion through the
human amniotic membrane: requirement for a Proteinase cascade.
Cell 1986; 47:487^198.
71. He C, Wilhelm SM, Pentland AP, et al. Tissue cooperation in a
proteolytic cascade activating human interstitial collagenase. Proc
Natl Acad Sei (USA) 1989; 86:2632-2636.
72. Birkedal-Hansen H, Lin H-Y, Birkedal-Hansen B, Windsor LT, Pierson MC. Degradation of collagen fibrils by live cells. Role of expression and activation of procollagenase. In: Birkedal-Hansen H, Werb
Z, Welgus HG, Van Wart HE, eds. Matrix Metalloproteinases and
Inhibitors. Stuttgart: Gustav Fischer Verlag; 1992:368-374.
73. Peppin GJ, Weiss SJ. Activation of the endogenous metalloproteinase, gelatinase, by triggered human neutrophils. Proc Natl Acad Sei
(USA) 1986; 83:4322^1326.
74. Weiss SJ, Peppin G, Ortiz X, Ragsdale C, Test ST. Oxidative autoactivation of latent collagenase by human neutrophils. Science 1985;
227:747-749.
75. Sottrup-Jensen L. Structure, shape, and mechanism of Proteinase
complex formation. J Biol Chem 1989; 264:11539-11542.
76. Sottrup-Jensen L, Sand O, Kristensen L, Fey GH. The ot-macroglobulin bait region. Sequence diversity and localization of cleavage

glucocorticoid hormone.

May

J Periodontol
1993

(Supplement)

sites for proteinases in five mammalian a-macroglobulins. J Biol

Chem 1989; 264:15781-15789.
77. Sottrup-Jensen L, Hansen HF, Christensen U. Generation and reactivity of "nascent" a2-macroglobulin: Localization of cross-links in
a2-macroglobulin-trypsin complex. Ann NY Acad Sei 1983; 421:188208.
78. DeClerck YA, Yean TD, Lu HS, Ting J, Langley KE. Inhibition of
autoproteolytic activation of interstitial procollagenase by recombinant metalloproteinase inhibitor MI/TIMP-2. / Biol Chem 1991;
266:3893-3899.
79. Howard EW, Bullen EC, Banda MJ. Regulation of the autoactivation
of human 72-kDa progelatinase by tissue inhibitor of metalloproteinases-2. J Biol Chem 1991; 266:13064-13069.
80. Carmichael DF, Sommer A, Thompson RC, et al. Primary structure
and cDNA cloning of human fibroblast collagenase inhibitor. Proc
Natl Acad Sei (USA) 1986; 83:2407-2411.
81. Docherty AJP, Lyons A, Smith BJ, et al. Sequence of human tissue
inhibitor of metalloproteinases and its identity to erythroid-potentiating activity. Nature 1985; 318:66-69.
82. Stetler-Stevenson WG, Krutzsch HC, Liotta LA. Tissue inhibitor of
metalloproteinase (TIMP-2). A new member of the metalloproteinase inhibitor family. J Biol Chem 1989; 264:17374-17378.
83. Boone TC, Johnson MJ, DeClerck YA, Langley KE. cDNA cloning
and expression of a metalloproteinase inhibitor related to tissue inhibitor of metalloproteinases. Proc Natl Acad Sei (USA) 1990;
87:2800-2804.
84. Murphy G, Docherty AJP, Hembry RM, Reynolds JJ. Metalloproteinases and tissue damage. BrJ Rheumatol 1991; 30:25-31.
85. Welgus HG, Bauer EA, Stricklin GP. Elevated levels of human
collagenase inhibitor in blister fluids of diverse etiology. J Invest
Dermatol 1986; 258:12252-12258.
86. Stricklin GP, Welgus HG. Human skin fibroblast collagenase inhibitor. Purification and biochemical characterization. / Biol Chem 1983;
258:12252-12258.
87. Lin H-Y. Production of Collagenase by Normal Human Keratinocytes. [Thesis]. Birmingham, Alabama: University of Alabama at
Birmingham, 1987:19-20.
88. Cawston TE, Murphy G, Mercer E, Galloway WA, Hazleman BL,
Reynolds JJ. The interaction of purified rabbit bone collagenase with
purified rabbit bone metalloproteinase inhibitors. Biochem J 1983;
211:313-318.
89. Welgus HG, Jeffrey JJ, Eisen AZ, Roswit WT, Stricklin GP. Human
skin fibroblast collagenase: Interaction with substrate and inhibitor.
Coll Relat Res 1985; 5:167-179.
90. Murphy G, Koklitis P, Carne AF. Dissociation of tissue inhibitor of
metalloproteinases (TIMP) from enzyme complexes yields fully active inhibitor. Biochem J 1989; 261:1031-1034.
91. Howard EW, Bullen EC, Banda MJ. Preferential inhibition of 72
and 92-kDa gelatinases by tissue inhibitor of metalloproteinases-2.
JBiol Chem 1991; 266:13070-13075.
92. DeClerck YA, Yean TD, Ratzkin BJ, Lu HS, Langley KE. Purification and characterization of two related but distinct metalloproteinase inhibitors secreted by bovine aortic endothelial cells. / Biol
Chem 1989; 264:17445-17453.
93. Stetler-Stevenson WG, Krutzsch HC, Liotta LA. Tissue inhibitor of
metalloproteinase (TIMP-2). A new member of the metalloproteinase inhibitor family. JBiol Chem 1989; 264:17374-17378.
94. Stetler-Stevenson WG, Brown PD, Onisto M, Levy AT, Liotta LA.
Tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA expression
in tumor cell lines and human tumor tissues. J Biol Chem 1990;
265:13933-13938.
95. Clark SD, Kobayashi DK, Welgus HG. Regulation of the expression
of tissue inhibitor of metalloproteinases and collagenase by retinoids
and glucocorticoids in human fibroblasts. J Clin Invest 1987; 80:12801288.
96. Coulombe B, Skup D. In vitro synthesis of the active tissue inhibitor

