MATRIX

METALLOPROTEINASES

AGENDA
INTRODUCTION
TYPES
STRUCTURE
SYNTHESIS
INHIBITORS

 The endopeptidases or proteinases are key

enzymes in tissue degradation.
matrix macromolecules can be degraded by
proteinases from the four major
classes(metallo,serine,cysteine,aspartic active site
residues)
Members of the metallo and serine family are
very important in the initial phases of
degradation

 Except in special circumstances such as

bone resorption or other situations where
the cell is in close apposition with its
matrix, extracellular degradation is usually
thought to occur at neutral ph values
Consequently proteinases of the metallo
and serine families are most responsible
for the initial phases of degradation

 The MMPs are a family of proteolytic enzymes

that mediate the degradation of extracellular
matrix including interstitial& basement
membrane
collagen,fibronectin,elastin,laminin&proteoglyca
ns(liott&stetier-stevensonb1990)
They are also known as matrixins and sometimes
incorrectly referred to as collagenases

 The Mmps require metal ions for their

catalytic activity namely calcium and zinc
and zinc being located in the active site of
the enzyme
The enzymes are secreted in latent form as
proenzymes that need to be activated in the
extracellular environment

 Activity of mmps is strictly regulated at the gene

transcription level and by endogenous activators
and inhibitors
The major cell types in inflamed and healthy
periodontal tissues 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.
An imbalance between levels of activated mmps
and their inhibitors leads to degradation in
pathological processes

 Source:
1.primarily by connective tissue cells like
fibroblasts
2.secondarily by hematopoietic cells including
monocytes,leucocytes,macrophages,keratinocytes
,endothelial cells and epithelial cells
3.many types of tumours

 Degradation of the ECM may involve as many as

4 apparently distinct pathways
Periodontal disease is initially characterised by
destruction of ecm subjacent to the junctional
epithelium including the basement membrane
followed by the breakdown of deeper connective
tissue including the periodontal ligament and
alveolar bone

 Hansen described the cascade of ecm degradation

which includes
1.intracellular pathway or phagocytic pathway
mediated by acid cathepsins
2.extracellular pathways include
a.plasminogen dependent pathway(serine
proteases)
b.matrix metalloproteinase pathway
c.osteoclastic pathway

TYPES OF MMP

COLLAGENASES:
COLLAGENASES 1
Known as mmp-1or fibroblast type collagenase.it
has a mol wt of 55kda
Synthesised by
fibroblasts,keratinocytes,endothelial
cells,osteoblasts,chondrocyt,monocytes,macroph
ages
The fib-cl enzyme is synthesised on demand by
initiating transcription of fib-cl gene.this process
causes a lag period of 6-12 hours before enzyme
can be detected in extracellular environment

COLLAGENASE 2
Known as mmp-8 or neutrophil
collagenase,molecular weight of 75kda
Synthesised Mostly by pmn and
occasionally in chondrocytes
The pmn-cl gene is expressed only by pmn
leukocytes and the pmn type collagenase is
rapidly released from specific granules
storage sites of triggered pmns

COLLAGENASE 3
Known as mmp-13,has been cloned from human
breast carcinoma and is expressed abundantly in
cancer tissues,has a mol wt of 53.8kda
Synthesised by chondrocytes
The collagenase subgroups are the principal
neutral proteinases capable of degrading native
fibrillar collagens in the extracellular space

 They cleave collagens at a specific site

generating N-terminal fragments of length and
c-terminal fragments of length
These become susceptible to degradation by
other mmps such as gelatinase A&B
The matrix substrate of all collagenases include
collagen type 1,2,3,7,8,10,gelatin,proteoglycan
core protein.

