269

Molecular and Cellular Biochemistry 253: 269–285, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Regulation of matrix metalloproteinases:An overview

Sajal Chakraborti, Malay Mandal, Sudip Das, Amritlal Mandal andTapati ChakrabortiDepartment of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, India

Abstract

Matrix metalloproteinases (MMPs) are a major group of enzymes that regulate cell-matrix composition. MMP genes show ahighly conserved modular structure. Ample evidence exists on the role of MMPs in normal and pathological processes, includ-ing embryogenesis, wound healing, inflammation, arthritis, cardiovascular diseases, pulmonary diseases and cancer. The ex-pression patterns of MMPs have interesting implications for the use of MMP inhibitors as therapeutic agents. Insights mightbe gained as to the preference for a general MMP inhibitor as opposed to an inhibitor designed to be specific for certain MMPfamily members as it relates to a defined disease state, and may give clues to potential side effects. The signalling pathwaysthat lead to induction of expression of MMPs are still incompletely understood, but certain patterns are beginning to emerge.Regarding inhibition of MMP expression at the level of kinase pathways, it is possible that selective chemical inhibitors fordistinct signalling pathways (e.g. MAPK, PKC) will hopefully, soon be available for initial clinical trials. Overexpression ofselective dual specificity MAPK phosphatases have been shown to prevent MMP promoter activation which could also be usedas a novel strategy to prevent activation of AP-1 and ETS transcription factors and MMP promoters in vivo. Interactions be-tween members of different transcription factors provide fine-tuning of the transcriptional regulation of MMP promoter activ-ity. MMPs play a crucial role in tumor invasion. Although the expression of MMPs in malignancies has been studied widely,the specific role of distinct MMPs in the progression of cancer may be more complex than has been assumed. For example, ithas recently been shown that MMP-3, MMP-7, MMP-9 and MMP-12 can generate angiostatin from plasminogen, indicatingthat their expression in peritumoral area may in fact serve to limit angiogenesis and thereby inhibit tumor growth and invasion.The recent view about the role of stromal cells in the progression of cancer cell growth and metastasis is particularly interest-ing, and additional studies about the regulation of MMP gene expression and activity in malignancies are needed to understandthe role and regulation of MMPs in tumor cell invasion. (Mol Cell Biochem 253: 269–285, 2003)

Key words: matrix metalloproteinases, tissue inhibitors of metalloproteinases, transcriptional regulation, transcription factors,gene expression, nitric oxide, mitogen activated protein kinases

Abbreviations: MMP – matrix metalloproteinase; TIMP – tissue inhibitor of metalloproteinase; TS2 – thrombospondin-2; LRP– lipoprotein receptor-related protein; uPA – urokinase type plasminogen activator; tPA – tissue type plasminogen activator;MT-MMP – membrane type matrix metalloproteinase; MAPK – mitogen activated protein kinase; PKC – protein kinase C;ERK – extracellular signal activated protein kinase; MEK – mitogen activated ERK activating kinase; SAPK – stress activatedprotein kinase; JNK – c-Jun activated protein kinase; NO – nitric oxide; eNOS – endothelial nitric oxide synthetase; DETA NONOate– (2,2′-hydroxy nitroso hydrazino) bis-ethanamine; Pln – plasminogen; SMCs – smooth muscle cells; MΦ – macrophages; HFC– human fibroblast collagenase; HNC – human neutrophil collagenase

Address for offprints: S. Chakraborti, Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India(E-mail: s_chakraborti@hotmail.com)

270

Introduction

The interaction of cells with extracellular matrix (ECM) arecritical for the normal development and function of organ-isms. Modulation of cell-matrix interactions occurs throughthe action of unique proteolytic systems responsible for hy-drolysis of a variety of ECM components. By regulating theintegrity and composition of the ECM structure, these enzymesystems play a pivotal role in the control of signals elicitedby matrix molecules, which regulate cell proliferation, dif-ferentiation, and cell death. The turnover and remodeling ofECM must be highly regulated since uncontrolled proteoly-sis contributes to abnormal development and to the generationof many pathological conditions characterized by excessivedegradation of ECM components [1–4]. Matrix metallo-proteases (MMPs) are a major group of enzymes that regu-late cell-matrix composition. The MMPs are zinc-dependentendopeptidases known for their ability to cleave one or sev-eral ECM constituents, as well as non matrix proteins. Theycomprise a large family of protease that share common struc-tural and functional elements and products of different genes[4]. Table 1 summarizes the classifications of the MMPs andshows their distribution within the human genome.

The MMPs are homogeneous enzymes, however, their struc-tures vary depending upon which domains are present [5–7].All members of this family contain a propeptide and a cata-lytic domain. The catalytic domain (~ 100 amino acids) con-tains the catalytic machinery including the zinc binding siteand a conserved methionine. This domain contains addi-tional zinc and calcium ions which maintain the three di-mensional structure of MMPs and are necessary for stabilityand enzymatic activities. Stromeolysin-1, stromeolysin-2and interstitial collagenase has an added hemopexin-like do-main on the C-terminal end. Gelatinases A and B have theC-terminal hemopexin-like domain between the active en-zyme and the zinc binding sites. Gelatinase B also has a typeV collagen-like domain between the zinc binding domain andthe hemopexin domain [8]. Substrate specificity differs amongthese enzymes. Substrate specificities, chromosomal localiza-tion and domain structure of MMPs are illustrated in Table 1and Fig. 1.

MMP genes show a highly conserved modular structure(Fig. 2). The collagenase (CL), stromelysin-1 (SL-1), andstromeolysin-2 (SL-2) genes each contain ten exons and nineintrons in 8–12 kbp of DNA [9, 10]. 72 kDa gelatinase (72 kDaGL) and 92 kDa GL genes are considerably larger (26–27 kbp)and contain three additional exons, which encodes the threefibronectin type II domains. The extended hinge region of 92kDa GL is encoded entirely in exon 6 [11]. The CL and SL-1genes are located on the long arm of chromosome 11 [12]; 72kDa GL gene is located on chromosome 16 (Fig. 2) [13].

Most cells synthesize and immediately secrete matrix met-alloproteases into the extracellular matrix [5]. Inflammatory

cells, however, store proteases of this class (i.e. neutrophilcollagenase and gelatinase B). Tissue distribution of theseproteases varies widely. Some are constitutively synthesized(e.g. 72 kDa gelatinase) by many cells, while others are syn-thesized mainly upon stimulation (e.g. collagenase) [14, 15].

Ample evidence exists on the role of MMPs in normal andpathological processes, including embryogenesis, woundhealing, inflammation, arthritis, cardiovascular diseases andcancer. For example, maintenance of the structural integrityof the major arteries, the aorta in particular, requires that thecollagen and elastin components of the vessel walls be pro-tected from degradation. Injury to the vessel (atherosclero-sis) results in inflammatory processes thereby generatingmetalloproteases that are able to degrade this componentsresulting in vessel wall dilation and an increase in the possi-bility of rupture [16]. Breakdown of the medial connectivetissue of arterial walls is a major factor in the developmentof aneurysms. It has been demonstrated that the 92 kDagelatinase is found in high levels in abdominal aortic aneu-rysms [17]. Certain defects in the structure of the arteries maybe the result of the abnormal collagen structure, either dueto irregularities in the structure of the collagen or due tochanges in the regulatory processes, conceivably affecting thebalance between MMPs and their inhibitors [18].

