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Involvement of heparanase in migration of microglial cells
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Biochimica et Biophysica Acta 1780 (2008) 709 – 715 www.elsevier.com/locate/bbagen
Involvement of heparanase in migration of microglial cells Hisaaki Takahashi a,⁎, Hiroaki Matsumoto b , Anna Smirkin a , Tomohide Itai a , Yoshio Nishimura c , Junya Tanaka a a
Department of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime University, Toon, Ehime 791-0295, Japan b Department of Neurosurgery, Graduate School of Medicine, Ehime University, Toon, Ehime 791-0295, Japan c Microbial Chemistry Research Center, Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan Received 27 August 2007; received in revised form 19 November 2007; accepted 20 December 2007 Available online 5 January 2008
Abstract Heparanase, a matrix-degrading enzyme that cleaves heparan sulfate side chains from heparan sulfate proteoglycans (HSPGs), has been shown to facilitate cell invasion, migration, and extravasation of metastatic tumor cells or immune cells. In this study, the expression and functions of heparanase were investigated using rat primary cultured microglia, the resident macrophages in the brain. The microglia were found to express heparanase mRNA and protein. Microglia treated with lipopolysaccharide (LPS) were activated, expressed induced nitric oxide synthase and elevated the expression of heparanase. Heparanase has two molecular weights: a 65 kDa latent form and an active 50 kDa. Both forms were expressed by LPS-treated activated microglia; however, untreated microglia primarily expressed the latent form. Cell lysates from microglia actually degraded Matrigel containing HSPG. Heparanase was colocalized with the actin cytoskeleton in microglial leading edges or ruffled membranes. Microglia transmigrated through a Matrigel-coated pored membrane. This process was inhibited by SF-4, a specific heparanase inhibitor, in a concentration-dependent manner. Degraded HSPG was generated when microglia transmigrated through the coated membrane, and this was also inhibited by SF-4. The results suggest the involvement of heparanase in the migration or invasion of microglia or brain macrophages across basement membrane around brain vasculature. © 2007 Elsevier B.V. All rights reserved. Keywords: Heparanase; Microglial cell; Migration
1. Introduction Heparan sulfate proteoglycans (HSPGs) are essential components of an intricate network of macromolecules constituting extracellular matrix (ECM) and basement membrane (BM). HSPGs have a variety of functions, such as the sequestration of bioactive molecules and enhancement of cell adhesion, and they act as co-receptors for many cell surface molecules [1]. Therefore, the regulated turnover or degradation of HSPGs in ECM/BM networks by matrix-degrading enzymes may be a critical process under physiological and pathological conditions [1,2]. Heparanase is an endo-β-D-glucuronidase that specifically cleaves the 1,4-linkage between glucronic acid and N-acetyl glucosamine of heparan sulfate (HS) side chains of HSPGs [3,4]. ⁎ Corresponding author. Tel.: +81 89 960 5241; fax: +81 89 960 5242. E-mail address: [email protected] (H. Takahashi). 0304-4165/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2007.12.014
Heparanase activity is implicated in the metastatic abilities of various tumor-derived cells [5–7]; however, its activity is also detected in a variety of normal cell types, including neutrophils, macrophages, endothelial cells, and astrocytes [8–12]. Cleavage of HS side chains by heparanase is thought to facilitate cell migration, invasion, and extravasation into tissues, particularly of immune cells [8]. This is partly because activation of heparanase may amplify immune reactions by releasing growth factors, cytokines, and chemokines that bind to HS. Microglia are the brain macrophages that share many characteristics with monocytes or peripheral macrophages [13,14]. Microglia have ramified long thin processes and small somata in the normal mature brain. However, even after a minor pathological incident, they rapidly become activated and stereotypical changes in morphology occur, characterized by shortened processes and enlarged somata, and the cells then migrate towards the damaged area to eliminate tissue debris or
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defend against pathogens [13,14]. Microglial cells accumulate within the core of amyloid plaques to eliminate toxic senile plaques in transgenic mouse models of Alzheimer's disease [15]. Furthermore, bone marrow-derived cells are said to cross the blood–brain barrier (BBB) and differentiate into fully functional microglia in the normal brain [16]. Indeed, transgender cells with microglial phenotypes were present in patients subjected to sex-mismatched bone marrow transplantation [17]. Thus, bone marrow-derived microglial precursors probably migrate through the intact BBB under normal and pathological processes to degrade many different molecules that constitute the BM around the brain vasculature. However, the molecules responsible for the disruption of the BM that are expressed by microglia and their precursors have not been fully elucidated. Among these molecules, heparanase is a probable candidate involved in microglial migration or differentiation. In this study, we investigated whether rat primary cultured microglial cells express heparanase and whether this enzyme is involved in their migration. Our results showed that microglial cells expressed heparanase and the enzyme was able to degrade HS side chains of HSPGs. Enzymatic activity was correlated with in vitro transmigration ability through an artificial BM/ ECM containing HSPGs and this ability was inhibited by SF-4, a heparanase-specific inhibitor. These results suggest that heparanase plays a significant role in the migration of microglia across the BM around brain vasculature. 2. Materials and methods 2.1. Microglial cell culture Mixed glial cell cultures were prepared from the forebrain cells of newborn Wistar rats as described previously [18]. Mixed glial cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in flasks for 12 days. Microglial cells were isolated by shaking flasks for 1 h, seeding on a suspension culture dish, and incubating for 10 min in a CO2 incubator to remove contaminating cells not of macrophage lineage. The purity of microglial cells was N98%, as determined by immunostaining with a microglia/macrophage marker, CD11b [19].
2.2. RT-PCR Total RNA was isolated from cells using ISOGEN (Nippon gene, Tokyo, Japan). cDNA was obtained from DNase I-treated RNA by reverse transcription using an oligo(dT) primer, according to the manufactures instructions. DNA fragments were amplified by GoTaq DNA polymerase (Promega, Madison, WI) using specific primers for heparanase, iNOS and β-actin. PCR conditions and primer sequences used in this study are described elsewhere [12,20].
2.4. Heparanase activity assay The heparanase activity assay was carried out by the enzyme-linked immunosorbent assay (ELISA) developed by Xu et al [22]. Microglial cells (1 × 106 cells) were lysed in 300 µl of heparanase assay buffer (0.1 M sodium acetate (pH 5.0), 0.1 mg/ml bovine serum albumin (BSA; Sigma), 0.01% Triton X-100 and protease inhibitor cocktail (Sigma)) and then sonicated. Cell lysates were spun down at 15,000 rpm for 30 min, and the supernatants were collected. Matrigel, an artificially reconstituted BM/ECM containing HSPGs, was used to coat a 96well ELISA plate, and was blocked with 5% BSA in PBS at room temperature for 1 h before cell lysates (50 µl) with or without a specific inhibitor of heparanase, SF-4, were added to each well. SF-4 is an uronic acid-type gemdiamine 1-N-iminosugar synthesized from siastatin B, a secondary metabolite of Streptomyces and was identified as a potent inhibitor of the activity of heparanase isolated from bacteria (Fig. 1) [23,24]. After overnight incubation at 37 ˚C, anti-HS IgM mAb (1:500, clone 10E4; Seikagaku Corp., Tokyo, Japan) was added and the plate was incubated at room temperature for 1 h, followed by the addition of horseradish peroxidase-conjugated goat anti-mouse IgM antibody (1:5000, Stressgen, CA) at room temperature for 1 h. Development of immunocomplex was performed by addition of 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABS; Sigma) substrate, and the OD405 absorbance was measured with a plate reader (Bio-Rad).
2.5. Immunofluorescent staining Microglial cells were seeded on poly-L-lysine (PLL)-coated glass coverslips for immunocytochemical analysis. Cells treated with or without 1 µg/ml LPS (Escherichia coli serotype 055:B5; Sigma) [25] for 6 h were fixed with 4% paraformaldehyde (PFA), permeabilized and blocked with Tris-buffered saline (TBS) containing 0.1% Tween 20 and 1 mg/ml BSA for 30 min. Microglial cells were then incubated with rabbit anti-mouse heparanase (1:2000). After washing with TBS containing 0.1% Tween 20, cells were incubated with a mixture of Cy3-labeled secondary antibodies (Chemicon, Temecula, CA) and FITC-labeled phalloidin (Invitrogen, Carlsbad, CA). Hoechst 33258 (Sigma) was used for nuclear staining. The immunostained specimens were observed under a BX-52 (Olympus, Tokyo, Japan) conventional microscope equipped with a CCD camera.
