Heparanase Induces Endothelial Cell Migration via Protein Kinase B/Akt Activation

Abstract

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intra-chain sites. Blood-borne neutrophils, macrophages, mast cells, and platelets exhibit heparanase activity that is thought to be stored in specific granules. The degranulated heparanase is implicated in extravasation of metastatic tumor cells and activated cells of the immune system. Degranulation and heparanase release in response to an inflammatory stimulus or platelet activation would facilitate cellular extravasation directly, by altering the composition and structural integrity of the extracellular matrix, or indirectly, by releasing HS-bound proinflammatory cytokines and chemokines. We hypothesized that in addition to such indirect effect, the released heparanase may also locally affect and activate neighboring cells such as endothelial cells. Here, we provide evidence that addition of the 65-kDa latent heparanase to endothelial cells enhances Akt signaling. Heparanase-mediated Akt phosphorylation was independent of its enzymatic activity or the presence of cell membrane HS proteoglycans and was augmented by heparin. Moreover, addition of heparanase stimulated phosphatidylinositol 3-kinase-dependent endothelial cell migration and invasion. These results suggest, for the first time, that heparanase activates endothelial cells and elicits angiogenic responses directly. This effect appears to be mediated by as yet unidentified heparanase receptor. Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intra-chain sites. Blood-borne neutrophils, macrophages, mast cells, and platelets exhibit heparanase activity that is thought to be stored in specific granules. The degranulated heparanase is implicated in extravasation of metastatic tumor cells and activated cells of the immune system. Degranulation and heparanase release in response to an inflammatory stimulus or platelet activation would facilitate cellular extravasation directly, by altering the composition and structural integrity of the extracellular matrix, or indirectly, by releasing HS-bound proinflammatory cytokines and chemokines. We hypothesized that in addition to such indirect effect, the released heparanase may also locally affect and activate neighboring cells such as endothelial cells. Here, we provide evidence that addition of the 65-kDa latent heparanase to endothelial cells enhances Akt signaling. Heparanase-mediated Akt phosphorylation was independent of its enzymatic activity or the presence of cell membrane HS proteoglycans and was augmented by heparin. Moreover, addition of heparanase stimulated phosphatidylinositol 3-kinase-dependent endothelial cell migration and invasion. These results suggest, for the first time, that heparanase activates endothelial cells and elicits angiogenic responses directly. This effect appears to be mediated by as yet unidentified heparanase receptor. Heparanase is an endo-β-d-glucuronidase capable of cleaving heparan sulfate (HS) 1The abbreviations used are: HS, heparan sulfate; BAEC, bovine aortic endothelial cells; CHO, Chinese hamster ovary; EC, endothelial cells; ECM, extracellular matrix; HSPG, heparan sulfate proteoglycans; HUVEC, human umbilical vein endothelial cells; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; LS, laminaran sulfate; PBS, phosphate-buffered saline; BSA, bovine serum albumin.1The abbreviations used are: HS, heparan sulfate; BAEC, bovine aortic endothelial cells; CHO, Chinese hamster ovary; EC, endothelial cells; ECM, extracellular matrix; HSPG, heparan sulfate proteoglycans; HUVEC, human umbilical vein endothelial cells; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; LS, laminaran sulfate; PBS, phosphate-buffered saline; BSA, bovine serum albumin. side chains at a limited number of sites, yielding HS fragments of still appreciable size (∼5-7 kDa) (1Vlodavsky I. Friedmann Y. J. Clin. Invest. 