Proteinases are generally classified into aspartic proteinases, cysteine proteinases, serine proteinases, and metalloproteinases according to their catalytic mechanism.
Because of the optimal acidic pH and intra-cellular localization within lysosomes, most of the aspartic proteinases and cysteine proteinases are involved in intra-cellular degradation of extra-cellular matrix (ECM) components.
Serine proteinases and metalloproteinases are proteinases that act at neutral pH and play a central role in extra-cellular degradation of ECM macromolecules.
ECM-degrading metalloproteinases are composed mainly of the MMP (matrix metalloproteinase) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene families.
Most endogenous proteinase inhibitors are proteinase class specific, whereas α2 macroglobulin inhibits the activities of all classes of proteinase.
The activities of ECM-degrading proteinases at the local tissues are regulated by the balance between the proteinases and their inhibitors, which may be determined by production rates of proteinases and inhibitors, their secretion, activation of proenzymes, and cell surface anchoring and recycling systems of the activated proteinases.
ProMMPs (precursors of MMPs) are activated via the extra-cellular, intra-cellular, and cell surface pathways depending on the enzymes.
Aggrecan and type II collagen, two major ECM components, may be degraded in articular cartilage by differential or complementary actions of the MMP and ADAMTS species in arthritides.
In rheumatoid arthritis, articular cartilage is destroyed by proteinases accumulated in synovial fluid, direct contact with proteolytic pannus, and proteinases derived from chondrocytes. Bone is resorbed by osteoclasts mainly by the action of cathepsin K and MMP-9 under acidic and hypercalcemic conditions in subosteoclastic compartments.
In osteoarthritis, chondrocyte-derived metalloproteinases, including the MMP and ADAMTS species, contribute primarily to the breakdown of articular cartilage.
Extra-cellular matrix (ECM) is an important component of multicellular organisms, including humans. It provides the tissue architecture, fills the gaps between the cells, separates tissue components, acts as a scaffolding for migrating cells, acts as a growth factor pool, and sends the signals directly to the cells. While the cell-ECM interaction regulates various fundamental cellular functions, including growth, differentiation, apoptosis, and migration, upon tissue remodeling or cell migration in tissue, ECM becomes a physical barrier that needs to be degraded. ECM degradation is achieved by proteolytic enzymes, termed endopeptidases or proteinases . In normal healthy conditions, activity of ECM-degrading proteinases is under tight control, keeping tissue homeostasis. On the other hand, in pathologic conditions, activity of these proteinases are either elevated excessively for tissue destructive diseases or decreased for fibrotic diseases. Thus, regulatory mechanisms of ECM degradation are important to understand to reveal pathogenesis of these diseases. In rheumatoid arthritis and osteoarthritis, activity of ECM-degrading proteinases are elevated, causing destruction of joint tissue, including cartilage and bone. It is getting clearer that these unbalanced ECM metabolisms are due not only to upregulation of proteinase genes, but also to other factors. This chapter provides up-to-date information about ECM-degrading proteinases and their regulations in rheumatoid arthritis and osteoarthritis.
Extra-cellular Matrix-Degrading Proteinases
ECM is a meshwork of large macromolecules, and thus their degradation is caused by endopeptidases or proteinases that cleave internal peptide bonds of polypeptide chains. The impact of exopeptidases that cleave a few amino acids either from N- or C-terminus in ECM integrity is unlikely to be significant even if they exist. There are four different classes of proteinases that have been implicated in ECM degradation: aspartic proteinases, cysteine proteinases, serine proteinases, and metalloproteinases, which are classified according to their catalytic machinery.
Most aspartic proteinases have two aspartic acid residues in their catalytic sites, where the nucleophile that attacks the scissile peptide bond is an activated water molecule. Mammalian aspartic proteinases include the digestive enzymes (pepsin and chymosin), the intra-cellular cathepsin D and cathepsin E, and rennin. Among the proteinases belonging to this group, cathepsin D is the major aspartic proteinase involved in ECM degradation. It exhibits proteolytic activity against most substrates such as aggrecan and collagen telopeptides with optimal pH between pH 3.5 and 5. Because of the acidic optimal pH and intra-cellular localization within lysosomes, cathepsin D is probably responsible for intra-cellular degradation of phagocytosed ECM fragments that previously were degraded in the extra-cellular spaces. However, a study on cartilage explant cultures using the aspartic proteinase inhibitor suggests the possibility that cathepsin D secreted extra-cellularly contributes to the degradation of aggrecan in articular cartilage.
