1. Introduction
1.4 Regulation of MMPs
1.4.1 Factors inducing and inhibiting MMPs
The expression of MMPs is positively regulated (stimulated) by various factors including cytokines (e.g., IL‐1, TNF). For instance,TNF stimulates the secretion of active MMP‐2 in a human organ culture model representing full‐thickness human skin and may also affect matrix remodeling during wound healing and other physiological and pathological processes (16).
Moreover,TNF (in combination with IL‐1‐α or IL‐1‐β) leads to a highly synergistic MMP‐3 increase (17). Other inducing factors are chemokines and growth factors including epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), platelet‐derived growth factor (PDGF), and the extracellular matrix metalloprotease inducer (EMMPRIN) (18) as well as physical agents such as ultraviolet‐B irradiation. On the other hand, expression of MMPs is negatively regulated by suppressive factors such as IL‐4, IL‐10, IL‐13, and TGF‐β (19).
This suppression was revealed in IL‐4 which suppresses MMP‐9 expression in human monocytes stimulated with (20). TGF‐β also reduces MMP‐9 expression induced by TNF in MonoMac‐6 (MM‐6) monocytic cells (21). In addition, IL‐10 inhibits MMP‐9 induction by ConA in human monocytes (22).
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several TF. As shown in MMP‐9 studies both ETS and AP‐1 sites were required for ras‐induced up‐regulation of MMP‐9 promoter activity (23). Moreover, synergistic up‐regulation of MMP‐9 is mediated through interactions of AP‐1 and NF‐κB (24), as shown by specific mutations in the AP‐1 and NF‐κB bindingsites of the MMP‐9 promoter (24). Equivalent results have been obtained in the promoters of MMP‐1 and MMP‐3 (25). The effect of estrogen receptor (ER)‐ coupled c‐fos on MMP‐9 activity was mediated by the proximal AP‐1 site of the promoter. Furthermore, suppression of AP‐1 is responsible for CADPE‐induced inhibition of MMP‐9 induction and cell invasion (26).
A recent study has shown that the inhibition of MMP‐9 expression by H.sabdariffa leaf extract may act through the suppression of the Akt/NF‐kB signaling pathway, which in turn led to the reduced invasiveness of the cancer cells (27). SP‐1 also binds to the MMP‐9 promoter to induce transcription, and the inhibition of SP‐1 leads to decreased MMP‐9 expression (28). Moreover, mutation of either SAF‐1 or AP‐1 binding sites greatly affects induction of the MMP‐9 promoter and reduces the ability of SAF‐1 and AP‐1 to activate transcription (29). In the promoters of other MMP family members, further regulating binding sites have been reported, e.g., for MMP‐2, ‐13,
‐14, and ‐19, polyoma virus enhancer‐A‐binding‐protein‐3 (PEA3) sites are present and in MMP‐1, ‐3, ‐9, 11, 13, and 19, TATA boxes are present in addition to CEBP which is present in MMP‐1, ‐11, and ‐14 (Fig. 1.3).
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are removed. This leads to a cascade of reactions resulting in conformational changes and the activation of the enzyme (31). This cleavage of cysteine’s through initiation of the cascade is called “cysteine–switch” (32). Most of the MMPs are activated either extracellular by other active MMPs or serine proteinases. For example, MMP‐3 activates pro‐MMP‐1, ‐7, ‐8, ‐9, and ‐ 13, whereas MMP‐2 activates pro‐MMP‐1, ‐9, and ‐13, and MMP‐7 can activate pro‐MMP‐8 and
‐9. In addition, the activation of MMP‐2 is regulated differently and occurs at the cell surface through a unique pathway, which involves MMP‐14 (MT1‐MMP) and TIMP‐2. In this process, pro‐MMP‐2 forms tight complex with TIMP‐2, which is essential for cell activation by MMP‐14 (33). MMP‐11, ‐14, and ‐28, however, can be activated intra‐cellular by furan‐like serine proteinases (34).
