Estefanía Ugarte-Berzal

α4β1 integrin and 190-kDa CD44v constitute a cell surface docking complex for gelatinase B/MMP-9 in chronic leukemic but not in normal B cells

As B-cell chronic lymphocytic leukemia (B-CLL) progresses, malignant cells extravasate and infiltrate lymphoid tissues. Several molecules, including gelatinase B/MMP-9, contribute to these processes. Although mainly a secreted protease, some MMP-9 is present at the B-CLL cell surface and the function, mode of anchoring, and interactions of this MMP-9 are unknown. Here we show that anti-MMP-9 antibodies immunoprecipitated a 190-kDa CD44v isoform and alpha4beta1 integrin from B-CLL cells, but not from normal B cells. Function-blocking antibodies to alpha4beta1 or CD44, or transfection with specific siRNAs, decreased cell-associated proMMP-9 and increased the secreted form. B-CLL cells attached to and bound proMMP-9 and active MMP-9, and this was inhibited by blocking the expression or function of alpha4beta1 or CD44. The MMP-9 hemopexin domain was critical in these interactions. alpha4beta1 and 190-kDa CD44v (but not CD44H) formed a complex at the cell surface, since they both coimmunoprecipitated with anti-alpha4, anti-beta1, or anti-CD44 antibodies. Immunofluorescence analyses confirmed that alpha4beta1 and CD44v colocalized with MMP-9. Binding of proMMP-9 inhibited B-CLL cell migration, and this required MMP-9 proteolytic activity. Thus, we have identified alpha4beta1 and CD44v as a novel proMMP-9 cell surface docking complex and show that cell-associated MMP-9 may regulate B-CLL cell migration and arrest.

Matrix Metalloproteinase-9 Promotes Chronic Lymphocytic Leukemia B Cell Survival through Its Hemopexin Domain

Matrix metalloproteinase-9 (MMP-9) is the major MMP produced by B-CLL cells and contributes to their tissue infiltration by degrading extracellular and membrane-anchored substrates. Here we describe a different function for MMP-9 in B-CLL, which involves the hemopexin domain rather than its catalytic function. Binding of soluble or immobilized (pro)MMP-9, a catalytically inactive proMMP-9 mutant, or the MMP-9 hemopexin domain to its docking receptors alpha4beta1 integrin and CD44v, induces an intracellular signaling pathway that prevents B-CLL apoptosis. This pathway is induced in all B-CLL cases, is active in B-CLL lymphoid tissues, and consists of Lyn activation, STAT3 phosphorylation, and Mcl-1 upregulation. Our results establish that MMP/receptor binding induces intracellular survival signals and highlight the role of (pro)MMP-9 in B-CLL pathogenesis.

VEGF/VEGFR2 interaction down-regulates matrix metalloproteinase–9 via STAT1 activation and inhibits B chronic lymphocytic leukemia cell migration

B-cell chronic lymphocytic leukemia (B-CLL) migration involves several molecules, including matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). We have studied whether VEGF regulates MMP-9. VEGF significantly reduced MMP-9 protein expression in a dose-dependent manner, measured by gelatin zymography. Blocking the VEGFR2 receptor restored MMP-9 levels, implicating this receptor in the observed effect. Down-regulation of MMP-9 by VEGF resulted in significant inhibition of B-CLL cell migration through Matrigel or human umbilical vein endothelial cells, confirming the crucial role of MMP-9 in these processes. Reverse-transcription polymerase chain reaction analyses revealed that VEGF regulated MMP-9 at the transcriptional level. Indeed, VEGF induced STAT1 tyrosine phosphorylation, and this was blocked by inhibiting VEGFR2. STAT1 was responsible for MMP-9 down-regulation, as STAT1 gene silencing restored MMP-9 production and B-CLL cell migration in the presence of VEGF. Thus, the levels of VEGF and MMP-9 influence B-CLL cell expansion and both molecules could constitute therapeutic targets for this disease.

