An ELISA method for the detection and quantification of human heparanase

Heparanase is a mammalian endo-beta-D-glucuronidase that cleaves heparan sulfate side chains at a limited number of sites. Heparanase enzymatic activity is thought to participate in degradation and remodeling of the extracellular matrix and to facilitate cell invasion associated with tumor metastasis, angiogenesis, and inflammation. Traditionally, heparanase activity was well correlated with the metastatic potential of a large number of tumor-derived cell types. More recently, heparanase upregulation was detected in an increasing number of primary human tumors, correlating, in some cases, with poor postoperative survival and increased tumor vascularity. The present study was undertaken to develop a highly sensitive ELISA suitable for the determination and quantification of human heparanase in tissue extracts and body fluids. The assay preferentially detects the 8+50 kDa active heparanase heterodimer vs. the latent 65 kDa proenzyme and correlates with immunoblot analysis of heparanase containing samples. It detects heparanase at concentrations as low as 200 pg/ml and is suitable for quantification of heparanase in tissue extracts and urine.     

Heparanase processing by lysosomal/endosomal protein preparation

Heparanase is an endo-beta-glucuronodase involved in cleavage of heparan sulfate side chains, activity that is strongly implicated in cell dissemination associated with tumor metastasis and inflammation. Heparanase is first synthesized as a latent 65 kDa precursor that is converted into an active enzyme upon proteolytic processing. Previously, we have reported that elevation of the lysosomal pH results in complete inhibition of heparanase processing, suggesting that lysosomal protease(s) and acidic pH conditions are required for heparanase processing. Here, we adopted a cell fractionation approach and provide evidence that incubation of the pro-enzyme with lysosome/endosome, but not with cytoplasmic fractions resulted in processing and activation of the 65 kDa latent heparanase. Moreover, while the water soluble lysosome/endosome fraction exhibited no apparent processing activity, heparanase processing by the water insoluble lysosome/endosome membrane fraction was readily detected and exhibited the expected pH dependency.     

Cloning,expression, and purification of mouse heparanase

Heparanase is an endoglucuronidase that plays an important role in tumor invasion and metastasis. A full-length heparanase gene was cloned from a mouse embryo cDNA library and determined to encode a protein of 535 amino acids that is 77% identical to human heparanase. The full-length mouse gene was stably expressed in NS0 myeloma cells. The recombinant mouse heparanase protein was purified to homogeneity from cell lysates by a combination of Con-A affinity chromatography, heparin affinity chromatography, and size exclusion chromatography. The purified protein consisted of a non-covalent heterodimer of 50- and 8-kDa polypeptides, similar to the human homolog. The protein was enzymatically active in assays using radiolabeled ECM and heparan sulfate as substrates. The maximum heparanase activity was observed at acidic conditions; however, significant activity was also detected at neutral pH. The enzymatic activity of mouse heparanase was blocked by known heparanase inhibitors.     

Involvement of heparanase in migration of microglial cells

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.     

Soluble Heparan Sulfate Fragments Generated by Heparanase Trigger the Release of Pro-Inflammatory Cytokines through TLR-4

Heparanase is a β-D-endoglucuronidase that cleaves heparan sulfate (HS), facilitating degradation of the extracellular matrix (ECM) and the release of HS-bound biomolecules including cytokines. The remodeling of the ECM by heparanase is important for various physiological and pathological processes, including inflammation, wound healing, tumour angiogenesis and metastasis. Although heparanase has been proposed to facilitate leukocyte migration through degradation of the ECM, its role in inflammation by regulating the expression and release of cytokines has not been fully defined. In this study, the role of heparanase in regulating the expression and release of cytokines from human and murine immune cells was examined. Human peripheral blood mononuclear cells treated ex vivo with heparanase resulted in the release of a range of pro-inflammatory cytokines including IL-1β, IL-6, IL-8, IL-10 and TNF. In addition, mouse splenocytes treated ex vivo with heparanase resulted in the release of IL-6, MCP-1 and TNF. A similar pattern of cytokine release was also observed when cells were treated with soluble HS. Furthermore, heparanase-induced cytokine release was abolished by enzymatic-inhibitors of heparanase, suggesting this process is mediated via the enzymatic release of cell surface HS fragments. As soluble HS can signal through the Toll-like receptor (TLR) pathway, heparanase may promote the upregulation of cytokines through the generation of heparanase-cleaved fragments of HS. In support of this hypothesis, mouse spleen cells lacking the key TLR adaptor molecule MyD88 demonstrated an abolition of cytokine release after heparanase stimulation. Furthermore, TLR4-deficient spleen cells showed reduced cytokine release in response to heparanase treatment, suggesting that TLR4 is involved in this response. Consistent with these observations, the pathway involved in cytokine upregulation was identified as being NF-κB-dependent. These data identify a new mechanism for heparanase in promoting the release of pro-inflammatory cytokines that is likely to be important in regulating cell migration and inflammation.     

