Figure 5.
CD44 ligands influence merlin activity. The soluble extracellular domain of CD44 sequesters different ligands from either logarithmically growing or confluent cells. Cells of clone 54 were seeded in triplicates into 24-well plates and treated with doxycycline to induce merlin expression. Either on day 1 (logarithmically growing cells) or on day 3 (confluent cells) 10 ng/mL of the soluble extracellular domain of CD44, produced in COS-7 cells as described (Bartolazzi et al. 1994), was added, either as wild-type form (solCD44wt) or as mutant-peptide defective in glycosaminoglycan binding (solCD44mut). As control, cells without doxycycline were treated identically (not shown). Both solCD44wt or solCD44mut activated merlin in logarithmically growing cells (left growth curve, seen as reduced growth rate), whereas merlin activity was blocked in confluent cultures (right growth curve, seen as reversal of growth inhibition by merlin), and this effect was dependent on the presence of the glycosaminoglycan-binding motif. Similar results were obtained with clone 67. The insert shows a Western blot (higher-resolution gel) detecting doxycycline-induced merlin (α-merlin C-18) from lysates of clone 54 cells at low cell density prior (0 min) or after treatment with 10 ng/mL of solCD44wt for 5 min. HA-dependent growth inhibition in logarithmically growing schwannoma cells overexpressing merlin. Cells of clone 67 were seeded in triplicates into 24-well plates. Doxycycline and HA (100 μg/mL) were added as indicated 1 d prior to the cell counts shown. HA addition to doxycycline-induced cells caused retardation of growth (left panel). Doxycycline alone did not affect growth during logarithmic phase (see also Fig. 2A). Similar results were obtained with clone 54. The insert shows a Western blot (higher resolution gel) detecting doxycycline-induced merlin from lysates of clone 67 cells at low density prior (0 min) or after treatment with 100 μg/mL of HA for 5 min.