Pilar Gonzalo

MT1-MMP: Universal or particular player in angiogenesis?

Tumorigenesis involves not only tumor cells that become transformed but also the peritumoral stroma which reacts inducing inflammatory and angiogenic responses. Angiogenesis, the formation of new capillaries from preexisting vessels, is an absolute requirement for tumor growth and metastasis, and it can be induced and modulated by a wide variety of soluble factors. During angiogenesis, quiescent endothelial cells are activated and they initiate migration by degrading the basement membranes through the action of specific proteases, in particular of matrix metalloproteinases (MMPs). Among these, the membrane type 1-matrix metalloproteinase (MT1-MMP) has been identified as a key player during the angiogenic response. In this review, we will summarize the role of MT1-MMP in angiogenesis and the regulatory mechanisms of this protease in endothelial cells. Since our recent findings have suggested that MT1-MMP is not universally required for angiogenesis, we hypothesize that the regulation and participation of MT1-MMP in angiogenesis may depend on the nature of the angiogenic stimulus. Experiments aimed at testing this hypothesis have shown that similarly to the chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12, lipopolysaccharide (LPS) seems to induce the formation of capillary tubes by human or mouse endothelial cells (ECs) in an MT1-MMP-independent manner. The implications of these findings in the potential use of MT1-MMP inhibitors in cancer therapy are discussed.

MT1-MMP collagenolytic activity is regulated through association with tetraspanin CD151 in primary endothelial cells

MT1-MMP plays a key role in endothelial function, as underscored by the angiogenic defects found in MT1-MMP deficient mice. We have studied the molecular interactions that underlie the functional regulation of MT1-MMP. At lateral endothelial cell junctions, MT1-MMP colocalizes with tetraspanin CD151 (Tspan 24) and its associated partner alpha3beta1 integrin. Biochemical and FRET analyses show that MT1-MMP, through its hemopexin domain, associates tightly with CD151, thus forming alpha3beta1 integrin/CD151/MT1-MMP ternary complexes. siRNA knockdown of HUVEC CD151 expression enhanced MT1-MMP-mediated activation of MMP2, and the same activation was seen in ex vivo lung endothelial cells isolated from CD151-deficient mice. However, analysis of collagen degradation in these experimental models revealed a diminished MT1-MMP enzymatic activity in confined areas around the cell periphery. CD151 knockdown affected both MT1-MMP subcellular localization and its inclusion into detergent-resistant membrane domains, and prevented biochemical association of the metalloproteinase with the integrin alpha3beta1. These data provide evidence for a novel regulatory role of tetraspanin microdomains on the collagenolytic activity of MT1-MMP and indicate that CD151 is a key regulator of MT1-MMP in endothelial homeostasis.

MT1-MMP Is Required for Myeloid Cell Fusion via Regulation of Rac1 Signaling

Cell fusion is essential for fertilization, myotube formation, and inflammation. Macrophages fuse under various circumstances, but the molecular signals involved in the distinct steps of their fusion are not fully characterized. Using null mice and derived cells, we show that the protease MT1-MMP is necessary for macrophage fusion during osteoclast and giant-cell formation in vitro and in vivo. Specifically, MT1-MMP is required for lamellipodia formation and for proper cell morphology and motility of bone marrow myeloid progenitors prior to membrane fusion. These functions of MT1-MMP do not depend on MT1-MMP catalytic activity or downstream pro-MMP-2 activation. Instead, MT1-MMP null cells show a decreased Rac1 activity and reduced membrane targeting of Rac1 and the adaptor protein p130Cas. Retroviral rescue experiments and protein binding assays delineate a signaling pathway in which MT1-MMP, via its cytosolic tail, contributes to macrophage migration and fusion by regulating Rac1 activity through an association with p130Cas.

Retinoid X receptor α controls innate inflammatory responses through the up-regulation of chemokine expression

The retinoid X receptor α (RXRα) plays a central role in the regulation of many intracellular receptor signaling pathways and can mediate ligand-dependent transcription by forming homodimers or heterodimers with other nuclear receptors. Although several members of the nuclear hormone receptor superfamily have emerged as important regulators of macrophage gene expression, the existence in vivo of an RXR signaling pathway in macrophages has not been established. Here, we provide evidence that RXRα regulates the transcription of the chemokines Ccl6 and Ccl9 in macrophages independently of heterodimeric partners. Mice lacking RXRα in myeloid cells exhibit reduced levels of CCL6 and CCL9, impaired recruitment of leukocytes to sites of inflammation, and lower susceptibility to sepsis. These studies demonstrate that macrophage RXRα plays key roles in the regulation of innate immunity and represents a potential target for immunotherapy of sepsis.

Matrix metalloproteinases : New routes to the use of MT1-MMP as a therapeutic target in angiogenesis-related disease

Angiogenesis, the formation of new vessels from pre-existing capillaries, is a fundamental physiological process which is also critical for the development of several pathological conditions; thus a diminished angiogenic response is related to ischemic disorders, whereas increased angiogenesis is associated with tumorigenesis and chronic inflammatory diseases. New ways of modulating angiogenesis therefore have potential in the treatment of these diseases. During angiogenesis, normally quiescent endothelial cells (ECs) become migratory and invade the surrounding tissue. To do this, they require a specific enzyme machinery to degrade the tissue barriers presented by the basement membranes and the interstitial matrix. This function is supplied by matrix metalloproteinase (MMP) proteins, a large family of enzymes responsible for degrading a variety of extracellular matrix (ECM) components and for modulating the bioactivity of transmembrane receptors and soluble factors. In this review we examine the participation of MMPs--in particular membrane type 1-matrix metalloproteinase (MT1-MMP)--in the different steps of angiogenesis, and discuss the mechanisms of regulation of MT1-MMP in ECs. Finally, we explore the potential use of MMP inhibitors (MMPI) in the treatment of angiogenesis-related disease, with especial emphasis on novel approaches to the inhibition of MT1-MMP activity in ECs.

