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• W2034908043 abstract "Heparanase is an endo-β-d-glucuronidase involved in extracellular matrix remodeling and degradation and implicated in tumor metastasis, angiogenesis, inflammation, and autoimmunity. The enzyme is synthesized as a latent 65-kDa protein and is processed in the lysosomal compartment to an active 58-kDa heterodimer, where it is stored in a stable form. In contrast, its heparan sulfate substrate is localized extracellularly, suggesting the existence of mechanisms that trigger heparanase secretion. Here we show that secretion of the active enzyme is mediated by the protein kinase A and C pathways. Moreover, secretion of active heparanase was observed upon cell stimulation with physiological concentrations of adenosine, ADP, and ATP, as well as by the noncleavable ATP analogue adenosine 5′-O-(thiotriphosphate). Indeed, heparanase secretion was noted upon cell stimulation with a specific P2Y1 receptor agonist and was inhibited by P2Y receptor antagonists. The kinetics of heparanase secretion resembled the secretion of cathepsin D, a lysosomal enzyme, indicating that the secreted heparanase is of lysosomal origin. We suggest that secretion of active heparanase is initiated by extracellular cues activating the protein kinase A and C signaling pathways. The secreted enzyme(s) then facilitate cell invasion associated with cancer metastasis, angiogenesis, and inflammation. Heparanase is an endo-β-d-glucuronidase involved in extracellular matrix remodeling and degradation and implicated in tumor metastasis, angiogenesis, inflammation, and autoimmunity. The enzyme is synthesized as a latent 65-kDa protein and is processed in the lysosomal compartment to an active 58-kDa heterodimer, where it is stored in a stable form. In contrast, its heparan sulfate substrate is localized extracellularly, suggesting the existence of mechanisms that trigger heparanase secretion. Here we show that secretion of the active enzyme is mediated by the protein kinase A and C pathways. Moreover, secretion of active heparanase was observed upon cell stimulation with physiological concentrations of adenosine, ADP, and ATP, as well as by the noncleavable ATP analogue adenosine 5′-O-(thiotriphosphate). Indeed, heparanase secretion was noted upon cell stimulation with a specific P2Y1 receptor agonist and was inhibited by P2Y receptor antagonists. The kinetics of heparanase secretion resembled the secretion of cathepsin D, a lysosomal enzyme, indicating that the secreted heparanase is of lysosomal origin. We suggest that secretion of active heparanase is initiated by extracellular cues activating the protein kinase A and C signaling pathways. The secreted enzyme(s) then facilitate cell invasion associated with cancer metastasis, angiogenesis, and inflammation. Heparanase is an endoglycosidase that specifically cleaves heparan sulfate (HS) 2The abbreviations and trivial name used are: HS, heparan sulfate; HSPG, heparan sulfate proteoglycans; ECM, extracellular matrix; PMA, phorbol 12-myristate 13-acetate; ATPγS, adenosine 5′-O-(thiotriphosphate); 2Me-SADP, 2-methylthioadenosine 5′-diphosphate; PPADS, pyridoxal phosphate-6-azophenyl-2′-4′-disulfonic acid; Bis, bisindolylmaleimide I; MRS 2179, 2′-deoxy-N6-methyladenosine-3′,5′-bisphosphate; PKC, protein kinase C; PKA, protein kinase A. 2The abbreviations and trivial name used are: HS, heparan sulfate; HSPG, heparan sulfate proteoglycans; ECM, extracellular matrix; PMA, phorbol 12-myristate 13-acetate; ATPγS, adenosine 5′-O-(thiotriphosphate); 2Me-SADP, 2-methylthioadenosine 5′-diphosphate; PPADS, pyridoxal phosphate-6-azophenyl-2′-4′-disulfonic acid; Bis, bisindolylmaleimide I; MRS 2179, 2′-deoxy-N6-methyladenosine-3′,5′-bisphosphate; PKC, protein kinase C; PKA, protein kinase A. side chains of heparan sulfate proteoglycans (HSPG) (1Vlodavsky I. Friedmann Y. J. Clin. Invest. 