2001; 108: 341-347Crossref PubMed Scopus (536) Google Scholar, 2Freeman C. Parish C.R. Biochem. J. 1998; 330: 1341-1350Crossref PubMed Scopus (169) Google Scholar, 3Pikas D.S. Li J.-P. Vlodavsky I. Lindhal U. J. Biol. Chem. 1998; 273: 18770-18777Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Participating in extracellular matrix (ECM) degradation and remodeling, heparanase activity has been traditionally correlated with the metastatic potential of tumor-derived cells (4Nakajima M. Irimura T. Nicolson G.L. J. Cell. Biochem. 1998; 36: 157-167Crossref Scopus (278) Google Scholar, 5Vlodavsky 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 (716) Google Scholar, 6Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: M99-M108PubMed Google Scholar, 7Bashkin P. Razin E. Eldor A. Vlodavsky I. Blood. 1990; 75: 2204-2212Crossref PubMed Google Scholar). Similarly, heparanase has been shown to facilitate cell invasion associated with angiogenesis, autoimmunity, and inflammation (6Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: M99-M108PubMed Google Scholar, 7Bashkin P. Razin E. Eldor A. Vlodavsky I. Blood. 1990; 75: 2204-2212Crossref PubMed Google Scholar, 8Vlodavsky I. Eldor A. Haimovitz-Friedman A. Matzner Y. Ishai-Michaeli R. Lider O. Naparstek Y. Cohen I.R. Fuks Z. Invasion Metastasis. 1992; 12: 112-127PubMed Google Scholar, 9Dempsey L.A. Brunn G.T. Platt J.L. Trends Biol. Sci. 2000; 25: 349-351Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Among the few cell types that express heparanase under normal physiological conditions, platelets possess a high heparanase activity and were used as a source for heparanase purification (2Freeman C. Parish C.R. Biochem. J. 1998; 330: 1341-1350Crossref PubMed Scopus (169) Google Scholar, 10Freeman C. Browne A.M. Parish C.R. Biochem. J. 1999; 342: 361-368Crossref PubMed Scopus (31) Google Scholar). In fact, serum heparanase is mainly derived from activated platelets (11Ihrcke N.S. Parker W. Reissner K.J. Platt J.L. J. Cell. Physiol. 1998; 175: 255-267Crossref PubMed Scopus (75) Google Scholar). Heparanase was localized to tertiary granules of neutrophils (12Matzner Y. Bar-Ner M. Yahalom J. Ishai-Michaeli R. Fuks Z. Vlodavsky I. J. Clin. Invest. 1985; 76: 1306-1316Crossref PubMed Scopus (175) Google Scholar, 13Mollinedo F. Nakajima M. Llorens A. Barbosa E. Callejo S. Gajate C. Fabra A. Biochem. J. 1997; 327: 917-923Crossref PubMed Scopus (91) Google Scholar) and mast cells (7Bashkin P. Razin E. Eldor A. Vlodavsky I. Blood. 1990; 75: 2204-2212Crossref PubMed Google Scholar) and was released upon tumor necrosis factor-α and calcium ionophore treatments, respectively. Heparanase release by degranulation has been implicated in diapedesis and extravasation of a number of immune cells, including neutrophils, macrophages, and lymphocytes (8Vlodavsky I. Eldor A. Haimovitz-Friedman A. Matzner Y. Ishai-Michaeli R. Lider O. Naparstek Y. Cohen I.R. Fuks Z. Invasion Metastasis. 1992; 12: 112-127PubMed Google Scholar, 14Bartlett M.R. Underwood P.A. Parish C.R. Immunol. Cell Biol. 1995; 73: 113-124Crossref PubMed Google Scholar, 15Parish C.R. Hindmarsh E.J. Bartlett M. Staykova M.A. Cowden W.B. Willenborg D.O. Immunol. Cell Biol. 1998; 76: 104-113Crossref PubMed Scopus (50) Google Scholar), while heparanase inhibitors exhibited an anti-inflammatory activity (15Parish C.R. Hindmarsh E.J. Bartlett M. Staykova M.A. Cowden W.B. Willenborg D.O. Immunol. Cell Biol. 1998; 76: 104-113Crossref PubMed Scopus (50) Google Scholar). Cleavage of HS side chains by degranulated heparanase during inflammation may facilitate the passage of blood-borne normal and malignant cells into tissues by altering the composition and structural integrity of the subendothelial ECM (1Vlodavsky I. Friedmann Y. J. Clin. Invest. 