Cysteine proteinases are endopeptidases in which the nucleophile of the catalytic site is the sulfhydryl group of a cysteine residue. The ECM-degrading cysteine proteinases include lysosomal cathepsins B, L, S, and K and the calpains ( Table 8.1 ). Cathepsins B and L digest the telopeptide regions of fibrillar types I and II collagen, the nonhelical regions of types IX and XI collagen, and aggrecan at acidic pH. Cathepsin S has a similar spectrum of substrates within a broad range of pH values. Cathepsin K, also previously called cathepsin O, O2, or X , is a collagenolytic cathepsin that cleaves type I collagen at the triple helical regions at pH values between 4.5 and 6.6. The proteinase also degrades gelatin and osteonectin. Cathepsin K is highly expressed in human osteoclasts, and inactivating mutations or deletion of the gene result in an osteopetrotic phenotype in humans and animals; cathepsin K is thought to play a key role in osteoclast-dependent bone resorption (see later discussion). Because cathepsins B, L, S, and K are expressed in synovium or articular cartilage or both in rheumatoid arthritis and osteoarthritis, they may also be involved in the cartilage destruction through degradation of the ECM macromolecules when the local environment has shifted to acidic condition.
|Neutrophil elastase||Neutrophils||α1 PI|
|Cathepsin G||Neutrophils||α1 Antichymotrypsin|
|Proteinase 3||Neutrophils||α1 PI, elafin|
|Tissue kallikrein||Glandular tissues||Aprotinin; kallistatin|
|tPA||Endothelial cells; chondrocytes||PAI-1; PAI-2|
|uPA||Fibroblasts; chondrocytes||PAI-1; PAI-2; PN-1|
|Chymase||Mast cells||α1 PI|
|MMPs||Tissue cells; inflammatory cells||TIMP-1, 2, 3, and 4; |
RECK for MMP-2, 7, 9, and 14
|ADAMs||Tissue cells; inflammatory cells||TIMP-3; RECK for ADAM10|
a For details of ADAMs, ADAMTSs, and MMPs, see Tables 8.2 and 8.3 .
Calpains are Ca 2+ -dependent, papain-like cysteine proteinases that are ubiquitously distributed among mammalian cells. The best-characterized members of the calpain superfamily are μ-calpain and m-calpain, which also are called conventional (μ-calpain) and classic (m-calpain) calpains . Calpains are involved in various pathologic conditions such as muscle dystrophy by acting intra-cellularly. They are present in the extra-cellular spaces and in osteoarthritic synovial fluid, and they can degrade aggrecan.
Serine proteinases require the hydroxyl group of a serine residue acting as the nucleophile that attacks the peptide bond. Serine proteinases include the largest number of proteinases with around 40 family members. Most serine proteinases can degrade ECM macromolecules. The major ECM-degrading serine proteinases in joint tissues will be described below (see Table 8.1 ).
Neutrophil Elastase and Cathepsin G
Neutrophil elastase and cathepsin G are serine proteinases that are synthesized as precursors in promyelocytes in bone marrow and subsequently stored in the azurophil granules of polymorphonuclear leukocytes as active enzymes. Mature leukocytes do not synthesize elastase, but they mobilize azurophil granules to the cell surface and release the proteinases in response to various stimuli. Monocytes have low levels of elastase but lose the enzyme expression during the differentiation into macrophages. Neutrophil elastase and cathepsin G are basic glycoproteins with isoelectric points larger than 9 (neutrophil elastase) and about 12 (cathepsin G). They can be readily trapped in cartilage matrix that has a negative charge.
Neutrophil elastase and cathepsin G cleave elastin; the telopeptide region of fibrillar collagen types I, II, and III; other collagen types IV, VI, VIII, IX, X, and XI; and other ECM components such as fibronectin, laminin, and aggrecan at neutral pH. These serine proteinases can also be involved indirectly in the breakdown of ECM by activating the zymogen of matrix metalloproteinases (proMMPs) and by inactivating endogenous proteinase inhibitors such as α2 antiplasmin, α1 antichymotrypsin, and tissue inhibitors of metalloproteinases (TIMPs).
Mast Cell Chymase and Tryptase
Chymase and tryptase are packaged in secretory granules together with histamine and other mediators in mast cells, which are infiltrated in rheumatoid synovium. Chymase is a chymotrypsin-like proteinase with a broad spectrum of activity against ECM components such as type VI collagen and aggrecan. It also activates proMMPs such as proMMP-1, 3, and 9. Although prochymase is activated intra-cellularly and stored in the granules, the activity in the granules is limited at low pH and becomes fully active when released extra-cellularly. Tryptase is a trypsin-like proteinase that degrades type VI collagen and fibronectin; it also activates proMMP-3.