1.4.4 Regulation by inhibiting proteins
A third form of MMP regulation is mediated by endogenous specific inhibitors such as TIMPs or α2‐macroglobulin (35). The latter is an abundant plasma protein representing the major inhibitor of MMPs in tissue fluids (36). α2‐macroglobulin plays an important role in the irreversible clearance of MMPs. Inhibitors like TIMPs, however, are more specific and inhibit MMPs in a reversible manner (5). The first TIMP was described in 1975 as a protein in culture medium of human fibroblasts and in human serum, which was able to inhibit collagenase activity (37).
Meanwhile, four different types of TIMPs have been identified (TIMP‐1, ‐2, ‐3, and
‐4).
The interaction between latent MMP‐9 and TIMP‐1 is mediated via the C‐terminal domains of both proteins, whereas the N‐terminal inhibitory domain of TIMP‐1 remains ready to inhibit active MMPs (38, 39). Additionally, latent MMP‐9 is bound to TIMP‐1 before secretion (40).
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Mainly TIMP‐2 is a bi‐functional protein that acts as an inhibitor of MMPs and also as an activator of pro‐MMPs (41). TIMP‐3, which is tightly bound to the ECM, is involved in cell proliferation, apoptosis, and angiogenesis (42, 43). TIMP‐3 is binds to both pro‐MMP‐2 and pro‐ MMP‐9 (43, 44). In heart, MMP‐9 and TIMP‐4 are predominantly involved in cardiac remodeling by which TIMP‐4 mediates the effect of MMP‐9, moreover, the inhibition of MMP‐9 by either TIMP‐4 or PAR‐1 antagonist or by knocking down the gene itself improved cardiac function (45).
1.4.4 Regulation by further agents
MMPs can be also inhibited by chelating agents (e.g. EDTA), inhibiting antibodies, specific synthetic drugs (e.g. Marimastat), well known drugs with novel MMP inhibitory properties (like bisphosphonates or doxycycline), and chemically modified tetracycline as well as small synthetic peptides (46, 47). The group ofnon‐selective synthetic inhibitors includes the terminal fragment of the pro‐collagen C‐terminal proteinase enhancer protein (PCOLCE) (48) and a GPI‐ anchored glycoprotein called RECK (reversion including cysteine rich protein with Kazai motifs), which is the only known membrane‐bound MMP activation inhibitor (49).
1.5 The biological roles of MMPs
MMPs are important regulators of many biological and pathological processes due to their capability to degrade ECM components. Since ECM remodelling is a critical step in processes such as tissue growth and morphogenesis, MMPs are thought to play important roles during embryonic development, angiogenesis, apoptosis, and morphogenesis of specific tissues (e.g., lung, kidney, and breast) (50).
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Especially for cell proliferation and angiogenesis, ECM remodelling appears to be important, since for both processes the breakdown of ECM seems to be a prerequisite.
MMPs are also considered to be involved in many pathological conditions such as cancer, cardiovascular diseases, arthritis, nephritis, neurological disease, breakdown of the blood brain barrier, periodontal disease, skin ulceration, gastric ulcer, corneal ulceration, liver fibrosis, emphysema, and fibrotic lung disease (51). For example, in some cardiovascular disease studies, the contribution of MMP‐9 and ‐14 was revealed using MMP‐9‐ and ‐14‐deficient mice which both showed impaired angiogenesis during development (52). MMPs are also key‐players in the precise regulation of many bioactive molecules, mainly by their proteolytic and protein processing activity (11). In this context, it could be shown that MMPs cangenerate growth‐
promoting signals, asdemonstrated by the decreased cell proliferation rates of tumor cells injected into MMP‐9 deficient mice when compared to wild‐type mice (53, 54). Moreover, the proliferation of malignant cells as detected by immunohistochemistry for proliferating cell nuclear antigen is restored by bone‐marrow transplantation with MMP‐9‐positive cells to MMP‐9‐deficient mice (54). Furthermore, MMPs also support metastasis as shown by the increased invasion of certain cell lines through collagen‐containing matrices, when MMP‐2, ‐3, ‐ 13 and –14 were overexpressed (55), (56) . In addition to metastasis, MMPs are involved in the development and progression of arthritis. For instance, MMP‐13 was characterized by an increased expression in ankle joints of WT mice during K/BxN serum‐induced arthritis. Although both K/BxN serum‐treated WT and MMP‐13 KO mice developed progressive arthritis with a similar onset, MMP‐13 KO mice showed significantly reduced disease over the whole arthritic
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period. These studies suggest that MMPs maybe applied as a potential therapeutic target in certain diseases (57, 58).