Volume 64
Number

5_BIRKEDAL-HANSEN

of metalloproteinases encoded by a complementary DNA from virusinfected murine fibroblasts. J Biol Chem 1988; 263:1439-1443.
97. Nomura S, Hogan BLM, Wills AJ, Heath JK, Edwards DR. Developmental expression of tissue inhibitor of metalloproteinases (TIMP)
RNA. Development 1989; 105:575-584.
98. Mawatari M, Kohno K, Mizoguchi H, et al. Effects of tumor necrosis factor and epidermal growth factor on cell surface morphology, cell surface receptors, and the production of tissue inhibitor of
metalloproteinases and IL-6 in human microvascular endothelial cells.
J Immunol 1989; 143:1619-1627.
99. Hibbs MS, Bainton DF. Human neutrophil gelatinase is a component
of specific granules. / Clin Invest 1989; 84:1395-1402.
100. Huhtala P, Chow LT, Tryggvason K. Structure of the human type
IV collagenase gene. J Biol Chem 1990; 265:11077-11082.
101. Huhtala P, Tuuttila A, Chow LT, Lohi J, Keski-Oja J, Tryggvason
K. Complete structure of the human gene for 92-kDa type IV
collagenase. Divergent regulation of expression for the 92- and 72kilodalton enzyme genes in HT-1080 cells. / Biol Chem 1991;
266:16485-16490.
102. Auble DT, Brinckerhoff CE. The AP-1 sequence is necessary but
not sufficient for phorbol induction of collagenase in fibroblasts.
Biochemistry 1991; 30:4629-4635.
103. Sirum-Connolly K, Brinckerhoff CE. Interleukin-1 or phorbol induction of the stromelysin promoter requires an element that cooperates with AP-1. Nucleic Acids Res 1991; 19:335-341.
104. Gutman A, Wasylyk B. The collagenase gene promoter contains a
TPA and oncogene-responsive unit encompassing the PEA3 and AP1 binding sites. EMBO J 1990; 9:2241-2246.
105. McDonnell SE, Kerr LD, Matrisian LM. Epidermal growth factor
stimulation of stromelysin mRNA in rat fibroblasts requires induction of proto-oncogenes c-fos and c-jun and activation of protein
kinase C. Mol Cell Biol 1990; 10:4284-4293.
106. Angel P, Karin M. Specific members of the Jun protein family
regulate collagenase expression in response to various extracellular
stimuli. In: Birkedal-Hansen H, Werb Z, Welgus HG, Van Wart
HE, eds. Matrix Metalloproteinases and Inhibitors. Stuttgart: Gustav Fischer Verlag; 1992:156-164.
107. McCarthy JB, Wahl SM, Rees J, Olsen CE, Sandberg AL, Wahl
LM. Mediation of macrophage collagenase production by 3'-5' cyclic
adenosine monophosphate. / Immunol 1980; 124:2405-2409.
108. Takahashi S, Ito A, Nagino Y, Mori Y, Xie B, Nagase H. Cyclic
adenosine 3',5'-monophosphate suppresses interleukin-1-induced
synthesis of matrix metalloproteinases but not tissue inhibitor of
metalloproteinases in human uterine cervical fibroblasts. J Biol Chem
1991; 166:19894-19899.
109. Masada MP, Persson R, Kenney JS, Lee SW, Page RC, Allison
AC. Measurement of interleukin-la and -I in gingival crevicular
fluid: Implications for the pathogenesis or periodontal disease. /
Periodont Res 1990; 25:156-163.
110. Offenbacher S, Heasman PA, Collins JG. Modulation of host PGE2
secretion as a determinant of periodontal disease expression. J Periodont 1993; 64(suppl.):432-444.
111. Rossomando EF, White LB. A novel method for the detection of
TNF-alpha in gingival crevicular fluid. / Periodontal 1993; 64(suppl):
445^149.
112. Nordlund L, Hormia M, Saxen L, Thesleff I. Immunolohistochemical localization of epidermal growth factor receptors in human gingival epithelia. J Periodont Res 1991; 26:333-338.
113. Woolley DE, Davies RM. Immunolocalization of collagenase in
periodontal tissues. / Periodont Res 1981; 16:292-297.
114. Gangbar S, Overall CM, McCulloch AG, Sodek J. Identification of
polymorphonuclear leukocyte collagenase and gelatinase activities
in mouthrinse samples: Correlation with periodontal disease activity
in adult and juvenile Periodontitis. J Periodont Res 1990; 25:257267.
115. Villela B, Cogen RB, Bartolucci AA, Birkedal-Hansen H. Collagenolytic activity in crevicular fluid from patients with chronic adult