Plasmin and Plasminogen Activators
Plasminogen is synthesized in the liver and secreted to plasma. It can bind to fibrin and to cells, and after activation by plasminogen activators, plasmin readily digests fibrin. Membrane-bound plasmin also degrades many ECM components, including proteoglycan, fibronectin, type IV collagen, and laminin. Other important functions of plasmin are to initiate the activation of proMMPs, activate latent cell-associated transforming growth factor (TGF)-β1, and act as proenzyme convertase. Plasmin is generated by activation of plasminogen by plasminogen activators, including tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The tPA is synthesized as a proenzyme of 70 kDa and is secreted into the circulating blood primarily by endothelial cells, fibroblasts, chondrocytes, and tumor cells. The uPA was first purified from urine as a proenzyme of 54 kDa. It is converted to the active form of two chains of 30 kDa and 24 kDa linked by a disulfide bond. Alternatively, a fully active form of 33 kDa can be generated by plasmin. The expression of uPA is found in various cells, including invasive cancer cells, migrating keratinocytes, and activated leukocytes in pathologic situations. Pro-uPA and two-chain uPA bind to a specific uPA receptor (uPAR), a glycosylphosphatidylinositol (GPI)–anchored glycoprotein expressed on the cell surface of fibroblasts, macrophages, and tumor cells. Receptor-bound uPA preferentially activates cell membrane–bound plasminogen into plasmin. Cell membrane–bound plasmin can activate receptor-bound pro-uPA. Despite the highly restricted substrate specificity of the plasminogen activators, uPA cleaves other proteins in vitro, including fibronectin, fibrinogen, diphtheria toxin, and possibly uPA itself.
Two types of kallikreins, plasma and tissue kallikreins, have been identified. Plasma kallikrein, with two disulfide-linked chains (36 kDa and 52 kDa), is generated from prokallikrein of 88 kDa by coagulation factor XIIa or by kallikrein itself. It activates kininogens to bradykinin and activates proMMP-1 and proMMP-3. Tissue kallikrein, which is synthesized in glandular tissues, releases Lys-bradykinin from kininogen and activates proMMP-8.
Similar to aspartic proteinases, metalloproteinases are endopeptidases in which the nucleophilic attack on a peptide bond is mediated by a water molecule. A divalent metal cation, usually zinc, activates the water molecule. Among the metalloproteinases, MMPs (matrix metalloproteinases), which are also designated as matrixins (a subfamily of the metzincin superfamily), are key ECM-degrading, zinc-dependent endopeptidases ( Table 8.2 ). In addition, some members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family, which is an MMP-related gene family within the metzincin family, are also responsible for the degradation of ECM, such as cartilage proteoglycan ( Table 8.3 ). Only a few members of the ADAM (a disintegrin and metalloproteinase) family have limited activity to ECM components ( Table 8.4 ).
|Enzymes||ECM Substrates||Non-ECM Substrates|
|MMP-1||Collagens I, II, III, VII, X; gelatins; aggrecan; link protein entactin; tenascin; perlecan||α2 Macroglobulin; IGF-BP-2, -3, and -5; (Interstitial Collagenase) |
α1 PI; α1 antichymotrypsin; pro-IL-1β; CTGF
|MMP-8 (Neutrophil collagenase)||Collagens I, II, and III; gelatins; aggrecan; link protein||α1 PI|
|MMP-13 (Collagenase-3)||Collagens I, II, III, IV, IX, X, XIV; aggrecan; Fn; tenascin||CTGF; pro-TGF-β; α1 antichymotrypsin|
|MMP-2 (Gelatinase A)||Gelatins; collagens IV, V, VII, XI; Ln; Fn; elastin; aggrecan; link protein||Pro-TGF-β; FGF receptor I; MCP-3; IGFBP-5; pro-IL-1β; galectin-3; plasminogen|
|MMP-9 (Gelatinase B)||Gelatins; collagens III, IV, V; aggrecan; elastin; entactin; link protein||Pro-TGF-β; IL-2 receptor α; Kit-L; IGF-BP-3; pro-IL-1β; α1 PI; galectin-3; ICAM-1 plasminogen|
|MMP-3 (Stromelysin-1)||Aggrecan; decorin; gelatins; collagens III, IV, IX, X; Fn; Ln; tenascin; link protein; perlecan||IGF-BP-3; pro-IL-1β; HB-EGF; CTGF; E-cadherin; α1 antichymotrypsin; α1 PI; α2 macroglobulin; plasminogen; uPA; proMMP-1, 7, 8, 9, 13|