1.6 Matrix metalloproteinase‐9
MMP‐9, also known as 92 kDa type IV collagenaseor gelatinase B, was first purified from human macrophages in 1974 (59).The main ability of MMP‐9 is the degradation of gelatin, elastin, collagens IV, V, VII, X, XI, XIV, and fibronectin (60). MMP‐9 is mainly produced by alveolar macrophages, polymorph nuclear leukocytes, osteoclasts, keratinocytes, and invading trophoblasts (61). During human embryonic development, MMP‐9 contributes to the regulation of normal physiological processes, e.g., the morphogenesis of specific tissues such as breast, kidney, and lung (62, 63). MMP‐9 is constitutively expressed in brain and bones, whereas during wound healing, the expression of MMP‐9 has been observed in macrophages, migrating leukocytes, and keratinocytes (64). MMP‐9 has also been described to assist in monocyte migration (65).
1.7 Structure and regulation of MMP‐9
1.7.1 Structure of MMP‐9
In most parts, MMP‐9 possesses the same basic structure like the other MMPs (66). However, several features distinguish MMP‐9 from other MMP family members since it has a gelatin‐
binding domain between the pro‐domain and the catalytic active site. Like other proteolytic enzymes, MMP‐9 is first synthesized as inactive enzyme (i.e., the zymogen) (4).
The included signal pre‐peptide on its N‐terminus is cleaved upon arrival to the endoplasmic reticulum (ER) (67). The remaining pro‐peptide domain contains a short stretch of 10 amino
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acids which, once attached, maintains enzyme latency by folding over the catalytic domain and interacting with the zinc ion that is responsible for MMP‐9’s enzymatic activity (68). In addition, three repeating fibronectin type‐II sequences inserted in the catalytic site are necessary for the selective binding and cleavage of collagen and elastin by MMP‐9 (69). The hpx‐domain is involved in TIMP‐1 and TIMP‐3 binding and some proteolytic activities (4) (Fig. 1.4).
Fig. 1.4: Schematic structure of MMP‐9 (according to Yabluchanskiy et.al. 2013 (29), modified. The catalytic site contains three essential zinc ion binding sites. At the zymogen stage, a cysteine residue within the pro‐domain interacts with zinc to prevent substrate binding. The haemopexin domain mediates interaction with enzyme substrates. Specific to the gelatinases is the fibronectin‐like domain, which further facilitates substrate binding. MMP, matrix metalloproteinase.
1.7.2 Regulation of MMP‐9
1.7.2.1 Cytokines and Growth Factors
The MMP‐9 mRNA expression is regulated by several cytokines and growth factors (Fig. 1.5).
Among the cytokines capable of regulating MMP‐9 expression, a major important role is assigned to TNF which triggers the production of MMP‐9 through the protein kinase C‐ (PKC‐)
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dependent signal transduction pathway (70). Additional results indicate that MMP‐9 synthesis and secretion were significantly induced after exposure to the cytokines TNF or IL‐1α, while MMP‐2 levels remained unchanged (71).
