Isolation and characterization of human monoclonal antibodies specific to MMP-1A, MMP-2 and MMP-3

Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are up-regulated in many diseases, but the use of MMP inhibitors for therapeutic purposes has often been disappointing, possibly for the limited specificity of the drugs used in clinical trials. In principle, individual MMPs could be specifically drugged by monoclonal antibodies, either by inhibition of their catalytic activity or by antibody-based pharmacodelivery strategies. In this article we describe the isolation and affinity maturation of recombinant antibodies (SP1, SP2, SP3) specific to the murine catalytic domains of MMP-1A, MMP-2 and MMP-3. These novel reagents allowed a systematic comparative immunofluorescence analysis of the expression patterns of their cognate antigens in a variety of healthy, cancerous and arthritic murine tissues. While all three MMPs were strongly expressed in tumor and arthritis specimens, MMP-1A was completely undetectable in the normal tissues tested, while MMP-2 and MMP-3 exhibited a weak expression in certain normal tissues (e.g., liver). The new antibodies may serve as building blocks for the development of antibody-based therapy strategies in mouse models of pathology.     

Opposite effects of high glucose on MMP-2 and TIMP-2 in human endothelial cells

Diabetes mellitus (DM) is a major risk factor for atherosclerosis and causes multiple cardiovascular complications. Although high glucose can induce matrix metalloproteinases (MMPs), its inhibitors and cell apoptosis, little is known about the roles of MMPs in regulating cell apoptosis in response to high glucose. To address this issue, we elucidated the relationship between MMPs, its inhibitors and cell apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with medium containing 5.5 mM or 33 mM of glucose in the presence or the absence of ascorbic acid and MMP inhibitors (GM6001 and endogenous tissue inhibitors of MMPs, TIMP-1, and TIMP-2). For detection of cell apoptosis, the cell death detection ELISA assay was used. The results revealed that high glucose-induced apoptosis could be suppressed by ascorbic acid, GM6001 and TIMP-2, but not by TIMP-1. The activities of MMP-2, MMP-9 and its inhibitors, TIMP-1, TIMP-2 after high glucose treatment, were also detected by ELISA method. We found that the activated form of MMP-2, but not MMP-9, was increased, while the level of TIMP-2, but not TIMP-1, was decreased. In Western blot and RT-PCR analysis, the expression of TIMP-2, but not TIMP-1, after high glucose treatment was downregulated, whereas the levels of MMP-2 and -9 proteins and mRNA were not changed. The present study indicated that oxidative stress induced by high glucose might be involved in the opposite effects on MMP-2 activation and TIMP-2 downregulation. This reactive oxygen species (ROS)-dependent MMP-2 activation in turn mediates high glucose-induced cell apoptosis in HUVECs.     

Activities and polymorphisms of MMP-2 and MMP-9, smoking,diabetes and risk of prostate cancer

Molecular Biology Reports - Matrix metalloproteinases (MMPs) are a group of zinc dependent enzymes that are involved in tumor cell invasion and metastasis. The role of MMP-2 and -9 genetic...     

CXCR4 promotes oral squamous cell carcinoma migration and invasion through inducing expression of MMP-9 and MMP-13 via the ERK signaling pathway

The increased migration and invasion of oral squamous cell carcinoma cells are key events in the development of metastasis to the lymph nodes and distant organs. Although the chemokine receptor CXCR4 and its ligand, stromal cell-derived factor-1α, have been found to play an important role in tumor invasion, its precise role and potential underlying mechanisms remain largely unknown. In this study, we showed that knockdown of CXCR4 significantly decreased Tca8113 cells migration and invasion, accompanied with the reduction of MMP-9 and MMP-13 expression. Inhibition of ligand binding to CXCR4 by a specific antagonist TN14003, also led to reduced cancer cell migration and invasion. Because the degradation of the extracellular matrix and the basement membrane by proteases, such as matrix metalloproteinases (MMP) is critical for migration and invasion of cancer cells, we investigated the expression of several MMPs and found that the expression of functional MMP-9 and MMP-13 was selectively decreased in CXCR4 knockdown cells. More importantly, decreased cell migration and invasion of CXCR4 knockdown cells were completely rescued by exogenous expression of MMP-9 or MMP-13, indicating that the two MMPs are downstream targets of CXCR4-mediated signaling. Furthermore, we found the level of phosphorylated extracellular signal-regulated kinase (ERK) was significantly decreased in CXCR4-silenced cells, suggesting that ERK may be a potential mediator of CXCR4-regulated MMP-9 and MMP-13 expression in Tca8113 cells. Taken together, our results strongly suggest the underlying mechanism of CXCR4 promoting Tca8113 migration and invasion by regulating MMP-9 and MMP-13 expression perhaps via activation of the ERK signaling pathway.     