H NMR spectroscopic studies establish that heparanase is a retaining glycosidase

Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains of proteoglycans in basement membranes and the extracellular matrix (ECM). Heparanase is implicated in several diverse pathological processes associated with ECM degradation such as metastasis, inflammation and angiogenesis and is thus an important target for anti-cancer and anti-inflammatory drug discovery. Heparanase has been classed as belonging to the clan A glycoside hydrolase family 79 based on sequence analysis, secondary structure predictions and mutagenic analysis, and thus it has been inferred that it is a retaining glycosidase. However, there has been no direct experimental evidence to support this conclusion. Herein we describe ¹H NMR spectroscopic studies of the hydrolysis of the pentasaccharide substrate fondaparinux by heparanase, and provide conclusive evidence that heparanase hydrolyses its substrate with retention of configuration and is thus established as a retaining glycosidase. Knowledge of the mechanism of hydrolysis may have implications for future design of inhibitors for this important drug target.     

Human heparanase. Purification, characterization, cloning, and expression.

Heparan sulfate and heparan sulfate proteoglycans are present in the extracellular matrix as well as on the external cell surface. They bind various molecules such as growth factors and cytokines and modulate the biological functions of binding proteins. Heparan sulfate proteoglycans are also important structural components of the basement membrane. Heparanase is an endo-beta-D-glucuronidase capable of cleaving heparan sulfate and has been implicated in inflammation and tumor angiogenesis and metastasis. In this study, we report the purification of a human heparanase from an SV40-transformed embryonic fibroblast cell line WI38/VA13 by four sequential column chromatographies. The activity was measured by high speed gel permeation chromatography of the degradation products of fluorescein isothiocyanate-labeled heparan sulfate. The enzyme was purified to homogeneity, yielding a peptide with an apparent molecular mass of 50 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Using the amino acid sequences of the N-terminal and internal heparanase peptides, a cDNA coding for human heparanase was cloned. NIH3T3 and COS-7 cells stably transfected with pBK-CMV expression vectors containing the heparanase cDNA showed high heparanase activities. The homology search revealed that no homologous protein had been reported.     

Heparanase modulation by Wingless/INT (Wnt)

Heparanase is an endo-beta-glucuronidase, the only enzyme in mammals capable of cleaving heparan sulfate/heparin chains from proteoglycans. The oligosaccharides generated by heparanase present extensive biological functions since such oligosaccharides interact with adhesion molecules, growth factors, angiogenic factors and cytokines, modulating cell proliferation, migration, inflammation, and carcinogenesis. However, the regulation of heparanase activity is not fully understood. It is known that heparanase is synthesized as an inactive 65 kDa isoform and that post-translation processing forms an active 50 kDa enzyme. In the present study, we are interested in investigating whether heparanase is regulated by its own substrate as observed with many other enzymes. Wild-type Chinese hamster (Cricetulus griséus) ovary cells (CHO-K1) were treated with different doses of heparin. Heparanase expression was analyzed by Real-time PCR and flow cytometry. Also, heparanase activity was measured. The heparanase activity assay was performed using a coated plate with biotinylated heparan sulfate. In the present assay, a competitive heparin inhibition scenario was set aside. Exogenous heparin trigged a cell signaling pathway that increased heparanase mRNA and protein levels. The Wnt/beta-catenin pathway, judged by TCF-driven luciferase activity, seems to be involved to enhance heparanase profile during treatment with exogenous heparin. Lithium chloride treatment, an activator of the Wnt/beta-catenin pathway, confirmed such mechanism of transduction in vivo using zebrafish embryos and in vitro using CHO-K1 cells. Taken together the results suggest that heparin modulates heparanase expression by Wnt/beta-catenin.

