Characterization of Mechanisms Involved in Secretion of Active Heparanase

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. 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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. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google by V. Delande N. Zhang Z. Coomans C. Roebroek A. Durr J. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Sci. 2004; PubMed Scopus Google an the and a the C that to form the active heparanase enzyme J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. M. J. M. R. P. A. D. G. S. S. D. D. R. J. M. R. C. Biochem. J. PubMed Scopus Google Scholar, F. Vlodavsky I. Ilan N. Biochem. Biophys. Res. PubMed Scopus Google Scholar). heparanase was noted to as and L. A. O. E. I. Ilan N. Vlodavsky I. J. Sci. PubMed Google Scholar, O. L. C. N. Ilan N. E. Vlodavsky I. Res. PubMed Scopus Google Scholar, E. R. Vlodavsky I. Ilan N. PubMed Scopus Google Scholar). a that the heparanase the 65-kDa latent enzyme, we that the active heparanase in a A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Sci. 2004; PubMed Scopus Google Scholar). we heparanase by E. R. Vlodavsky I. Ilan N. PubMed Scopus Google the as the A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Sci. 2004; PubMed Scopus Google and cathepsin cathepsin and as G. Z. R. S. Peretz T. Vlodavsky I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of heparanase in for a of A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Sci. 2004; PubMed Scopus Google Scholar, O. L. C. N. Ilan N. E. Vlodavsky I. Res. PubMed Scopus Google to that compartment as an enzyme that can secreted in to a a tightly extracellular We by heparanase secretion and activity in the cell medium in to we provide evidence that stimulation of cells with phorbol 12-myristate and the secretion of active heparanase in a and We further that physiological concentrations of ATP and the secretion of active heparanase and the and of these and was in the 65-kDa latent heparanase the medium of This was on protein A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Sci. 2004; PubMed Scopus Google Scholar). was by PMA, ATP, ATPγS, ADP, adenosine, 2-methylthioadenosine pyridoxal phosphate-6-azophenyl-2′-4′-disulfonic acid and The and bisindolylmaleimide in as and was to cell as a and and and and cells the in medium with and stable and cells transfected with the the heparanase the to the The is for efficient protein secretion by the signal peptide and and the protein C for by with for further and Heparanase and to by for in medium was and the cells or with the for an medium was and to heparanase activity or with to the and by under to and with the by and an enhanced substrate as (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, A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Sci. 2004; PubMed Scopus Google Scholar, F. G. M. D. Vlodavsky I. Ilan N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). transfected cells or with ATP for was then as A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Sci. 2004; PubMed Scopus Google Scholar, A. Bashenko Y. Vlodavsky I. Ilan N. Cancer Res. Google Scholar). cells with for with and in normal for by a with then with and with for and with and was by Heparanase of and of heparanase activity as in F. Vlodavsky I. Ilan N. Biochem. Biophys. Res. PubMed Scopus Google Scholar, I. Friedmann Y. Elkin M. R. Ishai-Michaeli R. M. O. Peretz T. I. L. I. PubMed Scopus Google Scholar). cells to on with to for medium was with and the cells in the or of the medium was The medium degradation was to on a with and their was in a of HS side chains HSPG the in medium for and on of cell medium was to the and the cells to for with and cells on the side of the by a cells with and under a and of activity is implicated in biological that ECM such as cancer metastasis, inflammation, and (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). however, heparanase was noted to as and A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Sci. 2004; PubMed Scopus Google Scholar, L. A. O. E. I. Ilan N. Vlodavsky I. J. Sci. PubMed Google Scholar, O. L. C. N. Ilan N. E. Vlodavsky I. Res. PubMed Scopus Google Scholar). heparanase secretion be in to its We by cells heparanase to PMA, by of the culture in of and cells with in a of the active heparanase in the culture In contrast, the latent 65-kDa protein was readily detected in the culture medium of cells due to its secreted (1Vlodavsky I. Friedmann Y. J. Clin. Invest. 2001; 108: 341-347Crossref PubMed Scopus (545) Google and its not upon In to the medium and cells was ECM, and heparanase activity was Heparanase activity was not detected in medium by cells not was clearly in medium by cells Heparanase activity was increased in the medium of cells in to with in with the of the heparanase detected by of and cells with not in a of heparanase secretion and activity not In contrast, of cells with a in secretion of the heparanase correlating with enhanced heparanase activity in the cell culture medium Since is a we the of to heparanase secretion by and cells or with in the or of or Heparanase secretion was by a in secretion of the heparanase an that was or reduced in the of the In contrast, the reduced the of PMA, as of of The was noted upon of cells with In these was to secretion of active heparanase and was inhibited by not by we the kinetics of heparanase secretion by and cells with for the medium was and heparanase secretion was by The heparanase was first detected in the culture medium and A was noted upon of cells with The in extracellular heparanase or is due to 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 heparanase was detected in A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Sci. 2004; PubMed Scopus Google we that the secreted heparanase in the culture medium with and such an In to support we the secretion kinetics of the lysosomal protein cathepsin with that of the heparanase secretion of the molecular of cathepsin D, processed and in was not by and resembled the kinetics of heparanase secretion suggesting that cellular of of and to the secretion of active heparanase as well as of a lysosomal enzyme, such as cathepsin D, the notion that heparanase is stored in the lysosomal compartment in a stable form A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. Vlodavsky I. Ilan N. J. 