Inhibition of MMP-3 or -9 suppresses lipopolysaccharide-induced expression of proinflammatory cytokines and iNOS in microglia

Recently, matrix metalloproteinases (MMPs) are emerging as important molecules in neuroinflammation as well as neuronal cell death. However, the role of MMPs in activated microglia remains unclear. In the present study, we found that expressions of MMP-1, -3, -8 and -9 were significantly induced by single or combined treatment of immunostimulants lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) in primary cultured microglia and BV2 microglial cells. Inhibition of MMP-3 or -9 significantly suppressed the expression of iNOS and pro-inflammatory cytokines and the activities of NF-κB, AP-1, and MAPK in LPS-stimulated microglia. The results suggest that MMP-3 and -9 both mediate LPS-induced inflammatory reactions. Inhibition of reactive oxygen species (ROS) by N-acetyl-cysteine or diphenylene iodonium significantly suppressed the expression of MMP-3, MMP-9, NO and TNF-α in LPS-stimulated microglia, suggesting that ROS is an early signaling inducer in LPS-stimulated microglial cells. MMP inhibitors also suppressed ROS production, suggesting a cross-talk between ROS and MMPs. Collectively, the present study demonstrates that MMP-3 and MMP-9 play a role as inflammatory mediators in activated microglia. Pharmacological intervention of MMPs especially MMP-3 and -9 would be a therapeutic strategy for the treatment of inflammatory diseases in the CNS caused by over-activation of microglial cells.     

Expression of genes for metalloproteinases (MMP-1, MMP-2, MMP-9, and MMP-12) associated with psoriasis

Using real-time polymerase chain reaction (RT-PCR), we measured mRNA amounts of matrix metalloproteinases (MMPs): MMP-1, MMP-2, MMP-9, and MMP-12 genes in psoriatic lesions and unaffected skin of the same patients. We observed significant (about 15-fold) increase in the expression level of matrix metalloproteinase MMP-1 and MMP-12 genes associated with psoriasis. The results of our studies of MMP gene expression in cultured primary human keratinocytes treated with interleukin (IL-17) have shown upregulation of MMP gene expression both in cultured keratinocytes and in psoriatic skin lesions. Therefore, upregulation of MMP genes in the skin affected by psoriasis could result from IL-17 effects on skin cells.     

Engineering of tissue inhibitor of metalloproteinases TIMP-1 for fine discrimination between closely related stromelysins MMP-3 and MMP-10

Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development. Here, we used a counter-selective screening strategy for directed evolution of yeast-displayed human TIMP-1 to obtain TIMP-1 variants highly selective for the inhibition of MMP-3 in preference over MMP-10. As MMP-3 and MMP-10 are the most similar MMPs in sequence, structure, and function, our results thus clearly demonstrate the capability for engineering full-length TIMP proteins to be highly selective MMP inhibitors. We show using protein crystal structures and models of MMP-3-selective TIMP-1 variants bound to MMP-3 and counter-target MMP-10 how structural alterations within the N-terminal and C-terminal TIMP-1 domains create new favorable and selective interactions with MMP-3 and disrupt unique interactions with MMP-10. While our MMP-3-selective inhibitors may be of interest for future investigation in diseases where this enzyme drives pathology, our platform and screening strategy can be employed for developing selective inhibitors of additional MMPs implicated as therapeutic targets in disease.     

Inhibition of matrix metalloproteinase 14 (MMP-14)-mediated cancer cell migration

Matrix metalloproteinases (MMPs) have been shown to be key players in both extracellular matrix remodeling and cell migration during cancer metastasis. MMP-14, a membrane-anchored MMP, in particular, is closely associated with these processes. The hemopexin (PEX) domain of MMP-14 has been proposed as the modulating region involved in the molecular cross-talk that initiates cell migration through homodimerization of MMP-14 as well as heterodimerization with the cell surface adhesion molecule CD44. In this study, minimal regions required for function within the PEX domain were investigated through a series of substitution mutations. Blades I and IV were found to be involved in cell migration. We found that blade IV is necessary for MMP-14 homodimerization and that blade I is required for CD44 MMP-14 heterodimerization. Cross-talk between MMP-14 and CD44 results in phosphorylation of EGF receptor and downstream activation of the MAPK and PI3K signaling pathways involved in cell migration. Based on these mutagenesis analyses, peptides mimicking the essential outermost strand motifs within the PEX domain of MMP-14 were designed. These synthetic peptides inhibit MMP-14-enhanced cell migration in a dose-dependent manner but have no effect on the function of other MMPs. Furthermore, these peptides interfere with cancer metastasis without affecting primary tumor growth. Thus, targeting the MMP-14 hemopexin domain represents a novel approach to inhibit MMP-14-mediated cancer dissemination.     

