Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation

Department of Pharmacology, Wayne State University, School of Medicine, Detroit, MI 48201, USA.
Experimental Cell Research (Impact Factor: 3.37). 05/2009; 315(7):1234-46. DOI: 10.1016/j.yexcr.2009.01.021
Source: PubMed

ABSTRACT Localization of proteases to the surface of endothelial cells and remodeling of the extracellular matrix (ECM) are essential to endothelial cell tube formation and angiogenesis. Here, we partially localized active cathepsin B and its cell surface binding partners, S100A/p11 (p11) of the annexin II heterotetramer (AIIt), to caveolae of human umbilical vein endothelial cells (HUVEC). Via a live-cell proteolysis assay, we observed that degradation products of quenched-fluorescent (DQ)-proteins (i.e. gelatin and collagen IV) colocalized intracellularly with caveolin-1 (cav-1) of HUVEC grown in either monolayer cultures or in vitro tube formation assays. Activity-based probes that bind covalently to active cysteine cathepsins and degradation products of DQ-collagen IV partially localized to intracellular vesicles that contained cav-1 and active cysteine cathepsins. Biochemical analyses revealed that the distribution of active cathepsin B in caveolar fractions increased during in vitro tube formation. Pro-uPA, uPAR, MMP-2 and MMP-14, which have been linked with cathepsin B to ECM degradation pathways, were also found to increase in caveolar fractions during in vitro tube formation. Our findings are the first to demonstrate through live-cell imaging ECM degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.

Download full-text


Available from: Matthew Bogyo, Aug 23, 2015
  • Source
    • "Such binding seems to facilitate conversion of procathepsin B to its active forms. Active CTSB imitates a cascade pericellular proteolytic activity at cancer cell surface [71] [72] [73]. CTSB is a member of the cysteine proteases family involved in various steps of cancer invasion, motility and dissemination by digestion of adhesion molecules, degradation of extracellular matrix and regulation of angiogenesis [64] [74]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Inflammatory breast cancer (IBC) is a highly metastatic and fatal form of breast cancer. In fact, IBC is characterized by specific morphological, phenotypic, and biological properties that distinguish it from non-IBC. The aggressive behavior of IBC being more common among young women and the low survival rate alarmed researchers to explore the disease biology. Despite the basic and translational studies needed to understand IBC disease biology and identify specific biomarkers, studies are limited by few available IBC cell lines, experimental models, and paucity of patient samples. Above all, in the last decade, researchers were able to identify new factors that may play a crucial role in IBC progression. Among identified factors are cytokines, chemokines, growth factors, and proteases. In addition, viral infection was also suggested to participate in the etiology of IBC disease. In this review, we present novel factors suggested by different studies to contribute to the etiology of IBC and the proposed new therapeutic insights.
    Journal of Advanced Research 06/2013; 5(5). DOI:10.1016/j.jare.2013.06.004
  • Source
    • "As has previously been remarked (Greenlee et al., 2007), because of their complex post-translational regulation, MMP assays that elucidate the activity of these enzymes, especially in vivo, are essential to the development of a coherent picture of the dynamics of ECM remodeling. Recently, approaches to this problem using activitybased MMP assays have been employed in tissue culture (Packard et al., 2009; Cavallo-Medved et al., 2009; Olson et al., 2009) and even in whole animal models (McIntyre et al., 2004; Chen et al., 2005; Crawford and Pilgrim, 2005; von Burstin et al., 2008; Scherer et al., 2008a, b). The zebrafish in particular, with its transparent, rapidly and externally developing embryo, provides an ideal experimental model for the application of this technique (dubbed 'in vivo zymography' or IVZ), because it allows for the high-resolution imaging of the fluorescent degradation products of the introduced substrates. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Investigations into the molecular mechanisms of, and cellular signaling pathways modulating ECM remodeling are especially challenging due to the complex post-translational regulation of the primary effectors of ECM catabolism - the matrix metalloproteinases (MMPs). Recently a variety of approaches to the detection of MMP activity have been developed, and the prospect of visualizing ECM remodeling activity in living tissues is now opening exciting avenues of research for matrix biologists. In particular the use of FRET-quenched MMP substrates, which generate a fluorescent signal upon hydrolysis, is becoming increasingly popular, especially because linkers with defined and/or restricted proteolytic sensitivity can be used to bind fluorophore-quencher pairs, making these probes useful in characterizing the activity of specific proteases. We have taken advantage of the transparency and amenability to reverse genetics of the zebrafish embryo, in combination with these fluorogenic MMP substrates, to develop a multiplex in vivo assay for MMP activity that we dub "differential in vivo zymography."
    Matrix biology: journal of the International Society for Matrix Biology 02/2011; 30(3):169-77. DOI:10.1016/j.matbio.2011.01.003 · 3.65 Impact Factor
  • Source
Show more