2001; 108: 341-347Crossref PubMed Scopus (545) Google Scholar, 2Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: 99-108PubMed Google Scholar). HSPG consist of a protein core to which HS side chains are covalently attached. These complex macromolecules are highly abundant in the extracellular matrix (ECM) and are thought to play an important structural role, contributing to ECM integrity and insolubility (3Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1674) Google Scholar). In addition, HS side chains can bind a variety of biological mediators, such as growth factors, cytokines, and chemokines, thus functioning as a readily available reservoir that can be liberated upon local or systemic cues. Moreover, HSPG on the cell surface participate in signal transduction cascades by potentiating the interaction between certain growth factors and their receptors (4David G. FASEB J. 1993; 7: 1023-1030Crossref PubMed Scopus (373) Google Scholar, 5Ornitz D.M. BioEssays. 2000; 22: 108-112Crossref PubMed Scopus (623) Google Scholar, 6Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2081) Google Scholar). HS-degrading activity is thus expected to affect several fundamental aspects of cell behavior under normal and pathological settings and should therefore be kept tightly regulated. Traditionally, heparanase activity was implicated in cellular invasion associated with angiogenesis, inflammation, and cancer metastasis (7Vlodavsky I. Fuks Z. Bar-Ner M. Ariav Y. Schirrmacher V. Cancer Res. 1983; 43: 2704-2711PubMed Google Scholar, 8Nakajima M. Irimura T. DiFerrante D. DiFerrante N. Nicolson G.L. Science. 1983; 220: 611-613Crossref PubMed Scopus (305) Google Scholar, 9Nakajima M. Irimura T. Di Ferrante N. Nicolson G.L. J. Biol. Chem. 1984; 259: 2283-2290Abstract Full Text PDF PubMed Google Scholar, 10Naparstek Y. Cohen I.R. Fuks Z. Vlodavsky I. Nature. 1984; 310: 241-244Crossref PubMed Scopus (253) Google Scholar, 11Matzner Y. Bar-Ner M. Yahalom J. Ishai-Michaeli R. Fuks Z. Vlodavsky I. J. Clin. Invest. 1985; 76: 1306-1313Crossref PubMed Scopus (176) Google Scholar). This notion gained further support by employing small interfering RNA and ribozyme technologies, clearly depicting heparanase-mediated HS cleavage and ECM remodeling as critical requisites for metastatic spread (12Edovitsky E. Elkin M. Zcharia E. Peretz T. Vlodavsky I. J. Natl. Cancer Inst. 2004; 96: 1219-1230Crossref PubMed Scopus (227) Google Scholar). Since the cloning of the heparanase gene and the availability of specific molecular probes, heparanase up-regulation was documented in an increasing number of primary human tumors, correlating with enhanced local and distant metastasis, increased microvessel density, and reduced postoperative survival of cancer patients. Collectively, these studies provide compelling evidence for the clinical relevance of the enzyme, making it an attractive target for the development of anti-cancer drugs (1Vlodavsky I. Friedmann Y. J. Clin. Invest. 2001; 108: 341-347Crossref PubMed Scopus (545) Google Scholar, 2Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: 99-108PubMed Google Scholar, 13Dempsey L.A. Brunn G.J. Platt J.L. Trends Biochem. Sci. 2000; 25: 349-351Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Similar to several other classes of enzymes, heparanase is first synthesized as a latent enzyme that appears as a ∼65-kDa protein when analyzed by SDS-PAGE. The 65-kDa latent enzyme is directed to the ER by a C terminal 35-amino acid signal peptide and is readily detected in the culture medium of transfected cells (14Gingis-Velitski S. Zetser A. Kaplan V. Ben-Zaken O. Cohen E. Levy-Adam F. Bashenko Y. Flugelman M.Y. Vlodavsky I. Ilan N. J. Biol. Chem. 2004; 279: 44084-44092Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The latent heparanase form does not accumulate extracellularly, however, due to an efficient cellular uptake (14Gingis-Velitski S. Zetser A. Kaplan V. Ben-Zaken O. Cohen E. Levy-Adam F. Bashenko Y. Flugelman M.Y. Vlodavsky I. Ilan N. J. Biol. Chem. 2004; 279: 44084-44092Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 15Vreys V. Delande N. Zhang Z. Coomans C. Roebroek A. Durr J. David G. J. Biol. Chem. 2005; 280: 33141-33148Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), followed