Jelena Gavrilovic

Mechanisms for pro matrix metalloproteinase activation

The activation of pro matrix metalloproteinases (MMPs) by sequential proteolysis of the propeptide blocking the active site cleft is regarded as one of the key levels of regulation of these proteinases. Potential physiological mechanisms including cell‐associated plasmin generation by urokinase‐like plasminogen activator, or the action of cell surface MT1‐MMPs appear to be involved in the initiation of cascades of pro MMP activation. Gelatinase A, collagenase 3 and gelatinase B may be activated by MT‐MMP based mechanisms, as evidenced by both biochemical and cell based studies. Hence the regulation of MT‐MMPs themselves becomes critical to the determination of MMP activity. This includes activation, assembly at the cell surfaces as TIMP‐2 complexes and subsequent inactivation by proteolysis or TIMP inhibition.

Proteolysis and cell migration: creating a path?

Both serine and metalloproteinases have been implicated in the complex integrated events underlying cell migration but no definitive single mechanism has emerged. Work over the past two years linking both membrane and soluble proteinases with integrins and other adhesion proteins and with intracellular signalling systems could herald the beginnings of a potential expansion of our understanding of the role and regulation of proteolysis in cell migration.

The Role of Plasmhogen Activators in the Regulation of Connective Tissue Metalloproteinasesa

The conversion of plasminogen to plasmin is a key event in many physiological and pathologal processes requiring regulated extracellular proteolysis. Direct focal degradation of proteins involved in cell-cell and cell-matrix interactions by plasmin has been described,js as well as activation of other degradative enzymes, notably the matrix rnetall~proteinases~ (MMPs). Complex control of the plasminogen activator cascade has been shown to be required for the movement and re-organization of cells and matrix in events such as ovulation, trophoblast implantation, embryogenesis, and angiogenesis, as well as in the invasion and metastasis of tumors. A further level of regulation is thought to be effected by the release by plasmin of matrix-sequestered growth factors, such as basic fibroblast growth factor and trdnsbrming growth factor /3,677 which are known to regulate the expression of both the plasminogen activatorinhibitor and the MMP-inhibitor systems2

Extracellular matrix and matrix metalloproteinases in sciatic nerve.

Although matrix metalloproteinases (MMPs) are increasingly being implicated in several pathologies of the nervous system, it is not yet clear what role they play in normal neurobiological processes. We review the expression of extracellular matrix (ECM) components as well as MMPs and tissue inhibitors of metalloproteinases (TIMPs) in the peripheral nervous system. We explore the expression of certain MMPs and the four TIMPs at the mRNA level in the postnatal mouse sciatic nerve. In addition, we have used substrate gel and in situ zymography to determine levels of MMP‐2 and ‐9 and TIMP activity in rat sciatic nerve after crush and during regeneration. A rapid and transient increase in MMP‐9 localised at and immediately distal to the site of injury was observed, whereas an increase in MMP‐2 activity was delayed, prolonged, and extended proximal and distal to the injury site. This activity coincides with periods of axonal elongation, suggesting that it could act to facilitate axonal extension along the nerve matrix. We also detected multiple species of gelatinolytic inhibitory activity, including TIMP‐1 and ‐3 in control and injured nerve. These activities probably act to prevent uncontrolled gelatinolytic activity, maintaining nerve integrity at the level essential for axonal regrowth.

Matrix metalloproteinase genes in Xenopus development

Matrix metalloproteinases (MMPs) are a large family of proteins in vertebrates, consisting of over 24 genes in humans, only a few of which have been identified in Xenopus. Three genes coding for MMPs in Xenopus have been identified and their expression studied during development. The membrane‐bound XMMP‐14 and ‐15 (XMT1‐MMP and XMT2‐MMP) both showed restricted expression patterns, the former principally localising to cranial neural crest tissues and the latter to the epidermis of the embryo. XMMP‐7 codes for an MMP that lacks the hemopexin‐like domain. It is expressed exclusively in macrophages or other myeloid cell types from early in development. Developmental Dynamics 231:214–220, 2004.

Distinct Functions of Natural ADAM-15 Cytoplasmic Domain Variants in Human Mammary Carcinoma

Adamalysins [a disintegrin and metalloproteinase (ADAM)] are a family of cell surface transmembrane proteins that have broad biological functions encompassing proteolysis, adhesion, and cell signal regulation. We previously showed that the cytoplasmic domain of ADAM-15 interacts with Src family protein tyrosine kinases and the adaptor protein growth factor receptor binding protein 2 (Grb2). In the present study, we have cloned and characterized four alternatively spliced forms of ADAM-15, which differ only in their cytoplasmic domains. We show that the four ADAM-15 variants were differentially expressed in human mammary carcinoma tissues compared with normal breast. The expression of the individual isoforms did not correlate with age, menopausal status, tumor size or grade, nodal status, Nottingham Prognostic Index, or steroid hormone receptor status. However, higher levels of two isoforms (ADAM-15A and ADAM-5B) were associated with poorer relapse-free survival in node-negative patients, whereas elevated ADAM-15C correlated with better relapse-free survival in node-positive, but not in node-negative, patients. The expression of ADAM-15A and ADAM-15B variants in MDA-MB-435 cells had differential effects on cell morphology, with adhesion, migration, and invasion enhanced by expression of ADAM-15A, whereas ADAM-15B led to reduced adhesion. Using glutathione S-transferase pull-down assays, we showed that the cytoplasmic domains of ADAM-15A, ADAM-15B, and ADAM-15C show equivalent abilities to interact with extracellular signal-regulated kinase and the adaptor molecules Grb2 and Tks5/Fish, but associate in an isoform-specific fashion with Nck and the Src and Brk tyrosine kinases. These data indicate that selective expression of ADAM-15 variants in breast cancers could play an important role in determining tumor aggressiveness by interplay with intracellular signaling pathways. (Mol Cancer Res 2008;6(3):383–94)