|MMP-10 (Stromelysin-2)||Aggrecan; Fn; Ln; collagens III, IV, V; link protein||ProMMP-1, 8, 10|
|MMP-7 (Matrilysin-1)||Aggrecan; gelatins; Fn; Ln; elastin; entactin; collagen IV; tenascin; link protein||Pro-α-defensin; Fas-L; β4 integrin; E-cadherin; pro-TNF; CTGF; HB-EGF; RANKL; IGF-BP-3; plasminogen|
|MMP-26 (Matrilysin-2)||Gelatin; collagen IV; Fn; fibrinogen||α1 PI; proMMP-9|
|MMP-11 (Stromelysin-3)||Fn; Ln; aggrecan; gelatins||α1 PI; α2 macroglobulin; IGF-BP-1|
|Other Secreted-Type MMPs|
|MMP-12 (Metalloelastase)||Elastin; Fn; collagen V; osteonectin||Plasminogen; apolipoprotein A|
|MMP-19 (RASI-1)||Collagen IV; gelatin; Fn; tenascin; aggrecan; COMP; Ln; nidogen||IGF-BP-3|
|MMP-20 (Enamelysin)||Amelogenin; aggrecan; gelatin; COMP||Unknown|
|Type I Transmembrane-Type MMPs|
|MMP-14 (MT1-MMP)||Collagens I, II, III; gelatins; aggrecan; Fn; Vn; Ln-1, -2, -4, -5; fibrin; perlecan;||ProMMP-2, -13; ADAM9; tTG; CD44; ICAM-1; LRP-1; syndecan 1; SLPI; CTGF; DR6, DJ-1; galectin-1, αV-integrin; C3b; EMMPRIN; ApoE; MICA; betaglycan; IL-8; Cyr61; dickkopf-1, KiSS-1, Dll1; peptidyl-prolyl cis-trans isomerase A|
|MMP-15 (MT2-MMP)||Fn; tenascin; nidogen; aggrecan; perlecan; Ln||ProMMP-2; tTG|
|MMP-16 (MT3-MMP)||Collagen III; Fn; gelatin||ProMMP-2; tTG|
|MMP-17 (MT4-MMP)||Gelatin; fibrinogen||Unknown|
|MMP-25 (MT6-MMP)||Gelatin; collagen IV; fibrin; Fn; Ln||ProMMP-2|
|ADAMTS||Other Names||Activity a||Functions||Tissue/Cell|
|ADAMTS||1C3-C5; METH1; KIAA1346||+||Digestion of aggrecan and versican; binding to heparin||Kidney; heart; cartilage|
|ADAMTS2||Procollagen N-proteinase||+||Processing of collagen I and II hPCPNI; PCINP N-propeptides||Skin; tendon|
|ADAMTS3||KIAA0366||+||Processing of collagen N-propeptides||Brain|
|ADAMTS4||KIAA0688; aggrecanase-1; ADMP-1||+||Degradation of aggrecan, brevican, and versican||Brain; heart; cartilage|
|ADAMTS5||ADAMTS11; aggrecanase-2; ADMP-2||+||Degradation of aggrecan||Uterus; placenta; cartilage|
|ADAMTS8||METH-2||+||Degradation of aggrecan; inhibition of angiogenesis||Lung; heart|
|ADAMTS9||KIAA1312||+||Digestion of aggrecan||Cartilage|
|ADAMTS13||VWFCP; C9orf8||+||Cleavage of von Willebrand factor||Liver; prostate; brain|
|ADAMTS14||—||+||Processing of collagen N-propeptides||Brain; uterus|
|ADAMTS15||—||+||Digestion of aggrecan||Liver (fetus); kidney (fetus)|
|ADAMTS16||—||+||Digestion of aggrecan||Prostate; brain; uterus|
|ADAMTS17||FLJ32769; LOC123271||—||—||Prostate; brain; liver|
|ADAMTS18||ADAMTS21; HGNC:16662||+||Digestion of aggrecan||Prostate; brain|
|ADAMTS20||—||+||Digestion of versican (and aggrecan)||Brain; testis|
|ADAM2||PH-30β; Fertilin-β α6β, and α9β||NP||Sperm/egg binding/fusion; binding to integrin αβ1||Sperm|
|ADAM7||EAP I; GP-83||NP||Binding to integrin α4β1, α4β7, and α9β1||Testis|
|ADAM8||MS2 (CD156)||P||Neutrophil infiltration; shedding of CD23||Macrophages; neutrophil|
|ADAM9||MDC9; MCMP; Meltrin-γ||P||Shedding of HB-EGF, TNF-p75 receptor, and APP; digestion of fibronectin and gelatin; binding to integrin α2β1, α6β1, α6β4, α9β1, and αVβ5||Various tissues|
|ADAM10||MDAM; Kuzbanian||P||Shedding of TNF, Delta, Delta-like 1, Jagged, N-cadherin, E-cadherin, VE-cadherin, Ephrin A2, Ephrin A5, Fas-l, IL-6R, APP, L1,CD44, and HB-EGF; digestion of collagen IV, gelatin, and myelin basic protein; presence of RRKR sequence||Kidney; brain; chondrocytes|
|ADAM11||MDC||NP||Tumor suppressor gene (?)||Brain|
|ADAM12||Meltrin-α; MCMP; MLTN; MLTNA||P||Muscle formation; presence of RRKR sequence; binding to integrin α4β1 and α9β1; digestion of IGF-BP-3 and -5; shedding of HB-EGF and epiregulin; digestion of collagen IV, gelatin, and fibronectin||Osteoblasts; muscle cells; chondrocytes; placenta|
|ADAM15||Metargidin; MDC15; AD56; CR II-7||P||Expression in arteriosclerosis; binding to integrin αvβ3, α5β1, and α9β1; digestion of collagen IV and gelatin; shedding of CD23||Smooth muscle cells; chondrocytes; endothelial cells; osteoclasts|
|ADAM17||TACE; cSVP||P||Shedding of TNF, TGF-β, TNF-p75 receptor, RANKL, amphiregulin, epiregulin, HB-EGF, APP, L-selectin, and CD44; presence of RRKR sequence; binding to integrin α5β1||Macrophages; various tissues; carcinoma|