Regulation of MMPs

MMP catabolism and clearance

An obvious means of regulating MMPs is via their ownproteolytic inactivation and physical clearance. Althoughconsiderable progress has been made in understanding theprogressive proteolytic processing of MMP propeptides, rela-tively little is known about the further autoproteolysis ofactive MMPs. Nevertheless, it is clear that some cleavagesinactivate MMPs whereas others, such as those that specifi-cally remove the hemopexin domain, can generate truncatedenzymes that lose their ability to cleave some substrates butretain their ability to cleave others [19]. Such processing canalso diminish their affinity for and ability to be inhibited byTIMPs, as has been found with C-terminally truncated MMP-2 [20]. Removal of the hemopexin like domain also cancelsthe ability of certain MMPs to localize to the cell surface. Inaddition, membrane type MMPs (MT-MMPs) can be secretedif they are cleaved at a juxtamembrane site before or after theyreach the cell surface [21]. Thus, factors that influence MMPdegradation can alter the steady state concentrations of MMPs,their substrate specificities, localization, and also their abil-ity to be activated or inhibited.

Thrombospondin (TS2), responsible for adhesion of mac-romolecules, has also been implicated in the clearance ofMMP2. Interestingly, TS2 deficient mice exhibit a number of

271

connective tissue abnormalities, and their fibroblasts have anadhesion defect that is the result of increased MMP2 levels[22]. The increased MMP-2 levels occur because TS2 nor-mally binds both latent and active MMP2 and TS2 is normallyendocytosed by the low density lipoprotein receptor-relatedprotein (LRP) which probably carries any bound MMP-2 withit [23]. The cellular internalization of TS2/MMP-2 complexesby the LRP scavenger receptor may, therefore, play an impor-tant role in regulating MMP-2 levels outside fibroblasts andother cells. It has been shown that MMP-13 is rapidly cleavedafter it binds to an MMP-13-specific 170 kDa high affinityreceptor present in various cell types [24]. The binding requiresCa2+, and the subsequent internalization and degradation ofMMP-13 requires LRP because LRP-null cells bind MMP-13but fail to internalize it. Moreover, the internalization of bothMMP-13 and TS2/MMP2 complexes is inhibited by the 39kDa receptor associated protein (RAP), which binds and in-hibits LRP. Thus, MMPs are tightly regulated by differentmechanisms during virtually every aspect of their life span,from their induction to their ultimate destruction [25].

Compartmentalization

An important concept is that cells do not indiscriminatelyrelease proteases. Proteinases, such as MMPs, are secretedand anchored to the cell membrane, thereby targeting theircatalytic activity to specific substrates within the pericellu-lar space. Specific cell-MMP interactions have been reportedin recent years, such as the binding of gelatinase A to theintegrin α

vβ

3 [26], binding of gelatinase B to CD44 [27], and

binding of matrilysin to surface proteoglycans [28]. Pro-gelatinase A also interacts with tissue inhibitor of metallo-proteinases TIMP-2 and MT1-MMP on the cell surface,and this trimeric complex is essential for activation of thisgelatinase [29, 30]. It is likely that other MMPs are also at-tached to cells via specific interaction to membrane proteins,and determining these anchors will lead to identifying acti-vation mechanisms and pericellular substrates.

Cells also rely on surface receptors to ‘sniff out’ the pres-ence and location of specific substrates. For matrix substrates,integrin-ligand contacts provide an unambiguous signal in-forming the cell of which protein it has encountered and,hence, which proteinase is needed and to where the enzymeshould be delivered and released. A clear example of this typeof spatial regulation is seen with collagenase 1 in human cu-taneous wounds. Collagenase 1 is induced in basal epider-mal cells (keratinocytes), in response to injury, as the cellmove off the basement membrane and contact type 1 colla-gen in the underlying dermis [31]. Only basal keratinocytesin contact with dermal type 1 collagen expressed collagenase-1, and this inductive response is specifically controlled by thecollagen binding integrin α

2β

1, which also directs secretion

of the enzyme to the points of cell-matrix contact [32]. Thissuggests that expression and activity of a specific MMP canbe confined to a specific location in an activated tissue.

Complex formation

The non-covalent bimolecular complexes formed betweenTIMP-1/TIMP-2 and catalytically active MMPs are entirelydifferent from that of the covalent complexes formed with a2-macroglobulins [33]. The complex formation has been foundto be blocked by small peptide inhibitors that act at the MMPactive sites [34]. By contrast, complexes formed with thelatent zymogens of the 72 and 92 kDa GL apparently do notinvolve the enzyme active site because the zymogens can befully activated by organomercurials while bound to the in-hibitor [35]. Based on the observation that TIMP complexeswith most activated MMP are stable in 0.1% SDS, Murphyet al. [36] and DeClerk et al. [37] developed a capture methodthat permits more detailed studies on the MMP intermediates.They demonstrated that TIMP-2 captures the nascent ‘switchopen’ form of 52 kDa proCL and prevents or retards its fur-ther autolytic conversion to the 42 kDa form. When Pln isused as the activator, 52 kDa proCL was converted to the 46kDa intermediate and was subsequently complexed with theinhibitor. Thus, further conversion to 42 kDa CL, which usu-ally follows formation of the 46 kDa intermediate, is blocked.

Activation

Degradation of extracellular matrix is a tightly controlledprocess under normal circumstances. Insufficient degrada-tion would prevent normal cell migration while excessivedegradation would result in loss of cell attachment to theECM as well as pathologic destruction of connective tissue.As the matrix metalloproteases are secreted as latent en-zymes, physiological activation becomes a critical controlpoint. Among others, plasmin and urokinase type plasmino-gen activator (uPA) and tissue-type plasminogen activator(tPA) were implicated as the important physiological acti-vators of MMPs [38].

The presence of uPA (Fig. 3) bound to a cell surface re-ceptor provides a mechanism for the cell to activate an arrayof proteases in close proximity to the cell surface with thepotential to restrict this activation to only a portion of the cellsurface. Interaction between the metalloproteinases exists andcan further enhance activity as has been suggested for strom-eolysin activation of interstitial collagenase and gelatinase B[39].

Like the plasmin/plasminogen activator system, gelatinaseA may be controlled by a cell surface associated activatorreceptor. This type of system would allow the cells to acti-

272

Tabl

e 1.

Sub

stra

te s

peci

fici

ties

,chr

omos

omal

loca

tion

s (h

uman

) an

d do

mai

n st

ruct

ure

of m

atri

x m

etal

lopr

otei

nase

s (M

MP

s)

Enz

yme

MM

PC

hrom

osom

al lo

cati

on*D

omai

nE

CM

Non

EC

MA

ctiv

ated

by

Act

ivat

or o

f(i

n hu

man

)st

ruct

ure

subs

trat

esu

bstr

ate

Col

lage

nase

s

Col

lage

nase

-1M

MP

-111

q22.

2-22

.3II

Col

lage

ns (

I, I

I, I

II, V

II, V

III

and

X),

α1-

PI,

IL

b-1,

pro

-TN

F, I

GF

BP

-3,

MM

P-3

,-10

MM

P-2

,

gela

tin,

pro

teog

lyca

n li

nk p

rote

in,

MM

P-2

, M

MP

-9pl

asm

in

aggr

ecan

, ve

risc

an,

tena

cin,

ent

acti

nka

llik

rein

,

chym

ase

Col

lage

nase

-2M

MP

-811

q22.