MMP‐9 may also be induced via induction by the vascular endothelial growth factor receptor (VEGF‐R1), as shown in lung‐specific metastasis (72). In addition, endothelial growth factor receptor (EGFR) activation also results in the induction of MMP‐9 (73‐75).
In contrast, MMP‐9 suppression is revealed through IL‐4 (20), TGF‐β (21), and IL‐10 (22) (see also chapter 1.1.4.1).
1.7.2.2 Signal Transduction
Mitogen‐activated protein kinases (MAPKs) including extracellular signal‐regulated kinase (ERK), c‐Jun N‐terminal kinase (JNKs), and p38 are part of critical signalling cascades converting upstream signals into biological responses such as cell proliferation, invasion, and transformation (76) (Fig. 1.5). MAPKs regulate MMP‐9 gene expression through transcription factors, e.g., c‐Jun is activated by p38 or JNK. The activated c‐Jun associates with its binding site(s) in the MMP‐9 promoter and subsequently recruits p38 as a cofactor to the promoter to induce its expression.
However, during metastasis of human colon cancer, this signalling event is also initiated by hyper‐expressed p38 that led to increased c‐Jun synthesis, MMP‐9 transcription, and MMP‐9‐dependent invasion through p38 interacting with c‐Jun. Thus, MAPK may act as both an activator and a cofactor of transcription factors to regulate MMP expression, finally leading to an
“invasive” response (77).
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Others have suggested that the combination of p38 and JNK are the most important regulatory signalling unit influencing MMPs expression and that both are required for the synergistic induction of MMPs by cytokines and growth factors (78). Indeed, other pathways have already been shown to regulate MMP‐9 expression individually (79) such as the ERK pathway which is mainly activated by growth factors and has been linked to cell proliferation, cell growth, and differentiation, and contributes to the transcriptional regulation of MMP‐9 in arterial smooth muscle cells (78).
Fig. 1.5: Signaling pathways involved in MMP gene transcription, and potential strategies for therapeutic intervention (according to Overall & Lopez‐Otin 2002 (11), modified. Compounds that are able to block the transcription of matrix metalloproteinase (MMP) genes at different levels are shown in red boxes. Extracellular
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factors, such as interferon‐ (IFN‐ ) inhibit MMP transcription via the JAK–STAT signalling pathway. Monoclonal antibodies against tumour necrosis factor‐ (anti‐TNF), soluble forms of the TNF receptor (sTNF‐R), natural antagonists of the interleukin (IL)‐1 receptor‐ >(IL‐1R ) or soluble forms of this receptor (sIL‐R) can block signalling pathways initiated by extracellular factors such as TNF‐ and IL‐1, which induce MMPs in cancer cells. Compounds such as manumycin A, SB203580, malolactomycin, SP600125, or PD98059 act at different levels to block the signal‐transduction pathways that are associated with MMP transcriptional induction in human tumours. Finally, there are several possibilities to target the nuclear factors that are responsible for MMP transcriptional up‐
regulation. Glucocorticoids, terpenoids, curcumin, nobiletin or NSAIDs (non‐steroidal anti‐inflammatory drugs) block the activity of transcription factors such as AP1 and NF‐ B, which regulate the transcription of several MMP genes.
Similarly, restoring the activity of transcription factors such as p53 and TEL, which negatively regulate MMP expression and the activity of which is lost in human tumours, could down regulate these genes. IFN‐ , interferon‐
; I B, inhibitor of B ; I BK; inhibitor of B kinase; JAK, JUN‐activated kinase; MAPK, mitogen‐activated protein kinase; MAPKK, mitogen‐activated protein kinase kinase; MAPKKK, mitogen‐activated protein kinase kinase kinase;
NF‐ B, nuclear factor of B; STAT, signal transducer and activator of transcription; TEL, translocation‐ETS‐ leukaemia.