GELATINASES
GELATINASE A
Known as mmp-2 or Mr 72k gelatinase,mol wt of
72kda
Most widely distributed of all mmps,synthesised
by fibroblasts,keratinocytes,endothelial
cells,monocytes/macrophages,osteoblasts,chondr
ocytes
It is present in a circulating form in plasma,it is
expressed constitutively by most cells in culture
and responds only moderately to growth factors
which induce the gelatinase B enzyme

MATRIX SUBSTRATE
bFGF,
collagens1,4,5,7,10,11
Elastin
Entactin
Fibrillin
Fibronectin
Galectin-2
Gelatin
Laminin-5

GELATINASE B
Known as Mr 92 k gelatinase,mol wt 92-95 kda
Synthesised mostly by pmn
,keratinocytes,monocytes/macrophages and
occasionally by fibroblasts
MATRIX SUBSTRATE
Collagen 1,4,5
Elastin,
Fibrillin
Galectin-3
Gelatin
Proteoglycan core protein

STROMELYSIN
STROMELYSIN-1
Known as mmp-3 or transin,molecular wt 55-

60kda
Synthesised by stromal cells either
constitutively
or after induction by growth
factors/cytokines(like IL-1,EGF,TNFa,PDGF)or phorbol esters
The enzyme does not appear to be expressed
by pmn leukocytes and keratinocytes in the
human

 MATRIX SUBSTRATE
Collagen 2,3,4,5,9,10,11
Elastin,fibrillin,fibronectin,gelatin,laminin5
Pro mmp-1,pro mmp-8
Pro mmp-g,pro mmp-13
Proteoglycan core
protein,tenascin,vitronectin

STROMELYSIN-2
Known as mmp-10,mol wt 55-60kda
It is expressed less abundantly than SL-1
It does not appear to respond to growth
factors(EGF&IL-1)or phorbol esters

 MATRIX SUBSTRATE
Collagen 2,3,4,5,9,10,11
Elastin,fibrillin,fibronectin,gelatin,laminin5
Pro mmp-1,pro mmp-8
Pro mmp-g,pro mmp-13
Proteoglycan core
protein,tenascin,vitronectin

STROMELYSIN-3
Known as mmp-11,mol wt 55-60kda
SL-3 transcripts are expressed by mesenchymal
cells of human mammary carcinomas often
adjacent to invading epithelial tumour cells
It is also induced in embryonic fibroblasts by
several growth factors
But it is not known whether it is produced by
cells of human periodontal tissues
MATRIX SUBSTRATE-alpha-1 antiprotease

MEMBRANE TYPE MMP

MT-1
It is also known as mmp-14,mol wt 65.9kda
Mmp-14 gene is expressed in stromal cells of
human colon,breast,head and neck carcinomas
and may also be expressed in fibroblasts
MATRIX SUSTRATE
Collagen-1,fibrillin,fibronectin,gelatin,laminin1,tenascin,pro mmp-2,pro mmp-13,proteoglycan
core protein

MT-2
Known as mmp-15,mol wt 75.8kda
MATRIX SUSTRATE
Fibronectin,laminin,pro-mmp 2,pro mmp
13,tenascin

MT-3
Known as mmp 16,mol wt 69.5kda
MATRIX SUBSTRATE
Collagen 3,fibronectin,gelatin,pro mmp-2

MT-4
Known as mmp-17,mol wt 61.7kda
Substrate unknown
The membrane type mmp have the
peculiarity of containing a transmembrane
domain and have been shown to induce the
processing of progelatinase A into its
activated form

NOVEL MMP
MMP-7
Known as matrilysin or PUMP
It is a punctuated metalloproteinase lacking a cterminal domain,expressed by mononuclear
phagocytes synovial cells,tumour cells,mol wt
29.7kda
Resembles the stromelysins in its proteinase
properties
MATRIX SUBSTRATE
Collagen-4,nidogen,fibronectin,pro-mmp1,gelatin,laminin,proteoglycan core protein

MMP-12
Known as macrophage metalloelastase,mol
wt 54kda
MATRIX SUBSTRATE
Collagen4,fibrillin,elastin,laminin,fibronectin,vitron
ectin

MMP-20
Known as enamelysin,mol wt 54.4kda
MATRIX SUBSTRATE
Amelogenin

MMP 18/19
Known as RASI-1,mol wt 57.4kda
MATRIX SUBSTRATE
unknown

MMP 26
Also known as endometase,matrilysin2
It is present in normal cells including the apical
epithelial conjunctiva cells of the human eye and
malignant cells of epithelial origin
Key regulators of wnt pathway(bcatenin,lymphoid enhanced binding factor/t cell
factor enhanced the transcriptional activity of
mmp26
It is present in humans and not in rodents

