Heparanase enhances nerve-growth-factor-induced PC12 cell neuritogenesis via the p38 MAPK pathway.

Heparanase is involved in the cleavage of the HS (heparan sulfate) chain of HSPGs (HS proteoglycans) and hence participates in remodelling of the ECM (extracellular matrix) and BM (basement membrane). In the present study we have shown that NGF (nerve growth factor) promoted nuclear enrichment of EGR1 (early growth response 1), a transcription factor for heparanase, and markedly induced heparanase expression in rat adrenal pheochromocytoma (PC12) cells. K252a, an antagonist of the NGF receptor TrkA (tyrosine kinase receptor A), decreased heparanase protein expression induced by NGF in PC12 cells. Suramin, a heparanase inhibitor, decreased heparanase in PC12 cells and blocked NGF-induced PC12 neuritogenesis. Stable overexpression of heparanase activated p38 MAPK (mitogen-activated protein kinase) by phosphorylation and enhanced the neurite outgrowth induced by NGF, whereas knock down of heparanase impaired this process. However, overexpression of latent pro-heparanase with a Y156A mutation still led to enhanced NGF-induced neurite outgrowth and increased p38 MAPK phosphorylation. Inhibition of p38 MAPK by SB203580 suppressed the promotion of NGF-induced neuritogenesis by the wild-type and mutant heparanase. The impaired differentiation by knock down of heparanase could be restored by transfection of wild-type or mutant heparanase in PC12 cells. The results of the present study suggest that heparanase, at least in the non-enzymatic form, may promote NGF-induced neuritogenesis via the p38 MAPK pathway. Abbreviations: BM, basement membrane; BrdU, bromodeoxyuridine; CNS, central nervous system; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; EGR1, early growth response 1; EMSA, electrophoretic mobility-shift assay; ERK, extracellular-signal-regulated kinase; GAG, glycosaminoglycan; GAP43, growth-associated protein 43; GFP, green fluorescent protein; HEK, human embryonic kidney; HS, heparan sulfate; HSPG, HS proteoglycan; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; M-MLV, Moloney murine leukaemia virus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NGF, nerve growth factor; RT, reverse transcription; SAPK, stress-activated protein kinase; shRNA, short hairpin RNA; VEGF, vascular endothelial growth factor