483

Periodontitis, localized juvenile Periodontitis and gingivitis, and from

healthy control subjects. J Periodont Res 1987; 22:381-389.

S, Kulkarni G, et al. Collagenase activity in recurrent

Periodontitis; Relationship to disease progression and doxycycline
therapy. J Periodont Res 1991; 26:479-485.
Sorsa T, Uitto V-J, Suomolainen M, Vauhkonen M, Lindy S. Comparison of interstitial collagenases from human gingiva, sulcular fluid
and polymorphonuclear leukocytes. J Periodont Res 1988; 23:386-

116. Lee W, Aitken

117.

393.
118. Uitto V-J, Suomolainen K, Sorsa T. Salivary collagenase. Origin,
characteristics and relationship to periodontal health. J Periodont
Res 1990; 25:135-142.
119. Cimasoni G. Crevicular Fluid Updated. In: Myers HM, ed. Monographs in Oral Science, vol. 12. Basel: S. Karger; 1983.
120. Schenkein HA, Genco RJ. Gingival fluid and serum in periodontal
diseases. I. Quantitative study of Immunoglobulins, complement
components, and other plasma proteins. J Periodontol 1977; 48:772-

777.
121. Tollefsen T, Saltvedt E. Comparative analysis of gingival fluid and
plasma by crossed immunoelectrophoresis. J Periodont Res 1980;
15:96-106.
122. Condacci I, Cimasoni G, Ahmad-Zadeh C. a2-macroglobulin in
sulci from healthy and inflamed human gingivae. Infect Immun 1982;
36:66-71.
123. Sengupta S, Lamster IB, Knocht A, Duffy TA, Gordon JM. The
effect of treatment on IgG, IgA, IgM and a-2-macroglobulin in
gingival crevicular fluid from patients with chronic adult Periodontitis. Arch Oral Biol 1988; 33:425-431.
124. Kryshtalskyj E, Sodek J. Nature of collagenolytic enzyme and inhibitor in crevicular fluid from healthy and inflamed periodontal
tissues of beagle dogs. / Periodont Res 1987; 22:264-269.
125. Kryshtalskyj E, Sodek J, Ferrier JM. Correlation of collagenolytic
enzymes and inhibitors in gingival crevicular fluid with clinical and
microscopic changes in experimental Periodontitis. Arch Oral Biol
1986; 31:21-31.
126. Larivee J, Sodek J, Ferrier JM. Collagenase and collagenase inhibitor activities in crevicular fluid of patients receiving treatment
for localized juvenile Periodontitis. J Periodont Res 1986; 21:702715.
127. Davis GA. Identification of an abundant latent 94kDa gelatin-degrading metalloproteinase in human saliva which is activated by acid
exposure: Implications for a role in digestion of collagenous proteins. Arch Biochem Biophys 1991; 286:551-554.
128. Cawston T, McLaughlin P, Coughlan R, Kyle V, Hazleman B.
Synovial fluids from infected joints contain metalloproteinase-tissue
inhibitor of metalloproteinase (TIMP) complexes. Biochim Biophys
Acta 1990; 1033:96-102.
129. Marucha PT, Zeff RA, Kreutzer DL. Regulation of IL-l gene
expression in human peripheral blood PMN. / Periodont Res 1991;
26:264-267.
130. Cogen RB, Heaven T, Jeffcoat M, Birkedal-Hansen H. Periodontal