2.6. Matrigel invasion assay and degradation of HSPGs An invasion assay was performed using transwell chamber membranes coated with Matrigel (8 µm pore size; BD Labware, Bedford, MA). Microglial cells (1 × 105 cells for transmigration assay and 5 × 103 for immunostaining of HS) in 500 µl of serum-free medium were seeded on the upper chamber, and the lower compartment was filled with 750 µl of DMEM or DMEM containing 10% FBS. FBS as a chemoattractant has been used for Matrigel invasion assay in many kinds of cells, including microglia lineage cells [26]. Various concentrations of SF-4 were added to the upper chambers at the same time. Cells were allowed to invade into a Matrigel at 37 °C for 24 h in a CO2 incubator. For the transmigration assay, cells that did not migrate were removed from the upper surface of the membrane with a cotton swab, and cells that migrated into the lower chamber were fixed with 4% PFA and stained with hematoxylin. For immunocytochemical analysis to detect degradation of HSPGs, the membrane was fixed with 4% PFA after wiping the lower surface with a cotton swab. After
2.3. Western blot analysis Cell lysates were prepared with Laemmli's sample solution. Equal amounts of total protein were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. The blots were probed with antibodies to heparanase (1:500, kindly gifted by Dr. Freeman, The Australian National University) [21]), iNOS (1:1000; BD Biosciences, San Jose, CA), and β-actin (clone AC15, 1:1000; Sigma Chemical Co., St Louis, MO). The anti-mouse heparanase antibody reacts with both latent (65 kDa) and active (50 kDa) forms as described elsewhere [12,21]. Immunoreacted bands were visualized using alkaline phosphatase-conjugated secondary antibodies (1:5000; Promega) followed by incubation with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as the enzyme substrates.
Fig. 1. Structure of SF-4, a specific heparanase inhibitor. SF-4 is an uronic acidtype gem-diamine 1-N-iminosugar synthesized from siastatin B, a secondary metabolite of Streptomyces [23].
H. Takahashi et al. / Biochimica et Biophysica Acta 1780 (2008) 709–715 blocking with TBS containing 1 mg/ml BSA, the Matrigel-coated membrane was incubated with anti-HS IgM mAb (1:200, clone 10E4) or anti-Δ HS IgM mAb (1:200, clone 3G10; Seikagaku Corp.) overnight at 4 °C. The anti-Δ HS IgM mAb reacted with a newly generated epitope, which was formed through degradation of HSPGs. The epitope contained desaturated hexuronate present at the non-reducing end of the heparan sulfate fragments of HSPGs. Membranes were further incubated with a biotinylated goat anti-mouse IgM antibody (1:1000, Chemicon) at room temperature for 1 h, and then with streptavidinperoxidase (Labvision, Fremont, CA) for 1 h. Immunoreaction was visualized with 3-amino-9-ethylcarbazole (AEC) as chromogen (Labvision). The membrane was excised, mounted onto a glass slide with mounting solution and then the total transmigrated cells were counted and images were captured under a light microscope.
2.7. Statistical analysis Values were expressed as mean ± standard deviation of means (SD). Significant differences between groups were analyzed by ANOVA with GraphPad InStat software (GraphPad Software, San Diego, CA) and significance was set at P b 0.05.
3. Results 3.1. Expression of heparanase in microglial cells Expression of mRNA-encoding heparanase by microglial cells was investigated by RT-PCR in the presence or the absence of lipopolysaccharide (LPS) (Fig. 2A). LPS-treated microglial cells expressed inducible NO synthase (iNOS)-mRNA, which implied that the microglia were activated (Fig. 2A). Although heparanase-mRNA was expressed in LPS-treated and non-treated microglial cells, LPS-treated cells upregulated the expression of heparanase mRNA. Heparanase protein was expressed by microglial cells, as revealed by Western blot analysis (Fig. 2B).
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A latent form of heparanase (65 kDa) was expressed by both types of microglia, but LPS-treated microglia expressed the protein at a higher level than the non-treated cells. Lysosomal proteases such as cathepsin D and L cleave the latent form to generate the activated enzyme of 50 kDa [27]. The active form was detected only in the lysate of LPS-treated microglia. The enzymatic activities of heparanase in the microglial lysates were assessed by detecting HS chain degradation of HSPGs with HS specific monoclonal antibody. LPS-treated microglial lysates significantly degraded more HS chains than non-treated lysates (Fig. 2C). Although the active form of heparanase was not detected in non-treated cells by Western blot analysis (Fig. 2B), the cell lysates elicited significant heparanase activity. Since microglial cells express various kinds of proteases, a heparanase inhibitor, SF-4, was used to rule out a possibility that the degraded HS chain was generated by some proteases that degrade Matrigel. SF4, which specifically inhibits endoglucuronidase activity of heparanase through binding to the active site of the enzyme [23], is significantly inhibited the HS chain degradation, indicating that heparanase plays a critical role in the HS chain degradation (Fig. 2C). 3.2. Intracellular localization of heparanase An immunocytochemical study was performed to determine the intracellular localization of heparanase. Fixed microglial cells grown on poly-L-lysine-coated glass coverslips were doublestained with anti-heparanase antibody and FITC labeledphalloidin to visualize the actin cytoskeleton. Heparanase was localized in the cytoplasm and nucleus in microglial cells (Fig. 3). In addition, the enzyme was strongly concentrated at the site of
Fig. 2. Induction of heparanase expression and enzymatic activity in LPS-treated microglial cells. RT-PCR (A) and Western blot (B) analysis on heparanase expression in non-treated (MG) and LPS-treated (MG + LPS) rat microglial cells and LPS-treated microglial cells. Activation of microglial cells was evaluated by detection of inducible NO synthase (iNOS). β-actin was used as an internal standard. (C) Enzymatic activity of heparanase in microglial cells. The cell lysates with or without SF-4 at the indicated concentrations were loaded to a Matrigel-coated ELISA plate and incubated overnight at 37 °C. Enzymatic activity of heparanase was investigated by an ELISA method, as described in Materials and methods. The value of control (data obtained in the absence of cell lysates) indicates the total amount of HS of HSPGs in Matrigel. Each column represents the mean ± standard deviation (SD) of three independent experiments. Asterisks represent a significant statistical difference between control vs MG, MG-LPS and MG-LPS + SF-4. ⁎P b 0.05, ⁎⁎P b 0.001. n.s. indicates no significance.
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Fig. 3. Localization of heparanase in microglial cells. Microglial cells were incubated with or without LPS for 6 h. Cells were fixed and stained with anti-mouse heparanase antibody (red; A and D). Cells were also stained with FITC-phalloidin to visualize actin filaments (green; B and E). Merged signals appear yellow (C and F). Arrowheads indicate cells with heparanase immunoreactivity at the site of membrane ruffles or leading edges. Insertion in Fig. 3D is a high-magnification view of the boxed area. Scale bar = 25 µm.
membrane ruffles and leading edges especially in non-treated microglial cells, both of which were characterized by an enriched actin cytoskeleton (Fig. 3A). On the contrary, in LPS-treated
microglial cells, the localization of heparanase in leading eddges became weaker, and a granular localization of heparanaseimmunoreactivity was more apparent (Fig. 3D).
Fig. 4. Involvement of heparanase in invasion of microglial cells. Microglial cells were plated onto Matrigel-coated inserts in the absence or presence of SF-4 at the indicated concentrations and placed in outer chambers containing DMEM with or without 10% FBS. (A–C) Representative photomicrographs of invaded microglial cells are shown. Transmigrated microglial cells were visualized by hematoxylin staining and counted after 24 h under a light microscope. Scale bar = 50 µm. (D) The mean value of transmigrated cells in 10% FBS condition is represented as 100%. Each column represents the mean ± SD of three independent experiments and statistical analysis of SF-4 0 mM vs others. ⁎P b 0.05, ⁎⁎P b 0.01, #P b 0.001.