2-22

.3II

Col

lage

ns (

I, I

I, I

II, V

, VII

, VII

I an

d X

),α

1-P

I, α

2-an

tipl

asm

in,

fibr

onec

tin

MM

P-3

,-10

,N

D

gela

tin,

agg

reca

n p

lasm

in,

Col

lage

nase

-3M

MP

-13

11q2

2.2-

22.3

IIC

olla

gens

(I,

II,

III

, IV

, IX

, X, X

IV),

MM

P-9

, pl

asm

inog

en a

ctiv

ator

MM

P-2

,-3,

-10,

MM

P-2

,-9

gela

tin,

agg

reca

n, p

erle

can,

lar

ge t

enas

cin-

C,

inhi

bito

r-2

-14,

-15,

pla

smin

fibr

onec

tin,

ost

eone

ctin

Col

lage

nase

-4M

MP

-18

___

_II

ND

ND

ND

ND

Gel

atin

ases

Gel

atin

ase

AM

MP

-216

q13

III

Col

lage

ns (

I, I

V, V

, VII

, X, X

I an

d X

IV),

IL-1

b, α

1-P

I, p

roly

syl

oxid

ase

MM

P-1

,-7,

-13,

MM

P-9

,-13

gela

tin,

ela

stin

, fi

bron

ecti

n, l

amin

in-1

,fu

sion

pro

tein

, M

MP

-1,

MM

P-9

-14,

-15,

-16,

-17

lam

inin

-5,

gale

ctin

-3,

aggr

ecan

, de

cori

n,M

MP

-13

-24,

-25,

try

ptas

e?

hyal

uron

idas

e-tr

eate

d ve

rsic

an,

prot

eogl

ycan

link

pro

tein

, os

teon

ecti

n

Gel

atin

ase

BM

MP

-920

q12-

13IV

Col

lage

ns (

IV, V

, VII

, X,a

nd X

IV),

gel

atin

,α

1-P

I, I

L-1

β, p

lasm

inog

enM

MP

-2,-

3,-1

3,N

D

elas

tin,

gal

ecti

n-3,

agg

reca

n, f

ibro

nect

in,

plas

min

hyal

uron

idas

e-tr

eate

d ve

rsic

an,

prot

eogl

ycan

link

pro

tein

, en

tact

in,

oste

onec

tin

Stro

mel

ysin

s

Stro

mel

ysin

-1M

MP

-311

q22.

2-22

.3II

Col

lage

ns (

III,

IV

, V a

nd I

X),

gel

atin

, agg

reca

n,α

1-P

I, a

ntit

hrom

bin-

III,

ovo

ssta

tin,

Pla

smin

,M

MP

-1,-

7,-8

,

vers

ican

, hy

alur

onid

ase-

trea

ted

vers

ican

, pe

rle-

subs

tanc

e P,

IL

-1β

, ser

um a

myl

oid

kall

ikre

in,

-9,-

13

can,

dec

orin

, pro

teog

lyca

n li

nk p

rote

in, l

arge

A, I

GF

BP

-3, f

ibri

noge

n an

d cr

oss-

chym

ase

ten

asci

n-C

, fi

bron

ecti

n, l

amin

in,

enta

ctin

,li

nked

fib

rin,

pla

smin

ogen

, M

MP

-tr

ypta

se

oste

onec

tin

1 ‘s

uper

acti

vati

on’,

MM

P-1

‘sup

erac

tiva

tion

’, M

MP

-2/T

IMP

-2

com

plex

, M

MP

-7,-

8,-9

,-13

Stro

mel

ysin

-2M

MP

-10

11q2

2.2-

22.3

IIC

olla

gens

(II

I, I

V a

nd V

), g

elat

in, c

asei

n,M

MP

-1,-

8E

last

ase,

MM

P-1

,-7,

-8,

aggr

ecan

, el

asti

n, p

rote

ogly

can

link

pro

tein

cath

epsi

n G

-9,-

13

aggr

ecan

, el

asti

n, p

rote

ogly

can

link

pro

tein

Stro

mel

ysin

-3M

MP

-11

22q1

1.2

VC

asei

n, l

amin

in,

fibr

onec

tin,

gel

atin

, co

llag

enα

1-P

I, c

asei

n, I

GF

BP

-1F

urin

ND

IV a

nd c

arbo

xym

ethy

late

d tr

ansf

erri

n

Mem

bran

e ty

pe M

MP

s

MT

1-M

MP

MM

P-1

414

q12.

2V

IC

olla

gens

(I,

II

and

III)

, cas

ein,

ela

stin

,α

1-P

I, M

MP

-2,-

13P

lasm

in,

furi

nM

MP

-2,-

13

fibr

onec

tin,

gel

atin

, la

min

in,

vitr

onec

tin,

larg

e te

nasc

in-C

, en

tact

in,

prot

eogl

ycan

s

MT

2-M

MP

MM

P-1

516

q12.

2V

IL

arge

ten

asci

n-C

, fi

bron

ecti

n, l

amin

in,

MM

P-2

ND

MM

P-2

,-13

enta

ctin

, ag

grec

an,

perl

ecan

MT

3-M

MP

MM

P-1

68q

21V

IC

olla

gen-

III,

gel

atin

, ca

sein

, fi

bron

ecti

nM

MP

-2N

DM

MP

-2

MT

4-M

MP

MM

P-1

712

q24

VI

ND

ND

ND

MM

P-2

MT

5-M

MP

MM

P-2

420

q11.

2V

IN

DN

DN

DM

MP

-2

MT

6-M

MP

MM

P-2

5?

ND

ND

ND

ND

MM

P-2

273

vate gelatinase A close to or actually bound to the cell mem-brane [39]. In fact, MMP-2 was found to be activated bymembrane type MMPs (MT-MMPs). MT-MMPs {MT1-MMP(MMP-14), MT2-MMP (MMP-15), MT3-MMP (MMP-16),and MT4-MMP (MMP-17)} are expressed at low levels inmany cell types. MT1-MMPs is the most predominant andthe most clearly regulated by cytokines and growth factors[40]. However, both MT2-MMP and MT3-MMP share theability to initiate the activation of pro-MMP-2 with MT1-MMP. In contrast, MT4-MMP has negligible proMMP-2 pro-cessing activity. Besides the activation of proMMP-2, it hasbeen shown that MT1-MMP may be responsible for the ac-tivation of proMMP-13, either directly or via MMP-2 ac-tivation [41]. In both cases, processing of the prodomain ofproMMP-13 occurs via a 56 kDa intermediate, yielding a fi-nal 48 kDa form. Disposition of activated cell surface asso-ciated gelatinase-A may take place in a similar manner to thatof activated plasminogen inhibitor to form an inactive com-plex. Whether cell surface associated activator receptor mech-anisms exist for gelatinase B or other matrix metalloproteasesremain to be determined [39].

Activation of SMCs and Mφ by proinflamatory moleculesgenerated in response to atherogenic stimuli has been shownto occur during various stages of atherosclerosis. Several re-cent studies suggested that ox-LDL may promote this proc-ess [42–44]. Exposure of cultured SMCs to proinflamatorycytokines and ox-LDL alters appreciably the steady-state lev-els of MT1-MMP mRNA. The augmented MT1-MMP mRNAcorrelated with increased plasma membrane-associated im-munoactive protein and catalytic function to precursorMMP-2, as demonstrated by Western blotting and gelatinzymography. These study provided a possible mechanismunderlying the findings that IL-1-or TNF-α-stimulated hu-man saphenous vein (smooth muscle cells) SMCs producesan increase in MMP-2 level [45]. It has been suggested thatox-LDL, directly or by inducing activators such as cytokines,may influence remodeling of the ECM in atherosclerosis.Reactive oxygen species were shown to activate MMPs indifferent systems [42, 44] which suggest that proinflamatorycytokines or ox-LDL could mediate the activation of MT1-MMP by generating highly reactive oxygen species.