     

Molecular properties and involvement of heparanase in cancer progression and normal development

Heparan sulfate proteoglycans (HSPGs) play a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Hence, cleavage of heparan sulfate (HS) affects the integrity and functional state of tissues and thereby fundamental normal and pathological phenomena involving cell migration and response to changes in the extracellular microenvironment. Here, we describe the molecular properties, expression and function of a human heparanase, degrading HS at specific intrachain sites. The enzyme is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and human tumor tissues. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also releases ECM-resident angiogenic factors in vitro and its overexpression induces an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, both critical steps in cancer progression. The enzyme is also involved in cell migration associated with inflammation and autoimmunity. The unexpected identification of a single predominant functional heparanase suggests that the enzyme is a promising target for drug development. In fact, treatment with heparanase inhibitors markedly reduces tumor growth, metastasis and autoimmune disorders in animal models. Studies are underway to elucidate the involvement of heparanase in normal processes such as implantation, embryonic development, morphogenesis, tissue repair, inflammation and HSPG turnover. Heparanase is the first functional mammalian HS-degrading enzyme that has been cloned, expressed and characterized. This may lead to identification and cloning of other glycosaminoglycan degrading enzymes, toward a better understanding of their involvement and significance in normal and pathological processes.     

Cell surface localization of heparanase on macrophages regulates degradation of extracellular matrix heparan sulfate

Extravasation of peripheral blood monocytes through vascular basement membranes requires degradation of extracellular matrix components including heparan sulfate proteoglycans (HSPGs). Heparanase, the heparan sulfate-specific endo-beta-glucuronidase, has previously been shown to be a key enzyme in melanoma invasion, yet its involvement in monocyte extravasation has not been elucidated. We examined a potential regulatory mechanism of heparanase in HSPG degradation and transmigration through basement membranes in leukocyte trafficking using human promonocytic leukemia U937 and THP-1 cells. PMA-treated cells were shown to degrade 35S-sulfated HSPG in endothelial extracellular matrix into fragments of an approximate molecular mass of 5 kDa. This was not found with untreated cells. The gene expression levels of heparanase or the enzyme activity of the amount of cell lysates were no different between untreated and treated cells. Immunocytochemical staining with anti-heparanase mAb revealed pericellular distribution of heparanase in PMA-treated cells but not in untreated cells. Cell surface heparanase capped into a restricted area on PMA-treated cells when they were allowed to adhere. Addition of a chemoattractant fMLP induced polarization of the PMA-treated cells and heparanase redistribution at the leading edge of migration. Therefore a major regulatory process of heparanase activity in the cells seems to be surface expression and capping of the enzyme. Addition of the anti-heparanase Ab significantly inhibited enzymatic activity and transmigration of the PMA-treated cells, suggesting that the cell surface redistribution of heparanase is involved in monocyte extravasation through basement membranes.     

Dissociation between mature phenotype and impaired transmigration in dendritic cells from heparanase-deficient mice

To reach the lymphatics, migrating dendritic cells (DCs) need to interact with the extracellular matrix (ECM). Heparanase, a mammalian endo-β-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and ECM. The role of heparanase in the physiology of bone marrow-derived DCs was studied in mutant heparanase knock-out (Hpse-KO) mice. Immature DCs from Hpse-KO mice exhibited a more mature phenotype; however their transmigration was significantly delayed, but not completely abolished, most probably due to the observed upregulation of MMP-14 and CCR7. Despite their mature phenotype, uptake of beads was comparable and uptake of apoptotic cells was more efficient in DCs from Hpse-KO mice. Heparanase is an important enzyme for DC transmigration. Together with CCR7 and its ligands, and probably MMP-14, heparanase controls DC trafficking.     

Heparanase is expressed in osteoblastic cells and stimulates bone formation and bone mass

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules. In bone, they are associated with cell surfaces and the extracellular matrix (ECM). The heparan sulfate (HS) chains of HSPGs bind a multitude of bioactive molecules, thereby controlling normal and pathologic processes. The HS-degrading endoglycosidase, heparanase, has been implicated in processes such as inflammation, vascularization associated with wound healing and malignancies, and cancer metastasis. Here we show progressive mRNA expression of the hpa gene (encoding heparanase) in murine bone marrow stromal cells undergoing osteoblastic (bone forming) differentiation and in primary calvarial osteoblasts. Bone marrow stromal cells derived from transgenic mice expressing recombinant human heparanase (rh-heparanase) and MC3T3 E1 osteoblastic cells exposed to soluble rh-heparanase spontaneously undergo osteogenic differentiation. In addition, the transgenic bone marrow stromal cells degrade HS chains. In wild-type (WT) and hpa-transgenic (hpa-tg) mice, heparanase is weakly expressed throughout the bone marrow with a substantial increase in osteoblasts and osteocytes, especially in the hpa-tg mice. Heparanase expression was absent in osteoclasts. Micro-computed tomographic and histomorphometric skeletal analyses in male and female hpa-tg versus WT mice show markedly increased trabecular bone mass, cortical thickness, and bone formation rate, but no difference in osteoclast number. Collectively, our data suggest that proteoglycans tonically suppress osteoblast function and that this inhibition is alleviated by HS degradation with heparanase.     

