Research ArticleLive-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation
Introduction
Angiogenesis, the formation of new blood vessels from the pre-existing vasculature, is a process in which stimulated endothelial cells remodel extracellular matrix (ECM), migrate through the ECM, proliferate, differentiate and eventually form endothelial tubules capable of blood transport [1]. Proteases of at least three classes (serine, cysteine, and metallo-) [including matrix metalloproteinases (MMPs), members of the urokinase plasmin(ogen) system and cysteine cathepsins] play crucial roles in angiogenesis (for review see [2]). Proteases participate in the angiogenic process by generating both pro- and anti-angiogenic factors from ECM proteins [3], [4], [5] and by processing growth factors and receptors, including integrins [6], [7]. For example, cleavage of type IV collagen by endothelial cell MMPs results in the exposure of cryptic αvβ3-integrin binding sites, thus promoting angiogenesis [7]. Urokinase plasminogen activator (uPA) converts plasminogen to plasmin which can be further processed to generate angiostatin, an anti-angiogenic compound [8]. Cathepsin B, a lysosomal cysteine protease, regulates the “angiogenic switch” [9] by initiating proteolytic cascades involved in ECM degradation that promote angiogenesis [10], or conversely, by cleaving collagen XVIII and generating endostatin, an anti-angiogenic factor [11]. Overall, the balance of protease activity and the interaction of proteases with the ECM contribute to regulation of the angiogenic process.
ECM remodeling and degradation by tumor cells are facilitated by the translocation of proteases to cell surfaces and their secretion into the extracellular milieu [12], [13], [14], [15]. Many proteases implicated in ECM remodeling and degradation are also associated with caveolae [12], a lipid-rich region of the plasma membrane involved in endocytosis, cholesterol transport and cell signaling events (for reviews, see [16], [17]). These proteases include uPA and its receptor uPAR, cathepsin B and its cell surface binding protein S100A10/p11 (p11) [the light chain of the annexin II heterotetramer (AIIt)], MMP-2 and MMP-14 [12]. In colorectal carcinoma cells, downregulation of caveolin-1 (cav-1), the main structural protein of caveolae, decreases distribution of cathepsin B and uPA to caveolae and ECM degradation by these cells [18]. The association of proteases with caveolae of endothelial cells is intriguing since caveolae are involved in angiogenesis (for review, see [19]). During endothelial cell migration, cav-1 localizes to the rear of migrating cells [20], [21], [22]. Moreover, downregulation of cav-1 impedes endothelial cell polarization and directional movement [20]. Indeed, knockdown of cav-1 suppresses tube formation by endothelial cells and also reduces vessel formation in the chicken chorioallantoic membrane assay [23]. Thus, we hypothesize that compartmentalization of proteases to caveolae in endothelial cells modulates cell migration, proteolysis of ECM proteins and tube formation.
In the present study, we identified active cathepsin B as well as its cell surface-binding partner, S100A10/p11 (p11), in caveolae of human umbilical vein endothelial cells (HUVEC): an association that is augmented during in vitro endothelial tube cell formation. Using a live-cell proteolysis assay in combination with in vitro tube formation assays and cysteine cathepsin activity-based probes, we observed extracellular degradation of ECM proteins (i.e., type IV collagen) by migrating HUVEC and intracellular colocalization of ECM degradation products with cav-1 in vesicles containing active cysteine cathepsins. These data suggest an association of active cathepsin B with caveolae and degradation and remodeling of the ECM during endothelial cell migration and tube formation.
Section snippets
Materials
M199 medium, heparin, N-Octyl β-d-glucopyranoside, 2-[N-morpholino]ethanesulfonic acid (MES), methyl-β-cyclodextrin (MβCD), and all other chemicals unless otherwise stated were from Sigma (St. Louis, MO); fetal bovine serum (FBS), Lipofectin reagent, dye-quenched fluorescent (DQ)-gelatin and DQ-collagen IV were from Invitrogen (Carlsbad, CA); bovine endothelial cell growth factor (bECGF) was from Roche Applied Science (Indianapolis, IN); polyclonal anti-caveolin (610059), monoclonal
Gelatin and collagen IV degradation products localize to vesicles containing cav-1 in HUVEC
Remodeling of the ECM is a process critical to the formation of tubular structures by endothelial cells. To analyze this process, we employed a well-established live-cell proteolysis assay [35] and compared HUVEC grown on coverslips coated with gelatin containing DQ-gelatin with HUVEC grown on coverslips coated with rBM containing DQ-collagen IV. Following 16 h of incubation, live cells were imaged by confocal microscopy for degradation products (green fluorescence) of the two DQ-protein
Discussion
Differentiation of endothelial cells during angiogenesis is a process that involves proteolytic enzymes, which degrade basement membrane and thereby facilitate cell migration, invasion and capillary tube formation. The accessibility of proteases to the ECM is enhanced by their localization on the cell membrane. Here, we explored one possible mechanism for cell surface association, i.e., the distribution of cathepsin B and other proteases associated with ECM degradation to caveolae on the cell
Acknowledgments
This work was supported by a National Institutes of Health (NIH) National Technology Center for Networks and Pathways Grant (U54-RR020843) and a DOD Breast Cancer Center of Excellence (DAMD17-02-1-0693). The Microscopy and Imaging Resources Laboratory is supported by the NIH National Technology Center Grant and NIH Center Grants P30-ES06639 and P30-CA22453.