Cysteine switch mechanism

The sequence data of matrix metalloproteases suggest thatthere is a highly conserved cysteine residue in the proenzymedomain of each enzyme. Van Wart’s group [46] proposed acysteine switch model that is illustrated in Fig. 4. For exam-ple, Cys73 of the latent human fibroblast collagenase is co-ordinated to the active site zinc atom in a fashion that blocksthe active site. All modes of activation lead to dissociationof Cys73 from the zinc atom with concomitant exposure of theTa

ble

1.C

onti

nued

Enz

yme

MM

PC

hrom

osom

al lo

cati

on*D

omai

nE

CM

Non

EC

MA

ctiv

ated

by

Act

ivat

or o

f(i

n hu

man

)st

ruct

ure

subs

trat

esu

bstr

ate

Oth

ers

Mat

rily

sin

MM

P-7

11q2

2.2-

22.3

IC

olla

gens

IV

and

X, g

elat

in, a

ggre

can,

MM

p-1,

-2,-

9M

MP

-3,-

10M

MP

2

deco

rin,

pro

teog

lyca

n li

nk p

rote

in,

MM

P-9

/TIM

P-1

com

plex

,pl

asm

in

fibr

onec

tin,

lam

inin

, in

solu

ble

fibr

onec

tin

α1-

PI,

pla

smin

ogen

fibr

ils,

ent

acti

n, l

arge

and

sm

all

tena

scin

-C,

oste

onec

tin,

β4

inte

grin

, el

asti

n, c

asei

n,

tran

sfer

rin

Mat

rily

sin-

2M

MP

-26

?I

Col

lage

n IV

, ge

lati

n, f

ibro

nect

inP

roM

MP

-9,

fibr

inog

en,

α1-

PI

ND

ND

Met

allo

elas

tase

MM

P-1

211

q22.

2-22

.3II

Col

lage

n IV

, ge

lati

n, e

last

in,

case

in,

α1-

PI,

fib

rino

gen,

fib

rin,

ND

ND

lam

inin

, pr

oteo

glyc

an m

onom

er,

fibr

in,

plas

min

ogen

, m

yeli

n ba

sic

fibr

onec

tin,

vit

rone

ctin

, en

acti

npr

otei

n

No

triv

ial

nam

eM

MP

-19

12q1

4II

Gel

atin

ND

Try

psin

ND

Ena

mel

ysin

MM

P-2

011

q22

IIA

mel

ogen

inN

DN

DN

D

No

triv

ial

nam

eM

MP

-23

1p36

VII

IN

DN

DN

DN

D

XM

MP

(X

enop

us)

MM

P-2

1–

VII

ND

ND

ND

ND

CM

MP

(C

hick

en)

MM

P-2

2–

IIN

DN

DN

DN

D

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active site. Accordingly, when Cys73 is ‘on’ the zinc, the ac-tivity of the enzyme is ‘off’. Thus, the dissociation of Cys73

from the zinc atom is viewed as the switch that leads to acti-vation. Activation of the enzyme with aminophenyl mercu-ric acetate (APMA) causes the cysteine to become dissociatedfrom the zinc (Fig. 4) and the proteinases then undergo au-tocatalytic cleavage [47].

The cysteine-switch mechanism allows the condensationof different theories that have been advanced into a single,integrated mechanism for the activation of MMPs. The earlyobservations that latent human fibroblast collagenase (HFC)could be activated by chaotropic ions and thiol blockingagents with a concomitant loss in molecular weight led to theproposal that they were enzyme-inhibitor complexes [48].This concept was supported by the observation that the in-

Fig. 1. Schematic representation of the domain structure of MMPs. S –signal peptide; P – propeptide; C – catalytic domain; F – fibronectin type IIdomain; CP – cysteine proline rich and IL-1 receptor like domain; L – link-age domain; H – hemopexin like domain; FR – furin recognition site; T –transmembrane domain; V – vitronectin like domain. Numbers in the fig-ure indicate domain structures.

Fig. 2. Exon structures of human FIB-CL, gelatinase A, gelatinase B andrat stromelysin-1 (taken from P.A. Huhtala, A. Tuuttila, L.T. Chou, J. Lohi,J. Keski-Oja, K. Tryggvason, J Biol Chem 266: 16487–16480, 1991 withpermission).

Fig. 3. Schematic representation of the potential role of the urokinase-typeplasminogen activator (u-PA), tissue type plasminogen activator (t-PA) andplasmin in the pericellular activation cascades for matrix metalloproteinases(MMPs). The majority of MMPs may be activated by the action of plasmingenerated at the cell surface by the juxtaposition of receptor bound u-PAand t-PA and membrane bound plasminogen. The plasmin-mediated acti-vation of stromelysin is central to the cascade and is able to productivelycleave plasmin-processed collagenase and progelagenase B to yield ac-tive forms. Other MMP interactions may also occur, leading to process-ing events. Progelatinase A follows a different pathway but this is apparentlycell membrane associated, involving specific binding and proteolytic pro-cessing that may be autocatalytic. PAIs – plasminogen activator inhibitors;α

2AP – α

2 antiplasmin; TIMP – tissue inhibitor of metalloproteinases; MT1-

MMP – membrane type-1 matrix metalloproteinase. (–→, activation/con-version; --→, inhibition).

active collagenase-α2macroglobulin complexes could bedissociated to reactivate the collagenase by treatment withtrypsin or sodium thiocyanate (NaSCN) [49]. The definitiveproof that latent collagenases are not enzyme inhibitor com-plexes was exemplified by the observation that HFC is se-creted as a single proenzyme protein chain [50] and that theloss in molecular weight on activation by organomercurialswas found to be due to autolysis rather than to release of aninhibitor [51]. Equally important was the observation thattreatment with organomercurials initially led to activationwithout a decrease in molecular weight, which also suggestthat a novel, non-proteolytic means exists for activation forsome MMPs [51].

Other evidence for non-proteolytic means of activation hascome from studies of latent human neutrophil collagenase(HNC). Macarty and Tschesche [52] have made the impor-tant observation that HNC could be activated by disulfidecontaining molecules by a disulfide-exchange mechanism.Their original view of the latent enzyme was that of a disulfidebonded enzyme-inhibitor complex where activation was be-lieved to release the inhibitor [53]. While the activation bydisulfide compounds has been confirmed [54], latent HNCis no longer believed to be an enzyme inhibitor complex [54,55], and it can be activated without a requisite reduction inmolecular weight [54]. In another series of experiments,

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Weiss et al. [56] have shown that both latent HNC and thelatent 92 kDa type IV collagenase released by neutrophils canbe activated oxidatively by HOCl that is produced from H

2O

2

and Cl– by myeloperoxidase during the respiratory burst.There are also reports of the activation of latent MMP bynonenzymic tissue factors [57, 58] by unknown mechanisms.These reports represent additional examples of MMP activa-tion that are not initiated proteolytically.

While the non-proteolytic means of activation exists, it isclear that latent MMPs can also be activated by differentproteases [49, 51]. The proteolytic mechanism for this acti-vation may be that found for the activation of latent HFC bytrypsin [51, 59]. The initial event is the hydrolysis of thepropeptide domain of the 52 kDa proenzyme to yield a 46kDa species that is still inactive. This species subsequentlyactivates autolytically via loss of the propeptide domain that

contains the cysteine switch residue (Fig. 4). The autolyticcleavage site in latent HFC is probably immobilized and pro-tected from autolysis in the intact zymogens. Cleavage bytrypsin within the propeptide domain apparently triggers theexposure of this site and facilitates autolysis. Similar eventsare presumably involved in the activation of the other MMPby proteases. The mechanism of the spontaneous auto-acti-vation of the MMP is not clearly known. However, this couldbe the consequence of a number of circumstances, rangingfrom the presence of traces of residual activating proteasesto a slow inherent autolytic activity, or to a slow molecularoxygen-catalyzed oxidation of the sulfhydryl group of thecysteine switch residue. The activation of the MMP by all ofthe proteolytic and nonproteolytic means known to date canbe accounted for the cysteine switch mechanism [47].