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3.3. Involvement of heparanase in migration of microglial cells To assess the contribution of heparanase to the migration activity of microglial cells, they were seeded on Transwell inserts coated with Matrigel containing HSPGs (Fig. 4). Based on the above results, microglia increased the expression of heparanase in response to LPS. However, prolonged incubation with LPS under the serum-free condition decreased the viability of microglia (data not shown), and lysates from microglia cultured in the absence of LPS has an ability to degraded HSPGs (Fig. 2). Therefore, the following migration assay was conducted in the absence of LPS. Serum-free medium was added to the upper chamber and medium containing 10% FBS was added to the lower chamber. Microglial cells on the inserts in the serum-free DMEM transmigrated through pores of the inserts coated with Matrigel to the lower chamber filled with the serum-supplemented DMEM (Fig. 4B). Transmigration was significantly less without the addition of the serum to the lower chamber (Fig. 4A). To evaluate the contribution of heparanase to the transmigration of microglial cells SF-4 was added to the medium in the upper chambers. SF-4 significantly inhibited transmigration through the Matrigel-coated inserts in a concentration-dependent manner (Fig. 4C and D). SF-4 dissolved in culture medium at 1 mM did not influence the viability or phagocytic activity of microglial cells (data not shown). To examine whether degraded HSPGs were generated when the microglial cells transmigrated through Matrigel, immunostaining with antibodies to HS or to degraded HSPGs devoid of the HS chain was performed (Fig. 5). Abundant immunoreactivity of the HS network and faint immunostaining of the degraded HSPGs were observed on the Matrigel-coated inserts in the serum-free condition, where microglial transmigration had scarcely occurred (Fig. 5A and D). No obvious differences
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in the HS staining pattern occurred between the serum-free and serum-supplemented conditions (Fig. 5A, B, C); however, degraded HSPGs were abundant only in the serum-supplemented chamber, where microglial cells actively transmigrated through the inserts (Fig. 5E). When SF-4 was added to the upper medium, the increase in degraded HSPGs was abolished, even in the presence of serum (Fig. 5F). Thus, the transmigration of microglia through the Matrigel depends on the activity of heparanase. 4. Discussion In response to even a minor pathological event in the brain, microglial cells become activated and migrate toward the site of the event [13,14]. Furthermore, during physiological development and pathological processes, precursors of microglia may be supplied from the circulation [16,28–30]. During these events, microglia must migrate across the BM/ECM containing HSPGs that act as a barrier for the migrating cells. Based on the present study, which showed that rat primary cultured microglial cells express heparanase mRNA and proteins, cells may utilize heparanase to degrade the HSPGs in the BM/ECM when they migrate across the barrier. Furthermore, the expression level of the enzyme increased when the cells were activated in response to LPS. Therefore, it is likely that activated microglia increase the expression of heparanase to aid their migration through the BM/ECM. Two molecular species with different molecular weights of 65 and 50 kDa were detected in the microglial lysates with the antibody to heparanase. The 65 kDa species is said to be the latent form of heparanase, which can be cleaved by lysosomal proteases such as cathepsin D or L to the active 50 kDa species [27]. In this study, Western blotting detected the active form in the lysate of LPS-treated microglia only. In fact, the lysate of
Fig. 5. Immunodetection of HS or digested HS chain in Matrigel. Matrigel chamber after transmigration assays were performed by immunostaining with HS (A–C) or degraded-HS (D–F) specific antibody. Representative photomicrographs are shown. The Matrigel was counterstained with hematoxylin to visualize the microglial cells. Note that the degraded HS chain was strongly detected in 10% FBS condition. Scale bar = 50 µm.
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LPS-treated microglia degraded HSPGs more significantly (Fig. 2). This result appears consistent with previous observations, which demonstrated an elevated expression of cathepsin B and L [31] or increased lysosomal enzyme activity [32] in activated microglia. Thus, the presence of the active form of heparanase might be a good marker of activated microglia in addition to the expression of iNOS or TNFα. Although the active form of heparanase could not be detected in the lysate of microglia cultured in the absence of LPS, the lysate significantly degraded HSPGs (Fig. 2C). The immunocytochemical analysis in this study showed that heparanase appeared to be located in ruffled membranes or leading edges of microglia. Furthermore, this observation appears to correspond with a previous study which demonstrated that the cell surface localization of heparanase in macrophages closely correlated to that of microglia in terms of cell lineage [9]. Conversely, the enzyme was more diffusely distributed through the cytoplasm of LPS-treated microglia. Accordingly, in the non-treated microglia heparanase and the lysosomal enzymes responsible for enzyme activation may be separately localized, and this may be the reason why the active form was undetectable in Western blotting experiments. Moreover, during preparation of the lysate from the non-treated microglia, heparanase may have been partially degraded by lysosomal proteases released through homogenization, which resulted in destruction of the lysosomal membrane. Such mechanisms may explain the discrepancy between the results obtained by Western blotting and the activity assay. It has been reported that the 65 kDa-species of heparanase may act both as a precursor of the active form and a cell adhesion molecule [33,34]. Heparanase-mediated cell adhesion to ECM results in integrin-dependent cell spreading or reorganization of the actin cytoskeleton, and this action is independent of its endoglucuronidase activity. The present immunocytochemical results showing the localization of the enzyme at the leading edges and ruffled membranes support the notion. Such localization was more apparent in non-treated microglia, suggesting that the latent form has a significant role in the adhesion and migration of microglia. In contrast, apparent granular localization of heparanase, suggesting lysosomal localization, in activated microglia may be required for its activation by processing and storage, as reported in other cell [35]. Primary cultured microglia isolated from mixed glial cell cultures are very different in terms of macrophage-like functions from matured ramified microglia in the brain, which are thought to be in a resting state. For example, CD68, a marker of phagocytes, is constitutively expressed by primary cultured microglia but not by microglia in the normal mature brain [36,37]. The cultured cells normally display amoeboid rather than ramified morphology, which is characterized by small somata and long thin processes. Such amoeboid morphology may resemble monocyte-like precursors of microglia in the brain. Therefore, the present results may reflect the nature of bone marrowderived microglial precursors rather than the microglia or macrophages in the brain parenchyma. In fact, microglia expressing heparanase were not detected in normal brain parenchyma (data not shown). Moreover, macrophages or activated microglia did