disease activity determined by digital subtraction radiography. / Dent
Res 1989; 68(spec. issue):(Abstr. 111).
131. Birkedal-Hansen H, Pierson M, Heaven T, Jeffcoat M, Cogen RB.
Bone loss, GCF collagenase and GCF flow in human Periodontitis.
J Dent Res 1989; 68(spec. issue):(Abstr. 1221).
132. Vaes G, Delaisse J-M, Eeckhout Y. Relative roles of collagenase
and lysosomal cysteine-proteinases in bone resorption. In: BirkedalHansen H, Werb Z, Welgus HG, Van Wart HE, eds. Matrix Metalloproteinases and Inhibitors. Stuttgart: Gustav Fischer Verlag;
1992:383-388.
133. Blair HC, Kahn AJ, Crouch EC, Jeffrey JJ, Teitelbaum SL. Isolated
osteoclasts resorb the organic and inorganic components of bone. /
Cell Biol 1986; 102:1164-1172.
134. Vaes G. Cellular biology and biochemical mechanism of bone resorption. Clin Orthop 1988; 231:239-271.
135. Gowen M, Mundy GR. Actions of recombinant interleukin 1, in-

J Periodontol
484

136.

137.
138.

139.

140.

May 1993 (Supplement)

MATRIX METALLOPROTEINASES

terleukin 2, and interferon--y on bone resorption in vitro. / Immunol

1986; 136:2478-2482.
Bertolini DR, Nedwin GE, Bringman TS, Smith DD, Mundy GR.
Stimulation of bone resorption and inhibition of bone formation in
vitro by human tumour necrosis factors. Nature 1986; 319:516-518.
Saklatvala J. Tumour necrosis factor a stimulates resorption and inhibits
synthesis of proteoglycan in cartilage. Nature 1986; 322:547-549.
Heath JK, Saklatvala J, Meikle MC, Atkinson SJ, Reynolds JJ. Pig
interleukin-1 (catabolin) is a potent stimulator of bone resorption in
vitro. Calcif Tissue Int 1985; 37:95-97.
Nagase H, Barrett AJ, Woessner JF, Jr. Nomenclature and glossary
of the matrix metalloproteinases. In: Birkedal-Hansen H, Werb Z,
Welgus HG, Van Wart HE, eds. Matrix Metalloproteinases and
Inhibitors. Stuttgart: Gustav Fischer Verlag; 1992:421-424.
Deutsch DG, Mertz ET. Plasminogen: Purification from human plasma
by affinity chromatography. Science 1970; 170:1095-1096.

141.

Hayakawa T, Yamashita K, Kodama S, Iwata H, Iwata K. Tissue

inhibitor of metalloproteinases and collagenase activity in syno-

vial fluid of human rheumatoid arthritis. Biomed Res 1991; 12:169

173.
142. Cawston TE, McLaughlin P, Hazleman BL. Paired serum and synovial fluid values of a2-macroglobulin and TTMP in rheumatoid
arthritis. BrJRheumatol 1987; 26:354-358.
143. Virca GD, Mallya RK, Pepys MB, Schnebli HP. Quantitation of
human leukocyte elastase, cathepsin G, ct-2-macroglobulin and ct1-proteinase inhibitor in Osteoarthritis and rheumatoid arthritis synovial fluids. Adv Exp Med Biol 1982; 167:345-353.
Send reprint requests to: Dr. Henning Birkedal-Hansen, Department of
Oral Biology, University of Alabama School of Dentistry, University of
Alabama at Birmingham, UAB Station, Birmingham, AL 35294.