Implications of the cysteine-switch mechanism forphysiological mechanisms of activation of MMPs

The cysteine-switch model allows flexibility in the way thatan individual MMP is activated. Thus, one MMP may be moresusceptible to activation by one mechanism than another. Forexample, while HFC is efficiently activated by trypsin [51,59], fibroblast 72 kDa type IV collagenase is poorly activatedby this proteolytic route [10]. This may have important physi-ological implications in that it may allow for the selectiveactivation of one or a small number of MMPs at certain sites.Alternatively, since each MMP may be activated by more thanone means, this could endow a given cell with flexibility inregard to the way activation is achieved. There may, in fact,be different activation mechanisms for the same MMP indifferent cells and tissues. The neutrophils are a prime exam-ple in that its oxidative burst, a key characteristic of itsphagocyte phenotype, is well suited to activate its latentMMP oxidatively. Interestingly, however, the neutrophilapparently has both oxidative and non-oxidative paths for theactivation of its 92 kDa type IV collagenase [60]. In other cellsor tissues, plasminogen activator dependent pathways may bemore appropriate. Until it can be established precisely how alatent MMP is activated under a given set of circumstances,all possible modes of activation should be considered.

Transcriptional regulation of MMPgenes

In intact organisms, degradative tasks are accomplishedboth by growth factor/cytokine-dependent and independentmechanisms. Among the members of the MMP gene family,two pairs of enzymes (PMN and FIB-CL, 72 and 92 kDaGL) have been identified with almost identical substratespecificity but with different transcriptional regulation. One

Fig. 4. Cysteine switch mechanism for activation of metalloproteinases.Cysteine in the proenzyme domain (Fig. 1) contacts zinc to maintain latencyof the enzymes. Physical agents such as sodium dodecyl sulfate (SDS) orchaotropic agents can unfold the structure to expose zinc. Reagents that re-act with sulfhydril groups N-ethylmaleimide (NEM), oxidized glutathione(GSSG), hypochlorous acid (HOCl) and organomercurials such as amino-phenyl mercuric acetate (APMA) will inactivate the cysteine. Alternatively,proteolytic enzymes can cleave the propeptide, even ahead of cysteine. In asecond step, then active forms can be autocatalytically cleaved by the acti-vated metalloproteinases to remove the propeptide and confer permanentactivity (taken from E.B. Springman, E.L. Angleton, H. Birkedel-Hansen, H.E.van Wart, Proc Natl Acad Sci USA 87: 364–368, 1990 with permission).

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member of each pair responds to growth factors and cytokineswhereas the other one does not. Growth factor-responsiveMMPs (FIB-CL, SL-1, SL-3, and 92 kDa GL) are regulatedby closely related mechanisms. In contrast, the 72 kDa GLis widely expressed by most cell types, and appears to be onlymarginally induced or repressed by the growth factors andcytokines [61, 62]. The regulation of expression of MMP inPMN is uniquely different from that of other cell types. Syn-thesis of PMN-CL and 92 kDa GL is already completed bythe time the PMN enters the vasculature, and any furtherregulation is mediated by granule release rather than by tran-scriptional events.

Stimulation or repression of growth factor and cytokine-responsive MMP genes in many cases results in 20–50 foldchanges in mRNA and protein levels. For example, transcrip-tion of the FIB-CL and SL-1 genes (and in some cells the SL-3 and 92 kDa GL genes as well) is induced by IL-1β, TNF-α,PDGF, TGF-α, EGF, bFGF, and nerve growth factor (NGF)and with few exceptions [61] abrogated by TGF-β.

Normal MMP gene expression

The dramatic over expression of members of the matrix de-grading metalloproteinase (MMP) family in pathologicalconditions characterized by connective tissue destruction, asevidenced by diseases such as arthritis, atherosclerosis, peri-odontitis, and cancer, have suggested that tight regulation ofMMP genes is critical for normal tissue homeostasis. An un-derstanding of the molecular mechanisms controlling MMPgene expression under normal and diseased conditions may,therefore, provide clues for the eventual rational therapeuticinterventions [63].

The regulation of MMP genes in normal tissues has yet tobe thoroughly examined, but initial studies point to complexand highly individualized patterns of expression for the vari-ous members of the MMP family. Examples of cell type- andtissue-specific regulation, inducible and constitutive expres-sion, discrepancies between in vitro and in vivo patterns ofexpression add to the complexity. Although the field is stillevolving, certain generalities are emerging from the availabledata [63].

Expression of most MMPs is normally low in tissues andis induced when remodeling of ECM is required. MMP geneexpression is primarily regulated at the transcriptional level,but there is also evidence about modulation of mRNA sta-bility of stromelysine, collagenase and gelatinase A in re-sponse to growth factors and cytokines [62, 64]. Analysis ofthe promoter sequence of several MMP genes (such as MMP-1, MMP-3, MMP-7, MMP-9, MMP-10, MMP12 and MMP-13) appeared to provide insights in to the similarities on theexpression patterns of different MMP family members (Fig.5) [63].

The human gelatinase A promoter has several of the char-acteristics of a housekeeping, or constitutive promoter [11](Fig. 5). This observation may help to explain the widespreadexpression of gelatinase A. Increased expression of otherMMPs, the promoter regions of which do not contain con-served cis elements (such as MMP-2 and MMP-11), have alsobeen observed in malignancies, indicating overlapping mech-anisms in the regulation of expression of these genes.

AP-1 transcription factors and MMP gene expression

Several growth factors and cytokines-mediated pathwaysconverge at the AP-1 binding site, which also constitutes thephorbol ester-responsive element (TRE) [65, 66]. AP-1 com-plexes are heterodimers of proteins of the two proto-oncogenefamilies (jun and fos) is found at approximately –70 bp in thepromoter region of each inducible MMP gene (Fig. 5). AP-1transcription factors are leucine zipper proteins that binds to a

Fig. 5. Regulatory elements of promoter regions of human MMP genes.Boxes indicate the following cis elements. AP-1 – activator protein-1; PEA3– polyoma enhancer A binding protein-3; TIE – TGF-β inhibitory element;GC – Sp-1 binding site; SBE – SIAT binding element; c/EBP-β – CCATT/enhancer binding protein-β; OSE-2 – osteoblast specific element-2; SPRE– stromelysin-1 PDGF responsive element; TRE – octamer binding protein;Sil – silencer sequence; NF-kβ – nuclear factor kβ; NF-1 – nuclear factor-1; RARE – retinoic acid responsive element (taken from J. Westermarck,V-M. Kahari, FASEB J 13: 781–792, 1999 with permission).

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consensus DNA sequence (5′-TGAG/CTCA-3′) as a dimericcomplex [67, 68]. AP-1 binding sequences have been iden-tified for the FIB-CL, SL-1 and 92 kDa GL genes but aremissing in the 72 kDa gene [66]. Different AP-1 dimers bindDNA with different affinities, which is thought to be at leastpartly responsible for the diverse biological effects of distinctAP-1 complexes. Oncogene and phorbol ester-mediated in-duction of FIB-CL proceeds along a c-fos dependent path-way [69] as does the induction of SL-1 by PDGF but not byEGF [70].

The proximal AP-1 element located between –72 and –66plays a major role in the transcriptional regulation of MMP-1 gene expression, as shown by the results that mutation ofthis element dramatically reduces the basal activity and re-sponsiveness of the MMP-1 promoter to external stimuli [71–73]. The importance of additional AP-1 elements found in thepromoters of MMP-1, -3, and -9 is not clear. It has been shownthat in the rabbit MMP-1 promoter, they bind fos and juncontaining AP-1 dimers and partially mediate the effect ofphorbol esters [74].

Sirum and Brinckerhoff have [75] identified a putativeAP-1 binding site in human fibroblast SL-2 promoter. Theyhave suggested that the single base change at position 5 (Aor G) and perhaps a substitution of T for A in position 9 im-mediately following the consensus sequence might accountfor the apparent lack of response of this promoter to TPAin fibroblasts. The promotor region of the human keratino-cyte SL-2 gene has been found to be highly responsive toTPA while the AP-1 site is necessary but not a sufficientelement for transcriptional activation of FIB-CL/SL-1 genes[76].