not express heparanase in ischemic lesions in rats with transient occlusion of the middle cerebral artery [12]. Instead, only reactive astrocytes located in the peri-ischemic lesions were identified as heparanase-expressing cells. HSPGs may be localized exclusively in BM around the brain vasculature, but not in the brain parenchyma [38]. Astrocyte end-feet intimately adhere to the BM around the brain blood vessels to induce formation of the BBB. After an ischemic insult, vascular reconstruction occurs and this may require the expression of heparanase in reactive astrocytes. On the other hand, since there are comparatively little amount of HSPGs in brain parenchyma, the parenchymal microglia or macrophages may not necessarily express the enzyme, even when they move their processes or actively migrate [39]. Alternatively, heparanase may be important for microglia or their precursors to invade into the brain parenchyma across the BM around brain vasculature from the circulation. During embryonic development, blood-derived precursor cells related to the monocyte-macrophage lineage invade the brain parenchyma across the BM, and then transform into ramified resting microglia upon maturation of the brain [28–30]. Such migration through the BM may occur even in the normal mature brain, based on reports demonstrating that replacement of ramified microglia with bone marrow-derived cells regularly occurs [17,40,41]. In the physiological process of microglial differentiation, heparanase may act as an adhesion molecule first for adherence to the BM while reorganizing the actin cytoskeletons of the precursor cells. Thereafter, heparanase may start degradation of the HSPGs that form the BM through its endoglucuronidase activity. Acknowledgements We thank Dr. C. Freeman (The Australian National University, Australia) for helpful comments and for providing the antibody to heparanase. This study was partly supported by grants from Ehime University and the Ministry of Education, Culture, Sports, Science, and Technology, Japan. References [1] M. Bernfield, M. Gotte, P.W. Park, O. Reizes, M.L. Fitzgerald, J. Lincecum, M. Zako, Functions of cell surface heparan sulfate proteoglycans, Annu. Rev. Biochem. 68 (1999) 729–777. [2] T.N. Wight, M.G. Kinsella, E.E. Qwarnstrom, The role of proteoglycans in cell adhesion, migration and proliferation, Curr. Opin. Cell Biol. 4 (1992) 793–801. [3] I. Vlodavsky, Y. Friedmann, M. Elkin, H. Aingorn, R. Atzmon, R. IshaiMichaeli, M. Bitan, O. Pappo, T. Peretz, I. Michal, L. Spector, I. Pecker, Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis, Nat. Med. 5 (1999) 793–802. [4] M.D. Hulett, C. Freeman, B.J. Hamdorf, R.T. Baker, M.J. Harris, C.R. Parish, Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis, Nat. Med. 5 (1999) 803–809. [5] I. Vlodavsky, O. Goldshmidt, E. Zcharia, S. Metzger, T. Chajek-Shaul, R. Atzmon, Z. Guatta-Rangini, Y. Friedmann, Molecular properties and involvement of heparanase in cancer progression and normal development, Biochimie 83 (2001) 831–839. [6] O. Goldshmidt, E. Zcharia, R. Abramovitch, S. Metzger, H. Aingorn, Y. Friedmann, V. Schirrmacher, E. Mitrani, I. Vlodavsky, Cell surface expression
H. Takahashi et al. / Biochimica et Biophysica Acta 1780 (2008) 709–715
[7]
[8] [9]
[10]
[11]
[12]
[13] [14] [15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
and secretion of heparanase markedly promote tumor angiogenesis and metastasis, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 10031–10036. N. Ilan, M. Elkin, I. Vlodavsky, Regulation, function and clinical significance of heparanase in cancer metastasis and angiogenesis, Int. J. Biochem. Cell Biol. 38 (2006) 2018–2039. C.R. Parish, C. Freeman, M.D. Hulett, Heparanase: a key enzyme involved in cell invasion, Biochim. Biophys. Acta 1471 (2001) M99–M108. N. Sasaki, N. Higashi, T. Taka, M. Nakajima, T. Irimura, Cell surface localization of heparanase on macrophages regulates degradation of extracellular matrix heparan sulfate, J. Immunol. 172 (2004) 3830–3835. S. Gingis-Velitski, A. Zetser, M.Y. Flugelman, I. Vlodavsky, N. Ilan, Heparanase induces endothelial cell migration via protein kinase B/Akt activation, J. Biol. Chem. 279 (2004) 23536–23541. D. Marchetti, J. Li, R. Shen, Astrocytes contribute to the brain-metastatic specificity of melanoma cells by producing heparanase, Cancer Res. 60 (2000) 4767–4770. H. Takahashi, H. Matsumoto, Y. Kumon, T. Ohnishi, C. Freeman, Y. Imai, J. Tanaka, Expression of heparanase in nestin-positive reactive astrocytes in ischemic lesions of rat brain after transient middle cerebral artery occlusion, Neurosci. Lett. 417 (2007) 250–254. G.W. Kreutzberg, Microglia: a sensor for pathological events in the CNS, Trends Neurosci. 19 (1996) 312–318. K. Nakajima, S. Kohsaka, Microglia: activation and their significance in the central nervous system, J. Biochem. (Tokyo) 130 (2001) 169–175. A.R. Simard, D. Soulet, G. Gowing, J.P. Julien, S. Rivest, Bone marrowderived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease, Neuron 49 (2006) 489–502. M.A. Eglitis, E. Mezey, Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 4080–4085. C.R. Cogle, A.T. Yachnis, E.D. Laywell, D.S. Zander, J.R. Wingard, D.A. Steindler, E.W. Scott, Bone marrow transdifferentiation in brain after transplantation: a retrospective study, Lancet 363 (2004) 1432–1437. J. Tanaka, H. Fujita, S. Matsuda, K. Toku, M. Sakanaka, N. Maeda, Glucocorticoid- and mineralocorticoid receptors in microglial cells: the two receptors mediate differential effects of corticosteroids, Glia 20 (1997) 23–37. A. Yokoyama, L. Yang, S. Itoh, K. Mori, J. Tanaka, Microglia, a potential source of neurons, astrocytes, and oligodendrocytes, Glia 45 (2004) 96–104. H. Matsumoto, Y. Kumon, H. Watanabe, T. Ohnishi, M. Shudou, C. Ii, H. Takahashi, Y. Imai, J. Tanaka, Antibodies to CD11b, CD68, and lectin label neutrophils rather than microglia in traumatic and ischemic brain lesions, J. Neurosci. Res. 85 (2007) 994–1009. J.A. Joyce, C. Freeman, N. Meyer-Morse, C.R. Parish, D. Hanahan, A functional heparan sulfate mimetic implicates both heparanase and heparan sulfate in tumor angiogenesis and invasion in a mouse model of multistage cancer, Oncogene 24 (2005) 4037–4051. X. Xu, R.M. Quiros, J.B. Maxhimer, P. Jiang, R. Marcinek, K.B. Ain, J.L. Platt, J. Shen, P. Gattuso, R.A. Prinz, Inverse correlation between heparan sulfate composition and heparanase-1 gene expression in thyroid papillary carcinomas: a potential role in tumor metastasis, Clin. Cancer Res. 9 (2003) 5968–5979. Y. Nishimura, E. Shitara, H. Adachi, M. Toyoshima, M. Nakajima, Y. Okami, T. Takeuchi, Flexible synthesis and biological activity of uronic
[24]
[25]
[26]
[27] [28] [29] [30] [31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
715
acid-type gem-diamine 1-N-iminosugars: a new family of glycosidase inhibitors, J. Org. Chem. 65 (2000) 2–11. T. Satoh, Y. Nishimura, S. Kondo, T. Takeuchi, A practical synthesis from siastatin B of (3S,4S,5R,6R)-4,5-dihydroxy-6-(trifruoroacetamido)piperidine-3-carboxylic acid having antimetastatic activity in mice, Carbohyd. Res. 286 (1996) 173–178. Y. Kuwabara, A. Yokoyama, L. Yang, K. Toku, K. Mori, I. Takeda, T. Shigekawa, B. Zhang, N. Maeda, M. Sakanaka, J. Tanaka, Two populations of microglial cells isolated from rat primary mixed glial cultures, J. Neurosci. Res. 73 (2003) 22–30. A.P. Wheeler, C.M. Wells, S.D. Smith, F.M. Vega, R.B. Henderson, V.L. Tybulewicz, A.J. Ridley, Rac1 and Rac2 regulate macrophage morphology but are not essential for migration, J. Cell Sci. 119 (2006) 2749–2757. I. Shafat, I. Vlodavsky, N. Ilan, Characterization of mechanisms involved in secretion of active heparanase, J. Biol. Chem. 281 (2006) 23804–23811. E.A. Ling, W.C. Wong, The origin and nature of ramified and amoeboid microglia: a historical review and current concepts, Glia 7 (1993) 9–18. K.D. Barron, The microglial cell. A historical review, J. Neurol. Sci. 134 (1995) 57–68. M.A. Cuadros, J. Navascues, The origin and differentiation of microglial cells during development, Prog Neurobiol 56 (1998) 173–189. R.B. Banati, G. Rothe, G. Valet, G.W. Kreutzberg, Detection of lysosomal cysteine proteinases in microglia: flow cytometric measurement and histochemical localization of cathepsin B and L, Glia 7 (1993) 183–191. J. Tanaka, K. Toku, S. Matsuda, S. Sudo, H. Fujita, M. Sakanaka, N. Maeda, Induction of resting microglia in culture medium devoid of glycine and serine, Glia 24 (1998) 198–215. O. Goldshmidt, E. Zcharia, M. Cohen, H. Aingorn, I. Cohen, L. Nadav, B.Z. Katz, B. Geiger, I. Vlodavsky, Heparanase mediates cell adhesion independent of its enzymatic activity, FASEB J. 17 (2003) 1015–1025. I. Sotnikov, R. Hershkoviz, V. Grabovsky, N. Ilan, L. Cahalon, I. Vlodavsky, R. Alon, O. Lider, Enzymatically quiescent heparanase augments T cell interactions with VCAM-1 and extracellular matrix components under versatile dynamic contexts, J. Immunol. 172 (2004) 5185–5193. O. Goldshmidt, L. Nadav, H. Aingorn, C. Irit, N. Feinstein, N. Ilan, E. Zamir, B. Geiger, I. Vlodavsky, B.Z. Katz, Human heparanase is localized within lysosomes in a stable form, Exp. Cell Res. 281 (2002) 50–62. D.C. Chugani, N.L. Kedersha, L.H. Rome, Vault immunofluorescence in the brain: new insights regarding the origin of microglia, J. Neurosci. 11 (1991) 256–268. J.A. Amat, H. Ishiguro, K. Nakamura, W.T. Norton, Phenotypic diversity and kinetics of proliferating microglia and astrocytes following cortical stab wounds, Glia 16 (1996) 368–382. J. van Horssen, L. Bo, C.M. Vos, I. Virtanen, H.E. de Vries, Basement membrane proteins in multiple sclerosis-associated inflammatory cuffs: potential role in influx and transport of leukocytes, J. Neuropathol. Exp. Neurol. 64 (2005) 722–729. A. Nimmerjahn, F. Kirchhoff, F. Helmchen, Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo, Science 308 (2005) 1314–1318. A. Flugel, M. Bradl, G.W. Kreutzberg, M.B. Graeber, Transformation of donor-derived bone marrow precursors into host microglia during autoimmune CNS inflammation and during the retrograde response to axotomy, J. Neurosci. Res. 66 (2001) 74–82. H. Xu, M. Chen, E.J. Mayer, J.V. Forrester, A.D. Dick, Turnover of resident retinal microglia in the normal adult mouse, Glia 55 (2007) 1189–1198.