Circular trimers of gelatinase B/matrix metalloproteinase-9 constitute a distinct population of functional enzyme molecules differentially regulated by tissue inhibitor of metalloproteinases-1.

Gelatinase B/matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) cleaves many substrates and is produced by most cell typesxa0as a zymogen, proMMP-9, in complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). Natural proMMP-9 occurs as monomers, homomultimers and heterocomplexes, but our knowledge about the overall structure of proMMP-9 monomers and multimers is limited. We investigated biochemical, biophysical and functional characteristics of zymogen and activated forms of MMP-9 monomers and multimers. In contrast with a conventional notion of a dimeric nature of MMP-9 homomultimers, we demonstrate that these are reduction-sensitive trimers. Based on the information from electrophoresis, AFM and TEM, we generated a 3D structure model of the proMMP-9 trimer. Remarkably, the proMMP-9 trimers possessed a 50-fold higher affinity for TIMP-1 than the monomers. Inxa0vivo, this finding was reflected in a higher extent of TIMP-1 inhibition of angiogenesis induced by trimers compared with monomers. Our results show that proMMP-9 trimers constitute a novel structural and functional entity that is differentially regulated by TIMP-1.

A novel CD44-binding peptide from the pro-matrix metalloproteinase-9 hemopexin domain impairs adhesion and migration of chronic lymphocytic leukemia (CLL) cells.

Background: proMMP-9 binds to CLL cells through the hemopexin domain (PEX9), contributing to disease progression. Results: A 20-residue sequence (P6) within PEX9 inhibits CD44-mediated CLL-proMMP-9 interaction and cell migration. P6 cooperates with the previously identified sequence P3, also located in PEX9. Conclusion: P6 is a CD44-binding site and impairs proMMP-9 effects on CLL. Significance: P6 may be a novel therapeutic target in CLL. (pro)MMP-9 binds to CLL cells through the PEX9 domain and contributes to CLL progression. To biochemically characterize this interaction and identify potential therapeutic targets, we prepared GST-PEX9 forms containing structural blades B1B2 or B3B4. We recently described a sequence in blade B4 (P3 sequence) that bound α4β1 integrin and partially impaired cell adhesion and migration. We have now studied the possible contribution of the B1B2 region to cell interaction with PEX9. CLL cells bound to GST-B1B2 and CD44 was the primary receptor. GST-B1B2 inhibited CLL cell migration as effectively as GST-B3B4. Overlapping synthetic peptides spanning the B1B2 region identified the sequence FDAIAEIGNQLYLFKDGKYW, present in B1 and contained in peptide P6, as the most effective site. P6 inhibited cell adhesion to PEX9 in a dose-dependent manner and with an IC50 value of 90 μm. P6 also inhibited cell adhesion to hyaluronan but had no effect on adhesion to VCAM-1 (α4β1 integrin ligand), confirming its specific interaction with CD44. Spatial localization analyses mapped P6 to the central cavity of PEX9, in close proximity to the previously identified P3 sequence. Both P6 and P3 equally impaired cell adhesion to (pro)MMP-9. Moreover, P6 synergistically cooperated with P3, resulting in complete inhibition of CLL cell binding to PEX9, chemotaxis, and transendothelial migration. Thus, P6 is a novel sequence in PEX9 involved in cell-PEX9/(pro)MMP-9 binding by interacting with CD44. Targeting both sites, P6 and P3, should efficiently prevent (pro)MMP-9 binding to CLL cells and its pathological consequences.

Glycosylation of matrix metalloproteases and tissue inhibitors: present state, challenges and opportunities.

Current knowledge about the glycosylation of matrix metalloproteinases (MMPs) and the inhibitors of metalloproteinases (TIMPs) is reviewed. Whereas structural and functional aspects of the glycobiology of many MMPs is unknown, research on MMP-9 and MMP-14 glycosylation reveals important functional implications, such as altered inhibitor binding and cellular localization. This, together with the fact that MMPs contain conserved and many potential attachment sites for N-linked and O-linked oligosaccharides, proves the need for further studies on MMP glycobiology.