2001; 108: 341-347Crossref PubMed Scopus (536) Google Scholar, 8Vlodavsky I. Eldor A. Haimovitz-Friedman A. Matzner Y. Ishai-Michaeli R. Lider O. Naparstek Y. Cohen I.R. Fuks Z. Invasion Metastasis. 1992; 12: 112-127PubMed Google Scholar, 14Bartlett M.R. Underwood P.A. Parish C.R. Immunol. Cell Biol. 1995; 73: 113-124Crossref PubMed Google Scholar). In addition, heparanase may facilitate the release of a multitude of HS-bound growth factors, cytokines and chemokines that would, in turn, amplify the immune reaction or activate the vascular endothelium (16Vaday G.G. Lider O. J. Leukocyte Biol. 2000; 67: 149-159Crossref PubMed Scopus (209) Google Scholar, 17Elkin M. Ilan N. Ishai-Michaeli R. Friedmann Y. Papo O. Pecker I. Vlodavsky I. FASEB J. 2001; 15: 1661-1663Crossref PubMed Scopus (277) Google Scholar). We hypothesized that in addition to this indirect effect mediated by HS-bound growth factors, degranulated heparanase may also activate endothelial cells directly. To study this hypothesis, human (HUVEC) and bovine (BAEC) endothelial cells (EC) were incubated with the latent 65-kDa heparanase protein. As noted for primary fibro-blasts (18Nadav L. Eldor A. Yacoby-Zeevi O. Zamir E. Pecker I. Ilan N. Geiger B. Vlodavsky I. Katz B.Z. J. Cell Sci. 2001; 115: 2179-2187Google Scholar) and tumor-derived cells (19Zetser A. Bashenko Y. Miao H.-Q. Vlodavsky I. Ilan N. Cancer Res. 2003; 63: 7733-7741PubMed Google Scholar), the exogenously added heparanase rapidly interacts with EC, followed by internalization and processing into the 50-kDa active form. Interestingly, heparanase uptake by EC was accompanied by Akt phosphorylation that preceded heparanase processing and exhibited time and dose dependence. Heparanase-mediated Akt phosphorylation was independent of its enzymatic activity and the presence of membrane HS proteoglycans (HSPG) and was augmented by heparin. Moreover, addition of heparanase stimulated phosphatidylinositol 3-kinase (PI3K)-dependent endothelial cell migration and invasion. These results suggest, for the first time, that heparanase activates EC and elicits angiogenic responses directly. This effect appears to be mediated by as yet unidentified heparanase receptor. Cells and Cell Culture—HUVEC were kindly provided by Dr. Neomi Lanir (Rambam Medical Center, Haifa, Israel) and were cultured in gelatin-coated dishes, as described previously (20Ilan N. Mahooti S. Madri J.A. J. Cell Sci. 1998; 111: 3621-3631Crossref PubMed Google Scholar). Adult BAEC were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 1 ng/ml basic fibroblast growth factor and antibiotics, as described previously (21Elkin M. Miao H.-Q. Nagler A. Aingorn E. Reich R. Hemo I. Dou H.L. Pines M. Vlodavsky I. FASEB J. 2000; 14: 2477-2485Crossref PubMed Scopus (100) Google Scholar). Mutant Chinese hamster ovary (CHO) cells (pgsA-745) deficient in xylosyltransferase and unable to initiate glycosaminoglycan synthesis, were kindly provided by Dr. J. Esko (University of California, San Diego, CA) and cultured in RPMI medium supplemented with 10% fetal calf serum and antibiotics. CHO K1 and HT-29 colon carcinoma cells (also known as WiDr) were purchased from the ATCC and cultured in Dulbecco's modified Eagle's medium, as above. HT-29 cells synthesize perlecan, a secreted proteoglycan, but no other proteoglycans (22Fuki I.V. Iozzo R.V. Williams K.J. J. Biol. Chem. 2000; 275: 25742-25750Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and are therefore a useful model for cell surface HS-deficient cells. In vitro tube-like structure formation on Matrigel-coated plates was performed as described (23Ilan N. Mahooti S. Rimm D.R. Madri J.A. J. Cell Sci. 1999; 112: 3005-3014Crossref PubMed Google Scholar). HEK 293 cells, stably transfected with the human heparanase cDNA construct in the mammalian pSecTag vector (Invitrogen), were kindly provided by Dr. H-Q. Miao (ImClone Systems Inc., New York). This plasmid vector provides the IgGκ signal peptide to ensure efficient protein secretion, together with Myc and His tags at the C terminus of the enzyme to enable easy detection and purification (19Zetser A. Bashenko Y. Miao H.