Amber force field implementation, molecular modelling study, synthesis and MMP-1/MMP-2 inhibition profile of (R)- and (S)-N-hydroxy-2-(N-isopropoxybiphenyl-4-ylsulfonamido)-3-methylbutanamides

Ab initio calculations (B3LYP/Lanl2DZ level of theory) were performed in this study to determine all the structural and catalytic zinc parameters required in order to study MMPs and their complexes with hydroxamate inhibitors by means of the AMBER force field. The parameters thus obtained were used in order to study the docking of some known MMPi (Batimastat, CGS 27023A and Prinomastat) and our previously described inhibitor a which had shown an inhibitory activity for MMP-1, and -2, with the aim of explaining the different selectivity. On this basis the two enantiomers (R)-b and (S)-b were designed and synthesized, as more potent MMP-2 inhibitors than our previously described inhibitor a. Between these two enantiomers the eutomer (R)-b proved to be 24.7 times and 15.3 times more potent than CGS 27023A and the parent compound a on MMP-2, maintaining a higher index of MMP-2/MMP-1 selectivity compared with CGS 27023A and the more potent inhibitor Prinomastat. The hydroxamate (R)-b can be considered as a progenitor of a new class of biphenylsulfonamido-based inhibitors that differ from compound a in the presence of an alkyl side chain on the C alpha atom, and show different potency and selectivity profiles on the two MMPs considered.     

Tissue mRNA expression in rat of newly described matrix metalloproteinases

Matrix metalloproteinases (MMPs) comprise a large group of endoproteinases that degrade all protein components of the extracellular matrix. Functionally, MMPs contribute to several different physiological as well as pathological conditions. The number of newly described MMPs has increased in recent years, although current knowledge about their expression pattern in various tissues remains incomplete. Here we analyzed the relative mRNA expression of the most recently described MMPs--MT5-MMP (MMP-24), MT6-MMP (MMP-25), MMP-27 and epilysin (MMP-28)--in a broad selection of rat tissues using real time-PCR. MMP-24 mRNA was found to be widely expressed with predominance in the central nervous system. MMP-25 mRNA, in contrast, exhibited peak expression levels in testis, kidney and skeletal muscle, differing from previously described distribution patterns in humans. mRNAs for MMP-27 and MMP-28 were generally expressed at a lower level. All four MMPs studied were detected at higher mRNA levels in bone and kidney, suggesting a possible role of these MMPs in physiological processes within these two organs. The present study highlights the differential distribution pattern of newly described MMPs among different tissues and underlines differences in the mRNA expression between different species.     

Investigating metalloproteinases MMP-2 and MMP-9 mechanosensitivity to feedback loops involved in the regulation of in vitro angiogenesis by endogenous mechanical stresses

Angiogenesis is a complex morphogenetic process regulated by growth factors, but also by the force balance between endothelial cells (EC) traction stresses and extracellular matrix (ECM) viscoelastic resistance. Studies conducted with in vitro angiogenesis assays demonstrated that decreasing ECM stiffness triggers an angiogenic switch that promotes organization of EC into tubular cords or pseudo-capillaries. Thus, mechano-sensitivity of EC with regard to proteases secretion, and notably matrix metalloproteinases (MMPs), should likely play a pivotal role in this switching mechanism. While most studies analysing strain regulation of MMPs used cell cultured on stretched membranes, this work focuses on MMP expression during self-assembly of EC into capillary-like structures within fibrin gels, i.e. on conditions that mimics more closely the in vivo cellular mechanical microenvironment. The activity of MMP-2 and MMP-9, two MMPs that have a pivotal role in capillaries formation, has been monitored in pace with the progressive elongation of EAhy926 cells that takes place during the emergence of cellular cords. We found an increase of the zymogen proMMP-2 that correlates with the initial stages of EC cords formation. However, MMP-2 was not detected. ProMMP-9 secretion decreased, with levels of MMP-9 kept at a rather low value. In order to analyse more precisely the observed differences of EAhy926 response on fibrin and plastic substrates, we proposed a theoretical model of the mechano-regulation of proMMP-2 activation in the presence of type 2 tissue inhibitor of MMPs (TIMP-2). Using association/dissociation rates experimentally reported for this enzymatic network, the model adequately describes the synergism of proMMP-2 and TIMP-2 strain activation during pseudo-capillary morphogenesis. All together, these results provide a first step toward a systems biology approach of angiogenesis mechano-regulation by cell-generated extracellular stresses and strains.     