A 17-residue Sequence from the Matrix Metalloproteinase-9 (MMP-9) Hemopexin Domain Binds α4β1 Integrin and Inhibits MMP-9-induced Functions in Chronic Lymphocytic Leukemia B Cells

Background: proMMP-9 binds to B-CLL cells and contributes to malignant cell migration/arrest and survival. Results: The VPLDTHDVFQ sequence from the proMMP-9 PEX9 domain inhibits α4β1 integrin-mediated proMMP-9-B-CLL interaction, transendothelial migration, and proMMP-9-induced survival signaling. Conclusion: The VPLDTHDVFQ sequence is an α4β1 integrin binding site in proMMP-9 and blocks proMMP-9 effects on B-CLL cells. Significance: The identified sequence may be a novel therapeutic target in B-CLL. We previously showed that pro-matrix metalloproteinase-9 (proMMP-9) binds to B chronic lymphocytic leukemia (B-CLL) cells and contributes to B-CLL progression by regulating cell migration and survival. Induction of cell survival involves a non-proteolytic mechanism and the proMMP-9 hemopexin domain (PEX9). To help design specific inhibitors of proMMP-9-cell binding, we have now characterized B-CLL cell interaction with the isolated PEX9. B-CLL cells bound soluble and immobilized GST-PEX9, but not GST, and binding was mediated by α4β1 integrin. The ability to recognize PEX9 was observed in all 20 primary samples studied irrespective of their clinical stage or prognostic marker phenotype. By preparing truncated forms of GST-PEX9 containing structural blades B1B2 or B3B4, we have identified B3B4 as the primary α4β1 integrin-interacting region within PEX9. Overlapping synthetic peptides spanning B3B4 were then tested in functional assays. Peptide P3 (FPGVPLDTHDVFQYREKAYFC), a sequence present in B4 or smaller versions of this sequence (peptides P3a/P3b), inhibited B-CLL cell adhesion to GST-PEX9 or proMMP-9, with IC50 values of 138 and 279 μm, respectively. Mutating the two aspartate residues to alanine rendered the peptides inactive. An anti-P3 antibody also inhibited adhesion to GST-PEX9 and proMMP-9. GST-PEX9, GST-B3B4, and P3/P3a/P3b peptides inhibited B-CLL cell transendothelial migration, whereas the mutated peptide did not. B-CLL cell incubation with GST-PEX9 induced intracellular survival signals, namely Lyn phosphorylation and Mcl-1 up-regulation, and this was also prevented by the P3 peptides. The P3 sequence may, therefore, constitute an excellent target to prevent proMMP-9 contribution to B-CLL pathogenesis.

Chemokine isoforms and processing in inflammation and immunity

The first dimension of chemokine heterogeneity is reflected by their discovery and purification as natural proteins. Each of those chemokines attracted a specific inflammatory leukocyte type. With the introduction of genomic technologies, a second wave of chemokine heterogeneity was established by the discovery of putative chemokine-like sequences and by demonstrating chemotactic activity of the gene products in physiological leukocyte homing. In the postgenomic era, the third dimension of chemokine heterogeneity is the description of posttranslational modifications on most chemokines. Proteolysis of chemokines, for instance by dipeptidyl peptidase IV (DPP IV/CD26) and by matrix metalloproteinases (MMPs) is already well established as a biological control mechanism to activate, potentiate, dampen or abrogate chemokine activities. Other posttranslational modifications are less known. Theoretical N-linked and O-linked attachment sites for chemokine glycosylation were searched with bio-informatic tools and it was found that most chemokines are not glycosylated. These findings are corroborated with a low number of experimental studies demonstrating N- or O-glycosylation of natural chemokine ligands. Because attached oligosaccharides protect proteins against proteolytic degradation, their absence may explain the fast turnover of chemokines in the protease-rich environments of infection and inflammation. All chemokines interact with G protein-coupled receptors (GPCRs) and glycosaminoglycans (GAGs). Whether lectin-like GAG-binding induces cellular signaling is not clear, but these interactions are important for leukocyte migration and have already been exploited to reduce inflammation. In addition to selective proteolysis, citrullination and nitration/nitrosylation are being added as biologically relevant modifications contributing to functional chemokine heterogeneity. Resulting chemokine isoforms with reduced affinity for GPCRs reduce leukocyte migration in various models of inflammation. Here, these third dimension modifications are compared, with reflections on the biological and pathological contexts in which these posttranslational modifications take place and contribute to the repertoire of chemokine functions and with an emphasis on autoimmune diseases.

