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. 2001 Apr 15;15(8):968-80.
doi: 10.1101/gad.189601.

The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44

H Morrison  1 , L S Sherman, J Legg, F Banine, C Isacke, C A Haipek, D H Gutmann, H Ponta, P Herrlich
Affiliations

The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44

H Morrison et al. Genes Dev. .

Abstract

The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin's growth-inhibitory activity depends on specific interaction with the cytoplasmic tail of CD44, a transmembrane HA receptor. At low cell density, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data indicate that merlin and CD44 form a molecular switch that specifies cell growth arrest or proliferation.

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Figures

Figure 1
Figure 1
Regulated tumor suppressor function of merlin expressed in the schwannoma cell line RT4-D6P2T. (A) At confluency, merlin expression is increased in primary rat Schwann cells but not in the RT4-D6P2T schwannoma cell line. Primary rat Schwann cells at early passage (2–3) were plated on poly-l-lysine-coated 100 mm dishes in DMEM + 10% FCS at high- and low-cell densities, RT4-D6P2T schwannoma cells on uncoated plates. After 24 h, lysates from density-matched cells were produced and resolved on a mini-gel followed by Western blotting using the antimerlin antibody C18 (αmerlin; merlin band at 70 kD). The equal loading of 25 μg of total protein was confirmed by an actin Western blot (αactin; apparent molecular mass 43 kD). (B) Doxycycline-inducible merlin expression clones. RT4-D6P2T cells were stably cotransfected with the r-tet regulator and doxycycline-inducible wild-type (clones 54 and 67) and mutant (L64P) merlin constructs. Cells at 70% confluency were harvested at 8 h after addition of doxycycline. Resolution of the 70 kD merlin band as in A. (C) Merlin reduces agar colony formation. Doxycycline-inducible clones expressing either wild-type merlin (clones 54 and 67), the mutant L64P, or vector control cells were placed in soft agar (see Material and Methods). Average number of colonies per well were plotted and standard errors indicated. (D) Merlin inhibits tumor growth in vivo. Subcutaneous tumor growth after injection of clone 54 cells into nude mice (see Material and Methods). Where indicated, doxycycline was added to the drinking water. The experiments also were performed with clone 67 with similar results.
Figure 2
Figure 2
Activation of merlin at high cell density. (A) Wild-type merlin reduces growth in culture dishes but only at high cell density. RT4-D6P2T clones 54, 67, and L64P and vector control cells were seeded at low density (triplicates in 24-well dishes) and cultivated in the presence or absence of doxycycline. Cells per well were counted at 24-h intervals and the numbers plotted starting at 24 h after seeding. Standard errors are indicated. The insert shows a Western blot using an anti-Rb antibody (and anti-actin loading control) demonstrating that at high cell density (day 3 of culture) in the presence doxycycline, Rb is hypophosphorylated. Apparent molecular weights of phosphorylated, hypophosphorylated Rb and of actin are 112, 110, and 43kD, respectively. (B) Merlin reduces foci formation in culture dishes. Photomicrographs of RT4-D6P2T clone 67 cells in culture dishes at day 5 after seeding at low cell density in the absence or presence of doxycycline. Arrows in the left panel indicate large foci. Note absence of focus formation in the right panel.
Figure 2
Figure 2
Activation of merlin at high cell density. (A) Wild-type merlin reduces growth in culture dishes but only at high cell density. RT4-D6P2T clones 54, 67, and L64P and vector control cells were seeded at low density (triplicates in 24-well dishes) and cultivated in the presence or absence of doxycycline. Cells per well were counted at 24-h intervals and the numbers plotted starting at 24 h after seeding. Standard errors are indicated. The insert shows a Western blot using an anti-Rb antibody (and anti-actin loading control) demonstrating that at high cell density (day 3 of culture) in the presence doxycycline, Rb is hypophosphorylated. Apparent molecular weights of phosphorylated, hypophosphorylated Rb and of actin are 112, 110, and 43kD, respectively. (B) Merlin reduces foci formation in culture dishes. Photomicrographs of RT4-D6P2T clone 67 cells in culture dishes at day 5 after seeding at low cell density in the absence or presence of doxycycline. Arrows in the left panel indicate large foci. Note absence of focus formation in the right panel.
Figure 3
Figure 3
High cell density triggers the activation of merlin and the functionally required association with CD44. A hypophosphorylated form of the endogenous (A) or doxycyclin-induced (B) merlin can be resolved in confluent cultures. Clone 54 cells were cultivated without doxycycline (endogenous merlin; A) or 54, 67, and L64P cells in the presence of doxycycline for 8 h (B). Equal cell numbers from either logarithmic or confluent (high cell density) growth conditions were lysed and the lysates subjected to a higher resolution gel and Western blotting using the anti-merlin antibody (C18) or anti-actin. Where indicated, merlin was immunoprecipitated prior to gel electrophoresis. “Control IP” defines a precipitation with Sepharose A beads alone. The exposure time in B was about 30 times shorter than in A. As indicated, lysates or immunoprecipitates were treated with calf intestinal phosphatase (CIP). The slower migrating band disappears upon digestion with CIP (shown for clone 54). L64P mutant merlin, however, remained phosphorylated even at cell confluency shown by the fact that the slower migrating band was converted to a faster migrating band upon digestion with CIP. Apparent molecular mass: 70 kD for the phosphorylated merlin form, 69 