Article Figures & Data
Figures
- FIG. 1.
Merlin regulates glial cell growth in vitro. (A) Immunoblot analysis of merlin expression in primary glial cell lysates demonstrates a >95% reduction in merlin expression in Nf2−/− glia (Ad5-Cre treatment) relative to that in WT glia (Ad-LacZ treatment), as assessed by scanning densitometry. α-Tub, α-tubulin. (B) Nf2−/− glia exhibited a 2.3-fold increase in proliferation relative to WT glia, as determined by [3H]thymidine incorporation. *, P < 0.001 (unpaired t test). (C) WT and Nf2−/− glial cells were seeded in fibronectin-coated plates, and the number of adherent cells was determined using a spectrophotometric assay. No differences in cell attachment were observed between WT and Nf2−/− glial cells. OD, optical density. (D) WT and Nf2−/− glial cells were immunostained with cleaved caspase-3 antibody in the presence and absence of staurosporine (5 nM). No differences in apoptosis were observed between WT and Nf2-deficient glial cells in either the presence or absence of staurosporine. (E) β-Catenin staining along cell contact junctions in WT and NF2−/− glial cells revealed similar patterns by immunofluorescence microscopy.
- FIG. 2.
Merlin regulates glial cell growth in vivo. (A) Immunoblot analysis of merlin expression in Nf2flox/flox and Nf2GFAPCKO mouse brain lysates also demonstrates a >90% reduction in merlin expression in Nf2GFAPCKO mice, as assessed by scanning densitometry. α-Tub, α-tubulin. (B) Representative immunofluorescence photomicrographs of hippocampi from BrdU-injected Nf2flox/flox and Nf2GFAPCKO mice labeled with both BrdU (green) and GFAP (red) antibodies. Nf2GFAPCKO mouse brains had 2.2-fold more BrdU-positive cells than Nf2flox/flox mouse brains. *, P < 0.001 (unpaired t test). (C) Representative photomicrographs of hippocampi from Nf2flox/flox and Nf2GFAPCKO mice labeled with GFAP antibodies. Nf2GFAPCKO mouse brains had a 2.4-fold increase in the number of GFAP-expressing cells compared to Nf2flox/flox mouse brains. *, P = 0.001 (unpaired t test). (D) Representative photomicrographs of in vivo TUNEL staining of hippocampi from Nf2flox/flox and Nf2GFAPCKO mouse brains, revealing no significant differences in the number of apoptotic cells between Nf2GFAPCKO and Nf2flox/flox mice.
- FIG. 3.
Expression of WT but not mutant merlin reduces Nf2−/− glial cell proliferation in vitro. (A) Immunoblot analysis of merlin expression in WT and Nf2−/− glia infected with empty MSCV, MSCV containing WT merlin (MSCV.NF2), or MSCV containing mutant merlin (MSCV.NF2.L64P). α-Tubulin (α-Tub) protein expression was used as an internal control for equal protein loading in all Western blot analyses. (B) Merlin reintroduction reduces the increased proliferation observed in Nf2−/− glia to WT levels, whereas the reintroduction of mutant (L64P) merlin has no effect. *, P < 0.003 (unpaired t test).
- FIG. 4.
Merlin regulates Src activation in Nf2−/− glial cells. (A) Rac1 activation assay shows similar Rac1 activities in WT and Nf2−/− glial cell lysates. Immunoblot analyses using phospho-specific MAPK and AKT antibodies demonstrated no change in MAPK or AKT activation in Nf2−/− glia compared to WT glia in vitro. Total Rac1, MAPK, and AKT expression levels were used as internal controls for equal protein loading. α-Tub, α-tubulin. (B) Immunoblot analysis of Src activation in WT and Nf2-deficient (Nf2−/−) glia, using phospho-specific Src antibodies (specific for phosphorylation at Y416, Y215, and Y527). Nf2−/− glia exhibited a 6.7-fold increase in Src activation at tyrosine 416 compared to WT glia, with no change in Src phosphorylation at tyrosine 215 or tyrosine 527. (C) Nf2GFAPCKO mouse brains also show increased activation of Src (2.9-fold) compared to Nf2flox/flox mouse brains, as assessed by immunoblot analyses and scanning densitometry. (D) In a Src kinase activity assay, Nf2−/− glia exhibited a 4.7-fold increase in Src activity relative to WT glia. *, P < 0.001 (unpaired t test). OD, optical density. (E) WT but not mutant merlin reduces Src activation in Nf2−/− glia.