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Since and was noted to heparanase secretion and we the of to a cell in to by the of active heparanase in the cell cell to cells to ATP and cells to adenosine and cells to receptor by cell of the heparanase in the culture medium was by increased heparanase activity in the and cells we the of ATP on heparanase secretion as a of and cells heparanase or with ATP the Heparanase secretion the culture medium was by in the ATP enhanced secretion of the heparanase A of was in ATP concentrations in the secretion of cathepsin further and the lysosomal compartment as the primary of these we the kinetics of heparanase secretion in to cells or with ATP for the and heparanase secretion was the 65-kDa latent heparanase was readily secreted by cells secretion of the protein was to cells with ATP The heparanase was first detected ATP addition, and to 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). secretion of the the secretion of the 65-kDa latent These in secretion kinetics the that the in the cell culture medium is the of extracellular of the 65-kDa latent thus that the form an and heparanase secretion upon ATP cells for medium was then and cells or with the of ATP in the of medium was and to with and cells for medium was and cells with ATP for the in the of The cell medium was analyzed by with cells for medium was and the cells as or with ATP for then and with and and by the and upon ATP we heparanase in and cells by of In heparanase was noted to in a in with studies A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Sci. 2004; PubMed Scopus Google Scholar, O. L. C. N. Ilan N. E. Vlodavsky I. Res. PubMed Scopus Google Scholar, O. Zcharia E. Z. R. I. I. E. Vlodavsky I. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, upon ATP a and in to the cell In addition, upon ATP due to a readily Heparanase by ATP and the P2Y1 and that heparanase secretion by and is mediated by the and signaling we a signaling is by of cells with ATP secretion of the protein in with and and with a of cathepsin secretion secretion of heparanase and cathepsin was by with and and the C of these cells with heparanase secretion that was by the Bis, by was These further by heparanase activity in the cell heparanase activity was increased upon reduced heparanase activity to the of and in an in heparanase activity correlating with the ATPγS, a ATP analogue that be by cells as an was as as ATP in heparanase secretion not the notion that heparanase secretion by ATP, and by other is a In to cell surface receptors that heparanase we specific P2Y receptor and antagonists. of cells with a P2Y1 agonist J. PubMed Scopus Google a of heparanase secretion in with the of ATP and on heparanase secretion and Moreover, the of was by PPADS, an of and and by an specific for P2Y1 clearly the P2Y1 receptors and as of heparanase secretion. Since heparanase activity with cellular we of extracellular heparanase activity upon stimulation cell ATP and enhanced and cell a cellular to the molecular and by HSPG are thought to play in biological cell inflammation, cancer metastasis, and (3Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1674) Google Scholar, G. FASEB J. 1993; 7: 1023-1030Crossref PubMed Scopus (373) Google Scholar, M. M. O. J. M. Annu. Rev. Biochem. PubMed Scopus Google Scholar, Esko J.D. Rev. PubMed Scopus Google Scholar). HSPG their several and The of and by HS I. Chem. PubMed Scopus Google Scholar, Biol. 2004; PubMed Scopus Google and the of heparanase to these active (1Vlodavsky I. Friedmann Y. J. Clin. Invest. 2001; 108: 341-347Crossref PubMed Scopus (545) Google Scholar, I. J. R. R. Ishai-Michaeli R. J. Klagsbrun M. Natl. Sci. U. S. A. PubMed Scopus Google that heparanase extracellular activity be kept tightly regulated. that heparanase are are expected to several Heparanase noted in an increasing number of human clearly gene as a Y. Elkin M. Peretz T. Vlodavsky I. 25: PubMed Scopus Google Scholar, T. M. S. S. T. T. L.A. M. M. R. PubMed Scopus Google Scholar, S. T. Hulett M.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the heparanase and extracellular implicated as a (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, A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. Vlodavsky I. Ilan N. J. Sci. 2004; PubMed Scopus Google Scholar, G. Z. R. S. Peretz T. Vlodavsky I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Heparanase is first as a enzyme an signal peptide that the protein to the ER and protein secretion. Indeed, the 65-kDa latent heparanase protein 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, 15Vreys V. Delande N. Zhang Z. Coomans C. Roebroek A. Durr J. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the protein does not accumulate to due to and efficient cellular uptake mediated by cell surface HSPG, the and the receptors (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. 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Full Text Full Text PDF PubMed Scopus Google by cell surface HS and was by the of not in with of HSPG as of 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). the availability of extracellular heparanase is tightly by that secretion of the enzyme and a that of the secreted enzyme by efficient cellular a of active heparanase in the extracellular The of the heparanase can be by extracellular of the 65-kDa latent however, does not to be the of the the of the 65-kDa in the kinetics studies for ATP Moreover, the of cathepsin to cell on the of the protein in the medium not in with the notion that heparanase V. Delande N. Zhang Z. Coomans C. Roebroek A. Durr J. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Levy-Adam F. Kaplan V. S. Bashenko Y. S. Flugelman M.Y. 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