The gelatinases,MMP-2 and MMP-9, as fine tuners of neuroinflammatory processes

This review focuses on the complementary roles of MMP-2 and MMP-9 in leukocyte migration into the brain in neuroinflammation, studied mainly in a murine model of experimental autoimmune encephalomyelitis (EAE) that has similarity to the human disease multiple sclerosis. We discuss the cellular sources of MMP-2/MMP-9 in EAE, their sites of activity, and how cleavage of the to-date identified MMP-2/MMP-9 substrates at the blood-brain barrier facilitate leukocyte filtration of the central nervous system (CNS). Where necessary, comparisons are made to inflammatory processes in the periphery and to other MMPs relevant to neuroinflammation. While the general principles concerning MMP-2 and MMP-9 function discussed here are relevant to all inflammatory situations, the details regarding substrates and molecular mechanisms of action are likely to be specific for neuroinflammation.     

Adenylyl cyclase-associated protein-1/CAP1 as a biological target substrate of gelatinase B/MMP-9

Matrix metalloproteinases (MMPs) are classically associated with the turnover of secreted structural and functional proteins. Although MMPs have been shown to process also a kaleidoscope of membrane-associated substrates, little is known about the processing of intracellular proteins by MMPs. Physiological and pathological cell apoptosis, necrosis and tumor lysis by chemotherapy, radiotherapy or immunological cytotoxicity, are examples of conditions in which an overload of intracellular proteins becomes accessible to the action of MMPs. We used a model system of dying human myelomonocytic cells to study the processing of intracellular protein substrates by gelatinase B/MMP-9 in vitro. Adenylyl cyclase-associated protein-1 or CAP1 was identified as a novel and most efficient substrate of gelatinase B/MMP-9. The presence of CAP1 in the extracellular milieu in vivo was documented by analysis of urine of patients with systemic autoimmune diseases. Whereas no active MMP-9 could be detected in urines of healthy controls, all urine samples of patients with clinical parameters of renal failure contained activated MMP-9 and/or MMP-2. In addition, in some of these patients indications of CAP1 cleavage are observed, implying CAP1 degradation in vivo. The high turnover rate of CAP1 by MMP-9, comparable to that of gelatin as the natural extracellular substrate of this enzyme, may be critical to prevent pathological conditions associated with considerable cytolysis.     

UVA-mediated downregulation of MMP-2 and MMP-9 in human epidermal keratinocytes

While human dermal fibroblasts increase the expression and secretion of distinct matrix metalloproteinases (MMPs) in response to ultraviolet (UV) irradiation, much less is known about regulation of MMPs with regard to normal human epidermal keratinocytes (NHEK). In this in vitro study, the effect of ultraviolet A (UVA) irradiation on gelatinase expression and secretion by NHEK was investigated. Irradiation of NHEK with non-toxic doses of UVA resulted in a dose-dependent downregulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). A single dose of 30JUVA/cm(2) lowered MMP-2 activity to 26% and MMP-9 activity to 33% compared with mock-irradiated cells at 24h after irradiation. Downregulation of MMP-2 and MMP-9 steady-state mRNA levels was observed at 4h after UVA irradiation. The inhibitory effect of UVA on gelatinases was mediated by UVA-generated singlet oxygen (1O(2)). These findings suggest an inverse response to UVA irradiation in NHEK than in fibroblasts.     

Inhibition of matrix metalloproteinase MMP-2 activates chloride current in human airway epithelial cells.

Matrix metalloproteinases (MMPs) are involved in the remodeling and degradation of the extracellular matrix. Recently, it has been found that MMPs also contribute to processes not directly related to tissue remodeling, such as platelet aggregation or degranulation of airway gland cells. Since mucus secretion is closely related to ion channel function, we investigated whether MMPs could also be involved in the regulation of ion channels. We used human airway submucosal cell line Calu-3 to study the effects of MMPs on whole-cell current and transepithelial short-circuit current (I(sc)). Phenanthroline, a specific inhibitor of MMPs, increased whole-cell current with the half-maximally effective dose of 5.2 microM, and reversibly activated I(sc) in transepithelial measurements. Current stimulated by phenanthroline displayed linear current-voltage relationships and had inhibitor pharmacology and ion selectivity consistent with cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity. Zymography and Western blot showed significant expression of MMP-2 in Calu-3 cells. Moreover, anti-MMP-2 antibodies (1 microg/mL) increased whole-cell current and I(sc), whereas human recombinant MMP-2 (10 ng/mL) reduced it. We also studied the expression of MMPs and the effects of phenanthroline on whole-cell current in A549 cells, which are derived from airway surface epithelium and do not express CFTR Cl- channels. While these cells also showed significant expression of MMP-2, inhibition of this enzyme with phenanthroline exerted no significant effect on whole-cell current. It is concluded that MMP-2 is involved in the regulation of CFTR Cl- channels in human airways.