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 α vβ 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 α vβ 3-integrin, GRGDSP (50 nM), or an inhibitor of protein synthesis, anisomycin (10 μg/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