1.7.2.3 Transcription Factors
The analysis of a 600 bp fragment of the MMP‐9 promoter in human shows several potential TF‐binding sites responsible for its regulation (80). As shown in Fig. 1.6, the promoter of the MMP‐9 gene mainly contains BS for NF‐κB, Sp1, AP‐1, and other TF (e.g. C/EBP, PEA3) (81, 82).
These binding sites are highly conserved in humans, rats, and mice.
Fig. 1.6: Regulatory elements in the promoter regions of MMP‐9. (according to Overall & Lopez‐Otin 2002 (11), modified). The promoters are shown in the direction 5'–3', with the transcription start sites indicated with a bent arrow, and the transcription‐factor‐binding sites placed within boxes. The relative positions of the different elements are not drawn to scale. Transcription‐factor‐binding sites include: the activator proteins (AP)‐1, the retinoblastoma control element (RCE), the keratinocyte differentiation‐factor responsive element (KRE), the transforming growth factor b inhibitory element (TIE), nuclear factor of B (NF‐ B) site, the polyomavirus enhancer‐A binding‐protein‐3 (PEA3) site, the TATA‐box (TATA).
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1.7.2.4 Activators and inhibitors
On the protein level, MMP‐9 zymogen is also activated via the “cysteine switch” in which the cysteine‐zinc interaction is abrogated by removal of the pro‐peptide domain (83). Upon activation, MMP‐9 is converted from a 92 kDa zymogen form to an 82 kDa active form (84).
MMP‐9 is activated by other MMPs, including MMP‐2, ‐3, ‐13, ‐17, and ‐26 (85‐88). For example, plasmin/MMP‐3 mediates an activation mechanism by which plasmin or MMP‐3 directly activates pro‐MMP‐9 (89) (Fig. 1.7).
Further regulation of MMP activity is offered by the TIMPs. Inhibition of MMP‐9 activation is performed by TIMP‐1 binding to the zymogen forms of the enzyme (90). Interestingly, it has been shown that TIMP‐1 is also able to bind and to inhibit the activity ofmature MMP‐9 (40, 43).
However, already activated MMP‐9 proteins are mainly controlled by TIMP‐3 (90, 91).
In circulation, α2macroglobulin inhibits MMP‐9 to prevent systemicMMP‐9 activation (29).
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also regulate inflammatory processes, e.g., by processing IL‐1β to its biologically active and mature form (95). It has also been suggested to play a role in the apoptotic process (96).
Additionally, MMP‐9 plays an important role in angiogenesis and neovascularization (97). MMP‐
9 has been shown to be importantly required for the recruitment of endothelial stem cells, a critical component of angiogenesis (97). Consequently, KO models of MMP‐9 resulted in delayed vascularization in addition to effects like apoptosis (98). Other studiesin MMP‐9 KO indicated that MMP‐9 plays a key role in cardiac rupture after myocardial infarction (99).
Moreover, neutrophil‐derived MMP‐9 appears to be elevated during acute myocardial infarction (MI), whereas macrophage‐secreted MMP‐9 is more important during subsequent tissue remodeling. Thus, MMP‐9 plays an important role in ventricular remodeling after acute MI and it has been concluded that plasma levels of MMP‐9 following MI may be predictive for this process.
High levels of MMP‐9 in patients with coronary artery disease have been recently reported (100) and high MMP‐9 levels also correlate with coronary artery ectasia (101) thus also serving as a predictor of increased mortality in patients with coronary artery disease (102). Serum levels of MMP‐9 have been reported to be elevated inpatients with MI and angina (103). Moreover, MMP‐9 plays an important role in animal models of both cerebral ischemia and human stroke. A critical role of MMP‐9 has been also shown for the development of abdominal aortic aneurysm using MMP gene deletion mice (104). Since MMP‐9 cleaves a variety of ECM molecules, recent studies on atherosclerotic plaque stability using a series of apoE/MMP‐9 double KO mice have indicated that MMP‐9 has a protective role by limiting plaque growth and enhancing plaque stability (105).