Biochimica et Biophysica Acta 1780 (2008) 709 – 715 www.elsevier.com/locate/bbagen
Involvement of heparanase in migration of microglial cells Hisaaki Takahashi a,⁎, Hiroaki Matsumoto b , Anna Smirkin a , Tomohide Itai a , Yoshio Nishimura c , Junya Tanaka a a
Department of Molecular and Cellular Physiology, Graduate School of Medicine, Ehime University, Toon, Ehime 791-0295, Japan b Department of Neurosurgery, Graduate School of Medicine, Ehime University, Toon, Ehime 791-0295, Japan c Microbial Chemistry Research Center, Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan Received 27 August 2007; received in revised form 19 November 2007; accepted 20 December 2007 Available online 5 January 2008
Abstract Heparanase, a matrix-degrading enzyme that cleaves heparan sulfate side chains from heparan sulfate proteoglycans (HSPGs), has been shown to facilitate cell invasion, migration, and extravasation of metastatic tumor cells or immune cells. In this study, the expression and functions of heparanase were investigated using rat primary cultured microglia, the resident macrophages in the brain. The microglia were found to express heparanase mRNA and protein. Microglia treated with lipopolysaccharide (LPS) were activated, expressed induced nitric oxide synthase and elevated the expression of heparanase. Heparanase has two molecular weights: a 65 kDa latent form and an active 50 kDa. Both forms were expressed by LPS-treated activated microglia; however, untreated microglia primarily expressed the latent form. Cell lysates from microglia actually degraded Matrigel containing HSPG. Heparanase was colocalized with the actin cytoskeleton in microglial leading edges or ruffled membranes. Microglia transmigrated through a Matrigel-coated pored membrane. This process was inhibited by SF-4, a specific heparanase inhibitor, in a concentration-dependent manner. Degraded HSPG was generated when microglia transmigrated through the coated membrane, and this was also inhibited by SF-4. The results suggest the involvement of heparanase in the migration or invasion of microglia or brain macrophages across basement membrane around brain vasculature. © 2007 Elsevier B.V. All rights reserved. Keywords: Heparanase; Microglial cell; Migration
1. Introduction Heparan sulfate proteoglycans (HSPGs) are essential components of an intricate network of macromolecules constituting extracellular matrix (ECM) and basement membrane (BM). HSPGs have a variety of functions, such as the sequestration of bioactive molecules and enhancement of cell adhesion, and they act as co-receptors for many cell surface molecules [1]. Therefore, the regulated turnover or degradation of HSPGs in ECM/BM networks by matrix-degrading enzymes may be a critical process under physiological and pathological conditions [1,2]. Heparanase is an endo-β-D-glucuronidase that specifically cleaves the 1,4-linkage between glucronic acid and N-acetyl glucosamine of heparan sulfate (HS) side chains of HSPGs [3,4]. ⁎ Corresponding author. Tel.: +81 89 960 5241; fax: +81 89 960 5242. E-mail address: [email protected] (H. Takahashi). 0304-4165/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2007.12.014
Heparanase activity is implicated in the metastatic abilities of various tumor-derived cells [5–7]; however, its activity is also detected in a variety of normal cell types, including neutrophils, macrophages, endothelial cells, and astrocytes [8–12]. Cleavage of HS side chains by heparanase is thought to facilitate cell migration, invasion, and extravasation into tissues, particularly of immune cells [8]. This is partly because activation of heparanase may amplify immune reactions by releasing growth factors, cytokines, and chemokines that bind to HS. Microglia are the brain macrophages that share many characteristics with monocytes or peripheral macrophages [13,14]. Microglia have ramified long thin processes and small somata in the normal mature brain. However, even after a minor pathological incident, they rapidly become activated and stereotypical changes in morphology occur, characterized by shortened processes and enlarged somata, and the cells then migrate towards the damaged area to eliminate tissue debris or
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defend against pathogens [13,14]. Microglial cells accumulate within the core of amyloid plaques to eliminate toxic senile plaques in transgenic mouse models of Alzheimer's disease [15]. Furthermore, bone marrow-derived cells are said to cross the blood–brain barrier (BBB) and differentiate into fully functional microglia in the normal brain [16]. Indeed, transgender cells with microglial phenotypes were present in patients subjected to sex-mismatched bone marrow transplantation [17]. Thus, bone marrow-derived microglial precursors probably migrate through the intact BBB under normal and pathological processes to degrade many different molecules that constitute the BM around the brain vasculature. However, the molecules responsible for the disruption of the BM that are expressed by microglia and their precursors have not been fully elucidated. Among these molecules, heparanase is a probable candidate involved in microglial migration or differentiation. In this study, we investigated whether rat primary cultured microglial cells express heparanase and whether this enzyme is involved in their migration. Our results showed that microglial cells expressed heparanase and the enzyme was able to degrade HS side chains of HSPGs. Enzymatic activity was correlated with in vitro transmigration ability through an artificial BM/ ECM containing HSPGs and this ability was inhibited by SF-4, a heparanase-specific inhibitor. These results suggest that heparanase plays a significant role in the migration of microglia across the BM around brain vasculature. 2. Materials and methods 2.1. Microglial cell culture Mixed glial cell cultures were prepared from the forebrain cells of newborn Wistar rats as described previously [18]. Mixed glial cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in flasks for 12 days. Microglial cells were isolated by shaking flasks for 1 h, seeding on a suspension culture dish, and incubating for 10 min in a CO2 incubator to remove contaminating cells not of macrophage lineage. The purity of microglial cells was N98%, as determined by immunostaining with a microglia/macrophage marker, CD11b [19].
2.2. RT-PCR Total RNA was isolated from cells using ISOGEN (Nippon gene, Tokyo, Japan). cDNA was obtained from DNase I-treated RNA by reverse transcription using an oligo(dT) primer, according to the manufactures instructions. DNA fragments were amplified by GoTaq DNA polymerase (Promega, Madison, WI) using specific primers for heparanase, iNOS and β-actin. PCR conditions and primer sequences used in this study are described elsewhere [12,20].
2.4. Heparanase activity assay The heparanase activity assay was carried out by the enzyme-linked immunosorbent assay (ELISA) developed by Xu et al [22]. Microglial cells (1 × 106 cells) were lysed in 300 µl of heparanase assay buffer (0.1 M sodium acetate (pH 5.0), 0.1 mg/ml bovine serum albumin (BSA; Sigma), 0.01% Triton X-100 and protease inhibitor cocktail (Sigma)) and then sonicated. Cell lysates were spun down at 15,000 rpm for 30 min, and the supernatants were collected. Matrigel, an artificially reconstituted BM/ECM containing HSPGs, was used to coat a 96well ELISA plate, and was blocked with 5% BSA in PBS at room temperature for 1 h before cell lysates (50 µl) with or without a specific inhibitor of heparanase, SF-4, were added to each well. SF-4 is an uronic acid-type gemdiamine 1-N-iminosugar synthesized from siastatin B, a secondary metabolite of Streptomyces and was identified as a potent inhibitor of the activity of heparanase isolated from bacteria (Fig. 1) [23,24]. After overnight incubation at 37 ˚C, anti-HS IgM mAb (1:500, clone 10E4; Seikagaku Corp., Tokyo, Japan) was added and the plate was incubated at room temperature for 1 h, followed by the addition of horseradish peroxidase-conjugated goat anti-mouse IgM antibody (1:5000, Stressgen, CA) at room temperature for 1 h. Development of immunocomplex was performed by addition of 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABS; Sigma) substrate, and the OD405 absorbance was measured with a plate reader (Bio-Rad).
2.5. Immunofluorescent staining Microglial cells were seeded on poly-L-lysine (PLL)-coated glass coverslips for immunocytochemical analysis. Cells treated with or without 1 µg/ml LPS (Escherichia coli serotype 055:B5; Sigma) [25] for 6 h were fixed with 4% paraformaldehyde (PFA), permeabilized and blocked with Tris-buffered saline (TBS) containing 0.1% Tween 20 and 1 mg/ml BSA for 30 min. Microglial cells were then incubated with rabbit anti-mouse heparanase (1:2000). After washing with TBS containing 0.1% Tween 20, cells were incubated with a mixture of Cy3-labeled secondary antibodies (Chemicon, Temecula, CA) and FITC-labeled phalloidin (Invitrogen, Carlsbad, CA). Hoechst 33258 (Sigma) was used for nuclear staining. The immunostained specimens were observed under a BX-52 (Olympus, Tokyo, Japan) conventional microscope equipped with a CCD camera.