Overexpression of progelatinase B/proMMP-9 affects migration regulatory pathways and impairs chronic lymphocytic leukemia cell homing to bone marrow and spleen

This study addresses the role of (pro)MMP‐9 overexpression in CLL cell migration. We have used primary CLL cells and CLL‐derived MEC‐1 cells transfected with empty (mock cells) or proMMP‐9‐encoding (MMP‐9 cells) lentiviral vectors. The constitutive (pro)MMP‐9 expression in mock cells and primary CLL cells was similar, whereas in MMP‐9 cells, expression resembled that of CLL cells incubated with proMMP‐9. In xenograft models, in NOD/SCID mice, MMP‐9‐MEC‐1 transfectants showed significantly reduced homing to bone marrow and spleen compared with mock cells. Likewise, incubation of primary CLL cells with proMMP‐9, before injection into mice, inhibited their homing to these organs. This inhibition was specific, dose‐dependent, and observed in all CLL tested, independently of prognostic markers or disease stage. Additionally, the MMP‐9 catalytic activity was only partially involved, as the inactive mutant proMMP‐9MutE had a partial effect. MMP‐9 cells also showed impaired migration in vitro, which was reverted by reducing (pro)MMP‐9 expression with siRNAs. CLL migration thus requires optimal (pro)MMP‐9 expression levels, below or above which migration is hampered. Biochemical analysis of the (pro)MMP‐9 effect indicated that MMP‐9 cells or primary CLL cells incubated with proMMP‐9 had reduced activation of migration regulatory molecules, including RhoAGTPase, Akt, ERK, and FAK. In contrast, p190RhoGAP (RhoA inhibitor) and PTEN (Akt/ERK/FAK inhibitor) were up‐regulated in MMP‐9 cells. Reduction of (pro)MMP‐9 expression by siRNAs restored RhoA activity and diminished PTEN levels. Our results reveal a novel function for (pro)MMP‐9 in modulating signaling pathways leading to CLL cell arrest. Therefore, local high (pro)MMP‐9 expression may contribute to malignant cell retention in lymphoid organs and disease progression.

The molecular biology of matrix metalloproteinases and tissue inhibitors of metalloproteinases in inflammatory bowel diseases

Abstract Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be predominant proteases and protease inhibitors involved in the pathogenesis of inflammatory bowel diseases (IBD) through their ability to remodel the extracellular matrix (ECM) in response to inflammatory stimuli and by their immunomodulating effects. An imbalance between MMPs and TIMPs has been linked with acute and chronic inflammation and aberrant tissue remodeling, as seen in IBD. Moreover, recurrent phases of tissue destruction and subsequent tissue repair can cause serious complications in IBD patients such as fistulas and fibrosis. The aims of this review are (i) to summarize current literature on genetic association, mRNA, and protein expression studies with regard to MMPs and TIMPs in IBD patients and various animal models, including those with transgenic and knockout mice; (ii) to compare biochemical and molecular biological data in humans with those obtained in animal model studies and (iii) to critically evaluate and translate how this knowledge may be used in practical terms to understand better the pathophysiology and mechanisms operating in IBD and to apply this for improvement of clinical outcomes at diagnostic, prognostic and therapeutic levels.