MMP 27
Recently discovered and primarily located
in extracellular spaces in extracellular
matrix and maps to chromosome 11q24
Very little information is available about
sources or size of this protein

STRUCTURE OF MMPS
Most members of the MMP family are
organised into three basic distinctive and
well conserved domains based on
structural considerations
1.aminoterminal propeptide
2.catalytic domain
3.haemopexin like domain at the
carboxyterminal

 SIGNAL PEPTIDE:
Functions as a signal to facilitate secretion
of the MMP into the extracellular space via
the endoplasmic reticulum
MMP-17 does not possess a signal
peptide(puente et al 1996)

 PROPEPTIDE:
Near the c-terminal portion of this segment is a
highly conserved cysteine that has the importance
in preserving the latency of proforms
Physiologic activation of mmps is probably
initiated by proteases that cleave specific sites
within the propeptide but final processing to the
active mmp requires inter molecular
autoproteolytic cleavage by the target mmp

 CATALYTIC DOMAIN:
It includes the binding site of two zinc ions and
atleast one calcium ion(massova et al 1998)
One zinc ion(catalytic zinc)is present in the
active site while the second zinc ion (structural
zinc) and the calcium ion reside together in a
different site on the same domain
The catalytic zinc ion is essential for the
proteolytic activity of the mmps

 HAEMOPEXIN:
They contain a disulfide bridge and are
structurally related to hemopexin and vitronectin
It is highly conserved among the mmps except
for mmp-7 where it is absent
It is not essential for mmp catalytic activity
although binding of the inhibitors of mmp
activity (TIMPs)is facilitated by the presence of
this domain
This contributes to substrate specificity of mmp-1
and the stromelysins

 HINGE REGION:
All mmps except for matrilysin have a
proline rich hinge region that connects the
catalytic domain with adjacent haemopexin
domain
The hinge region is absent in mmp-7

 FIBRONECTIN LIKE REPEATS:

The gelatinases alone have this domain
associated with the catalytic domain
There are three repeats of the fibronectin type 2
domain inserted in the catalytic domain just
ahead of the zinc binding region
These residues aid in the binding of enzyme to
gelatin

 MEMBRANE INSERTION EXTENSION:

The four membrane type mmps have an
extension that allows insertion of these
proteases into the cell membrane
They are added onto the c-terminal domain

 FURIN LIKE CLEAVAGE INSERTS:

Furin is an enzyme that is associated with the
golgi apparatus within cells
Three of the membrane associated mmps
(mmp14-16)along with mmp-11 are further
distinguished by a short sequence inserted
between the propeptide domain and the catalytic
domain
This site enables intracellular activation by furin
like convertases

SYNTHESIS OF MMP
RECEPTOR TYROSINE KINASE
It is one of the major cell surface receptor
The ligands for RTK are nerve growth
factor,PDGF,FGF,EGF and insulin
Binding of a ligand to this receptor stimulates the
receptors intrinsic protein tyrosine kinase activity
which stimulates a signal transduction cascade
leading to changes in cellular physiology

 Binding of a ligand causes autophosphorylation

to occur in the cytosolic domain
Which then activates ras which belongs to the
GTPase superfamily
In unstimulated cells most Ras is in the inactive
form with bound GDP,binding of a growth factor
to the RTK leads to formation of active Ras GTP

 Activated Ras binds to N-terminal domain

of Raf
Raf then phosphorylates MEK which
phosphorylates MAPK
Activation mapk is a major step in
translating extracellular signals into
various cellular functions

 Phosphorylation of MAPK promotes not only its

catalytic activity but also its dimerization
The dimeric form of MAPKinase can be
translocated to the nucleus where it regulates the
activity of transcription factors
Activated MAPK induces expression of c-fos
&phosphorylation of c-jun

 The fos and jun proteins are induced by growth

factors&agents such as TNF-a,IL1,leukoregulin,chemical tumour protector etc
The fos and jun proteins bind to form the AP-1
transcription factor
Multiple stimuli like growth factors,cytokines T
cell activators,neurotransmitters and UV
radiation can activate AP-1