kD for the hypophosphorylated form. Marker proteins were run with each gel and the sizes confirmed in all experiments. Doxycycline-induced (C) and endogenous (D) hypophosphorylated merlin, but not ezrin and moesin, coprecipitated with CD44 from lysates of high cell density cultures. For coimmunoprecipitation experiments, lysates of high cell density cultures were incubated overnight with the CD44 antibody 5G8 and then treated with protein A sepharose. “Control IP” as in B. Western blotting was performed using either an anti-merlin antibody or an anti-ezrin antibody that also recognizes moesin, showing ezrin at a molecular weight of 80 kD and moesin at a molecular weight of 76 kD. Mutant merlin L64P is not precipitated with CD44. Sequestering of merlin by binding to overexpressed, soluble CD44 cytoplasmic tails abolishes merlin function (E). The doxycycline-inducible merlin expressing clone 54 was stably transfected with expression constructs encoding either a wild-type or mutated CD44 cytoplasmic tail fused to GST or GST alone. The mutant form was chosen for its inability to bind ezrin (see text and Material and Methods). Subclones with high expression levels (see Western blot with an anti-CD44 tail-specific antibody [αCD44tail]) were selected, placed in soft agar, and their colony forming ability (see Material and Methods) was determined in the presence or absence of doxycycline. By pull-down experiments (F) the association of merlin with the wild type, but not the mutant cytoplasmic tail of CD44, was confirmed. To this end, cells of clone 67 transfected with either wild-type or mutant CD44 tail constructs, were plated at high cell density. Eight hours after doxycycline addition, the cells were lysed and the CD44 tails enriched by GSH agarose and protein resolved by mini-gel. Western blotting was performed using either anti-merlin (C-18), anti-ezrin, or anti-CD44 tail antibodies. Note that the resolution of the gels differs from those in A through D.
Figure 4
Figure 4
Ezrin, moesin, and phosphorylated merlin are complexed with CD44 at low cell density. Ezrin, moesin, and doxycycline-induced (A) or endogenous (B) phosphorylated merlin are coprecipitated with CD44. For coimmunoprecipitation experiments, clone 67 cells and parental RT4-D6P2T were seeded at low cell density. All procedures were as in Fig. 3C and D. Lysates of clone 67 cells at high cell density (conf) were included to control for the resolution of merlin modifications. Overexpressed wild-type cytoplasmic tail of CD44 pulls down merlin, ezrin, and moesin from lysates of cells at low cell density (C). Cells of clone 67 transfected with either wild-type or mutant CD44 tail constructs, were plated at low cell density. Eight hours after doxycycline addition, the cells were lysed and the CD44 tails enriched by GSH agarose. Procedures as in Fig. 3F. Phosphorylated merlin coprecipitates with ezrin and moesin at low cell density (D). Clone 67 cells were plated at low or high density and treated with doxycycline for 8 h. Coimmunoprecipitation was performed as described in A, except for the use of an antiezrin/moesin antibody (C-19) for the precipitation. Lysates were subjected to a higher resolution gel. Western blotting as in A.
Figure 5
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.
Figure 6
Figure 6
CD44-dependent merlin activation and growth inhibition in several cell lines. (A) The CD44-specific antibody IM7 activates merlin. Cells from clones 54 and 67 were seeded into 8-well chamber slides at low density. Either IM7 or Hermes-3 (control antibody) and doxycycline were added where indicated and incubated for 12 h (control cells were without antibody). Cells then were labeled with BrdU for 60 min and stained for incorporation using a biotinylated BrdU antibody. The mean percent cells that incorporated BrdU, are plotted and standard errors are indicated. The insert shows a Western blot (higher resolution gel) detecting doxycycline-induced merlin (α-merlin C-18) from lysates of clone 67 cells at low density prior (0 min) or after treatment with IM7 for 5 min. (B) CD44-dependent activation of merlin and inhibition of growth in several types of cells. All cells were seeded into 8-well chamber slides at low density and incubated with HA or IM7 where indicated, for 12 h. BrdU incorporation and detection as in A. The CD44 antibody IM7 (as well as KM81, not shown) inhibited proliferation of NIH3T3 cells similarly to the result in A. HA activated merlin in mouse fibroblasts (NIH3T3 cells), in dog kidney epithelial cells (MDCK), and rat kangaroo epithelial cells (Ptk2). In addition cells negative for CD44 when stably transfected with a construct encoding full-length CD44s but not CD44 mutated in the ERM-binding domain, were also growth-inhibited in response to HA treatment. (C) Addition of HA induces dephosphorylation of merlin in several types of cells. NIH3T3 and MDCK cells as in B were harvested untreated (a) or after treatment with 100 μg/mL of HA (b) for 12 h (similar dephosphorylation after 5 min, not shown). Cell lysates were treated with CIP (c) prior to Western blotting (higher-resolution gel) as in A.
Figure 7
Figure 7
Model of CD44 action in logarithmic and confluent growth conditions. Specific ligands determine two functional states of CD44 that influence cytoplasmic complexes. The ligands of the growth mode have not been defined. It is, however, known that CD44, and particularly the larger-splice variants, serve as platform for the activation of growth factors (GF; Bourguignon et al. 1997; Sherman et al. 1998; Van der Voort et al. 1999; Sherman et al. 2000; V. Orian-Rousseau, L. Chen, P. Herrlich, and H. Ponta, unpubl.). (GFR) growth-factor receptor; (PP) protein phosphatase. Additional components are likely associated with the CD44-bound complexes (gray boxes).
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