References (63)
- et al.
Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9)
J. Biol. Chem.
(1997) - et al.
Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA)
J. Biol. Chem.
(1991) - et al.
Generation and degradation of human endostatin proteins by various proteinases
FEBS Lett.
(2000) - et al.
Cell-surface cathepsin B: understanding its functional significance
Curr. Top. Dev. Biol.
(2003) - et al.
Involvement of caveolae and caveolae-like domains in signalling, cell survival and angiogenesis
Cell Signal.
(2002) - et al.
Loss of caveolin-1 polarity impedes endothelial cell polarization and directional movement
J. Biol. Chem.
(2005) - et al.
Knockdown of caveolin-1 by antisense oligonucleotides impairs angiogenesis in vitro and in vivo
Biochem. Biophys. Res. Commun.
(2000) - et al.
Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains
J. Biol. Chem.
(1996) - et al.
Glycosaminoglycans facilitate procathepsin B activation through disruption of propeptide-mature enzyme interactions
J. Biol. Chem.
(2007) - et al.
Mutant K-ras regulates cathepsin B localization on the surface of human colorectal carcinoma cells.
Neoplasia
(2003)
Cell surface complex of cathepsin B/annexin II tetramer in malignant progression
Biochim. Biophys. Acta
Multiple functions of caveolin-1
J. Biol. Chem.
MMP-2 colocalizes with caveolae on the surface of endothelial cells
Exp. Cell Res.
Identification of a novel domain at the N terminus of caveolin-1 that controls rear polarization of the protein and caveolae formation
J. Biol. Chem.
Caveolin-1 expression enhances endothelial capillary tubule formation
J. Biol. Chem.
Endostatin associates with lipid rafts and induces reorganization of the actin cytoskeleton via down-regulation of RhoA activity
J. Biol. Chem.
Src-mediated tyrosine phosphorylation of caveolin-1 induces its association with membrane type 1 matrix metalloproteinase
J. Biol. Chem.
uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV
Exp. Cell Res.
Imaging proteolysis by living human breast cancer cells
Neoplasia
CA074 methyl ester: a proinhibitor for intracellular cathepsin B
Arch. Biochem. Biophys.
The mannose receptor family
Biochim. Biophys. Acta
Mechanisms of angiogenesis
Nature
Pericellular proteases in angiogenesis and vasculogenesis
Arterioscler. Thromb. Vasc. Biol.
Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis
Nat. Cell Biol.
Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis
Arterioscler. Thromb. Vasc. Biol.
Role of plasminogen activator-plasmin system in tumor angiogenesis
Cell. Mol. Life Sci.
Molecular crosstalk between adhesion receptors and proteolytic cascades in vascular remodelling
Thromb. Haemost.
Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo
J. Cell Biol.
Cathepsin B regulates the intrinsic angiogenic threshold of endothelial cells
Mol. Biol. Cell
Cell surface association of matrix metalloproteinase-9 (gelatinase B)
Cancer Metastasis Rev.
Cysteine cathepsins: multifunctional enzymes in cancer
Nat. Rev. Cancer
Cited by (97)
Cancer – Proteases in Progression and Metastasis
2022, Encyclopedia of Cell Biology: Volume 1-6, Second EditionMolecular probes for selective detection of cysteine cathepsins
2021, Organic and Biomolecular ChemistryCathepsin B inhibitors: Further exploration of the nitroxoline core
2018, Bioorganic and Medicinal Chemistry LettersDevelopment of 3D culture models of plexiform neurofibroma and initial application for phenotypic characterization and drug screening
2018, Experimental NeurologyCitation Excerpt :The ability to invade the matrix is promoted by tumor cell secretion of matrix metalloproteinases (MMPs). This class of proteases is located at the cell surface (Sevenich and Joyce, 2014) and at the leading edge of tumors (Cavallo-Medved et al., 2009) enabling the processing of substrates. Although it is the work of multiple proteinases interacting with companion proteins that create a favorable environment for invasion and metastasis, the degradation of collagen IV is part of the process.
Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance sensor
2017, Journal of Pharmaceutical and Biomedical Analysis