- FIG. 5.
Src inhibition restores Nf2−/− glial cell proliferation to WT levels in vitro. (A) The PP2 Src inhibitor blocks Src activation in Nf2−/− glia. (B) PP2 treatment eliminates the growth advantage observed in Nf2−/− glia, as measured by [3H]thymidine incorporation. *, P < 0.001 (unpaired t test). (C) Immunoblot analysis of Src expression and activation in WT and Nf2−/− glia following infection with Src.shRNA or vector (pLK01-GFP) demonstrates >90% inhibition of Src expression and activation after Src.shRNA treatment, as assessed by scanning densitometry. Total Src or tubulin (α-Tub) protein expression was used as an internal control for equal protein loading. (D) Src.shRNA treatment eliminates the growth advantage observed in Nf2−/− glia, as measured by [3H]thymidine incorporation *, P < 0.001 (unpaired t test).
- FIG. 6.
Merlin regulates phosphorylation of the Src effectors FAK and paxillin. (A) Immunoblot analyses of WT and Nf2−/− glial lysates following phosphotyrosine (pTyr) affinity binding with antibodies against p130CAS, Ack1, and CASPR show no change in the phosphorylation state of these Src effectors. Merlin and α-tubulin (α-Tub) expression levels are included as internal controls. (B) Nf2-deficient (Nf2−/−) glia demonstrated a 4.9-fold increase in FAK (Y576/577) phosphorylation and a 5.5-fold increase in paxillin (Pxn) phosphorylation (Y118) (left). Nf2GFAPCKO mouse brains also showed increased activation of FAK (2.2-fold) and paxillin (2.5-fold) compared to Nf2flox/flox mouse brains, as assessed by immunoblot analyses and scanning densitometry (right). (C) WT but not mutant merlin inhibits both FAK and paxillin phosphorylation in Nf2−/− glia. (D) The Src inhibitor PP2 (left) as well as Src.shRNA silencing (right) reverses the hyperphosphorylation of both FAK and paxillin in Nf2−/− glia.
- FIG. 7.
Merlin growth regulation requires FAK and paxillin signaling. (A) (Top) Echistatin inhibits the phosphorylation of both FAK and paxillin but has no effect on Src activation. (Bottom) Echistatin treatment eliminates the growth advantage observed in Nf2−/− glia, as determined by [3H]thymidine incorporation. *, P < 0.001 (unpaired t test). (B) (Top) FAK.shRNA inhibits the expression of FAK (>92% reduction) as well as the phosphorylation of both FAK and paxillin. No effect on Src activation was observed. (Bottom) FAK.shRNA treatment eliminates the growth advantage observed in Nf2−/− glia, as assessed by [3H]thymidine incorporation. *, P < 0.003 (unpaired t test). (C) (Top) Pxn.shRNA treatment inhibits the expression of paxillin (>95% reduction) as well as the phosphorylation of paxillin but has no effect on FAK and Src activation. Total tubulin, Src, FAK, and paxillin expression levels were used as internal controls for equal protein loading. (Bottom) Pxn.shRNA treatment eliminates the growth advantage observed in Nf2−/− glia, as determined by [3H]thymidine incorporation. *, P < 0.001 (unpaired t test).
- FIG. 8.
Merlin regulation of glial cell growth is Src dependent in vivo. (A) Representative immunofluorescence photomicrographs of hippocampi from BrdU-injected Nf2flox/flox and Nf2GFAPCKO mice, with or without treatment with the Src inhibitor PP2 (2 mg/kg). Images were labeled with both BrdU (green) and GFAP (red) antibodies. A twofold decrease in the number of BrdU-positive cells was observed in the PP2-treated Nf2GFAPCKO brains compared to vehicle-treated mouse brains. *, P = 0.001 (unpaired t test). (B) Representative photomicrographs of hippocampi labeled with GFAP antibodies. PP2-treated Nf2GFAPCKO mice exhibited a twofold decrease in the number of GFAP-labeled cells compared to vehicle-treated Nf2GFAPCKO littermates. No effect of PP2 treatment on Nf2flox/flox mice was seen. *, P < 0.002 (unpaired t test). (C) Immunoblot analysis demonstrates amelioration of the increased Src, FAK, and paxillin activation following PP2 treatment. The expression levels of tubulin, total Src, FAK, and paxillin were used as internal controls for equal protein loading.