-Q. Vlodavsky I. Ilan N. Cancer Res. 2003; 63: 7733-7741PubMed Google Scholar). Antibodies and Reagents—The following antibodies were purchased from Santa Cruz Biotechnology: anti-Erk 2 (sc-154), anti-phospho-ERK (sc-7383), anti-Akt (sc-5298), and anti-Myc epitope tag (sc-40). Anti-phospho-Akt antibody was purchased from Cell Signaling Technology (Beverly, MA). Anti-heparanase antibody (number 1453) was raised against the entire 65-kDa heparanase precursor isolated from the conditioned medium of heparanase-transfected 293 cells. This antibody was affinity-purified on 50-kDa protein A. F. S. Bashenko Y. S. Vlodavsky I. Ilan N. J. Cell Sci. Google Scholar). sulfate was purchased from H.-Q. Elkin M. Aingorn E. Ishai-Michaeli R. C. Vlodavsky I. J. 1999; PubMed Scopus Google Scholar). The was purchased from and was in of the was as a Heparanase uptake the 65-kDa heparanase precursor was added to cell at a of 1 under the time the medium was cells were with PBS, and cell were as described Heparanase uptake and processing were by with anti-Myc and (number 1453) Cell and were with 1 for at with 1 and into a of inhibitors were to as described previously (19Zetser A. Bashenko Y. Miao H.-Q. Vlodavsky I. Ilan N. Cancer Res. 2003; 63: 7733-7741PubMed Google Scholar, N. Mahooti S. Madri J.A. J. Cell Sci. 1998; 111: 3621-3631Crossref PubMed Google Scholar). Cell were to in plates and were with the of a were with to cells and incubated with growth medium, and cell migration into the was followed for in the presence or of exogenously added heparanase Invasion was performed modified with Matrigel-coated membrane as described previously (19Zetser A. Bashenko Y. Miao H.-Q. Vlodavsky I. Ilan N. Cancer Res. 2003; 63: 7733-7741PubMed Google Scholar). was that were with Heparanase and was with and were and the were with for 1 at were with were following with and were or were in as and cell were as described above. Heparanase of and of heparanase activity were performed as described in (12Matzner Y. Bar-Ner M. Yahalom J. Ishai-Michaeli R. Fuks Z. Vlodavsky I. J. Clin. Invest. 1985; 76: 1306-1316Crossref PubMed Scopus (175) Google Scholar, I. J. M. J. J. in Cell New Scholar). heparanase was incubated with from ECM The medium was to on a were with and in a To the enzymatic activity of was and following was to as described above. were at with Akt activation is as of at independent by Heparanase to at the C physiological vascular EC are to be locally to of To study the effect of heparanase on EC, human (HUVEC) and bovine (BAEC) EC were incubated with exogenously added latent 65-kDa heparanase and heparanase uptake was followed by of As in heparanase rapidly interacts with primary EC in with the anti-Myc epitope antibody a signal and following heparanase Interestingly, the anti-Myc antibody a protein that to the added 65-kDa heparanase while a protein was at time heparanase processing was and with the anti-Myc antibody rapidly and preceded heparanase processing that by and was mainly by 2 and This may that in addition to the processing at and that the and the 50-kDa heparanase E. M. J. M. R. P. A. S. S. H. R. J. R. C. Biochem. J. 2003; PubMed Scopus Google Scholar, A.M. J.L. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), heparanase is also to processing at the C in of the tag Heparanase Akt that heparanase in stably transfected cells a in Akt phosphorylation that correlated with an cellular (19Zetser A. Bashenko Y. Miao H.