Cell Surface Localization of Heparanase On Macrophages Regulates Degradation of Extracellular Matrix Heparan Sulfate
Cell Surface Localization of Heparanase On Macrophages Regulates Degradation of Extracellular Matrix Heparan Sulfate
Cell Surface Localization of Heparanase On Macrophages Regulates Degradation of Extracellular Matrix Heparan Sulfate
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The Journal of Immunology
M acrophages and related cells play essential roles in the HS. The HS moieties play essential roles in the interaction of
immune system, such as inflammation, defense against HSPG with a wide range of molecules including ECM components
microbial infection, immunity to foreign substances, (collagen, laminin, fibronectin, and others), cytokines (basic fibro-
wound healing, and angiogenesis. Monocytes circulate throughout blast growth factor, platelet-derived growth factor, hepatocyte
the body, extravasate through the endothelial lining of the blood growth factor, and others), and enzymes (lipoprotein lipase and
vessel wall, and enter the underlying tissue in response to local others) (1–3). Degradation of HS causes loss of mechanical integ-
inflammation. During the process, monocytes should pass through rity of basement membrane and release of soluble mediators.
the vascular basement membrane that supports the structure and The first indication that HS maintains the mechanical integrity
survival of endothelial cells and also prevents the vessels from of basement membrane came from a work in which heparitinase
mechanical destruction. The basement membrane mainly consists digestion of glomerular basement membranes resulted in a loss of
of type IV collagen, laminin, and heparan sulfate (HS)4 proteo- function (4). The ability of tumor cells to degrade basement mem-
glycans (HSPGs). Degradation of these basement membrane com-
brane was shown to be due to a HS-specific endo--glucuronidase
ponents results in disintegration of the structure and it is conceiv-
(5, 6). cDNA cloning and expression of human heparanase have
able that such processes are a regulatory step for the extravasation.
recently been reported by four groups (7–10). The cDNA encodes
HSPGs are ubiquitous in extracellular matrices (ECMs) including
a unique protein of 543 amino acids that contains a potential signal
basement membranes, and consist of diverse core polypeptides and
peptide sequence and six putative N-linked glycosylation sites. It
was predicted that the 543-aa polypeptide formed a proenzyme
*Laboratory of Cancer Biology and Molecular Immunology, Graduate School of that was processed to be a mature 50 kDa active enzyme after
Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; and †Tsukuba Research removal of 157 N-terminal amino acids. The active enzyme has
Institute, Novartis Pharma, Tsukuba, Japan
been claimed to be a heterodimer that comprises the 50-kDa
Received for publication August 26, 2003. Accepted for publication January 7, 2004. polypeptide and a short fragment of 8-kDa peptide derived from
The costs of publication of this article were defrayed in part by the payment of page the N terminus of heparanase proenzyme (11). The active enzyme
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact. shows at least 100-fold HS degradation activity in comparison to
1
This work was supported by Grants-in-Aid from the Ministry of Education, Science, the proenzyme (8), therefore this processing could be one of the
Sports and Culture of Japan (11557180, 11672162, and 12307054), and from the critical regulatory steps of heparanase. Although it was assumed
Program for Promotion of Basic Research Activities for Innovative Biosciences.
that secreted or membrane-associated heparanase is responsible for
2
Current address: Division of Cell Biology, Institute of Life Science, Soka Univer- the degradation of ECM, the mechanisms involved in translocation
sity, Tokyo 192-8577, Japan.
3
of the enzyme have not been elucidated.
Address correspondence and reprint requests to Dr. Tatsuro Irimura, Graduate
School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Heparanase activity was reported in platelets, neutrophils,
Tokyo 113-0033, Japan. E-mail address: irimura@mol.f.u-tokyo.ac.jp monocytes, macrophages, Langerhans cells, astrocytes, activated
4
Abbreviations used in this paper: HS, heparan sulfate; HSPG, HS proteoglycan; (but not resting) rat T lymphocytes, and umbilical vein endothelial
ECM, extracellular matrix; FL-HS, fluoresceinamine-labeled HS; SVBCE, simian
virus bovine corneal endothelial cell; EDC, 1-ethyl-3-(3-dimethylaminopropyl)car- cells or smooth-muscle cells (12–19). It is likely that heparanase is
bodiimide hydrochloride; MMP, matrix metalloproteinase. required for extravasation of the cells in the immune system, and
the activity should be under tight regulation to avoid tissue dam- X-100, 1 mM PMSF, 0.2 mM AEBSF, 10 g/ml leupeptin, 10 g/ml
age. In the present study, we asked whether the heparanase is in- pepstatin A, 1 g/ml aprotinin, pH 7.5) on ice for 30 min, followed by
volved in macrophage extravasation by use of macrophage-like centrifugation at 15,000 rpm for 10 min. Protein concentrations of the
supernatants were determined with bicinchoninic acid protein assay using
cell lines. Regulation of heparanase activity during macrophage BSA as a standard (Pierce). For preparation of fluoresceinated HS as the
differentiation and attachment to basement membranes was also heparanase substrate, HS (sodium salt, 1 mg), EDC (0.2 mg), and fluores-
investigated. A major regulatory process seems to be its unique ceinamine (FL; Fluka, Tokyo, Japan) (5 g) were dissolved in water, and
spatial distribution. stirred for 1 h at room temperature, followed by dialysis overnight with
water. The solution was then concentrated with a Centricon 30 concentrator
(Amicon, Bedford, MA). The ratio of attached fluorescein to unmodified
Materials and Methods carboxyl group was determined using the carbazole-sulfuric acid method.