Several co-transfection studies have shown that over ex-pression of jun and fos proteins enhance MMP-1 promoteractivity. Furthermore, it has been shown that simultaneousinduction of c-jun and jun-B mRNAs precedes induction ofMMP-1 gene expression by several types of stimuli [74, 77].c-jun is capable of inducing the minimal MMP-1 promoteractivity as a Jun/Jun homodimer, whereas induction of mini-mal (76 bp) MMP-1 promoter by jun-B requires the presenceof several AP-1 elements [78, 79]. However, in HT-1080 fi-brosarcoma cells, in which c-jun expression is not inducedby tumor promoter okadaic acid, jun-B containing AP-1 com-plexes mediate the activation of the full-length (3.8 kb) MMP-1 promoter [80]. It has also been shown that in NIH-3T3fibroblasts, over expression of junB alone does not stimulateMMP-1 promoter activity [73]. C-Jun has been determinedto be an independent activator of MMP-1 gene expression,whereas trans-activation of MMP-1 promoter by jun-B, andpossibly by other AP-1 transcription factors, is dependent onthe interaction with other transcription factors binding toadditional regulatory cis-elements in the 5′-flanking regionof the MMP-1 gene.

Recent studies with c-fos knockout mice revealed that c-fos is necessary for malignant and invasive progression ofskin papillomas and for induction of mouse MMP-3 and MMP-13 gene expression by platelet-derived growth factor and epi-dermal growth factor, but not by phorbol esters [81, 82]. Inaddition, over-expression of c-fos in transgenic mice under aninterferon-inducible promoter was shown to induce expressionof mouse MMP-13 in thymus, spleen, and predominantly inbone indicating that the capacity of c-fos to activate mouseMMP-13 gene expression in vivo is cell type specific [83]. Thefinding that neither mouse MMP-9, MMP-3, nor MMP-10expression was affected by c-fos over expression suggests thatc-fos differently regulates expression of distinct MMP genesin vivo and that the AP-1 element alone does not determine theinducibility of the MMP promoter by c-fos in vivo [83].

Little is known about the regulation of AP-1 activity inmalignant tumors in vivo. Increased expression of AP-1 geneshas been reported during growth of malignant tumors, butthere is no consistent pattern of AP-1 complexes that wouldserve as a marker for increased invasion or malignancy. De-creased expression of c-jun, junB, and c-fos genes were ob-served in human lung carcinomas as compared with normaltissue [84]. As the expression of different AP-1 componentsat sites of MMP expression during tumor invasion is notknown, determination of the specific AP-1 complex patternresponsible for induction of MMP gene expression in vivowould be important in developing specific approaches toprevent tumor invasion and metastasis [85].

Dexamethasone-induced suppression of alveolar macro-phage metalloproteinases was found to occur at a pre-trans-lational level. It has been shown that the transcriptionallycontrolled steroid regulates stromelysin expression at thetranscriptional level in human fibroblasts [86]. The mecha-nism of glucocorticoid induced transcriptional suppressionhas been suggested to be involved in binding the glucocorti-coid receptor complex to the activating protein jun [87–89].Thus, jun is unable to bind to AP-1 site on the collagenasepromoter region and consequently is unable to stimulate tran-scription of collagenase mRNA. In addition, it has been foundthat dexamethasone-mediated inhibition of metalloproteinasebiosynthesis was readily reversible after a 24-h washout pe-riod of the drug. It has, therefore, been suggested that gluco-corticoids inhibit expression of metalloproteinases and thusmay significantly limit tissue damage associated with in-flammatory and other activating stimuli. However, gluco-corticoids also down-regulate matrix production by residentcells of connective tissues, especially fibroblasts [90, 91]. Infact, the net effect of glucocorticoids is to diminish extracel-lular matrix accumulation. Thus, although glucocorticoidsmay reduce extracellular matrix damage associated with in-flammation, this effect is achieved at considerable expenseto the subsequent healing process.

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ETS transcription factors in the regulation of MMPexpression

Conserved polyoma enhancer A binding protein-3 (PEA3)elements that bind members of ETS transcription factors havebeen found in almost all inducible MMP promoters; with theexception of the MMP-12 promoter, they are located adja-cent to at least one AP-1 element (Fig. 5). ETS transcriptionfactors are helix-turn-helix proteins that share a modular do-main structure characterized by a highly conserved ETSdomain, which recognizes the purine rich PEA3 element A/CGGAA/T [92]. Although ETS proteins were shown to trans-active artificial promoter constructs containing only thePEA3 element, they do not usually dimerize and bind toDNA alone, but prefer to form complexes with other tran-scription factors, e.g. AP-1, for which they function as co-activators [92, 93].

The functional interplay between AP-1 and ETS factorsin the regulation of MMP gene expression revealed that invivo interactions between the distinct transcription factorsmay modulate the response of MMP promoters especiallyin situations where simultaneous induction of the expres-sion of both of them occurs, such as tumor cell growth andinvasion [85].

Second messenger signaling

The signaling pathways that lead to induction of expressionof MMPs are still incompletely understood, but certain pat-terns are beginning to emerge. Recent studies have suggestedthat protein kinase C (PKC), the major cellular receptor forphorbol esters such as TPA, acts as an important messengerfor the transcriptional regulation of growth factor-responsiveMMP genes [94]. However, PKC does not appear to be in-volved in all cases. For instance, SL-1 induction by nervegrowth factor (NGF) in rat PC12 pheochromocytoma cellsrequires multiple protein kinases acting on a number of postreceptor steps but probably not PKC [95]. Moreover, okadaicacid, a non TPA-type tumor promoter that does not activatePKC but induces ‘apparent’ activation of protein kinases byinhibition of protein phosphatases, also induces FIB-CL ex-pression through an AP-1-dependent pathway [96]. The roleof 3′-5′ cyclic adenosine monophosphate (cAMP) remainsenigmatic in that a rise in cytoplasmic cAMP in some sys-tems leads to stimulation of MMP expression and in othersto repression.

Recent studies suggest that cAMP alter activities of a dif-ferent transcriptional machinery than either TPA or growthfactors/cytokines. A rise in intracellular cAMP induces tran-sient expression of jun-B, whereas both c-jun and FIB-CL arerepressed. JunB, like c-jun, is capable of binding to AP-1 sitebut fails to activate the FIB-CL promoter [97].

Mitogen activated protein kinases

DNA binding and trans-activation capacity of both AP-1 andETS transcription factors are regulated by phosphorylationby mitogen-activated protein kinases (MAPKs) and serine/threonine kinases that mediate signals from cell membranereceptors triggered by growth factors, cytokines, hormones,and cell-cell and cell-matrix interactions [98–100]. ThreeMAPK pathways have been characterized in detail (Fig. 6).The ERK1,2 pathways are activated by a large variety ofmitogens and by growth factors whereas the ERK5,6 path-ways are mainly stimulated by oxidative and osmotic stresses[99, 100]. In contrast, environmental stresses and inflam-matory cytokines predominantly triggers JNK/SAPK andp38MAPK pathways (Fig. 6).

It is thought that the balance between distinct MAPKpathways regulates cell growth, differentiation, survival,and death. Constitutively active mutants of Raf-1 and MEK1have been shown to transform fibroblasts in vitro [101, 102].Activation of ERK1,2 pathways has been observed in renaland breast carcinomas [103]. Furthermore, the full transfor-mation capacity of the Ras oncogene was shown to requireactivation of stress-activated MAPKs indicating a functionalinterplay between mitogen and stress-activated MAPK path-ways during the transformation process [104].

Transcriptional activity and c-jun protein stability is in-creased by phosphorylation of serines 63 and 73 by JNK/SAPK, whereas phosphorylation of threonines 91 and 93 hasbeen shown to cause an increase in c-jun DNA binding [98].