by intracellular proteolytic processing (15Vreys V. Delande N. Zhang Z. Coomans C. Roebroek A. Durr J. David G. J. Biol. Chem. 2005; 280: 33141-33148Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 16Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar), yielding an 8-kDa polypeptide at the N terminus and a 50-kDa polypeptide at the C terminus that heterodimerize to form the active heparanase enzyme (17Fairbanks M.B. Mildner A.M. Leone J.W. Cavey G.S. Mathews W.R. Drong R.F. Slightom J.L. Bienkowski M.J. Smith C.W. Bannow C.A. Heinrikson R.L. J. Biol. Chem. 1999; 274: 29587-29590Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 18McKenzie E. Young K. Hircock M. Bennett J. Bhaman M. Felix R. Turner P. Stamps A. McMillan D. Saville G. Ng S. Mason S. Snell D. Schofield D. Gong H. Townsend R. Gallagher J. Page M. Parekh R. Stubberfield C. Biochem. J. 2003; 373: 423-435Crossref PubMed Scopus (104) Google Scholar, 19Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar). Likewise, heparanase was noted to reside primarily intracellularly within endocytic vesicles identified as endosomes and lysosomes (20Nadav L. Eldor A. Yacoby-Zeevi O. Zamir E. Pecker I. Ilan N. Geiger B. Vlodavsky I. Katz B.Z. J. Cell Sci. 2002; 115: 2179-2187Crossref PubMed Google Scholar, 21Goldshmidt O. Nadav L. Aingorn H. Irit C. Feinstein N. Ilan N. Zamir E. Geiger B. Vlodavsky I. Katz B.Z. Exp. Cell Res. 2002; 281: 50-62Crossref PubMed Scopus (73) Google Scholar, 22Cohen E. Atzmon R. Vlodavsky I. Ilan N. FEBS Lett. 2005; 579: 2334-2338Crossref PubMed Scopus (37) Google Scholar). Applying a polyclonal antibody (number 733) that preferentially recognizes the 50-kDa heparanase subunit versus the 65-kDa latent enzyme, we have demonstrated that the 50-kDa active heparanase subunit similarly resides in endocytic vesicles, assuming a perinuclear localization (16Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar). More recently, we have demonstrated heparanase processing by endosomal/lysosomal preparation (22Cohen E. Atzmon R. Vlodavsky I. Ilan N. FEBS Lett. 2005; 579: 2334-2338Crossref PubMed Scopus (37) Google Scholar), identified the lysosome as the heparanase-processing organelle (16Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar), and identified cathepsin family members, mainly cathepsin D and L, as heparanase-activating proteases (23Abboud-Jarrous G. Rangini-Guetta Z. Aingorn H. Atzmon R. Elgavish S. Peretz T. Vlodavsky I. J. Biol. Chem. 2005; 280: 13568-13575Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Accumulation of heparanase in endocytic vesicles for a relatively long period of time (16Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar, 21Goldshmidt O. Nadav L. Aingorn H. Irit C. Feinstein N. Ilan N. Zamir E. Geiger B. Vlodavsky I. Katz B.Z. Exp. Cell Res. 2002; 281: 50-62Crossref PubMed Scopus (73) Google Scholar) led us to hypothesize that this compartment may serve as an intracellular enzyme pool that can get secreted in response to a proper stimulus, ensuring a tightly regulated extracellular enzymatic function. We investigated this hypothesis by examining heparanase secretion and activity in the cell conditioned medium in response to exogenous stimuli. Here, we provide evidence that stimulation of tumor-derived cells with phorbol 12-myristate 13-acetate (PMA) and forskolin markedly enhances the secretion of active heparanase in a time- and dose-responsive manner. We further demonstrate that physiological concentrations of ATP and ADP similarly enhance the secretion of active heparanase and discuss the significance and possible implications of these findings. Antibodies and Reagents—Antibody 1453 was raised in rabbits against the entire 65-kDa latent heparanase isolated from the conditioned medium of heparanase-transfected 293 cells. This antibody was affinity-purified on immobilized, bacterially expressed 50-kDa heparanase-glutathione S-transferase fusion protein (16Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar). Monoclonal anti-heparanase antibody was kindly provided by Dr. Hua-Quan Miao (ImClone