Osteopontin increases CD44 expression and cell adhesion in RAW 264.7 murine leukemia cells

Background: Osteopontin (OPN) is an inducible cell attachment protein which binds alpha(v)beta(3)-integrin and CD44 receptors to promote tumor metastasis. We hypothesized that OPN alters expression of its CD44 receptor to promote neoplastic cell migration. Methods: RAW264.7 cells were stimulated with OPN (0-10 nM) for 0-12 hours to determine the time- and concentration-dependence of CD44 protein and mRNA expression. In selected instances, a competitive ligand for the alpha(v)beta(3)-integrin, GRGDSP (50 nM), or an inhibitor of protein synthesis, anisomycin (10 mug/ml), was added. Cell adhesion to hyaluronan was assayed with the crystal violet assay. Results: OPN upregulates plasma membrane total CD44 protein in a concentration-(ANOVA P = 0.001) and time-dependent (ANOVA P = 0.001) fashion. CD44v6 is not altered. Cell adhesion to hyaluronate increases in parallel with CD44 expression. Steady state mRNA levels for CD44 are not altered by OPN. 5 nM OPN increases CD44 protein half-life from 105 +/- 11 minutes to 278 +/- 15 minutes. (P < 0.03) Blockade of either alpha(v)beta(3)-integrin ablates the OPN-dependent increase in CD44. Conclusions: These data indicate that OPN increases plasma membrane CD44 expression and cell adhesion by binding to its alpha(v)beta(3)-integrin receptor. We conclude that OPN may promote tumor metastatic behavior by CD44 expression. (C) 2004 Elsevier B.V. All rights reserved.