|ADAM19||Meltrin-β; FKSG34||P||Formation of neuron; shedding of neuregulin, and RANKL; binding to integrin α4β1 and α5β1||Testis|
|ADAM20||—||P||Formation of sperm||Testis|
|ADAM23||MDC3||NP||Binding to integrin αvβ3||Brain; heart|
|ADAM28||e-MDC II; MDC-Lm; MDC-Ls||P||Digestion of VWF, IGF-BP-3, and CTGF; shedding of CD23; binding to integrin α4β1, α4β7, and α9β1||Epididymis; lung; stomach; pancreas|
|ADAM33||—||P||Mutation in bronchial asthma patients; shedding of APP and KL-1; digestion of insulin B chain; binding to integrin α4β1, α5β1, and α9β1||Lung (fibroblasts, smooth muscle cells)|
|ADAMDEC1||—||P||—||Lymphatic system; gastrointestinal system|
The human MMP family comprises 23 different members that have MMP designations (numbered according to a sequential numbering system) and common names coined by the authors of the published reports (see Table 8.2 ). On the basis of the biochemical properties provided by the domain structures and on their substrate specificity, these family members are classified into two major subgroups: secreted-type MMPs and membrane-anchored MMPs. MMP-4, 5, and 6 are excluded from the list because they are identical to other known MMPs (i.e., MMP-3 and 2). MMP-18 and 22 also are missing in Table 8.2 because they are assigned to Xenopus collagenase-4 and chicken MMP. Many of the secreted-type MMPs are composed of three basic domains—the prodomain, catalytic domain, and hemopexin-like domain—that are preceded by hydrophobic signal peptides ( Fig. 8.1 ). The N-terminal prodomain has one unpaired cysteine in the conserved sequence of PRCGXPD. The cysteine residue in the sequence interacts with the catalytic zinc atom in the catalytic domain to maintain the proenzyme in an inactive state by preventing it from binding the water molecule to interact for the catalysis. The catalytic domain has the zinc-binding motif HEXGHXXGXXH, in which three histidines bind to and hold the catalytic zinc atom. The hemopexin-like domain, which is connected to the catalytic domain by the proline-rich hinge region, is considered as a molecular interaction interface and plays a role in determining the substrate specificity in some MMPs. In addition to these basic domains, gelatinases have additional insertions of three repeats of fibronectin type II domain in the catalytic domain (see Fig. 8.1 ), which provides them with collagen-binding properties. Matrilysins are the smallest MMP member, lacking the hemopexin-like domains. Furin-activated MMPs contain insertions of a basic amino acid motif of Arg-Xxx-Lys-Arg (RXKR) at the C-terminus of the prodomain that is recognized and cleaved by proprotein convertases, including furin (see Fig. 8.1 ). These enzymes are thus secreted to extra-cellular milieus as an active form. MMP-23 is synthesized as a type II transmembrane type MMP having type II transmembrane domain in the N-terminus of prodomain. It contains the RRRR sequence at the end of the prodomain that can be cleaved by proprotein convertases during secretion, which makes the enzyme a soluble enzyme. The domain structure of MMP-23 is also unique among MMP members as it has a cysteine array and an immunoglobulin-like domain instead of a hemopexin-like domain (see Fig. 8.1 ).
There are two types of membrane-type MMPs: type-I transmembrane type (MMP-14, -15, -16, and -24/MT1-, MT2-, MT3-, and MT5-MMPs) and GPI-anchored type (MMP-17 and -25/MT4- and MT6-MMPs). Type I transmembrane-type MMPs have a stalk region, the transmembrane domain, and a short cytoplasmic tail downstream of the hemopexin domain in addition to the common domain composition (prodomain, catalytic domain, hinge, and hemopexin-like domain). GPI-anchored types are synthesized with hydrophobic GPI anchoring the signal peptide sequence following the stalk region at their C-terminus. This GPI-anchoring signal peptide is cleaved off and the ectodomain transferred to the de-novo synthesized GPI moiety by transamidase in endoplasmic reticulum (see Fig. 8.1 ).