Systems Inc., New York, NY). Monoclonal anti-cathepsin D antibody, forskolin, PMA, ATP, ATPγS, ADP, adenosine, 2-methylthioadenosine 5′-diphosphate (2Me-SADP), pyridoxal phosphate-6-azophenyl-2′-4′-disulfonic acid (PPADS), and 2′-deoxy-N6-methyladenosine-3′,5′-bisphosphate (MRS 2179) were purchased from Sigma. The inhibitory compounds H89, U-73122, and bisindolylmaleimide I (Bis) were purchased from Calbiochem. Inhibitors were dissolved in Me2SO as stock solutions, and Me2SO was added to cell cultures as control, without a noticeable effect. Cell Culture and Transfection—Human MDA-MB-231, MDA-MB-435, and MDA-MB-468 breast carcinoma and HCT116 and HT29 colon carcinoma cells were purchased from the ATCC. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics. For stable transfection, subconfluent MDA-468, MDA-435, MDA-231, and HT29 cells were transfected with the pSecTag2 vector (Invitrogen) containing the full-length heparanase cDNA, using the FuGENE 6 reagent, according to the manufacturer's instructions (Roche Applied Science). The pSecTag2 vector is designed for efficient protein secretion driven by the IgGκ signal peptide and contains c-Myc and His tags at the protein C terminus. Transfection proceeded for 48 h, followed by selection with Zeocin (Invitrogen) for 2 weeks. Stable transfectant pools were further expanded and analyzed. Heparanase Secretion and Immunoblotting—Cells were grown to confluence, followed by incubation for 20 h in serum-free medium. Fresh serum-free medium was added, and the cells were incubated without or with the indicated reagent for an additional 20 h, unless stated otherwise. Conditioned medium was collected and applied onto 35S-labeled ECM-coated dishes to evaluate heparanase enzymatic activity (see below) or preabsorbed with concavalin A-Sepharose beads to concentrate the samples and reduce nonspecific reactivity, followed by SDS-PAGE under reducing conditions using 10% gels. After electrophoresis, proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad) and probed with the appropriate antibody followed by horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) and an enhanced chemiluminescent substrate (Pierce), as described (14Gingis-Velitski S. Zetser A. Kaplan V. Ben-Zaken O. Cohen E. Levy-Adam F. Bashenko Y. Flugelman M.Y. Vlodavsky I. Ilan N. J. Biol. Chem. 2004; 279: 44084-44092Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 16Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar, 24Levy-Adam F. Abboud-Jarrous G. Guerrini M. Beccati D. Vlodavsky I. Ilan N. J. Biol. Chem. 2005; 280: 20457-20466Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Immunocytochemistry—Heparanase transfected HT29 colon carcinoma cells were left untreated or incubated with ATP (10 μm) for 2 h. Indirect immunofluorescence staining was then performed essentially as described (16Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar, 25Zetser A. Bashenko Y. Miao H.-Q. Vlodavsky I. Ilan N. Cancer Res. 2003; 63: 7733-7741PubMed Google Scholar). Briefly, cells were fixed with cold methanol for 10 min, washed with phosphate-buffered saline, and subsequently incubated in phosphate-buffered saline containing 10% normal goat serum for 1 h at room temperature, followed by a 2-h incubation with monoclonal anti-heparanase antibodies. Cells were then extensively washed with phosphate-buffered saline and incubated with Cy2-conjugated secondary antibody (Jackson ImmunoResearch) for 1 h, washed, and mounted (Vectashield, Vector, Burlingame, CA). Nuclei were counterstained with propidium iodide (Vector), and staining was visualized by confocal microscopy. Heparanase Activity—Preparation of ECM-coated dishes and determination of heparanase activity were performed as described in detail elsewhere (19Levy-Adam F. Miao H.Q. Heinrikson R.L. Vlodavsky I. Ilan N. Biochem. Biophys. Res. Commun. 2003; 308: 885-891Crossref PubMed Scopus (102) Google Scholar, 26Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Bitan M. Pappo O. Peretz T. Michal I. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (725) Google Scholar). Briefly, cells (5 × 105 to 2 × 106) were plated on 35-mm dishes coated with 35S-labeled ECM. Cells were allowed to adhere for 30 min, medium was replaced with serum-free Dulbecco's modified Eagle's medium, and the cells were incubated (4-20 h, 37 °C) in the absence or presence of the indicated reagents. Alternatively, conditioned medium was applied directly onto 35S-ECM-coated dishes. The incubation medium containing sulfate-labeled degradation fragments was subjected to gel filtration on a Sepharose CL-6B column. Fractions (0.2 ml) were eluted with phosphate-buffered saline, and their radioactivity was counted in a β-scintillation counter. Degradation fragments of HS side chains were eluted at 0.5 < Kav < 0.8 (peak II, fractions 15-30). Nearly intact HSPG were eluted just after the Vo (Kav < 0.2, peak I, fractions 3-15). Cell Migration—Cells were incubated in serum-free medium for 20 h and applied on top of fibronectin-coated cell inserts (8 μm; Costar). Growth medium containing 10% fetal calf serum was added to the lower compartment, and the cells were allowed to migrate for 4 h. Inserts were subsequently fixed with 4% paraformaldehyde, and cells remaining on the upper side of the filter were removed by a cotton swab. Migrating cells were stained with crystal violate, visualized, and counted under a light microscope. PMA and Forskolin Induce Secretion of Active Heparanase—Heparanase activity is strongly implicated in biological processes that require ECM remodeling, such as cancer metastasis, inflammation, and angiogenesis (1Vlodavsky I. Friedmann Y. J. Clin. Invest. 2001; 108: 341-347Crossref PubMed Scopus (545) Google Scholar, 2Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: 99-108PubMed Google Scholar, 13Dempsey L.A. Brunn G.J. Platt J.L. Trends Biochem. Sci. 2000; 25: 349-351Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Interestingly, however, heparanase was noted to reside primarily intracellularly within endocytic vesicles identified as endosomes and lysosomes (16Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar, 20Nadav L. Eldor A. Yacoby-Zeevi O. Zamir E. Pecker I. Ilan N. Geiger B. Vlodavsky I. Katz B.Z. J. Cell Sci. 2002; 115: 2179-2187Crossref PubMed Google Scholar, 21Goldshmidt O. Nadav L. Aingorn H. Irit C. Feinstein N. Ilan N. Zamir E. Geiger B. Vlodavsky I. Katz B.Z. Exp. Cell Res. 2002; 281: 50-62Crossref PubMed Scopus (73) Google Scholar). Thus, heparanase secretion from intracellular pools may be required in order to exert its enzymatic function extracellularly. We examined this possibility by exposing cells overexpressing heparanase to PMA, followed by immunoblot analysis of the culture medium. As demonstrated in Fig. 1, treatment of MDA-468 (Fig. 1A), MDA-435 (Fig. 1B), HT29 (Fig. 1C), and 293 cells (Fig. 1D) with PMA resulted in a marked increase of the 50-kDa active heparanase subunit in the culture medium. In contrast, the latent 65-kDa protein was readily detected in the culture medium of control untreated cells (Con) due to its secreted nature (1Vlodavsky I. Friedmann Y. J. Clin. Invest. 2001; 108: 341-347Crossref PubMed Scopus (545) Google Scholar), and its levels were not significantly changed upon PMA treatment. In order to confirm the immunoblotting results, conditioned medium from control and PMA-treated HT29 cells was applied onto 35S-labeled ECM, and heparanase enzymatic activity was evaluated. Heparanase activity was not detected in medium conditioned by nontransfected HT29 cells (data not shown) but was clearly evident in medium conditioned by heparanase-transfected cells (Fig. 1E, Con). Heparanase activity was markedly increased in the conditioned medium of heparanase-transfected HT29 cells in response to treatment with PMA (Fig. 1E, PMA), in agreement with the elevated levels of the 50-kDa heparanase subunit detected by immunoblotting (Fig. 1C). Treatment of heparanase-transfected MDA-468, MDA-435, and HT29 cells with forskolin did not result in a significant increase of heparanase secretion (Fig. 1, A-C) and activity (data not shown). In contrast, treatment of heparanase-transfected 