Involvement of Fibronectin Type II Repeats in the Efficient Inhibition of Gelatinases A and B by Long-chain Unsaturated Fatty Acids

Abstract

The matrix metalloproteinases gelatinase A (MMP-2) and gelatinase B (MMP-9) are implicated in the physiological and pathological breakdown of several extracellular matrix proteins. In the present study, we show that long-chain fatty acids (e.g. oleic acid, elaidic acid, and cis-and trans-parinaric acids) inhibit gelatinase A as well as gelatinase B with K i values in the micromolar range but had only weak inhibitory effect on collagenase-1 (MMP-1), as assessed using synthetic or natural substrates. The inhibition of gelatinases depended on fatty acid chain length (with C18 > C16, C14, and C10), and the presence of unsaturations increased their inhibitory capacity on both types of gelatinase. Ex vivo experiments on human skin tissue sections have shown that micromolar concentrations of a long-chain unsaturated fatty acid (elaidic acid) protect collagen and elastin fibers against degradation by gelatinases A and B, respectively. In order to understand why gelatinases are more susceptible than collagenase-1 to inhibition by longchain fatty acids, the possible role of the fibronectin-like domain (a domain unique to gelatinases) in binding inhibitory fatty acids was investigated. Affinity and kinetic studies with a recombinant fibronectin-like domain of gelatinase A and with a recombinant mutant of gelatinase A from which this domain had been deleted pointed to an interaction of long-chain fatty acids with the fibronectin-like domain of the protease. Surface plasmon resonance studies on the interaction of longchain fatty acids with the three individual type II modules of the fibronectin-like domain of gelatinase A revealed that the first type II module is primarily responsible for binding these compounds. Matrix metalloproteinases (MMPs)1 compose a family of at least 23 related zinc-dependent endopeptidases (1) that are collectively able to degrade extracellular matrix proteins such as collagens, laminins, fibronectin, elastin, and proteoglycans. They are consequently implicated in physiological remodeling of connective tissue occurring in embryonic development and repair (2-4). Most of them are secreted as inactive proenzymes and are then extra-or pericellularly activated by other MMPs or serine proteinases (5). Their catalytic activities are strictly controlled by endogenous specific inhibitors designated as tissue inhibitors of metalloproteinases (TIMPs) (6) and also ␣ 2 -macroglobulin (7). The balance between activated MMPs and TIMPs determines the overall MMP proteolytic activity and consequently the extent of extracellular matrix degradation. Local disruption of the MMP-TIMP balance can lead to pathological degradative processes including rheumatoid arthritis, atherosclerosis, tumor growth, and metastasis (8 -10). MMPs are multidomain enzymes containing propeptide, catalytic and, except matrilysin (MMP-7), MMP-23, and endometase/matrilysin-2 (MMP-26), hemopexin-like domains. Gelatinase A (MMP-2) and gelatinase B (MMP-9) contain in addition three tandem copies of a 58-amino acid fibronectin type II (FN-II) modul...

Gelatinase-mediated migration and invasion of cancer cells

“…Long-chain fatty acids with 10 to 18 carbon atoms inhibit both gelatinases, but only weakly other MMPs as their binding site is in the collagen-binding domain (Berton et al, 2001). In general, the long and unsaturated fatty acids appear to be more potent than the short saturated ones (Berton et al, 2001).…”

“…In general, the long and unsaturated fatty acids appear to be more potent than the short saturated ones (Berton et al, 2001). However, the long-chain fatty acids are not gelatinase selective gelatinase, as they also inhibit other proteinases including neutrophil elastase and plasmin (Ashe and Zimmerman, 1977;Higazi et al, 1994).…”

“…Other extra domains that are not common to all members of MMPs include the collagenbinding domain (CBD) of gelatinases and the transmembrane domains of MT-MMPs. The CBD domain is composed of three fibronectin type II like repeats and is involved in binding of collagenous substrates and elastin (Steffensen et al, 1995), fatty acids (Berton et al, 2001) and thrombospondins (Bein and Simons, 2000). Although most of the MMPs are secreted proteins, six of them contain a transmembrane domain that is used to anchor these proteins on the cell surface.…”

Low Density Lipoprotein Receptor-related Protein Mediates Endocytic Clearance of Pro-MMP-2·TIMP-2 Complex through a Thrombospondin-independent Mechanism

“…Pro-MMP-2⅐TIMP-2 complex was purified from conditioned medium of human uterine cervical fibroblasts, as described (34). CBD 123 protein, the recombinant collagen-binding domain (CBD) comprising the three fibronectin type II-like modules of human MMP-2, was expressed in Escherichia coli and purified by gelatin-Sepharose 4B chromatography as reported previously (32,35). Purified full-length human LRP (36) was kindly provided by Dr. S. K. Moestrup (University of Aarhus, Aarhus, Denmark).…”

Pharmacomodulation of Broad Spectrum Matrix Metalloproteinase Inhibitors Towards Regulation of Gelatinases

“…For that purpose, long chain-unsaturated fatty acids, as oleic acid, have been described to inhibit HLE (Hornebeck et al, 1985;Shock et al, 1990;Tyagi & Simon, 1990) and to impede plasmin-mediated prostromelysin-1 activation (Huet et al, 2004) as well as gelatinases activities (Berton et al, 2001). To that respect, we envisaged the synthesis of a double-headed protease-MMP inhibitor (d-hPI) able to block elastase and MMP activities.…”