Collagenases (MMP-1, MMP-8, and MMP-13)
The classic collagenases include MMP-1 (interstitial collagenase, collagenase-1), MMP-8 (neutrophil collagenase, collagenase-2), and MMP-13 (collagenase-3). These MMPs attack triple helical regions of interstitial collagen types I, II, and III at a specific single site after the Gly residue of the partial sequences Gly-(Ile or Leu)-(Ala or Leu), located about three-fourths of the distance from the N-terminus. This cleavage generates fragments approximately three-fourths and one-fourth of the size of the collagen molecules. A biochemical study has disclosed the molecular mechanism of the cleavage: MMP-1 unwinds the triple helical structure by interacting with the α2(I) chain of type I collagen and cleaves the three α chains in succession. MMP-13 is unique in that it cleaves α chains of type II collagen at two sites of the Gly 906 -Leu 907 and Gly 909 -Gln 910 bonds. All of these collagenases degrade the interstitial collagens, but their specific activities against the collagens are different; MMP-1, 8, and 13 relatively digest types III, I, and II collagen better than others, respectively. Although rodents such as mice were originally thought to have only two collagenases ( MMP-8 and MMP-13 ) and to lack the MMP-1 gene, rodent homologues of the human MMP-1 gene were cloned and named mouse collagenase A and B ( Mcol-A and Mcol-B ). In addition to the interstitial fibrillar collagens, MMP-1, 8, and 13 degrade many other ECM macromolecules. MMP-1 digests entactin, collagen X, gelatins, perlecan, aggrecan, and cartilage link protein (see Table 8.2 ). MMP-8 digests aggrecan, gelatins, and cartilage link protein (see Table 8.2 ). MMP-13 hydrolyzes aggrecan; types IV, IX, X, and XIV collagens; fibronectin; and tenascin. Non-ECM substrates of MMP-1, 8, and 13 include α2 macroglobulin, α1 anti-proteinase inhibitor, α1 antichymotrypsin, insulin-like growth factor binding protein (IGF-BP)-2 and IGF-BP-3, connective tissue growth factor (CTGF), and pro-TGF-β (see Table 8.2 ).
Gelatinases (MMP-2 and MMP-9)
MMP-2 (gelatinase A) and MMP-9 (gelatinase B) belong to the gelatinase subgroup. Both MMPs readily digest gelatins and cleave types IV and V collagen. Elastin, aggrecan, and cartilage link protein also are substrates of the gelatinases. Although MMP-2 and 9 share such substrates, they have different activities on several ECM macromolecules. MMP-2, but not MMP-9, digests fibrillar type I and II collagens at the same site as collagenases, fibronectin, and laminin, and type III collagen and α2 chains of type I collagen are degraded only by MMP-9. The gelatinases also process directly TGF-β into an active ligand (see Table 8.2 ). MMP-2 and 9 cleave fibroblast growth factor receptor type I and interleukin (IL)-2 receptor type α (see Table 8.2 ). MMP-9 also releases soluble Kit-ligand. MMP-2 processes monocyte chemoattractant protein (MCP)-3 into an MCP-3 fragment deleting the N-terminal four amino acids, which can bind to CC-chemokine receptors and act as a general chemokine antagonist.
Stromelysins (MMP-3 and MMP-10)
The subgroup of stromelysins consists of MMP-3 (stromelysin-1) and MMP-10 (stromelysin-2). They share 78% identity in amino acid sequence and have similar enzymatic properties. The enzymes hydrolyze numerous ECM macromolecules, including aggrecan, fibronectin, laminin, and collagen IV (see Table 8.2 ). Types III, IX, and X collagen and telopeptides of types I, II, and XI collagen also are digested by MMP-3. In addition to the ECM components, MMP-3 is active on IGF-BP-3, IL-1β, heparin-binding epidermal growth factor (HB-EGF), CTGF, E-cadherin, α1 antichymotrypsin, and α1 proteinase inhibitor (see Table 8.2 ). MMP-3 also activates many proMMPs. A similar activator function has been identified for MMP-10.
Matrilysins (MMP-7 and MMP-26)
Matrilysins include MMP-7 (matrilysin-1) and MMP-26 (matrilysin-2), which are the smallest of the MMPs, having only the prodomain and catalytic domain. The substrate specificity of MMP-7 is similar to that of stromelysins, digesting numerous ECM components, including aggrecan; gelatins; fibronectin; laminin; elastin; entactin; types III, IV, V, IX, X, and XI collagen; fibrin/fibrinogen; vitronectin; tenascin; and link protein (see Table 8.2 ). Although these substrates overlap with the substrates of other MMPs, the specific activity of MMP-7 to most substrates is highest among the MMPs. Non-ECM molecules such as α-defensin, Fas ligand, β4 integrin, E-cadherin, plasminogen, TNF, and CTGF also are the substrates for MMP-7 (see Table 8.2 ). MMP-26 degrades gelatin, type IV collagen, fibronectin, fibrinogen, and α1 proteinase inhibitor, but information about other substrates is still limited.
