We have recently shown that the degradation products of hyaluronan of 3 to 10 disaccharides (o-HA), but not native high molecular weight hyaluronan, can induce angiogenesis in vivo and, as such, o-HA is an important regulator of the neovascularization process. As a continuation of this work, we have studied the cytoplasmic signal transduction pathways responsible for o-HA-activated endothelial cell proliferation. We show that the addition of o-HA (1 microg/ml) to bovine aortic endothelial 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 observed. Pretreatment of cells with an anti-CD44-receptor antibody (5 microg/ml) or the tyrosine kinase inhibitor genistein (10 microM) inhibited both o-HA-induced proliferation (p < 0.05) and protein tyrosine phosphorylation. In comparison, native hyaluronan had little effect on tyrosine 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 proliferation (p < 0.05). Examination by Western blotting showed that only the betaI and epsilon isoforms remained translocated to the membrane for at least 24 hours. These isoforms seem to be involved in modulating the proliferative effects of o-HA, because the transient translocation of PKC isoforms by PDBu was not sufficient to induce mitogenesis. Furthermore, we show that PKC activation of the cytoplasmic kinase cascade (Raf-1 kinase, MAP kinase kinase [MEK-1], and extracellular signal-regulated kinase [ERK-1]) by o-HA culminated in the nuclear translocation of ERK-1. This pathway is essentially linear, as shown by the ability of specific enzyme inhibitors (PDBu and PD98059) to prevent both activation of ERK-1- and o-HA-induced proliferation. We conclude that phosphorylation of the CD44 receptor results in an increase in tyrosine phosphorylation, leading to the activation of a cytoplasmic cascade and cell proliferation; this concurs with previous work, which showed that o-HA-induced proliferation of endothelial cells is CD44-receptor-mediated and accompanied by early response gene activation.