Heparan sulfate proteoglycans interact with many extracellular matrix constituents, growth factors and enzymes. Degradation of heparan sulfate by endoglycosidic heparanase cleavage affects a variety of biological processes. We have purified a 50-kDa heparanase from human hepatoma and placenta, and now report cloning of the cDNA and gene encoding this enzyme. Expression of the cloned cDNA in insect and mammalian cells yielded 65-kDa and 50-kDa recombinant heparanase proteins. The 50-kDa enzyme represents an N-terminally processed enzyme, at least 100-fold more active than the 65-kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and specimens of human breast, colon and liver carcinomas. Low metastatic murine T-lymphoma and melanoma cells transfected with the heparanase cDNA acquired a highly metastatic phenotype in vivo, reflected by a massive liver and lung colonization. This represents the first cloned mammalian heparanase, to our knowledge, and provides direct evidence for its role in tumor metastasis. Cloning of the heparanase gene enables the development of specific molecular probes for early detection and treatment of cancer metastasis and autoimmune disorders.

We have generated homozygous transgenic mice (hpa-tg) overexpressing human heparanase (endo-beta-D-glucuronidase) in all tissues and characterized the involvement of the enzyme in tissue morphogenesis, vascularization, and energy metabolism. Biochemical analysis of heparan sulfate (HS) isolated from newborn mice and adult tissues revealed a profound decrease in the size of HS chains derived from hpa-tg vs. control mice. Despite this, the mice appeared normal, were fertile, and exhibited a normal life span. A significant increase in the number of implanted embryos was noted in the hpa-tg vs. control mice. Overexpression of heparanase resulted in increased levels of urinary protein and creatinine, suggesting an effect on kidney function, reflected also by electron microscopy examination of the kidney tissue. The hpa-tg mice exhibited a reduced food consumption and body weight compared with control mice. The effect of heparanase on tissue remodeling and morphogenesis was best demonstrated by the phenotype of the hpa-tg mammary glands, showing excess branching and widening of ducts associated with enhanced neovascularization and disruption of the epithelial basement membrane. The hpa-tg mice exhibited an accelerated rate of hair growth, correlated with high expression of heparanase in hair follicle keratinocytes and increased vascularization. Altogether, characterization of the hpa-tg mice emphasizes the involvement of heparanase and HS in processes such as embryonic implantation, food consumption, tissue remodeling, and vascularization.

The human heparanase gene, an endo-beta-glucuronidase that cleaves heparan sulfate at specific intrachain sites, has recently been cloned and shown to function in tumor progression and metastatic spread. Antisense digoxigenin-labeled heparanase RNA probe and monoclonal anti-human heparanase antibodies were used to examine the expression of the heparanase gene and protein in normal, dysplastic, and neoplastic human colonic mucosa. To our knowledge, this is the first systematic study of heparanase expression in human colon cancer. Both the heparanase gene and protein were expressed at early stages of neoplasia, already at the stage of adenoma, but were practically not detected in the adjacent normal-looking colon epithelium. Gradually increasing expression of heparanase was evident as the cells progressed from severe dysplasia through well-differentiated to poorly differentiated colon carcinoma. Deeply invading colon carcinoma cells showed the highest levels of the heparanase mRNA and protein associated with expression of both the gene and enzyme by adjacent desmoplastic stromal fibroblasts. A high expression was also found in colon carcinoma metastases to lung, liver, and lymph nodes, as well as in the accompanying stromal fibroblasts. Moreover, extracts derived from tumor tissue expressed much higher levels of the heparanase protein and activity as compared to the normal colon tissue. In all specimens, the heparanase gene and protein exhibited the same pattern of expression. These results suggest a role of heparanase in colon cancer progression and may have both prognostic and therapeutic applications.

Heparanase is an endo-beta-D-glucuronidase that cleaves heparan sulfate and is implicated in diverse physiological and pathological processes. In this study we report on a novel direct involvement of heparanase in cell adhesion. We demonstrate that expression of heparanase in nonadherent lymphoma cells induces early stages of cell adhesion, provided that the enzyme is expressed on the cell surface. Heparanase-mediated cell adhesion to extracellular matrix (ECM) results in integrin-dependent cell spreading, tyrosine phosphorylation of paxillin, and reorganization of the actin cytoskeleton. The surface-bound enzyme also augments cell invasion through a reconstituted basement membrane. Cell adhesion was augmented by cell surface heparanase regardless of whether the cells were transfected with active or point mutated inactive enzyme, indicating that heparanase functions as an adhesion molecule independent of its endoglycosidase activity. The combined feature of heparanase as an ECM-degrading enzyme and a cell adhesion molecule emphasizes its significance in processes involving cell adhesion, migration, and invasion, including embryonic development, neovascularization, and cancer metastasis.

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