Furin-Activated Matrix Metalloproteinases (MMP-11 and MMP-28)
MMP-11 (stromelysin-3) and MMP-28 (epilysin) contain an RKRR sequence at the end of the prodomain, which is a unique motif for intra-cellular processing of proproteins to mature molecules by furin and other proprotein convertases. ProMMP-11 was activated during secretion by furin. MMP-11 shows only weak proteolytic activity against gelatin, laminin, fibronectin, and aggrecan, but it has respectable catalytic action in digesting α1 proteinase inhibitor, α2 macroglobulin, and IGF-BP-1 (see Table 8.2 ). MMP-28 can degrade casein, but its natural substrates are unknown.
Other Soluble MMP Enzymes (MMP-12, MMP-19, MMP-20, MMP-21, MMP-23, and MMP-27)
MMP-12 (metalloelastase), MMP-19 (RASI-1), MMP-20 (enamelysin), MMP-21, and MMP-27 have structural characteristics similar to those of collagenases and stromelysins. These MMPs are not classified into the previously mentioned subgroups because their substrates and other biochemical characters are not fully examined at present. MMP-12, also called metalloelastase , digests elastin, fibronectin, collagen V, osteonectin, and plasminogen (see Table 8.2 ). MMP-19, which was originally reported as MMP-18 but renamed as MMP-19, cleaves type IV collagen, laminin, fibronectin, gelatin, tenascin, entactin, fibrin/fibrinogen, aggrecan, and cartilage oligomeric matrix protein (COMP; see Table 8.2 ). MMP-20 also degrades amelogenin, aggrecan, and COMP. Substrates of MMP-21 and 27 are unknown, however.
MMP-23 (cysteine array–MMP, MIFR) is unique among MMP enzymes as it is synthesized as a type II transmembrane-protein, but it becomes a soluble enzyme upon activation. MMP-23 is able to degrade gelatin, but no information about other substrates is available (see Table 8.2 ). A unique aspect of MMP-23 is that this MMP is expressed in only the reproductive organs of both males and females, such as the endometrium, ovary, testis, and prostate. However, its biologic functions are not understood.
Membrane-Type Matrix Metalloproteinases (MMP-14, MMP-15, MMP-16, MMP-17, MMP-24, and MMP-25, or MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP)
There are two types of MT-MMPs: type I transmembrane-type and GPI-anchored-type, and these MT-MMPs are unique in that they are expressed on the cell surface as an active form and function on the cell surface. Type-I transmembrane type includes MMP-14 (MT1-MMP), MMP-15 (MT2-MMP), MMP-16 (MT3-MMP), and MMP-24 (MT5-MMP). All of these MT-MMPs can activate proMMP-2, but MT1-MMP is considered to be the major in vivo activator of proMMP-2 in various tissues (see later discussion). MT1-MMP also degrades a fibrillar collagen on the cell surface. Like other collagen-degrading MMP enzymes (MMP-1, -2, -8, and -13), it cleaves the triple helical part of collagen at ¾ from the N-terminus. MT1-MMP also degrades other ECM components, including fibronectin, laminin, aggrecan, and gelatin (see Table 8.2 ). MT2-MMP digests fibronectin, tenascin, nidogen, aggrecan, perlecan, and laminin. MT3-MMP cleaves type III collagen, fibronectin, and gelatins. MMP-17 (MT4-MMP) and MMP-25 (MT6-MMP) are GPI-anchored MMPs. MT4-MMP and MT6-MMP can digest gelatin and fibrin/fibrinogen (see Table 8.2 ).