2.6. Matrigel invasion assay and degradation of HSPGs An invasion assay was performed using transwell chamber membranes coated with Matrigel (8 µm pore size; BD Labware, Bedford, MA). Microglial cells (1 × 105 cells for transmigration assay and 5 × 103 for immunostaining of HS) in 500 µl of serum-free medium were seeded on the upper chamber, and the lower compartment was filled with 750 µl of DMEM or DMEM containing 10% FBS. FBS as a chemoattractant has been used for Matrigel invasion assay in many kinds of cells, including microglia lineage cells [26]. Various concentrations of SF-4 were added to the upper chambers at the same time. Cells were allowed to invade into a Matrigel at 37 °C for 24 h in a CO2 incubator. For the transmigration assay, cells that did not migrate were removed from the upper surface of the membrane with a cotton swab, and cells that migrated into the lower chamber were fixed with 4% PFA and stained with hematoxylin. For immunocytochemical analysis to detect degradation of HSPGs, the membrane was fixed with 4% PFA after wiping the lower surface with a cotton swab. After
2.3. Western blot analysis Cell lysates were prepared with Laemmli's sample solution. Equal amounts of total protein were separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. The blots were probed with antibodies to heparanase (1:500, kindly gifted by Dr. Freeman, The Australian National University) [21]), iNOS (1:1000; BD Biosciences, San Jose, CA), and β-actin (clone AC15, 1:1000; Sigma Chemical Co., St Louis, MO). The anti-mouse heparanase antibody reacts with both latent (65 kDa) and active (50 kDa) forms as described elsewhere [12,21]. Immunoreacted bands were visualized using alkaline phosphatase-conjugated secondary antibodies (1:5000; Promega) followed by incubation with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as the enzyme substrates.
Fig. 1. Structure of SF-4, a specific heparanase inhibitor. SF-4 is an uronic acidtype gem-diamine 1-N-iminosugar synthesized from siastatin B, a secondary metabolite of Streptomyces [23].
H. Takahashi et al. / Biochimica et Biophysica Acta 1780 (2008) 709–715 blocking with TBS containing 1 mg/ml BSA, the Matrigel-coated membrane was incubated with anti-HS IgM mAb (1:200, clone 10E4) or anti-Δ HS IgM mAb (1:200, clone 3G10; Seikagaku Corp.) overnight at 4 °C. The anti-Δ HS IgM mAb reacted with a newly generated epitope, which was formed through degradation of HSPGs. The epitope contained desaturated hexuronate present at the non-reducing end of the heparan sulfate fragments of HSPGs. Membranes were further incubated with a biotinylated goat anti-mouse IgM antibody (1:1000, Chemicon) at room temperature for 1 h, and then with streptavidinperoxidase (Labvision, Fremont, CA) for 1 h. Immunoreaction was visualized with 3-amino-9-ethylcarbazole (AEC) as chromogen (Labvision). The membrane was excised, mounted onto a glass slide with mounting solution and then the total transmigrated cells were counted and images were captured under a light microscope.
2.7. Statistical analysis Values were expressed as mean ± standard deviation of means (SD). Significant differences between groups were analyzed by ANOVA with GraphPad InStat software (GraphPad Software, San Diego, CA) and significance was set at P b 0.05.
3. Results 3.1. Expression of heparanase in microglial cells Expression of mRNA-encoding heparanase by microglial cells was investigated by RT-PCR in the presence or the absence of lipopolysaccharide (LPS) (Fig. 2A). LPS-treated microglial cells expressed inducible NO synthase (iNOS)-mRNA, which implied that the microglia were activated (Fig. 2A). Although heparanase-mRNA was expressed in LPS-treated and non-treated microglial cells, LPS-treated cells upregulated the expression of heparanase mRNA. Heparanase protein was expressed by microglial cells, as revealed by Western blot analysis (Fig. 2B).
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A latent form of heparanase (65 kDa) was expressed by both types of microglia, but LPS-treated microglia expressed the protein at a higher level than the non-treated cells. Lysosomal proteases such as cathepsin D and L cleave the latent form to generate the activated enzyme of 50 kDa [27]. The active form was detected only in the lysate of LPS-treated microglia. The enzymatic activities of heparanase in the microglial lysates were assessed by detecting HS chain degradation of HSPGs with HS specific monoclonal antibody. LPS-treated microglial lysates significantly degraded more HS chains than non-treated lysates (Fig. 2C). Although the active form of heparanase was not detected in non-treated cells by Western blot analysis (Fig. 2B), the cell lysates elicited significant heparanase activity. Since microglial cells express various kinds of proteases, a heparanase inhibitor, SF-4, was used to rule out a possibility that the degraded HS chain was generated by some proteases that degrade Matrigel. SF4, which specifically inhibits endoglucuronidase activity of heparanase through binding to the active site of the enzyme [23], is significantly inhibited the HS chain degradation, indicating that heparanase plays a critical role in the HS chain degradation (Fig. 2C). 3.2. Intracellular localization of heparanase An immunocytochemical study was performed to determine the intracellular localization of heparanase. Fixed microglial cells grown on poly-L-lysine-coated glass coverslips were doublestained with anti-heparanase antibody and FITC labeledphalloidin to visualize the actin cytoskeleton. Heparanase was localized in the cytoplasm and nucleus in microglial cells (Fig. 3). In addition, the enzyme was strongly concentrated at the site of
Fig. 2. Induction of heparanase expression and enzymatic activity in LPS-treated microglial cells. RT-PCR (A) and Western blot (B) analysis on heparanase expression in non-treated (MG) and LPS-treated (MG + LPS) rat microglial cells and LPS-treated microglial cells. Activation of microglial cells was evaluated by detection of inducible NO synthase (iNOS). β-actin was used as an internal standard. (C) Enzymatic activity of heparanase in microglial cells. The cell lysates with or without SF-4 at the indicated concentrations were loaded to a Matrigel-coated ELISA plate and incubated overnight at 37 °C. Enzymatic activity of heparanase was investigated by an ELISA method, as described in Materials and methods. The value of control (data obtained in the absence of cell lysates) indicates the total amount of HS of HSPGs in Matrigel. Each column represents the mean ± standard deviation (SD) of three independent experiments. Asterisks represent a significant statistical difference between control vs MG, MG-LPS and MG-LPS + SF-4. ⁎P b 0.05, ⁎⁎P b 0.001. n.s. indicates no significance.
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Fig. 3. Localization of heparanase in microglial cells. Microglial cells were incubated with or without LPS for 6 h. Cells were fixed and stained with anti-mouse heparanase antibody (red; A and D). Cells were also stained with FITC-phalloidin to visualize actin filaments (green; B and E). Merged signals appear yellow (C and F). Arrowheads indicate cells with heparanase immunoreactivity at the site of membrane ruffles or leading edges. Insertion in Fig. 3D is a high-magnification view of the boxed area. Scale bar = 25 µm.
membrane ruffles and leading edges especially in non-treated microglial cells, both of which were characterized by an enriched actin cytoskeleton (Fig. 3A). On the contrary, in LPS-treated
microglial cells, the localization of heparanase in leading eddges became weaker, and a granular localization of heparanaseimmunoreactivity was more apparent (Fig. 3D).
Fig. 4. Involvement of heparanase in invasion of microglial cells. Microglial cells were plated onto Matrigel-coated inserts in the absence or presence of SF-4 at the indicated concentrations and placed in outer chambers containing DMEM with or without 10% FBS. (A–C) Representative photomicrographs of invaded microglial cells are shown. Transmigrated microglial cells were visualized by hematoxylin staining and counted after 24 h under a light microscope. Scale bar = 50 µm. (D) The mean value of transmigrated cells in 10% FBS condition is represented as 100%. Each column represents the mean ± SD of three independent experiments and statistical analysis of SF-4 0 mM vs others. ⁎P b 0.05, ⁎⁎P b 0.01, #P b 0.001.