Fig. 6. Schematic presentation of mammalian mitogen activated proteinkinase pathways for MMP expression and activity. ECM – extracellularmatrix; PKC – protein kinase C; MAPK – mitogen-activated protein kinase;MAPK2 – mitogen-activated protein kinase kinase; MAPK3 – mitogen-ac-tivated protein kinase kinase kinase; ERK – extracellular signal-regulatedkinase; MEK – MAP/ERK kinase; SAPK – stress-activated protein kinase;JNK – stress activated jun N-terminal kinase; MMP – matrix metallo-proteinase; TIMP – tissue inhibitor of metalloproteinase; LIMP – large in-hibitor of metalloproteinase, IMP – inhibitor of metalloproteinase. (–→ ,activation/conversion; --→ , inhibition).

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Activation of ERK1,2 has been shown to induce c-Jun ex-pression and phosphorylation, indicating cross-talk betweenERK1,2 and JNK/SAPK pathways in the regulation of c-Junactivity [105]. Although p38 MAPK has not been found todirectly phosphorylate c-jun, it contributes to enhanced AP-1 activity by activation of transcription factors such as ATF-2, Elk-1 and SAP-1 that up-regulate c-jun and c-fos promoteractivities [98]. Phosphorylation of c-fos, for example, byERK2 has been shown to stabilize the c-fos protein andcaused an increase in trans-activation and transformationcapacity of c-fos. Furthermore, the activity and DNA bind-ing of ETS transcription factors, for example, SAP-1 whichparticipate in c-fos promoter activation through serum re-sponsive element, has been found to be regulated by ERK1,2/JNK/SAPK, and p38MAPK pathways [99, 100]. Activationof Ras has been shown to trans-activate ETS-1 and ETS-2,and ERK1,2 and JNK/SAPK pathways that coordinatelymediate activation of ETS factor PEA3 with Ras [106].

Thus, both AP-1 and ETS transcription factors are sub-ject to regulation by mitogen- and stress activated MAPKsignaling pathways. Recently, specific adapter proteins havebeen identified which connects MAPK kinase cascades un-der some specific situations [107, 108]. These signal transduc-tion cascades allow strict control of amplification, feedback,cross-talk, and branching of the initial signals triggered fromthe cell membrane resulting in a precise regulation of geneexpression in situations such as tumor cell invasion.

Role of MAPKs in the regulation of MMP gene expression

The role of phosphorylation in the regulation of the AP-1 andETS transcription factors is fairly well established. However,reports regarding the role of distinct phosphorylation-depend-ent signaling pathways in the regulation of MMP gene ex-pression has been determined only recently. Increased serine/threonine phosphorylation as a result of inhibition of serine/threonine phosphatase (PP2A) by tumor promoter okadaicacid induces expression of MMP-1 and MMP-3 at the tran-scriptional level [80, 96]. Evidence for the role of MAPKsin the transcriptional regulation of MMP gene expression wasdetermined by that blocking the ERK pathways which abro-gates Ras, serum, and TPA-mediated induction of 72 bp ofMMP-1 promoter, harboring the proximal AP-1 element[109]. Protein kinase C (PKC) has been shown to activateERK1,2 signaling pathway, and has recently been shown thatselective over-expression of PKC isoforms PKCδ, PKCε, andPKCζ activate the full-length MMP-1 promoter [110]. Innormal skin fibroblasts, an increase in MMP-1 expression byTNFα and the IL-1-induced lipid second messenger ceramideis mediated by coordinated activation of ERK1,2/5,6, JNK/SAPK, and p38 MAPK pathways [111]. The ERK1,2/5,6,JNK/SAPK and p38MAPK activities are required for induc-

tion of MMP-1 expression and promoter activity by the tumorpromoter okadaic acid [112]. The involvement of stress-ac-tivated MAPK pathways in the regulation of MMP gene ex-pression is also supported by the findings that inhibition ofp38MAPK activity by the specific chemical inhibitorSB203580 blocks the IL-1 elicited induction of MMP-1 andMMP-3 expression in human fibroblasts and vascular en-dothelial cells [113]. These recent reports indicate that mi-togen activated ERK1,2/5,6 pathways are the major activatorof MMP-1 gene expression.

Nitric oxide (NO)

Recent studies have demonstrated that in response to vascu-lar injury, levels of MMP-2 proteins and its activation aresignificantly increased, whereas TIMP-2 levels are signifi-cantly decreased, in balloon-injured rat carotid artery [114].In addition, the level of cardiovascular system-specific MMPinhibitor TIMP- 4 has been shown to be increased 2 weeksafter vascular injury and that can be correlated temporallywith cessation of SMC migration and onset of collagen depo-sition, indicating an important role for MMPs in promotingSMC migration in response to vascular injury [115]. It has,therefore, been suggested that MMPs are important for SMCmigration in response to injury and atherosclerosis.

Analysis of MMP activity by zymography demonstratedthat SMCs expressing the endothelial nitric oxide synthetase(eNOS) gene showed a decrease in MMP-2 activity underbasal and IL-1β-stimulated conditions. Also, pro-MMP-9 ac-tivity was decreased in conditioned medium collected fromeNOS gene-transfected cells stimulated with IL-1β. Inhibi-tion of MMP-2 activation and decrease in pro-MMP-9 activ-ity were also observed in cells treated with the nitric oxidedonor, DETA NONOate and the cGMP amplifier,8-bromo-cGMP which indicated that eNOS gene transfer-mediatedinhibition of MMP activity is due to NO and may involve theNO/cGMP pathway. Inhibition of MMP-2 and MMP-9 byNO has been shown to be associated with an increase inTIMP-2 secretion under basal and IL-1β-stimulated condi-tions. Thus, a decrease in MMP-2 and MMP-9 activities andan increase in TIMP-2 level has been suggested to contrib-ute to the decrease in the migration of SMC that has beenfound under NO/cGMP signalling phenomenon [116]. Themechanisms for eNOS gene transfer-mediated inhibition ofMMP-9 secretion in IL-1β-stimulated SMCs is not clear atpresent. However, It has been suggested that an increase inthe production of NO by eNOS gene transfer may inhibitactivation of the NF-κB signaling pathway [116].

Thus, endothelial cell-mediated inhibition of SMC migra-tion and proliferation could occur by NO. Although NO hasbeen shown to decrease MMP-9 synthesis/secretion while in-creasing TIMP-2 secretion, direct evidence on the effect of

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eNOS gene transfer on the transcriptional regulation of MMP-2, MMP-9, and TIMP-2 need to be demonstrated to clearly as-certain the role of NO/cGMP pathway in the transcriptionalregulation MMPs and TIMPs gene expression [116].

NO could also directly regulate activation of MMPs. Di-rect modulation of MMP activity by NO donors via MMPperoxynitrite formation and nitrosylation has been demon-strated [42, 117]. An increase in MMP-2 activation by per-oxynitrite in smooth muscle cells of different origins havebeen demonstrated [1, 42]. In contrast, Owens et al. [117]showed a decrease in MMP-2 activity by NO donors whencells were incubated with conditioned medium collectedfrom rat pulmonary fibroblast. The mechanism by whichNO/cGMP pathway regulates MMPs expression and activ-ity is currently unclear.

MMPs regulation by inhibitors

Matrix metalloproteinases are inhibited by two types of ambi-ent proteinase inhibitors tissue inhibitors of metalloproteinases,TIMPs (TIMP-1, TIMP-2, TIMP-3 and TIMP-4) and the in-hibitors of metalloproteinases (IMPs). The general proteinaseinhibitors α2-macroglobulins also inhibit matrix metallo-proteinases.

All active forms of matrix metalloproteinases are inhibited byTIMPs. They form complexes with matrix metalloproteinasesand interact with the active site plus a site in the carboxyl termi-nal hemopexin-like region [118, 119]. The C-terminal region ofTIMPs interacts with the C-terminal region of the enzyme, in-creasing the rate of association by many folds.

