Michael Stefanidakis

Activation of NMDA receptors promotes dendritic spine development through MMP-mediated ICAM-5 cleavage

Matrix metalloproteinase (MMP)-2 and -9 are pivotal in remodeling many tissues. However, their functions and candidate substrates for brain development are poorly characterized. Intercellular adhesion molecule-5 (ICAM-5; Telencephalin) is a neuronal adhesion molecule that regulates dendritic elongation and spine maturation. We find that ICAM-5 is cleaved from hippocampal neurons when the cells are treated with N-methyl-d-aspartic acid (NMDA) or α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA). The cleavage is blocked by MMP-2 and -9 inhibitors and small interfering RNAs. Newborn MMP-2– and MMP-9–deficient mice brains contain more full-length ICAM-5 than wild-type mice. NMDA receptor activation disrupts the actin cytoskeletal association of ICAM-5, which promotes its cleavage. ICAM-5 is mainly located in dendritic filopodia and immature thin spines. MMP inhibitors block the NMDA-induced cleavage of ICAM-5 more efficiently in dendritic shafts than in thin spines. ICAM-5 deficiency causes retraction of thin spine heads in response to NMDA stimulation. Soluble ICAM-5 promotes elongation of dendritic filopodia from wild-type neurons, but not from ICAM-5–deficient neurons. Thus, MMPs are important for ICAM-5–mediated dendritic spine development.

Identification of a negatively charged peptide motif within the catalytic domain of progelatinases that mediates binding to leukocyte beta 2 integrins.

The αMβ2 integrin of leukocytes can bind a variety of ligands. We screened phage display libraries to isolate peptides that bind to the αM I domain, the principal ligand binding site of the integrin. Only one peptide motif, (D/E)(D/E)(G/L)W, was obtained with this approach despite the known ligand binding promiscuity of the I domain. Interestingly, such negatively charged sequences are present in many known β2 integrin ligands and also in the catalytic domain of matrix metalloproteinases (MMPs). We show that purified β2 integrins bind to pro-MMP-2 and pro-MMP-9 gelatinases and that that the negatively charged sequence of the MMP catalytic domain is an active β2 integrin-binding site. Furthermore, a synthetic DDGW-containing phage display peptide inhibited the ability of β2 integrin to bind progelatinases but did not inhibit the binding of cell adhesion-mediating substrates such as intercellular adhesion molecule-1, fibrinogen, or an LLG-containing peptide. Immunoprecipitation and cell surface labeling demonstrated complexes of pro-MMP-9 with both the αMβ2 and αLβ2 integrins in leukocytes, and pro-MMP-9 colocalized with αMβ2 in cell surface protrusions. The DDGW peptide and the gelatinase-specific inhibitor peptide CTTHWGFTLC blocked β2 integrin-dependent leukocyte migration in a transwell assay. These results suggest that leukocytes may move in a progelatinase-β2 integrin complex-dependent manner.

Intracellular and Cell Surface Localization of a Complex between αMβ2 Integrin and Promatrix Metalloproteinase-9 Progelatinase in Neutrophils

We have recently demonstrated that promatrix metalloproteinases (proMMPs), particularly proMMP-9, are potent ligands of the leukocyte β2 integrins. We studied here the complex formation between proMMP-9 and αMβ2, the major MMP and integrin of neutrophils. On resting neutrophils, the proMMP-9/αMβ2 complex was primarily detected in intracellular granules, but after cellular activation it became localized to the cell surface, as demonstrated by immunoprecipitation and double immunofluorescence. Further indication of the complex formation was that neutrophils and αMβ2-transfected L cells, but not the wild-type L cells or leukocyte adhesion deficiency cells, bound to immobilized proMMP-9 or its recombinant catalytic domain in a β2 integrin-dependent manner. Peptides that bound to the αM integrin-I domain and inhibited its complex formation with proMMP-9 prevented neutrophil migration in a transendothelial assay in vitro and in a thioglycolate-elicited peritonitis in vivo. These results suggest that the translocating proMMP-9/αMβ2 complex may be part of the cell surface machinery guiding neutrophil migration.

Role of leukemia cell invadosome in extramedullary infiltration

Acute myelogenous leukemias (AMLs) are characterized by medullary and extramedullary invasion. We hypothesized that a supramolecular complex, the leukemia-cell invadosome, which contains certain integrins, matrix metalloproteinases (MMPs), and other as-yet unidentified proteins, is essential for tissue invasion and may be central to the phenotypic diversity observed in the clinic. Here we show that the specific binding of MMP-9 to leukocyte surface beta(2) integrin is required for pericellular proteolysis and migration of AML-derived cells. An efficient antileukemia effect was obtained by the hexapeptide HFDDDE, a motif of the MMP-9 catalytic domain that mediates integrin binding: HFDDDE prevented proMMP-9 binding, transmigration through a human endothelial cell layer, and extracellular matrix degradation. Notably, the functional protein anchorage between beta(2) integrin and proMMP-9 described in this study does not involve the enzymatic active sites targeted by known MMP inhibitors. Taken together, our results provide a biochemical working definition for the human leukemia invadosome. Disruption of specific protein complexes within this supramolecular target complex may yield a new class of anti-AML drugs with anti-invasion (rather than or in addition to cytotoxic) attributes.

Peptide-mediated delivery of therapeutic and imaging agents into mammalian cells

Modern molecular targeting provides new opportunities for imaging, diagnosis and treatment of diseases. Small molecular weight peptides have the potential for enhancing targeting of compounds, and they may also have therapeutic effects by themselves. The limiting step for successful molecular targeting is the development of efficient peptide delivery systems. This review will focus on peptides developed by phage display and combinatorial chemistry for the delivery of pharmaceuticals, radioactive compounds and gene expression vectors. Target cell-specific delivery can be improved by peptides that penetrate the cell membrane or alternatively induce receptor-mediated endocytosis. In addition, peptides that contain endosomal escape signals or nuclear localization motifs may help trafficking of therapeutics to appropriate locations inside the cell. Small molecule radiolabelled peptides are the preferred agents for targeting and for diagnostic imaging of various organs as they are easily synthesized, effectively penetrate tissues, and are rapidly cleared from the circulation. Such peptides have been tested in animals and humans in the fields of cancer, cardiology, neurology, inflammation/infection, atherosclerosis and thrombosis.