293 cells with forskolin stimulated a significant increase in secretion of the 50-kDa heparanase subunit (Fig. 1D), correlating with enhanced heparanase activity in the cell culture medium (Fig. 1F). Since PMA is a strong PKC inducer, we next examined the ability of PKC inhibitors to block heparanase secretion induced by PMA. To this end, heparanase-transfected HT29 (Fig. 1G, upper panel) and MDA-435 (Fig. 1G, lower panel) cells were left untreated (-) or stimulated with PMA in the absence or presence of PKC (Bis) or PKA (H89) inhibitors. Heparanase secretion was examined by immunoblotting. PMA treatment elicited a marked increase in secretion of the 50-kDa heparanase subunit, an increase that was practically blocked (Fig. 1G, top) or markedly reduced (Fig. 1G, bottom) in the presence of the PKC inhibitor Bis. In contrast, the PKA inhibitor H89 only slightly reduced the effect of PMA, indicating, as expected, activation of PKC rather than of PKA. The inverse situation was noted upon treatment of heparanase-transfected 293 cells with forskolin. In these cells, forskolin was found to effectively induce secretion of active heparanase (Fig. 1D), and this effect was significantly inhibited by H89 but not by Bis (not shown). Next, we examined the kinetics of heparanase secretion elicited by PMA and forskolin (Fig. 2). Heparanase-transfected MDA-435 cells were incubated with PMA for the time indicated, conditioned medium was collected, and heparanase secretion was examined by immunoblotting. The 50-kDa heparanase subunit was first detected in the culture medium 1 h following PMA stimulation, peaked at 2 h, and gradually decreased (Fig. 2A, top). A similar kinetic was noted upon treatment of heparanase-transfected 293 cells with forskolin (Fig. 2B, top). The decline in extracellular heparanase levels at later time points following PMA or forskolin treatment is most probably due to HS-mediated heparanase uptake (14Gingis-Velitski S. Zetser A. Kaplan V. Ben-Zaken O. Cohen E. Levy-Adam F. Bashenko Y. Flugelman M.Y. Vlodavsky I. Ilan N. J. Biol. Chem. 2004; 279: 44084-44092Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Since the active 50-kDa heparanase subunit was mainly detected in endocytic vesicles (16Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar), we rationalized that the secreted heparanase found in the culture medium following treatment with PMA and forskolin originated from such an intracellular pool. In order to support this notion, we compared the secretion kinetics of the lysosomal protein cathepsin D with that of the 50-kDa heparanase subunit. Notably, secretion of the low molecular weight forms of cathepsin D, typically processed and residing in lysosomes, was not only induced by PMA and forskolin but also resembled the kinetics of heparanase secretion (Fig. 2, lower panels), suggesting that both proteins indeed originate from similar cellular compartments. Nucleotides Induce Secretion of Enzymatically Active Heparanase—The ability of PMA and forskolin to induce the secretion of enzymatically active heparanase as well as of a typical lysosomal enzyme, such as cathepsin D, supports the notion that heparanase is stored in the lysosomal compartment in a stable form (16Zetser A. Levy-Adam F. Kaplan V. Gingis-Velitski S. Bashenko Y. Schubert S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Cell Sci. 2004; 117: 2249-2258Crossref PubMed Scopus (190) Google Scholar, 21Goldshmidt O. Nadav L. Aingorn H. Irit C. Feinstein N. Ilan N. Zamir E. Geiger B. Vlodavsky I. Katz B.Z. Exp. Cell Res. 2002; 281: 50-62Crossref PubMed Scopus (73) Google Scholar) and is secreted in response to local or systemic cues, thus maintaining its extracellular availability tightly regulated. Since both PMA and forskolin are not considered physiological, we sought inducers more relevant to biological systems in which heparanase activity is implicated, mainly cancer metastasis and inflammation. The intracellular role of ATP is well recognized as a key energy source utilized for cellular metabolism. In addition, purines and pyrimidines, mainly ATP, ADP, UTP, and adenosine, have the ability to function extracellularly and to initiate signal transduction cascades mediated by a family of purinergic receptor that appears to play important roles in development, differentiation, and cell proliferation (27Gordon J.L. Biochem. J. 1986; 233: 309-319Crossref PubMed Scopus (1404) Google Scholar, 28Abbracchio M.P. Burnstock G. Jpn. J. Pharmacol. 