Two other types of metalloproteinase inhibitors have beenidentified. These are the smaller inhibitors of metalloproteinase(IMPs) and the large inhibitor of metalloproteinase (LIMP).LIMP is a complex composed of TIMP-2 and progelatinaseA [120]. This complex inhibits MMPs such as collagenase,gelatinase A and stromelysin. The ability of TIMP2-pro-gelatinase complex to inhibit these enzymes indicates that theinhibitory site is exposed with TIMP-2 molecule.

Active collagenase and stromelysin bind to TIMP-1 veryslowly. These enzymes bind at a faster rate to α2-macro-globulins.

α2-Macroglobulins inactivate susceptible proteinases byentrapment following cleavage of the bait region [121, 122].The proteinase cleaves one or more bonds in the 40 residuebait region and thereby initiates a conformational change thatleads to entrapment of the proteinase. In all α-macroglobulinsidentified so far, this conformational change leads to hydroly-sis of one internal thiol ester bond [–C (= O)–S–] per subunitand to generation of a highly reactive glutamyl residue [121].

The nascent glutamyl residue reacts with a lysyl side chainexposed on the surface of the attacking proteinase to covalentlycross-link the proteinase to the inhibitor by an ε-lysyl-γ-glutamyl bond thereby initiating a conformational change inthe large tetrameric macroglobulin that irreversibly traps theenzyme. Although the catalytic activity of the MMPs are notinhibited per se, their physical entrapment keeps the enzymefrom interacting with natural substrates, and the α2-mac-roglobulin/MMP-2 complex is eventually endocytosed andpermanently cleaved [122].

Conclusion and future direction

The expression patterns of MMPs have interesting implica-tions for the use of metalloproteinase inhibitors as therapeu-tic agents. Insights might be gained as to the preference for ageneral MMP inhibitor as opposed to an inhibitor designedto be specific for certain MMP family members as it relatesto a defined disease state, and may give clues to potentialside effects. For example, the expression of matrilysine,stromelysin-1 and stromelysin-3 in late-stage colon cancersuggests that the most efficacious inhibitor may be one de-signed to limit the activity of all of these enzymes. However,limiting the activity of the 72-kDa gelatinase B may pro-duce side effects related to the housekeeping functions ofthis enzyme. Inhibitors of matrilysin and stromelysin-3 mayhave dramatic effects on the female reproductive cycle be-cause of the apparent functions of these enzymes in prolif-erative endometrium. Although clearly the real answers tothese questions can only be determined empirically, furtherexamination of the expression patterns of the MMP genes innormal and pathological conditions can contribute signifi-cantly to the rational design of anti-MMP therapy [63].

Inhibition of specific MMPs in disease states and theregulation of each MMP gene will be an useful effort fortherapeutic purpose. An understanding of the molecularmechanism regulating the induction and repression of a spe-cific MMP, as compared to other family members, may pro-vide valuable insights for developing therapeutic agentswith specificities not achievable by more conventional ap-proach [63].

Pro-Tyr-Gly-Cys-Gly-Glu-Glu-Asn-Met-Val

S C=O

Pro-Tyr-Gly-Cys-Gly-Glu-Glu-Asn-Met-Val

SH C=O

NH

Proteinase

281

MMP expression, for example, during cancer cell inva-sion is regulated by multiple extracellular factors includingcytokines, growth factors, and interactions with adjacent cellsand ECM. Every level of regulation of MMP expression andactivity can be considered as target for the therapeutic inter-vention (Fig. 6). In this respect, it is important to understandthe mechanisms of the regulation of MMP expression in mal-ignant tumors in vivo. For example, it has recently been shownthat overexpression of transforming growth factor β (TGF-β) under keratin 6 promoter inhibited formation of benignskin tumors in transgenic mice but enhanced progression toinvasive carcinomas in mouse skin multistage carcinogenesismodel [123]. This indicated that different cell populationsmay alter their response to factors such as TGF-β. Further-more, it has been shown in the recent past that TNF-αstimulates MMP-1 expression in fibroblasts through 55 kDaTNF-α receptors [124]. Thus, complete characterization ofgrowth factors and cytokine receptors responsible for acti-vation of signaling pathways would help in designing recep-tor antagonists.

Regarding inhibition of MMP expression at the level ofkinase pathways, it is possible that selective chemical inhibi-tors for distinct signaling pathways (e.g. MAPK, PKC) will,hopefully, soon be available for initial clinical trials. It will,therefore, be important to identify growth and/or invasionspecific signalling cascades that could serve as targets forchemical inhibitors or dominant negative kinase mutants andantisense oligonucleotides [125]. Over-expression of selectivedual-specificity MAPK phosphatases have been shown to pre-vent MMP promoter activation [111, 112, 126] which couldalso be used as a novel strategy to prevent activation of AP-1and ETS transcription factors and MMP promoters in vivo.

Interactions between members of different transcriptionfactors provide fine-tuning of the transcriptional regulationof MMP promoter activity. It is possible that constitutiveexpression of specific transcription factors as a result of trans-formation may modulate the response of MMP promoter toextracellular signals. For example, over expression of sev-eral ETS factors has been observed in hematological malig-nancies as a result of proviral insertion into the ETS gene orby chromosomal translocation, possibly resulting in poten-tiation of AP-1 induced up-regulation of MMP expression,as shown recently in vitro [73, 127]. It has recently been shownthat peritoneally injected, double-stranded AP-1 oligomers po-tently prevent AP-1 dependent activation of MMP gene ex-pression in mouse arthrosis model [128]. Thus, it appearspossible to block transcriptional activation of MMP geneexpression in vivo by eliminating binding of the transcriptionfactors. Also, novel retinoids that potently inhibit cell pro-liferation selectively abrogates AP-1 mediated gene expres-sion [129]. Besides the treatment strategies targeted to inhibitMMP promoters activation, degradation of MMP mRNA byantisense RNA or ribozyme techniques may also provide

efficient and specific tool to prevent tumor cell invasion asshown in vitro and in vivo [130, 131].

The myocardial ECM is under constant remodeling byMMPs that in turn is regulated by various factors. Althoughsignificant advancement has been made in understanding theroles of MMPs, TIMPs and their regulators in the cardiovas-cular system, there is still much to be learned about the in-teraction of MMPs and their regulators in the developmentof myocardial fibrosis and the heart failure phenotype. Modu-lation of MMPs in the failing heart directly or through fac-tors that affect MMP activity may alter the ECM remodelingprocess and that may eventually affect progression of heartfailure. Thus, understanding the mechanisms of regulation ofMMPs and TIMPs may provide an important therapeutictarget for the discovery of new drugs for treating heart fail-ure [132].

An increase in the expression of different MMPs in can-cer tissues suggested that MMPs play a crucial role in tumorcell invasion. Although the expression of MMPs in malignan-cies has been studied widely, the specific role of distinctMMPs in the progression of cancer may be more complexthan has been assumed. For example, it has recently beenshown that MMP-3, MMP-7, MMP-9, and MMP-12 cangenerate angiostatin from plasminogen, indicating that theirexpression in peritumoral area may in fact serve to limitangiogenesis and thereby inhibit tumor growth and invasion[85]. The recent view about the role of stromal cells in theprogression of cancer cell growth and metastasis is particu-larly interesting, and additional studies about the regulationof MMP gene expression and activity in malignancies areneeded to understand the role and regulation of MMPs intumor cell invasion.

Acknowledgements

Financial assistance from the Indian Council of MedicalResearch (New Delhi) and the Department of Biotechnology(Government of India) is gratefully acknowledged. Thanksare due to Professor Kasturi Datta (School of Environmen-tal Sciences, Jawaharlal Nehru University, New Delhi) for herinterest in this work.

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