MT1-MMP and integrins: Hand-to-hand in cell communication

Integrins are transmembrane adhesion receptors essential for cell communication with the environment and in particular with the extracellular matrix (ECM). ECM components can be processed by several enzymes; one of the largest families involved in this task being matrix metalloproteinases (MMPs). MT1‐MMP (membrane type 1‐matrix metalloproteinase) is a membrane‐anchored MMP with important roles in processes such as tissue development, tumor invasion, and angiogenesis. In addition to its catalytic‐dependent functions, MT1‐MMP can interact, via its cytosolic tail, with intracellular components, and trigger signaling pathways that impact cell decisions. These features make MT1‐MMP similar to integrins, because both are able to integrate events in the extracellular and intracellular milieus. Accordingly, it is probably no coincidence that MT1‐MMP often associates and functionally cooperates with distinct integrins at specific cellular compartments. In this review, we discuss aspects of the molecular and functional interplay between MT1‐MMP and integrins in distinct cellular and biological contexts.

The protease MT1-MMP drives a combinatorial proteolytic program in activated endothelial cells

The mechanism by which proteolytic events translate into biological responses is not well understood. To explore the link of pericellular proteolysis to events relevant to capillary sprouting within the inflammatory context, we aimed at the identification of the collection of substrates of the protease MT1‐MMP in endothelial tip cells induced by inflammatory stimuli. We applied quantitative proteomics to endothelial cells (ECs) derived from wild‐type and MT1‐MMP‐null mice to identify the substrate repertoire of this protease in TNF‐α‐activated ECs. Bioinformatics analysis revealed a combinatorial MT1‐MMP proteolytic program, in which combined rather than single substrate processing would determine biological decisions by activated ECs, including chemotaxis, cell motility and adhesion, and vasculature development. MT1‐MMP‐deficient ECs inefficiently processed several of these substrates (TSP1, CYR61, NID1, and SEM3C), validating the model. This novel concept of MT1‐MMP‐driven combinatorial proteolysis in angiogenesis might be extendable to proteolytic actions in other cellular contexts.—Koziol, A., Gonzalo, P., Mota, A., Pollán, Á., Lorenzo, C., Colomé, N., Montaner, D., Dopazo, J., Arribas, J., Canals, F., Arroyo, A. G. The protease MT1‐MMP drives a combinatorial proteolytic program in activated endothelial cells. FASEB J. 26, 4481–4494 (2012). www.fasebj.org

Site-specific cellular functions of MT1-MMP.

The response to environmental cues such as inflammatory stimuli requires coordinated cellular functions. Certain proteins have functions on both sides of the plasma membrane to allow coordination between the extracellular and intracellular milieus. The membrane-anchored matrix metalloproteinase MT1-MMP is well positioned to sense and modify the extracellular environment by processing matrix components, transmembrane proteins and soluble factors. Recent findings show, however, that MT1-MMP also plays unexpected intracellular roles in macrophages through its location at the plasma membrane, the Golgi or the nucleus, impacting cell motility, metabolism and gene transcription. MT1-MMP is thus an example of the evolutionary diversification of protein function, allowing optimal coordination between extracellular stimuli and cellular responses. It remains to be determined whether these new MT1-MMP functions are specific to macrophages, professional phagocytes involved in inflammation, or are present in other inflammation-responsive cells. In this review, we will summarize these site-specific MT1-MMP functions in macrophages and comment on the possible conservation of these functions in endothelial cells.

An EMMPRIN/γ-catenin/Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions

ABSTRACT Cell–cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with &ggr;-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly.

MT1-MMP: A novel component of the macrophage cell fusion machinery.

Mice deficient in the matrix metalloproteinase MT1-MMP display defects in tissue development and angiogenesis, together with a complex bone phenotype characterized by several skeletal abnormalities and osteopenia. OCs and giant cells are multinucleated cells arising from the fusion of myeloid progenitors/macrophages that specialize respectively in bone resorption and engulfment of pathogens and foreign bodies. Our work identifies MT1-MMP as a novel component of the macrophage fusion machinery during OC and giant cell formation in vitro and in vivo. MT1-MMP is required for the proper lamellipodia formation and motility required to achieve proximity between fusion-competent myeloid cells; and roles of MT1-MMP in subsequent steps of the fusion process cannot be ruled out. For example, MT1-MMP might exert additional functions at fusion sites by forming molecular complexes with CD44 or tetraspanin proteins. Interestingly, the contribution of MT1-MMP to macrophage motility and fusion does not involve its catalytic activity. Instead, the MT1-MMP-cytosolic tail, in particular Tyr573, is required to bind the adaptor protein p130Cas and regulate localized Rac1 activity in myeloid progenitors. Modulation of this novel MT1-MMP–p130Cas–Rac1 signaling pathway in macrophages might have potential in the treatment of disorders involving increased OC activity or uncontrolled giant cell formation.