- FIG. 9.
Merlin regulates ErbB2 activation in glial cells. (A) Immunoblot analysis of WT and Nf2−/− glial lysates, using a phospho-specific EGFR antibody (Y845), demonstrates no change in EGFR activation. Total EGFR expression was used as an internal control for equal protein loading. (B) Immunoblot analyses of WT and Nf2−/− glial lysates following phosphotyrosine (pTyr) affinity binding with antibodies against ErbB4 and ErbB3 show no change in phosphorylation state for the other two ErbB family members. Merlin and tubulin expression levels were included as internal controls. (C) Nf2−/− glia exhibited a 7.2-fold increase in ErbB2-Y877 activation compared to WT controls in vitro, with no change in ErbB2-Y1248 phosphorylation. (D) Nf2GFAPCKO mouse brains exhibited a 2.3-fold increase in ErbB2 (Y877) activation compared to WT controls in vivo, as assessed by immunoblotting analysis and scanning densitometry. (E) In an ErbB2 kinase activity assay, Nf2−/− glia exhibited a 3.2-fold increase in ErbB2 activity relative to WT glia. *, P < 0.001 (unpaired t test). OD, optical density. (F) WT but not mutant merlin expression reduces ErbB2 activation in Nf2−/− glia.
- FIG. 10.
ErbB2 activation functions upstream of Src/FAK/paxillin in the merlin glial control growth pathway. (A) PP2 treatment of Nf2−/− glial cells has no effect on ErbB2 hyperactivation in vitro or in vivo. (B) Echistatin treatment of Nf2−/− glial cells has no effect on ErbB2 hyperactivation in vitro. Genetic inhibition of Src (C), FAK (D), or paxillin (E) in Nf2−/− glial cells, using Src.shRNA, FAK.shRNA, or Pxn.shRNA, respectively, has no effect on ErbB2 hyperactivation. Total tubulin and total ErbB2 expression levels were used as internal loading controls.
- FIG. 11.
Merlin regulation of glial cell growth requires ErbB2 activation. (A) (Top) Inhibition of ErbB2 activation with AG825 in Nf2−/− glia reduces Src, FAK, and paxillin hyperactivation to WT levels. (Bottom) AG825 treatment of Nf2−/− glia reduces cell proliferation to WT levels, as assessed by [3H]thymidine incorporation. No effect of AG825 treatment was observed in WT glia. *, P < 0.001 (unpaired t test). (B) (Top) Inhibition of ErbB2 expression and activation with ErbB2.shRNA (>98% reduction) reduces the hyperactivation of Src, FAK, and paxillin in Nf2−/− glia to WT levels. Total tubulin, Src, FAK, and ErbB2 expression levels were used as internal loading controls. (Bottom) Treatment with ErbB2.shRNA but not with vector eliminates the growth advantage observed in Nf2−/− glia, as assessed by [3H]thymidine incorporation. No effect of ErbB2.shRNA was observed in WT glia. *, P < 0.002 (unpaired t test).
- FIG. 12.