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1.9 MMP‐9 as a biomarker
Biomarkers are precious allies for research in the field of in vitro diagnostics which can be used to diagnose and monitor aetiology and progression of diseases and guide therapeutic decisions (106). Many clinical studies have established a relationship between MMP‐9 and certain diseases, making MMP‐9 a feasible candidate to add to the multiple biomarker lists. For example, a recent study has established a relationship between MMP‐9 and post‐MI remodeling and mortality thus making MMP‐9 a proximal biomarker for cardiac remodeling and a distal biomarker for inflammation (107). Additionally, some other studies showed the relationship between MMP‐9 and different types of cancers. For example, MMP‐9 levels in serum, plasma, and urine are significantly elevated in patients with breast cancer (108) and stage III or IV lung cancer (109). Urinary MMP‐9 levels also correlate with presence stage, and grade of bladder cancer (110). In addition, serum levels of MMP‐9 are significantly higher in patients with pancreatic ductal adenocarcinoma than in healthy controls (111). Both latent and activated forms of MMP‐9 have been detected in the cerebrospinal fluid of patients with brain tumors (112). Moreover, salivary MMP‐9 could be considered as a sensitive and specific diagnostic &
prognostic biomarker in the detection of oral lichen planus (OLP) (113).
It is important to note that the measurement of MMP‐9 in body fluids, in particular serum or plasma, can be influenced by the type of fluid and method of collection and storage. For example, basal MMP‐9 levels in serum/plasma can be influenced by the use of EDTA or heparin (114), a problem that can be alleviated by using sodium citrate instead (115). Another issue to be considered is that of sample storage. For example, it has been reported that plasma MMP‐9 is unstable and degrades rapidly even when stored at −80°C (116).
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However, the influence of direct and indirect effects of different anti‐coagulants and their impact on MMP‐9 expression is still poorly elucidated. Thus, further studies are needed to assess MMP‐9 as a biomarker due to controversial results and non‐standardized procedures in pre/post‐analytical.
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1.10 Aims of the study:
In this study, the effect of different anticoagulants on the regulation of MMP‐9 will be characterized, including the identification of molecular mechanism mediating these effects to assess the impact of the respective substances on the suitability of MMP‐9 as a biomarker.
The specific aims of the present study are:
To identify the effect of different anticoagulants on MMP‐9 expression. To identify the major MMP‐9 expressing cell type(s) in the blood.
To identify the mediator(s) inducing MMP‐9 production.
To elucidate the molecular mechanism(s) regulating MMP‐9 expression.
To characterize further cellular effects shown by the respective cell type(s) under MMP‐9‐ inducing conditions.
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2 Materials
2.1.1 Chemical Reagents:
Reagents Company/Origin
Acrylamide solution Roth, Karlsruhe, D
Agar Gibco Life Technologies, Karlsruhe, D
Agarose Biozym, Oldendorf, D
Ampicillin Sigma Aldrich, Darmstadt, D
Bovine serum albumin, fatty acid free Sigma Aldrich
Cell culture media: RPMI Gibco Life Technologies
Ethidium bromide Sigma Aldrich
EDTA Sigma Aldrich
Fetal serum albumin Biochrome, Berlin, D
Glycine Merck, Darmstadt, D
PBS buffer (pH 7.4) Biochrome
5x passive lysis buffers Promega, Heidelberg, D
Heparin Ratio pharm, ULM, D
Citrate Sarstadt, Numbrecht, D
Low molecular weight heparin Sanofi, Paris, France
2.1.2 Enzymes
Enzymes Company/Origin
Taq DNA‐Polymerase Qiagen, Hilden, D
Superscript ™ II Reverse Transcriptase Invitrogen, Karlsruhe, D
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2.1.3 DNA size markers
DNA size markers Company/Origin
DNA size markers Company/Origin