The main hyaluronan receptor, CD44, is linked to the actin cytoskeleton through its interactions with actin-binding proteins, especially the phosphorylated form of ERM (pERM). pERM and CD44–pERM interaction are required for proper actin organization. In vertebrates, hyaluronan is degraded by several hyaluronidases, among which Hyal2. Hyal2 is a GPI-anchored ubiquitous protein that is suggested to play important roles in tumor growth and inflammation by cleaving extracellular hyaluronan into smaller fragments. Various tumors and cancer cell lines were found to overexpress Hyal2. However, the actual functions of this enzyme are not elucidated. We have developed several cell models to explore the influence of Hyal2 on cellular behavior.

The current experiments were performed in the rat fibroblastic cell line Rat-1. As the rate of endogenous expression of Hyal2 in these cell lines is low, stable transfectants of Hyal2

(Rat1Hy2+) and a mock-transfected cell line (Rat1mock) were established. The expression of Hyal2 was checked using immunoprecipitation (IP) with a polyclonal antibody and real time PCR. Then, cell phenotype was studied using phase contrast and scanning electron microscopy. Hyal2 overexpression induced plasma membrane protrusions such as microspikes and microvilli, while decreasing overall cell size and the "adherent surface". Rat1mock cells did not differ from control cells. In Rat1Hy2+ cells, the actin filaments organization was severely distorted and mostly replaced by actin bundles. In addition, Hyal2 overexpression disrupted CD44(ERM and CD44(ERM(actin interactions as assessed using co-IP. The amount of ERM was not significantly diminished in Rat1Hy2+ cells but the amount of pERM (active form) was reduced and both CD44(pERM and actin(pERM interactions were severed.

Cell motility was increased two-fold by Hyal2 overexpression (12.41.0 vs 26.61.2 cells crossing an arbitrary line in 8 h for Rat1mock and Rat1Hy2+ cells, respectively; p<0.001). Hyal2 also increased cellular proliferation (assessed using ³H-thymidine incorporation) by 29% (n=3, P<0.05) and induced anchorage-independent growth in soft agar.

In summary, Hyal2 overexpression prevents ERM phosphorylation which is required for CD44 linkage to the actin cytoskeleton and for actin organization. Consequently, CD44(pERM(actin interactions are disrupted, the actin network is reorganized, cell motility increases, and the malignant phenotype is favored. We have developed a Hyal2 knockout mouse model and are poised to gauge the relevance of these events in vivo.