-Q. Vlodavsky I. Ilan N. Cancer Res. 2003; 63: 7733-7741PubMed Google Scholar). of the latent 65-kDa heparanase protein in a Akt phosphorylation in Akt activation heparanase addition and to Moreover, Akt phosphorylation was dose a effect at 1 as by This Akt activation was specific as the phosphorylation of other such as was or results were with EC for Akt and efficient uptake of heparanase by cell types is thought to be at in by the cell membrane (18Nadav L. Eldor A. Yacoby-Zeevi O. Zamir E. Pecker I. Ilan N. Geiger B. Vlodavsky I. Katz B.Z. J. Cell Sci. 2001; 115: 2179-2187Google Scholar). To the of in Akt heparanase was added to CHO cells that exhibit HS and sulfate Cell Biol. PubMed Scopus Google Scholar). of heparanase to HS-deficient cells in Akt in and to that in EC This heparanase was by the with HT-29 cells. This human colon tumor-derived cell perlecan, a secreted mainly in the ECM, but no other of (22Fuki I.V. Iozzo R.V. Williams K.J. J. Biol. Chem. 2000; 275: 25742-25750Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). of heparanase to HT-29 cells in Akt activation the that Akt activation As was noted for EC Akt activation and phosphorylation was by heparanase addition a in Akt phosphorylation upon heparanase addition to EC and to HS-deficient cells Akt activation was also following addition of heparanase to EC These that cell surface other are in Akt Akt of of Akt activation was following heparanase addition and preceded its processing into the 50-kDa active that was first its addition that Akt activation is independent of heparanase enzymatic To this CHO cells were incubated with heparanase in the presence or of the heparanase We previously that this heparanase activity and H.-Q. Elkin M. Aingorn E. Ishai-Michaeli R. C. Vlodavsky I. J. 1999; PubMed Scopus Google Scholar). of heparanase to CHO cells in a of Akt in with results Interestingly, addition of heparanase to CHO cells together with at a that heparanase enzymatic activity H.-Q. Elkin M. Aingorn E. Ishai-Michaeli R. C. Vlodavsky I. J. 1999; PubMed Scopus Google Scholar), Akt phosphorylation and the addition of heparanase and in a of Akt phosphorylation and is that HS-bound growth by of the fibroblast growth factor HS or to a with and a effect 2000; PubMed Scopus Google Scholar, A. 2000; PubMed Scopus Google Scholar, L. Biol. 2001; PubMed Scopus Google Scholar). a we the of Akt activation by be by heparin. To this C and cells were or incubated with heparanase or with heparin. As shown in Akt activation by heparanase was augmented by in a and with C and while no effect Similarly, heparanase Akt activation in CHO K1 cells and to a to high Akt phosphorylation in cells the addition of augmented Akt phosphorylation by heparanase and as was noted for primary endothelial cells C and These the that Akt activation is mediated by a heparanase and that may be for the 50-kDa active heparanase enzyme was in 65-kDa heparanase by of a heparanase activity was the ECM is therefore that Akt activation was by this of the active 50-kDa heparanase form. To the of heparanase activity for Akt heparanase was to the under high of active heparanase to the enzyme In addition, and in to the the heparanase protein is to uptake and an in heparanase from the The heparanase protein an in EC with the that under physiological heparanase may cell (19Zetser A. Bashenko Y. Miao H.