Overexpression of CD44 in pl85(neu)-transfected NIH3T3 cells promotes an up-regulation of hyaluronic acid-mediated membrane-cytoskeleton interaction and cell adhesion

Abstract

CD44 is a transmembrane glycoprotein known to bind hyaluronic acid (HA) in its extracellular domain and to contain at least one ankyrin-binding site in its cytoplasmic domain. In this study we have examined CD44 expression in a mouse fibroblast cell line transfected with the pl85(neu) oncogene cDNA. The results of RT-PCR and Southern blot analyses reveal that CD44s (CD44 standard form) transcript is expressed in both pl85(neu)-transfected cells and untransfected cells. Using surface iodination, anti-CD44 immunoprecipitation and immuno-binding assays, we have found that the number of CD44s molecules expressed on the surface of pl85(neu)-transfected cells are at least 4.5-fold higher than those detected on untransfected cells. Overexpression of surface CD44s in pl85(neu)-transfected cells results in a dramatic enhancement of HA-mediated cell adhesion. Scatchard plot analysis indicates that CD44s in pl85(neu) transfected cells binds directly and specifically to ankyrin. The binding affinity between CD44s and ankyrin in p185(neu)-transfected cells approximately 0.19 nM) appears to be somewhat higher than that found in the untransfected cells (K(p) approximately 0.30 nM). Double immunofluorescence staining and confocal microscopic analyses indicate that HA induces the HA receptor (i.e. CD44s) to form adhesion plaque-like structures, and causes an accumulation of intracellular ankyrin directly underneath HA receptor (CD44s)-adhesion plaque-like structures in pl85(neu)-transfected cells (but not in untransfected cells). These findings suggest that overexpression of CD44s and up-regulation of CD44s-ankyrin interaction by pl85(neu) oncogene may be one of the pre-requisite steps in regulating tumor cell behavior.