Thrombin enhances degradation of heparan sulfate in the extracellular matrix by tumor cell heparanase.

The ability of normal and malignant blood-borne cells to extravasate correlates with the activity of an endo-beta-D-glucuronidase (heparanase) which degrades heparan sulfate (HS) in the subendothelial extracellular matrix (ECM). The association of malignancy with different types of coagulopathies prompted us to study the effect of thrombin (EC 3.4.21.5), a serine protease elaborated during activation of the clotting cascade, on the ability of heparanase to degrade the ECM-HS. The circulating zymogen form of thrombin, prothrombin, was converted to proteolytically active thrombin during incubation with ECM. Thrombin generation by the ECM was time and dose dependent, reaching maximal conversion by 6 h incubation at 3 U/ml of prothrombin. Heparanase-mediated release of low Mr HS cleavage products from sulfate-labeled ECM was stimulated four- to sixfold in the presence of alpha-thrombin, but there was no effect on degradation of soluble HS. Similar results were obtained with heparanase preparations derived from mouse lymphoma and human hepatoma cell lines and from human placenta. Incubation of ECM with alpha-thrombin alone resulted in release of nearly intact high-Mr labeled proteoglycans. Thrombin stimulation of heparanase action was dose and time dependent, reaching a maximal value at 24 h incubation with 1 microM alpha-thrombin. The effect of modified thrombin preparations correlated with their proteolytic activity. Catalytically blocked preparations of thrombin (e.g., DIP-alpha-thrombin, MeSO2-alpha-thrombin) failed to facilitate heparanase action, while catalytically modified preparations (e.g., gamma-thrombin, NO2-alpha-thrombin) exerted only a slight enhancement. Antithrombin III (ATIII) and hirudin both inhibited thrombin-stimulated heparanase degradation of ECM-bound HS. Heparanase action was also facilitated by ECM-immobilized thrombin to an extent which was similar to that induced by soluble thrombin. This result implies that thrombin sequestered by the subendothelial ECM and protected from interaction with its natural inhibitor ATIII (Bar-Shavit et al., 1989, J. Clin. Invest. 84, 1096-1104) may participate locally in cellular invasion during tumor metastasis, inflammation, and autoimmunity.     

Modulation of the heparanase-inhibiting activity of heparin through selective desulfation, graded N-acetylation, and glycol splitting

Heparanase is an endo-beta-glucuronidase that cleaves heparan sulfate (HS) chains of heparan sulfate proteoglycans on cell surfaces and in the extracellular matrix (ECM). Heparanase, overexpressed by most cancer cells, facilitates extravasation of blood-borne tumor cells and causes release of growth factors sequestered by HS chains, thus accelerating tumor growth and metastasis. Inhibition of heparanase with HS mimics is a promising target for a novel strategy in cancer therapy. In this study, in vitro inhibition of recombinant heparanase was determined for heparin derivatives differing in degrees of 2-O- and 6-O-sulfation, N-acetylation, and glycol splitting of nonsulfated uronic acid residues. The contemporaneous presence of sulfate groups at O-2 of IdoA and at O-6 of GlcN was found to be non-essential for effective inhibition of heparanase activity provided that one of the two positions retains a high degree of sulfation. N-Desulfation/ N-acetylation involved a marked decrease in the inhibitory activity for degrees of N-acetylation higher than 50%, suggesting that at least one NSO3 group per disaccharide unit is involved in interaction with the enzyme. On the other hand, glycol splitting of preexisting or of both preexisting and chemically generated nonsulfated uronic acids dramatically increased the heparanase-inhibiting activity irrespective of the degree of N-acetylation. Indeed N-acetylated heparins in their glycol-split forms inhibited heparanase as effectively as the corresponding N-sulfated derivatives. Whereas heparin and N-acetylheparins containing unmodified D-glucuronic acid residues inhibited heparanase by acting, at least in part, as substrates, their glycol-split derivatives were no more susceptible to cleavage by heparanase. Glycol-split N-acetylheparins did not release basic fibroblast growth factor from ECM and failed to stimulate its mitogenic activity. The combination of high inhibition of heparanase and low release/potentiation of ECM-bound growth factor indicates that N-acetylated, glycol-split heparins are potential antiangiogenic and antimetastatic agents that are more effective than their counterparts with unmodified backbones.     

