ANGIOGENIC OLIGOSACCHARIDES OF HYALURONAN INDUCE PROTEIN-TYROSINE KINASE-ACTIVITY IN ENDOTHELIAL-CELLS AND ACTIVATE A CYTOPLASMIC SIGNAL-TRANSDUCTION PATHWAY RESULTING IN PROLIFERATION

We have recently shown that the degradation products of hyaluronan of 3 to 10 disaccharides (o-HA), but not native high molecular weight hya luronan, can induce angiogenesis in vivo and, as such, o-HA is an impo rtant regulator of the neovascularization process. As a continuation o f this work, we have studied the cytoplasmic signal transduction pathw ays responsible for o-HA-activated endothelial cell proliferation. We show that the addition of o-HA (1 mu g/ml) to bovine aortic endothelia l cells induces tyrosine phosphorylation of multiple proteins within 1 minute and that the activity remains above basal levels for at least 24 hours. Increased phosphorylation of the CD44 receptor was also obse rved. Pretreatment of cells with an anti-CD44-receptor antibody (5 mu g/ml) or the tyrosine kinase inhibitor genistein (10 mu M) inhibited b oth o-HA-induced proliferation (p < 0.05) and protein tyrosine phospho rylation. In comparison, native hyaluronan had little effect on tyrosi ne phosphorylation across the same time period. Protein kinase C (PKC) activity was increased 2- to 3-fold in the membranes of cells treated with o-HA, and a pretreatment with phorbol 12,13-dibutyrate (PDBu) to down-regulate PKC significantly inhibited o-HA-induced cell prolifera tion (p < 0.05). Examination by Western blotting showed that only the beta(1) and epsilon isoforms remained translocated to the membrane for at least 24 hours. These isoforms seem to be involved in modulating t he proliferative effects of o-HA, because the transient translocation of PKC isoforms by PDBu was not sufficient to induce mitogenesis. Furt hermore, we show that PKC activation of the cytoplasmic kinase cascade (Raf-1 kinase, MAP kinase kinase [MEK-1], and extracellular signal-re gulated kinase [ERK-1]) by o-HA culminated in the nuclear translocatio n of ERK-1. This pathway is essentially linear, as shown by the abilit y of specific enzyme inhibitors (PDBu and PD98059) to prevent both act ivation of ERK-1- and o-HA-induced proliferation. We conclude that pho sphorylation of the CD44 receptor results in an increase in tyrosine p hosphorylation, leading to the activation of a cytoplasmic cascade and cell proliferation; this concurs with previous work, which showed tha t o-HA-induced proliferation of endothelial cells is CD44-receptor-med iated and accompanied by early response gene activation.