Chemicals An enzymatic reaction was conducted in a 100 l mixture containing 25
mM sodium acetate buffer (pH 5.5), 5 g of FL-HS, 20 mM D-saccharic
RPMI 1640 medium was purchased from Nissui Pharmaceuticals (Tokyo,
acid 1,4-lactone (Sigma-Aldrich), and cell lysates at 37°C for 24 h. The
Japan), FCS from BioWhittaker (Walkersville, MD), PMSF from Wako
reaction was stopped by the addition of 100 g of heparin and subsequent
Biochemicals (Osaka, Japan), PMA (P8139), heparin (H3400), 4-(2-ami-
boiling for 5 min, followed by addition of solution (5 M NaCl, 250 mM
noethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), leupeptin, pep-
Tris-HCl, 0.5% Triton X-100, pH 7.4). The mixture was then centrifuged
statin A, aprotinin, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
at 1000 rpm for 5 min to precipitate the insoluble material. The products of
(EDC) from Sigma-Aldrich (St. Louis, MO), HS (sodium salt) obtained
FL-HS yielded by this reaction were analyzed by high-speed gel perme-
from bovine kidney from Seikagaku Kogyo (Tokyo, Japan), normal mouse
ation chromatography. Briefly, a 50 –100 l aliquot of the supernatant was
IgG1 from Zymed Laboratories (South San Francisco, CA), and FITC-
injected into Superose 12 HR 10/30 column (Amersham Biosciences)
conjugated goat anti-mouse IgG(Fc) Ab F(ab⬘)2 from Sigma-Aldrich. Pu-
Results
PMA-treated U937 and THP-1 cells degrade ECM HS
Promonocytic leukemia cell lines U937 and THP-1 before or after
PMA treatment were used in this study. Size distribution of re-
leased 35S-sulfated materials from endothelial ECM was deter-
mined after incubation with these cells. The released radioactivity
from ECM after incubation with untreated U937 or THP-1 cells
eluted at the void volume fraction (Fig. 1, A and B). The size
or heparin. This would be due to nonproteolytic migration reported 14. Matzner, Y., M. Bar-Ner, J. Yahalom, R. Ishai-Michaeli, Z. Fuks, and
I. Vlodavsky. 1985. Degradation of heparan sulfate in the subendothelial extra-
recently with U937 cells (33). cellular matrix by a readily released heparanase from human neutrophils: possible
Heparanase was previously shown to be localized primarily in role in invasion through basement membranes. J. Clin. Invest. 76:1306.
perinuclear acidic endosomal and lysosomal granules, and in the 15. Sewell, R. F., P. E. Brenchley, and N. P. Mallick. 1989. Human mononuclear
cells contain an endoglycosidase specific for heparan sulphate glycosaminogly-
tertiary granules of human neutrophils together with MMP-9 (34, can demonstrable with the use of a specific solid-phase metabolically radiola-
35). We examined heparanase localization in U937 cells differen- belled substrate. Biochem. J. 264:777.
tiated into macrophage-like cells by immunostaining with anti- 16. Naparstek, Y., I. R. Cohen, Z. Fuks, and I. Vlodavsky. 1984. Activated T lym-
phocytes produce a matrix-degrading heparan sulphate endoglycosidase. Nature
heparanase mAb. Intracellular heparanase distributed in the gran- 310:241.
ule-like structure in both PMA-treated and untreated cells (Fig. 4). 17. Marchetti, D., J. Li, and R. Shen. 2000. Astrocytes contribute to the brain-met-
astatic specificity of melanoma cells by producing heparanase. Cancer Res.
In PMA-treated cells, heparanase was distributed on the cell sur- 60:4767.
face and subsequently capped in an adhesion-dependent manner 18. Bernard, D., B. Mehul, C. Delattre, L. Simonetti, A. Thomas-Collignon, and
(Fig. 4). After the appearance on the cell surface, heparanase R. Schmidt. 2001. Purification and characterization of the endoglycosidase
heparanase 1 from human plantar stratum corneum: a key enzyme in epidermal
seems to behave as a membrane-associated protein, as the super- physiology? J. Invest. Dermatol. 117:1266.
natant of PMA-treated U937 cells scarcely showed heparanase ac- 19. Freeman, C., and C. R. Parish. 1997. A rapid quantitative assay for the detection
tivity (data not shown). Heparanase is a transmembrane protein of mammalian heparanase activity. Biochem. J. 325:229.
20. Gohji, K., H. Hirano, M. Okamoto, S. Kitazawa, M. Toyoshima, J. Dong,
with a hydrophobic stretch of 20 aa near its C terminus (7–9). Y. Katsuoka, and M. Nakajima. 2001. Expression of three extracellular matrix
Alternatively, heparanase may bind putative cell surface receptors. degradative enzymes in bladder cancer. Int. J. Cancer 95:295.
21. Kramer, R. H., K. G. Vogel, and G. L. Nicolson. 1982. Solubilization and deg-
A 300-kDa mannose phosphate receptor on activated T lympho-