ADAM and ADAMTS Families
Two ADAM (a disintegrin and metalloproteinase) gene families exist: the enzymes with the transmembrane domain (ADAM) and secreted-type ADAM with thrombospondin motifs (ADAMTS; see Fig. 8.1 ). The active sites in the catalytic domains of most members of both gene families contain a common sequence of HEXGHXXGXXHD with the “Met-turn,” which also is present in MMP members. The ADAMTS family includes 19 members. Although information about substrates and biologic functions is still limited, ADAMTS1-5, 8-9, 14-16, 18, and 20 are all ECM-degrading proteinases (see Table 8.3 ). ADAMTS1, 4, 5, 9, and 15 can preferentially cleave aggrecan at the five Glu-X bonds, including the Glu 373 -Ala 374 bond (the aggrecanase site). Because ADAMTS4 and ADAMTS5 are characterized as the first two aggrecanases, they are also named aggrecanase-1 and aggrecanase-2 , respectively ; versican is also digested by these proteinases, and brevican is cleaved by ADAMTS4 (see Table 8.3 ). The C-terminus–truncated ADAMTS4 also degrades fibromodulin and decorin. ADAMTS16, 18, and 20 also appear to have weak aggrecanase activity. ADAMTS2 and 3 process the N-terminal prodomain of type I and II collagens and are named procollagen N-proteinase . Activity of procollagen N-proteinase also is known with ADAMTS14. ADAMTS13 is a von Willebrand factor–cleaving proteinase, and its mutation causes thrombotic thrombocytopenic purpura. Proteinase activities of other ADAMTS species are still unknown. The human genome contains 25 ADAM genes, including 4 pseudogenes, and thus the human ADAM family is composed of 21 members (see Table 8.4 ). Among the ADAMs, ADAM8-10, 12, 15, 17, 19-21, 28, 30, 33, and ADAM-like decysin 1 (ADAMDEC1) exhibit proteolytic activity (i.e., they are proteinase-type ADAMs; see Table 8.4 ). Although ADAM10, 12, and 15 degrade type IV collagen, the main substrates of these ADAMs are considered to be various membrane proteins, which include precursors of cytokines and growth factors such as TNF, HB-EGF, and neuregulin; IGF-BPs; receptors such as p75 TNF receptor; IL-1 receptor II; and other membrane proteins related to development such as Notch ligand and ephrin (see Table 8.4 ). According to these data, a major function of the ADAMs is the membrane protein shedding. ADAM17 processes proTNF (type II transmembrane molecule) and releases the soluble TNF, and is thus called TNF-converting enzyme (TACE). ADAM17 is also involved in release of L-selectin, TGF-α, and p75 TNF receptor. ADAM9, 12, and 17 can shed HB-EGF from its precursor. ADAM12 and 28 cleave IGF-BP-3 and IGF-BP-5. CD23 is shed by ADAM8, 15, and 28. Other functions of ADAMs include binding to integrins, cell-cell interaction, cell migration, and signal transduction (see Table 8.4 ).
Regulation of Proteinase Activity
The activities of ECM-degrading proteinases in tissues are regulated by different means, including their gene expression, activation of zymogen form, and inhibition by their endogenous inhibitors. Depending on the enzyme, some enzymes are also regulated by cell surface binding, endocytosis, and recycling.
Under physiologic conditions, cells express only limited levels of MMPs or TIMPs in tissues. However, under inflammatory conditions, expression of these genes are stimulated by cytokines and other factors. Neutrophils synthesize MMP-8 and MMP-9 during the differentiation and store them within the granules of the differentiated cells. Macrophages upon treatment with LPS or phorbormyristate acetate (12-O-tetradecanoylphorbol-13-acetate, PMA) express MMP-1, MMP-9, MT1-MMP and TIMP-1. Tumor cells express many MMPs such as MMP-1, 7, 9, 10, and MMP-14 (MT1-MMP), as well as TIMP-1, predominantly by oncogenic stimuli. The gene expression of MMPs and TIMPs in the tissue cell are regulated by numerous factors, including cytokines, growth factors, and chemical and physical stimuli. Much information is available for regulators of MMP-1 and MMP-3 , which are coordinately expressed in many cell types upon stimulation with cytokines and growth factors, factors acting at the cell surface, and chemical agents ( Table 8.5 ). The upregulated production of MMP-1 and MMP-3 is suppressed by retinoic acid, TGF-β, and glucocorticoid. The gene expression of MMP-7 and MMP-9 is regulated by similar factors, but the regulation is stricter and fewer factors modulate the expression (see Table 8.5 ). MMP-14 expression is upregulated by PMA, concanavalin A, fibrillar collagen, basic fibroblast growth factor, and TNF, and it is downregulated by glucocorticoids in various cells. TNF and IL-1α was reported to stimulate osteoarthritic chondrocytes to express the MMP-14 gene. In contrast to these MMPs, MMP-2 and TIMP-2 are unique in that factors capable of enhancing the production of MMP-1, MMP-3, and TIMP-1 are inactive. TIMP-1 expression is enhanced or suppressed in response to many factors, including cytokines, growth factors, and oncogenic transformation (see Table 8.5 ). Effects of these stimulatory factors are common to the gene expression of MMPs , but they are regulated independently. TGF-β, retinoic acid, progesterone, and estrogen enhance TIMP-1 expression in fibroblasts, but they suppress the expression of MMP-1 and 3 . Although information about stimulating and suppressive factors of TIMP-1, 2 , and 3 is available ( Table 8.5 ), factors controlling the gene expression of TIMP-4 are not well known. Previous studies have identified the elements in the promoters of MMPs and TIMPs , which are related to or responsible for the stimulation or suppression of the gene expression with various factors. Regulation of the gene expression is generally explained by the structural characteristics of the promoters.