Macrophage migration and invasion is regulated by MMP10 expression.

This study was designed to identify metalloproteinase determinants of macrophage migration and led to the specific hypothesis that matrix metalloproteinase 10 (MMP10/stromelysin-2) facilitates macrophage migration. We first profiled expression of all MMPs in LPS-stimulated primary murine bone marrow-derived macrophages and Raw264.7 cells and found that MMP10 was stimulated early (3 h) and down-regulated later (24 h). Based on this pattern of expression, we speculated that MMP10 plays a role in macrophage responses, such as migration. Indeed, using time lapse microscopy, we found that RNAi silencing of MMP10 in primary macrophages resulted in markedly reduced migration, which was reversed with exogenous active MMP10 protein. Mmp10 −/− bone marrow-derived macrophages displayed significantly reduced migration over a two-dimensional fibronectin matrix. Invasion of primary wild-type macrophages into Matrigel supplemented with fibronectin was also markedly impaired in Mmp10 −/− cells. MMP10 expression in macrophages thus emerges as an important moderator of cell migration and invasion. These findings support the hypothesis that MMP10 promotes macrophage movement and may have implications in understanding the control of macrophages in several pathologies, including the abnormal wound healing response associated with pro-inflammatory conditions.

The role of plasminogen in cell-mediated collagen degradation.

The ability of VX2 tumour cells and chondrocytes to degrade radiolabelled collagen films was shown to be dependent on the presence of the serum proteinase plasminogen. Degradation of collagen films in the presence of plasminogen was inhibited by addition of exogenous TIMP indicating that such lysis was mediated by collagenase. VX2 cells required ten times less plasminogen than chondrocytes to effect comparable degradation; this result was probably related to the observation that VX2 cells did not synthesize the specific tissue inhibitor of metalloproteinases, TIMP.

Inhibition of type I collagen film degradation by tumour cells using a specific antibody to collagenase and the specific tissue inhibitor of metalloproteinases (TIMP)

Rabbit VX2 tumour cells in culture produced a collagenolytic activity which was shown to be immunologically identical to collagenase from rabbit articular chondrocytes and bone. VX2 cells degraded type I collagen films spontaneously and did not produce detectable levels of the tissue inhibitor of metalloproteinases (TIMP). Chondrocytes, however, required both stimulation of collagenase synthesis and activation to effect lysis and were observed to make appreciable amounts of TIMP. The degradation of type I collagen films by VX2 tumour cells was significantly inhibited by both a specific antibody to rabbit collagenase and by purified TIMP, thus demonstrating the unequivocal role of collagenase in this model system.

Plasma matrix metalloproteinases, low density lipoprotein oxidisability and soluble adhesion molecules after a glucose load in Type 2 diabetes

BackgroundAcute hyperglycaemia is an independent cardiovascular risk factor in Type 2 diabetes which may be mediated through increased oxidative damage to plasma low density lipoprotein, and in vitro, high glucose concentrations promote proatherogenic adhesion molecule expression and matrix metalloproteinase expression.MethodsWe examined these atherogenic risk markers in 21 subjects with Type 2 diabetes and 20 controls during an oral 75 g glucose tolerance test. Plasma soluble adhesion molecule concentrations [E-selectin, VCAM-1 and ICAM-1], plasma matrix metalloproteinases [MMP-3 and 9] and plasma LDL oxidisability were measured at 30 minute intervals.ResultsIn the diabetes group, the concentrations of all plasma soluble adhesion molecules fell promptly [all p < 0.0001] related principally to glycaemic excursions, but such changes also occurred in the control group. Plasma MMP-3 and -9 concentrations were lower [p < 0.05], and LDL oxidisability greater [p < 0.01] in the diabetes group but did not change in either group. There was a direct relationship between plasma MMP-9 and s ICAM-1 in the controls [r = 0.62; p = 0.006] perhaps suggesting a functional relationship between s ICAM-1 shedding and MMP-9.ConclusionsA glucose load leads to a rapid fall in plasma soluble adhesion molecule concentrations in Type 2 diabetes and controls, perhaps reflecting reduced generation of soluble from membrane forms during enhanced leukocyte – endothelial adhesion or increased hepatic clearance, without changes in plasma matrix metalloproteinase concentrations or low density lipoprotein oxidisability. These in vivo findings are in contrast with in vitro data.