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.

Kallikreins

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.

Matrix Metalloproteinases

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 ).

Schematic representation of domain structure of metalloproteinases. MMP can be divided into two major groups: soluble MMPs and membrane-type MMPs, and they can be further classified into six subgroups in soluble MMPs and two subgroups in membrane-type MMPs according to their structures. MMP-11, 21, 28, and 23, and MT-MMPs have a basic motif of RXKR that is recognized and cleaved by proprotein convertases to activate the enzymes by removing their prodomain. ADAMTS enzymes also have a conserved domain structure and differ in the number of thrombospondin motifs (TS) at their C-terminus. ADAMTS-4 is the smallest, without a C-terminal TS, and ADAMTS-5 and 8 have two. Other members have 2 to 14 repeats. ADAM enzymes have a similarly conserved domain structure. ADAMTS and ADAM enzymes have an RXKR motif at the C-terminus of their propeptide from activation by proprotein convertases. C, Cysteine; CA, cysteine array; Cat, catalytic domain; CP, cytoplasmic domain; CysR, cysteine-rich domain; DITG, disintegrin-like domain; EGF, EGF-like domain; FN-II, fibronectin type II repeats; GPI, GPI-anchoring signal; Hpx, hemopexin domain; IgG, IgG-like domain; L, linker or hinge region; L1, linker 1 or hinge region; L2, linker 2 or stalk region; Pro, prodomain; Sig, signal peptide; Spacer, spacer domain; TM, transmembrane domain; Type II TM, type II transmembrane domain.

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 ).

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 ).

Matrix Metalloproteinases

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.

TABLE 8.5

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