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3.3. Involvement of heparanase in migration of microglial cells To assess the contribution of heparanase to the migration activity of microglial cells, they were seeded on Transwell inserts coated with Matrigel containing HSPGs (Fig. 4). Based on the above results, microglia increased the expression of heparanase in response to LPS. However, prolonged incubation with LPS under the serum-free condition decreased the viability of microglia (data not shown), and lysates from microglia cultured in the absence of LPS has an ability to degraded HSPGs (Fig. 2). Therefore, the following migration assay was conducted in the absence of LPS. Serum-free medium was added to the upper chamber and medium containing 10% FBS was added to the lower chamber. Microglial cells on the inserts in the serum-free DMEM transmigrated through pores of the inserts coated with Matrigel to the lower chamber filled with the serum-supplemented DMEM (Fig. 4B). Transmigration was significantly less without the addition of the serum to the lower chamber (Fig. 4A). To evaluate the contribution of heparanase to the transmigration of microglial cells SF-4 was added to the medium in the upper chambers. SF-4 significantly inhibited transmigration through the Matrigel-coated inserts in a concentration-dependent manner (Fig. 4C and D). SF-4 dissolved in culture medium at 1 mM did not influence the viability or phagocytic activity of microglial cells (data not shown). To examine whether degraded HSPGs were generated when the microglial cells transmigrated through Matrigel, immunostaining with antibodies to HS or to degraded HSPGs devoid of the HS chain was performed (Fig. 5). Abundant immunoreactivity of the HS network and faint immunostaining of the degraded HSPGs were observed on the Matrigel-coated inserts in the serum-free condition, where microglial transmigration had scarcely occurred (Fig. 5A and D). No obvious differences
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in the HS staining pattern occurred between the serum-free and serum-supplemented conditions (Fig. 5A, B, C); however, degraded HSPGs were abundant only in the serum-supplemented chamber, where microglial cells actively transmigrated through the inserts (Fig. 5E). When SF-4 was added to the upper medium, the increase in degraded HSPGs was abolished, even in the presence of serum (Fig. 5F). Thus, the transmigration of microglia through the Matrigel depends on the activity of heparanase. 4. Discussion In response to even a minor pathological event in the brain, microglial cells become activated and migrate toward the site of the event [13,14]. Furthermore, during physiological development and pathological processes, precursors of microglia may be supplied from the circulation [16,28–30]. During these events, microglia must migrate across the BM/ECM containing HSPGs that act as a barrier for the migrating cells. Based on the present study, which showed that rat primary cultured microglial cells express heparanase mRNA and proteins, cells may utilize heparanase to degrade the HSPGs in the BM/ECM when they migrate across the barrier. Furthermore, the expression level of the enzyme increased when the cells were activated in response to LPS. Therefore, it is likely that activated microglia increase the expression of heparanase to aid their migration through the BM/ECM. Two molecular species with different molecular weights of 65 and 50 kDa were detected in the microglial lysates with the antibody to heparanase. The 65 kDa species is said to be the latent form of heparanase, which can be cleaved by lysosomal proteases such as cathepsin D or L to the active 50 kDa species [27]. In this study, Western blotting detected the active form in the lysate of LPS-treated microglia only. In fact, the lysate of
Fig. 5. Immunodetection of HS or digested HS chain in Matrigel. Matrigel chamber after transmigration assays were performed by immunostaining with HS (A–C) or degraded-HS (D–F) specific antibody. Representative photomicrographs are shown. The Matrigel was counterstained with hematoxylin to visualize the microglial cells. Note that the degraded HS chain was strongly detected in 10% FBS condition. Scale bar = 50 µm.
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LPS-treated microglia degraded HSPGs more significantly (Fig. 2). This result appears consistent with previous observations, which demonstrated an elevated expression of cathepsin B and L [31] or increased lysosomal enzyme activity [32] in activated microglia. Thus, the presence of the active form of heparanase might be a good marker of activated microglia in addition to the expression of iNOS or TNFα. Although the active form of heparanase could not be detected in the lysate of microglia cultured in the absence of LPS, the lysate significantly degraded HSPGs (Fig. 2C). The immunocytochemical analysis in this study showed that heparanase appeared to be located in ruffled membranes or leading edges of microglia. Furthermore, this observation appears to correspond with a previous study which demonstrated that the cell surface localization of heparanase in macrophages closely correlated to that of microglia in terms of cell lineage [9]. Conversely, the enzyme was more diffusely distributed through the cytoplasm of LPS-treated microglia. Accordingly, in the non-treated microglia heparanase and the lysosomal enzymes responsible for enzyme activation may be separately localized, and this may be the reason why the active form was undetectable in Western blotting experiments. Moreover, during preparation of the lysate from the non-treated microglia, heparanase may have been partially degraded by lysosomal proteases released through homogenization, which resulted in destruction of the lysosomal membrane. Such mechanisms may explain the discrepancy between the results obtained by Western blotting and the activity assay. It has been reported that the 65 kDa-species of heparanase may act both as a precursor of the active form and a cell adhesion molecule [33,34]. Heparanase-mediated cell adhesion to ECM results in integrin-dependent cell spreading or reorganization of the actin cytoskeleton, and this action is independent of its endoglucuronidase activity. The present immunocytochemical results showing the localization of the enzyme at the leading edges and ruffled membranes support the notion. Such localization was more apparent in non-treated microglia, suggesting that the latent form has a significant role in the adhesion and migration of microglia. In contrast, apparent granular localization of heparanase, suggesting lysosomal localization, in activated microglia may be required for its activation by processing and storage, as reported in other cell [35]. Primary cultured microglia isolated from mixed glial cell cultures are very different in terms of macrophage-like functions from matured ramified microglia in the brain, which are thought to be in a resting state. For example, CD68, a marker of phagocytes, is constitutively expressed by primary cultured microglia but not by microglia in the normal mature brain [36,37]. The cultured cells normally display amoeboid rather than ramified morphology, which is characterized by small somata and long thin processes. Such amoeboid morphology may resemble monocyte-like precursors of microglia in the brain. Therefore, the present results may reflect the nature of bone marrowderived microglial precursors rather than the microglia or macrophages in the brain parenchyma. In fact, microglia expressing heparanase were not detected in normal brain parenchyma (data not shown). Moreover, macrophages or activated microglia did