1998; 78: 113-145Crossref PubMed Scopus (385) Google Scholar). The P1 and P2Y subgroups of the purinergic receptors family are G-protein-coupled receptors that activate phospholipase C and modulate cAMP levels, leading to PKC- and PKA-mediated signal transduction (28Abbracchio M.P. Burnstock G. Jpn. J. Pharmacol. 1998; 78: 113-145Crossref PubMed Scopus (385) Google Scholar). Since PKA and PKC activation was noted to enhance heparanase secretion (Figs. 1 and 2), we examined the ability of nucleotides to elicit a similar response. All cell types included in this study responded to nucleotides by elevating the levels of active heparanase in the cell conditioned medium, yet each cell type exclusively responded to different nucleotides (Fig. 3). Thus, whereas 293 cells preferentially responded to ATP and ADP (Fig. 3A), MDA-435 cells responded to adenosine (ADO; Fig, 3B), and MDA-231 cells responded primarily to ADP (Fig. 3C), possibly reflecting different receptor profiles expressed by each cell line. Importantly, elevation of the 50-kDa heparanase subunit in the culture medium was accompanied by increased heparanase activity in the nucleotide-treated 293, MDA-435, and MDA-231 cells (Fig. 3, D-F, respectively). Next, we examined the effect of ATP on heparanase secretion as a function of concentration and incubation time. HEK293 cells stably overexpressing heparanase were left untreated (0) or stimulated with ATP at the indicated concentrations. Heparanase secretion into the culture medium was evaluated by immunoblotting. As shown in Fig. 4A, lowering the ATP concentration markedly enhanced secretion of the 50-kDa heparanase protein. A concentration of 1 μm was applied in subsequent experiments. Interestingly, low ATP concentrations were also more effective in stimulating the secretion of cathepsin D (Fig. 4A, bottom), further supporting endocytic vesicles and the lysosomal compartment as the primary source of these enzymes. Next, we examine the kinetics of heparanase secretion in response to ATP. To this end, heparanase-transfected 293 cells were left untreated or stimulated with 1 μm ATP for the indicated time, and heparanase secretion was evaluated. Whereas the 65-kDa latent heparanase was readily secreted by control, untreated cells (Fig. 4B, left, Con), secretion of the 50-kDa protein was restricted to cells treated with ATP (Fig. 4B, right, ATP). The 50-kDa heparanase subunit was first detected 20 min following ATP addition, peaked at 1 h, and declined thereafter to undetectable levels (16 h), possibly due to efficient cellular uptake of the secreted protein (14Gingis-Velitski S. Zetser A. Kaplan V. Ben-Zaken O. Cohen E. Levy-Adam F. Bashenko Y. Flugelman M.Y. Vlodavsky I. Ilan N. J. Biol. Chem. 2004; 279: 44084-44092Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Importantly, secretion of the 50-kDa subunit precedes the secretion of the 65-kDa latent heparanase. These differences in secretion kinetics rule out the possibility that the 50-kDa subunit found in the cell culture medium is the result of extracellular processing of the 65-kDa latent heparanase, thus strongly implying that the 50-kDa form originated from an intracellular pool.FIGURE 4Dose- and time-responsive heparanase secretion upon ATP stimulation. A" @default.
• W2034908043 created "2016-06-24" @default.
• W2034908043 creator A5007258222 @default.
• W2034908043 creator A5037824025 @default.
• W2034908043 creator A5051815910 @default.
• W2034908043 date "2006-08-01" @default.
• W2034908043 modified "2023-10-03" @default.
• W2034908043 title "Characterization of Mechanisms Involved in Secretion of Active Heparanase" @default.
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