 The AP-1 complex regulates genes involved with

cell proliferation
It activates genes such as
collagenase,stromelysin,IL-2 and TGF-b1
The transcription factor AP-1 binds to the TATA
box which is present in the promoter region of
the gene of all the secreted MMPs with the
exception of gelatinase A which leads to the
production of MMP

SYNTHESIS OF MMP
PROENZYME ACTIVATION
Mmps are synthesised as inactive
proenzymes,the agents that activate these
enzymes in vitro are
trypsin,plasmin,stromelysin&organomercurials
Conversion of the proenzyme to active enzyme
occurs through proinflammatory cytokine
stimulation,in addition degranulation of
phagocytic cells cause release of reduced oxygen
species which are effective in conversion of the
proenzyme to active enzyme state

 The mechanism of this action is not clearly

understood but may be through the switching of
cysteine switch mechanism
Latency of mmp precursors appears to be
maintained by a bond which links the cysteine
residue in the propeptide to the active site zn++
Disruption of the cys-zn++ bond is a prerequisite
to activation

 MMPs 11,14,15&16 are activated prior to their

secretion from the cell by furin like proteases
present on the cell golgi apparatus(nagase 1997)
Remaining mmps are secreted as an inactive
higher molecular wt proform(zymogen)with
activation occuring in the extracellular space
through proteolysis resulting in a lower mol wt
species either by
1.plasminogen cascade system(atkinson et al
1995)
2.by another member of the mmp family(fridman
et al 1992)

INHIBITORS OF MMPs
TYPES
1.endogenous inhibitors:
2.exogenous inhibitors:

 EXOGENOUS INHIBITORS
1.EDTA
2.phenanthroline
3.phosphorus containing peptides
4.sulphur based inhibitors
5.peptidyl hydroxamic acid derivatives
6.biphosphonates

 ENDOGENOUS INHIBITORS
1.alpha2 macroglobulin
2.TIMP

 ALPHA 2 MACROGLOBULIN
Major physiological plasma proteinase inhibitor
Restricted in its site of activity owing to its large
size
inactivates susceptible proteinase by trapping
them after cleavage of a peptide bond by a unique
venus fly trap mechanism

 TISSUE INHIBITOR OF MATRIX

METALLOPROTEINASE
They inhibit active forms of all the mmps
TIMPs control the extracellular collagen
degradation,they are key inhibitors in local
tissues
4 timps(1,2,3,4)are known
The local synthesis of mmps&timps must be
precisely balanced during tissue remodelling
under physiologic conditions(denharat DT
feng1993)

 TIMP is a sialoglycoprotein with mol wt of

28kda
fibroblasts,keratinocytes,monocytes/macro
phages,endothelial cells &osteoblasts
express TIMPs
TIMP acts by inhibition of proteinase and
blockage of autocatalytic MMP activation

 Inhibition represents a final level of control of

mmp activity and as such is a therapeutic
target(vincenti MP 1994)
TIMP 1&2 are secreted by many cell types in
culture&are found in body fluids&tissue extracts
TIMP3 is found in the ECM and it is insoluble
TIMP4is expressed by stromal cells of breast
tissue

 TIMP1 more efficiently inhibits

collagenase(mmp1)
TIMP2 is 10 fold more effective than
timp1 against gelatinases
A number of growth factors& cytokines
upregulate the expression of TIMP1

CONCLUSION
Although many questions remain unanswered
about MMPS & their inhibitors their
involvement in the maintenance & remodelling
of connective tissue seems well established
The cellular localisation of the new subfamily
MT-MMPS & the discovery of intracellular
processing of gelatinaseA in normal cells raise
the possibility of implicating MMPS in
cytoskeletal rearrangements & other cellular
events

 The development of specific inhibitors will

be an important tool in evaluating the roles
of different mmps in both physiologic &
pathologic processes
These specific inhibitors could lead to new
classes of drugs useful in the treatment of
many diseases in which mmps are
involved

THANK YOU