Merlin directly regulates the interaction between ErbB2 and Src. (A) Immunoprecipitation of ErbB2 from WT and Nf2−/− glia shows that the association between ErbB2 and Src as well as ErbB2 and p-Src (Y416) occurs only in Nf2-deficient glia. (B) WT but not mutant merlin expression inhibits ErbB2 and Src interaction in Nf2−/− glia. (C) AG825 treatment inhibits the association between ErbB2 and Src in Nf2-deficient glia. (D) PP2 treatment has no effect on the association between ErbB2 and Src in Nf2-deficient glia. Total ErbB2 was used as an internal control for ErbB2 precipitation. (E) Src and merlin directly associate with ErbB2 following immunoprecipitation of [35S]methionine-radiolabeled merlin (NF2.WT or NF2.L64P) or Src proteins with recombinant His-tagged ErbB2 (ErbB2.His). (Top) No binding to ErbB2 was observed with mutant merlin. (Middle) In the absence of the ErbB2 recombinant protein, there was no binding of radiolabeled NF2.WT, NF2.L64P, or Src protein to the His affinity gel. (Bottom) Input levels of radiolabeled NF2.WT, NF2.L64P, and Src proteins in the ErbB2.His immunoprecipitation analysis were used as normalization controls in scanning densitometric analyses to determine the percentages of bound proteins. A total of 32% of radiolabeled NF2.WT and 28% of Src proteins were bound to recombinant ErbB2 protein, whereas <1% of NF2.L64P was bound to ErbB2. (F) (Top) ErbB2-Src binding was decreased 67% in the presence of equal amounts of merlin and completely eliminated in the presence of a fivefold excess of merlin. (G) (Top) ErbB2-merlin binding was decreased 52% in the presence of equal amounts of Src, decreased 94% in the presence of a 5-fold excess of Src, and completely eliminated in the presence of a 10-fold excess of Src (top). (Bottom [F and G]) Immunoblotting was used to verify the amounts of nonradiolabeled merlin and Src added to each reaction mix.
- FIG. 13.
Merlin interacts with both ErbB2 and Src. (A) Immunoprecipitation of Src from WT and Nf2−/− glia shows that the association between Src and ErbB2 occurs only in Nf2-deficient glia. (B) WT but not mutant merlin expression inhibits Src and ErbB2 interaction in Nf2−/− glia. (C) AG825 treatment inhibits the association between Src and ErbB2 in Nf2-deficient glia. (D) PP2 treatment has no effect on the association between Src and ErbB2 in Nf2-deficient glia. Total Src was used as an internal control for Src precipitations. In WT glia, merlin and Src associate; however, this binding is not affected by SrbB2 or Src activation.
- FIG. 14.
Merlin binding to ErbB2 precludes the association of Src with ErbB2. (A) In an in vitro binding assay, [35S]methionine-radiolabeled full-length merlin containing residues 1 to 595 (NF2.WT), NF2.C-term containing residues 300 to 595, and NF2.C-term containing residues 300 to 557, bound to recombinant His-tagged ErbB2 (ErbB2.His). In contrast, no binding to ErbB2 was observed using NF2.N-term containing residues 1 to 300. Input levels for the reactions are shown and were used as normalization controls in scanning densitometric analyses to determine the percentages of bound proteins. Thirty percent of radiolabeled NF2.WT, 25% of NF2.C-term, and 24% of NF2.300-557 bound to recombinant ErbB2 protein, whereas <1% of NF2.N-term bound to ErbB2. (B) In an in vitro binding assay, [35S]methionine-radiolabeled full-length merlin containing residues 1 to 595 (NF2.WT), and NF2.C-term containing residues 300 to 595, bound to recombinant His-tagged ErbB2 (ErbB2.His). In contrast, no binding to ErbB2 was observed using either NF2.C-term containing residues 300 to 557, or NF2.N-term containing residues 1 to 300. Input levels for the reactions are shown and were used as normalization controls in scanning densitometric analyses to determine the percentages of bound proteins. Thirty-one percent of radiolabeled NF2.WT and 19% of NF2.C-term bound to recombinant Src protein, whereas <1% of NF2.N-term and NF2.300-557 bound to Src. (C) (Top) ErbB2-Src binding was decreased 48% in the presence of equal amounts of NF2.300-557, decreased 96% in the presence of a 5-fold excess of NF2.300-557, and completely eliminated in the presence of a 10-fold excess of NF2.300-557. (Bottom) Coomassie staining was used to verify the amount of nonradiolabeled NF2.300-557 added to each reaction mix. (D) (Top) NF2.300-557-ErbB2 binding was not altered by equal or excess amounts of Src. (Bottom) Immunoblotting was used to verify the amount of nonradiolabeled Src added to the reaction mixtures. (E) Potential molecular model for merlin growth regulation in glial cells. In WT cells, merlin associates with ErbB2 and precludes Src binding to ErbB2. In the absence of functional merlin, Src binds to activated ErbB2, leading to ErbB2-mediated Src phosphorylation/activation, Src effector protein phosphorylation, and increased glial cell growth.