not express heparanase in ischemic lesions in rats with transient occlusion of the middle cerebral artery [12]. Instead, only reactive astrocytes located in the peri-ischemic lesions were identified as heparanase-expressing cells. HSPGs may be localized exclusively in BM around the brain vasculature, but not in the brain parenchyma [38]. Astrocyte end-feet intimately adhere to the BM around the brain blood vessels to induce formation of the BBB. After an ischemic insult, vascular reconstruction occurs and this may require the expression of heparanase in reactive astrocytes. On the other hand, since there are comparatively little amount of HSPGs in brain parenchyma, the parenchymal microglia or macrophages may not necessarily express the enzyme, even when they move their processes or actively migrate [39]. Alternatively, heparanase may be important for microglia or their precursors to invade into the brain parenchyma across the BM around brain vasculature from the circulation. During embryonic development, blood-derived precursor cells related to the monocyte-macrophage lineage invade the brain parenchyma across the BM, and then transform into ramified resting microglia upon maturation of the brain [28–30]. Such migration through the BM may occur even in the normal mature brain, based on reports demonstrating that replacement of ramified microglia with bone marrow-derived cells regularly occurs [17,40,41]. In the physiological process of microglial differentiation, heparanase may act as an adhesion molecule first for adherence to the BM while reorganizing the actin cytoskeletons of the precursor cells. Thereafter, heparanase may start degradation of the HSPGs that form the BM through its endoglucuronidase activity. Acknowledgements We thank Dr. C. Freeman (The Australian National University, Australia) for helpful comments and for providing the antibody to heparanase. This study was partly supported by grants from Ehime University and the Ministry of Education, Culture, Sports, Science, and Technology, Japan. References [1] M. Bernfield, M. Gotte, P.W. Park, O. Reizes, M.L. Fitzgerald, J. Lincecum, M. Zako, Functions of cell surface heparan sulfate proteoglycans, Annu. Rev. Biochem. 68 (1999) 729–777. [2] T.N. Wight, M.G. Kinsella, E.E. Qwarnstrom, The role of proteoglycans in cell adhesion, migration and proliferation, Curr. Opin. Cell Biol. 4 (1992) 793–801. [3] I. Vlodavsky, Y. Friedmann, M. Elkin, H. Aingorn, R. Atzmon, R. IshaiMichaeli, M. Bitan, O. Pappo, T. Peretz, I. Michal, L. Spector, I. Pecker, Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis, Nat. Med. 5 (1999) 793–802. [4] M.D. Hulett, C. Freeman, B.J. Hamdorf, R.T. Baker, M.J. Harris, C.R. Parish, Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis, Nat. Med. 5 (1999) 803–809. [5] I. Vlodavsky, O. Goldshmidt, E. Zcharia, S. Metzger, T. Chajek-Shaul, R. Atzmon, Z. Guatta-Rangini, Y. Friedmann, Molecular properties and involvement of heparanase in cancer progression and normal development, Biochimie 83 (2001) 831–839. [6] O. Goldshmidt, E. Zcharia, R. Abramovitch, S. Metzger, H. Aingorn, Y. Friedmann, V. Schirrmacher, E. Mitrani, I. Vlodavsky, Cell surface expression
H. Takahashi et al. / Biochimica et Biophysica Acta 1780 (2008) 709–715
[7]
[8] [9]
[10]
[11]
[12]
[13] [14] [15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
and secretion of heparanase markedly promote tumor angiogenesis and metastasis, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 10031–10036. N. Ilan, M. Elkin, I. Vlodavsky, Regulation, function and clinical significance of heparanase in cancer metastasis and angiogenesis, Int. J. Biochem. Cell Biol. 38 (2006) 2018–2039. C.R. Parish, C. Freeman, M.D. Hulett, Heparanase: a key enzyme involved in cell invasion, Biochim. Biophys. Acta 1471 (2001) M99–M108. N. Sasaki, N. Higashi, T. Taka, M. Nakajima, T. Irimura, Cell surface localization of heparanase on macrophages regulates degradation of extracellular matrix heparan sulfate, J. Immunol. 172 (2004) 3830–3835. S. Gingis-Velitski, A. Zetser, M.Y. Flugelman, I. Vlodavsky, N. Ilan, Heparanase induces endothelial cell migration via protein kinase B/Akt activation, J. Biol. Chem. 279 (2004) 23536–23541. D. Marchetti, J. Li, R. Shen, Astrocytes contribute to the brain-metastatic specificity of melanoma cells by producing heparanase, Cancer Res. 60 (2000) 4767–4770. H. Takahashi, H. Matsumoto, Y. Kumon, T. Ohnishi, C. Freeman, Y. Imai, J. Tanaka, Expression of heparanase in nestin-positive reactive astrocytes in ischemic lesions of rat brain after transient middle cerebral artery occlusion, Neurosci. Lett. 417 (2007) 250–254. G.W. Kreutzberg, Microglia: a sensor for pathological events in the CNS, Trends Neurosci. 19 (1996) 312–318. K. Nakajima, S. Kohsaka, Microglia: activation and their significance in the central nervous system, J. Biochem. (Tokyo) 130 (2001) 169–175. A.R. Simard, D. Soulet, G. Gowing, J.P. Julien, S. Rivest, Bone marrowderived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease, Neuron 49 (2006) 489–502. M.A. Eglitis, E. Mezey, Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 4080–4085. C.R. Cogle, A.T. Yachnis, E.D. Laywell, D.S. Zander, J.R. Wingard, D.A. Steindler, E.W. Scott, Bone marrow transdifferentiation in brain after transplantation: a retrospective study, Lancet 363 (2004) 1432–1437. J. Tanaka, H. Fujita, S. Matsuda, K. Toku, M. Sakanaka, N. Maeda, Glucocorticoid- and mineralocorticoid receptors in microglial cells: the two receptors mediate differential effects of corticosteroids, Glia 20 (1997) 23–37. A. Yokoyama, L. Yang, S. Itoh, K. Mori, J. Tanaka, Microglia, a potential source of neurons, astrocytes, and oligodendrocytes, Glia 45 (2004) 96–104. H. Matsumoto, Y. Kumon, H. Watanabe, T. Ohnishi, M. Shudou, C. Ii, H. Takahashi, Y. Imai, J. Tanaka, Antibodies to CD11b, CD68, and lectin label neutrophils rather than microglia in traumatic and ischemic brain lesions, J. Neurosci. Res. 85 (2007) 994–1009. J.A. Joyce, C. Freeman, N. Meyer-Morse, C.R. Parish, D. Hanahan, A functional heparan sulfate mimetic implicates both heparanase and heparan sulfate in tumor angiogenesis and invasion in a mouse model of multistage cancer, Oncogene 24 (2005) 4037–4051. X. Xu, R.M. Quiros, J.B. Maxhimer, P. Jiang, R. Marcinek, K.B. Ain, J.L. Platt, J. Shen, P. Gattuso, R.A. Prinz, Inverse correlation between heparan sulfate composition and heparanase-1 gene expression in thyroid papillary carcinomas: a potential role in tumor metastasis, Clin. Cancer Res. 9 (2003) 5968–5979. Y. Nishimura, E. Shitara, H. Adachi, M. Toyoshima, M. Nakajima, Y. Okami, T. Takeuchi, Flexible synthesis and biological activity of uronic
[24]
[25]
[26]
[27] [28] [29] [30] [31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
715
acid-type gem-diamine 1-N-iminosugars: a new family of glycosidase inhibitors, J. Org. Chem. 65 (2000) 2–11. T. Satoh, Y. Nishimura, S. Kondo, T. Takeuchi, A practical synthesis from siastatin B of (3S,4S,5R,6R)-4,5-dihydroxy-6-(trifruoroacetamido)piperidine-3-carboxylic acid having antimetastatic activity in mice, Carbohyd. Res. 286 (1996) 173–178. Y. Kuwabara, A. Yokoyama, L. Yang, K. Toku, K. Mori, I. Takeda, T. Shigekawa, B. Zhang, N. Maeda, M. Sakanaka, J. Tanaka, Two populations of microglial cells isolated from rat primary mixed glial cultures, J. Neurosci. Res. 73 (2003) 22–30. A.P. Wheeler, C.M. Wells, S.D. Smith, F.M. Vega, R.B. Henderson, V.L. Tybulewicz, A.J. Ridley, Rac1 and Rac2 regulate macrophage morphology but are not essential for migration, J. Cell Sci. 119 (2006) 2749–2757. I. Shafat, I. Vlodavsky, N. Ilan, Characterization of mechanisms involved in secretion of active heparanase, J. Biol. Chem. 281 (2006) 23804–23811. E.A. Ling, W.C. Wong, The origin and nature of ramified and amoeboid microglia: a historical review and current concepts, Glia 7 (1993) 9–18. K.D. Barron, The microglial cell. A historical review, J. Neurol. Sci. 134 (1995) 57–68. M.A. Cuadros, J. Navascues, The origin and differentiation of microglial cells during development, Prog Neurobiol 56 (1998) 173–189. R.B. Banati, G. Rothe, G. Valet, G.W. Kreutzberg, Detection of lysosomal cysteine proteinases in microglia: flow cytometric measurement and histochemical localization of cathepsin B and L, Glia 7 (1993) 183–191. J. Tanaka, K. Toku, S. Matsuda, S. Sudo, H. Fujita, M. Sakanaka, N. Maeda, Induction of resting microglia in culture medium devoid of glycine and serine, Glia 24 (1998) 198–215. O. Goldshmidt, E. Zcharia, M. Cohen, H. Aingorn, I. Cohen, L. Nadav, B.Z. Katz, B. Geiger, I. Vlodavsky, Heparanase mediates cell adhesion independent of its enzymatic activity, FASEB J. 17 (2003) 1015–1025. I. Sotnikov, R. Hershkoviz, V. Grabovsky, N. Ilan, L. Cahalon, I. Vlodavsky, R. Alon, O. Lider, Enzymatically quiescent heparanase augments T cell interactions with VCAM-1 and extracellular matrix components under versatile dynamic contexts, J. Immunol. 172 (2004) 5185–5193. O. Goldshmidt, L. Nadav, H. Aingorn, C. Irit, N. Feinstein, N. Ilan, E. Zamir, B. Geiger, I. Vlodavsky, B.Z. Katz, Human heparanase is localized within lysosomes in a stable form, Exp. Cell Res. 281 (2002) 50–62. D.C. Chugani, N.L. Kedersha, L.H. Rome, Vault immunofluorescence in the brain: new insights regarding the origin of microglia, J. Neurosci. 11 (1991) 256–268. J.A. Amat, H. Ishiguro, K. Nakamura, W.T. Norton, Phenotypic diversity and kinetics of proliferating microglia and astrocytes following cortical stab wounds, Glia 16 (1996) 368–382. J. van Horssen, L. Bo, C.M. Vos, I. Virtanen, H.E. de Vries, Basement membrane proteins in multiple sclerosis-associated inflammatory cuffs: potential role in influx and transport of leukocytes, J. Neuropathol. Exp. Neurol. 64 (2005) 722–729. A. Nimmerjahn, F. Kirchhoff, F. Helmchen, Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo, Science 308 (2005) 1314–1318. A. Flugel, M. Bradl, G.W. Kreutzberg, M.B. Graeber, Transformation of donor-derived bone marrow precursors into host microglia during autoimmune CNS inflammation and during the retrograde response to axotomy, J. Neurosci. Res. 66 (2001) 74–82. H. Xu, M. Chen, E.J. Mayer, J.V. Forrester, A.D. Dick, Turnover of resident retinal microglia in the normal adult mouse, Glia 55 (2007) 1189–1198.