-Q. Vlodavsky I. Ilan N. Cancer Res. 2003; 63: 7733-7741PubMed Google Scholar, R. I. L. Vlodavsky I. Lider O. J. Med. 1995; PubMed Scopus Google Scholar, O. E. Cohen M. Aingorn H. Cohen I. L. Katz Geiger B. Vlodavsky I. FASEB J. 2003; PubMed Scopus Google Scholar). to the heparanase in a of Akt phosphorylation and that Akt activation is independent of heparanase Interestingly, under conditions, was also activated and that were Heparanase EC the effect of exogenously added heparanase on EC were on of Matrigel-coated conditions, EC into tube-like (23Ilan N. Mahooti S. Rimm D.R. Madri J.A. J. Cell Sci. 1999; 112: 3005-3014Crossref PubMed Google Scholar) in a that of exhibited a limited formation In addition of heparanase in a EC of EC EC formation on Matrigel-coated was and EC migration EC is to be in this To the effect of heparanase on EC were with the of a and cell migration into the was in or with heparanase As shown in addition of heparanase stimulated a response with migration in response to heparanase addition were and by migration on conditions, addition of heparanase in a in migration C and addition of heparanase together with the the effect of heparanase C and while the heparanase no effect on EC migration C and in with that heparanase enzymatic activity is for Akt activation and Moreover, heparanase stimulated a in EC invasion Matrigel-coated and this effect was by and These that heparanase EC This effect is is mediated by Akt and may a heparanase receptor. Heparanase activity has been to metastatic and immune cells capable of the ECM 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 (716) Google Scholar, 7Bashkin P. Razin E. Eldor A. Vlodavsky I. Blood. 1990; 75: 2204-2212Crossref PubMed Google Scholar, 8Vlodavsky I. Eldor A. Haimovitz-Friedman A. Matzner Y. Ishai-Michaeli R. Lider O. Naparstek Y. Cohen I.R. Fuks Z. Invasion Metastasis. 1992; 12: 112-127PubMed Google Scholar). Blood-borne neutrophils, macrophages, mast cells, and platelets exhibit heparanase activity that is thought to be stored in specific granules (1Vlodavsky I. Friedmann Y. J. Clin. Invest. 2001; 108: 341-347Crossref PubMed Scopus (536) Google Scholar, M. Irimura T. Nicolson G.L. J. Cell. Biochem. 1998; 36: 157-167Crossref Scopus (278) Google Scholar, 5Vlodavsky 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. 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Degranulation and heparanase release in response to a inflammatory or upon platelet may facilitate cellular extravasation by altering the composition and of the ECM or by releasing HS-bound proinflammatory cytokines and chemokines (16Vaday G.G. Lider O. J. Leukocyte Biol. 2000; 67: 149-159Crossref PubMed Scopus (209) Google Scholar). In addition, the released heparanase may also locally affect and activate neighboring cells such as endothelial cells. exogenously added heparanase stimulated Akt phosphorylation in and EC while other such as the were and The and efficient cellular and uptake of heparanase to be mediated by the cell membrane (18Nadav L. Eldor A. Yacoby-Zeevi O. Zamir E. Pecker I. Ilan N. Geiger B. Vlodavsky I. Katz B.Z. J. Cell Sci. 2001; 115: 2179-2187Google Scholar). Akt phosphorylation in and were in HS-deficient CHO and HT-29 cells that heparanase with cell surface other The of Akt phosphorylation by C and may the of a specific heparanase to growth factor to A. 2000; PubMed Scopus Google Scholar, L. J. L. G.L. P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, C. U. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to this heparanase are in of evidence that Akt activation is independent of heparanase enzymatic In of Akt phosphorylation were following heparanase addition and and preceded heparanase processing following heparanase In addition, Akt phosphorylation in response to heparanase was by the heparanase but was augmented and This is in with the of to Akt activation by heparanase C and for a mediated by of heparanase to in of the heparanase under high of heparanase enzymatic activity but also heparanase response mediated by cell membrane The heparanase This is in with that A.M. J.L. J. Biol. 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