Cell surface-expressed cation-independent mannose 6-phosphate receptor (CD222) binds enzymatically active heparanase independently of mannose 6-phosphate to promote extracellular matrix degradation

Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate, an important structural component of the extracellular matrix (ECM) and vascular basement membrane (BM). The cleavage of heparan sulfate by heparanase-expressing cells, such as activated leukocytes, metastatic tumor cells, and proliferating endothelial cells, facilitates degradation of the ECM/BM to promote cell invasion associated with inflammation, tumor metastasis, and angiogenesis. In addition to its enzymatic function, heparanase has also recently been shown to act as a cell adhesion and/or signaling molecule upon interaction with cell surfaces. Despite the obvious importance of the mechanisms for the binding of heparanase to cell surfaces, the receptor(s) for heparanase remain poorly defined. In this study, we identify the 300-kDa cation-independent mannose 6-phosphate receptor (CIMPR) as a cell surface receptor for heparanase. Purified platelet heparanase was shown to bind the human CIMPR expressed on the surface of a transfected mouse L cell line. Optimal binding was determined to be at a slightly acidic pH (6.5-7.0) with heparanase remaining on the cell surface for up to 10 min at 37 degrees C. In contrast, mouse L cells or Chinese hamster ovary cells expressing the cation-dependent mannose 6-phosphate receptor (CDMPR) showed no binding of heparanase. Interestingly, the binding of heparanase to CIMPR was independent of Man-6-P moieties. Significantly, primary human T cells upon activation were shown to dramatically up-regulate levels of cell surface-expressed CIMPR, which showed a concomitant increase in their capacity to bind heparanase. Furthermore, the tethering of heparanase to the surface of cells via CIMPR was found to increase their capacity to degrade an ECM or a reconstituted BM. These data suggest an important role for CIMPR in the cell surface presentation of enzymatically active heparanase for the efficient passage of T cells into an inflammatory site and have implications for the use of this mechanism by other cell types to enhance cell invasion.     

Heparanase Regulates Secretion,Composition, and Function of Tumor Cell-derived Exosomes

Emerging evidence indicates that exosomes play a key role in tumor-host cross-talk and that exosome secretion, composition, and functional capacity are altered as tumors progress to an aggressive phenotype. However, little is known regarding the mechanisms that regulate these changes. Heparanase is an enzyme whose expression is up-regulated as tumors become more aggressive and is associated with enhanced tumor growth, angiogenesis, and metastasis. We have discovered that in human cancer cells (myeloma, lymphoblastoid, and breast cancer), when expression of heparanase is enhanced or when tumor cells are exposed to exogenous heparanase, exosome secretion is dramatically increased. Heparanase enzyme activity is required for robust enhancement of exosome secretion because enzymatically inactive forms of heparanase, even when present in high amounts, do not dramatically increase exosome secretion. Heparanase also impacts exosome protein cargo as reflected by higher levels of syndecan-1, VEGF, and hepatocyte growth factor in exosomes secreted by heparanase-high expressing cells as compared with heparanase-low expressing cells. In functional assays, exosomes from heparanase-high cells stimulated spreading of tumor cells on fibronectin and invasion of endothelial cells through extracellular matrix better than did exosomes secreted by heparanase-low cells. These studies reveal that heparanase helps drive exosome secretion, alters exosome composition, and facilitates production of exosomes that impact both tumor and host cell behavior, thereby promoting tumor progression.     

Downregulating the expression of heparanase inhibits the invasion, angiogenesis and metastasis of human hepatocellular carcinoma

Invasion and metastasis are key features of human hepatocellular carcinoma (HCC). Heparanase is an endoglycosidase that can degrade extracellular matrix by cleaving heparan sulfate chains of heparan sulfate proteoglycan, thus playing important roles in the invasion and metastasis of human cancers. Heparanase has been detected in various human cancers and regarded as a prospective target in human cancer treatments. However, the effects of inhibiting the expression of heparanase on human HCC have not been fully evaluated. In this article we show that downregulating the expression of heparanase either by antisense oligodeoxynucleotide or by RNA interferencing can significantly reduce the expression of heparanase in SMMC7721 human HCC cells, leading to inhibition of the invasiveness, metastasis, and angiogenesis of HCC cells both in vitro and in vivo. Our results suggest that genetic downregulation of the expression of heparanase may serve as an efficient cancer therapeutic for human HCC.