The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression

Abstract

Inactivation of the NF2 tumor suppressor gene has been observed in certain benign and malignant tumors. Recent studies have demonstrated that merlin, the product of the NF2 gene, is regulated by Rac/PAK signaling. However, the mechanism by which merlin acts as a tumor suppressor has remained obscure. In this report, we show that adenovirus-mediated expression of merlin in NF2-deficient tumor cells inhibits cell proliferation and arrests cells at G1 phase, concomitant with decreased expression of cyclin D1, inhibition of CDK4 activity, and dephosphorylation of pRB. The effect of merlin on cell cycle progression was partially overridden by ectopic expression of cyclin D1. RNA interference experiments showed that silencing of the endogenous NF2 gene results in upregulation of cyclin D1 and S-phase entry. Furthermore, PAK1-stimulated cyclin D1 promoter activity was repressed by cotransfection of NF2, and PAK activity was inhibited by expression of merlin. Interestingly, the S518A mutant form of merlin, which is refractory to phosphorylation by PAK, was more efficient than the wild-type protein in inhibiting cell cycle progression and in repressing cyclin D1 promoter activity. Collectively, our data indicate that merlin exerts its antiproliferative effect, at least in part, via repression of PAK-induced cyclin D1 expression, suggesting a unifying mechanism by which merlin inactivation might contribute to the overgrowth seen in both noninvasive and malignant tumors.

FIG. 1.

Downregulation of cyclin D1 expression by merlin. (A) Immunofluorescence demonstrating that adenovirus-mediated expression of merlin in NF2-deficient Meso 17 and Meso 35 cells is localized predominantly at the cytoplasmic membrane. (B) Meso 17 cells were infected with either AdCtrl or Ad-NF2. After 24 h, total RNA was isolated, reverse transcribed, labeled, and hybridized to microarrays consisting of nearly 40,000 cDNAs. Among the differences in the gene expression patterns, a 3.8-fold decrease of cyclin D1 expression was observed in cells infected with AdNF2 compared to those infected with AdCtrl. A section of the microarray slide that includes the cyclin D1 spot is shown. The color scale shows the magnitude of the difference from the mean, with red and green indicating transcript levels below and above the median, respectively. (C) Total RNA isolated from Meso 17 cells mock infected or infected with AdCtrl or AdNF2 was subjected to real-time PCR analysis with cyclin D1- and β-actin-specific primers. The histogram depicts the range of cyclin D1 expression. The data are normalized to β-actin and presented as fold changes in cyclin D1 levels relative to those of the mock-infected sample.

FIG. 2.

Analysis of cell cycle regulators in Meso 17 and Meso 35 cells after adenovirus-mediated expression of merlin. (A) Cell lysates were electrophoresed and immunoblotted with the indicated antibodies. Infection with AdNF2 or AdNF2 A518 decreased cyclin D1 and increased p27 expression levels, which were accompanied by dephosphorylation of pRB. (B) Total cellular protein was immunoprecipitated with CDK4 antibody, and kinase assays were performed using glutathione S-transferase-Rb as a substrate, as described in Materials and Methods. Reexpression of AdNF2 or AdNF2 A518 inhibits CDK4 kinase activity (upper panel). Immunoblot analyses of immunoprecipitates with anti-CDK4 antibody demonstrate equivalent loading in each lane (lower panel). IgG-LC, Ig light chain.

FIG. 3.

Induction G₁ cell cycle arrest and inhibition of cell proliferation by reexpression of merlin. (A) After adenoviral infection for 24 h, Meso 17 and Meso 35 cells were stained with propidium iodide and subjected to FACS analysis to demonstrate cell cycle distribution based on DNA content. The experiments were conducted three times with similar results. (B) Expression of merlin inhibits S-phase entry. Meso 17 and Meso 35 cells were mock infected or infected with AdCtrl, Ad-NF2, or Ad-NF2 A518 for 18 h and incubated for an additional 6 h in the presence of 20 μM BrdU. Red staining depicts BrdU-labeled nuclei. DAPI staining (blue) was used as the counterstain. Histograms indicate percentages of BrdU-positive cells, which were calculated based on counts of 800 to 900 cells per treatment. Bars are means ± standard deviations of three independent experiments. (C) Equal numbers of Meso 17 cells were seeded in individual culture dishes. The cells were then mock infected or infected with AdCtrl, AdNF2, or AdNF2 A518. Viable cell counts (as determined by exclusion of trypan blue stain) were performed for 5 days at 24-h intervals. This experiment is representative of three independent experiments.

FIG. 4.

Overexpression of cyclin D1 attenuates merlin's effect on cell cycle progression. (A) Meso 17 cells were transfected with pcDNA3 empty plasmid or cyclin D1 expression plasmid pRC-D1 and selected with G418. The pooled pcDNA3 or pRC-D1 transfectants were mock infected or infected with AdCtrl, AdNF2, or AdNF2 A518. After 24 h, cells were lysed and total cellular protein was electrophoresed and immunoblotted with the indicated antibodies. The pooled pRC-D1 transfectants exhibited a moderate increase in cyclin D1 protein level compared to pcDNA3 control transfectants (compare the lane of mock-infected pcDNA3 transfectants with that of mock-infected pRC-D1 transfectants). Infection of pcDNA3 transfectants with AdNF2 or AdNF2 A518 resulted in pRB dephosphorylation and p27 upregulation, as was seen with the parental cells (Fig. 2A). These regulatory effects of merlin, however, were markedly impeded in pRC-D1 transfectants. (B) Cells were treated as above and subjected to FACS analyses. Infection of pcDNA3 transfectants with AdNF2 or AdNF2 A518 resulted in an accumulation of G₁ cells and a decrease in the population in S phase, whereas infection of pRC-D1 transfectants with either AdNF2 or AdNF2 A518 attenuated the change in cell cycle distribution.

FIG. 5.

Repression of PAK1-stimulated cyclinD1 promoter by merlin. HeLa cells were transiently cotransfected with the cyclin D1 luciferase reporter plasmid (−1745D1-Luc) and PAK1 wild type (WT) or the constitutively active form of PAK1 (PAK1 T423E), in the presence or absence of pcDNA3-HA-NF2 or pcDNA3-HA-NF2 A518. The arbitrary units of luciferase content were normalized with total protein concentration and plotted relative to the pcDNA3 control.

FIG. 6.

Inhibition of PAK activity by merlin. (A) Two-dimensional gel analysis of PAK1 activity. Figures depict Western blot analyses of PAK1 in Meso 17 cells mock infected or infected with AdCtrl, AdNF2, or AdNF2 A518. The altered phosphorylated form of PAK1 is indicated by arrows. (B) Meso 17 cells were infected in the same way as for panel A, the cell lysates were immunoblotted with anti-phospho-ERK or global ERK antibody, and no obvious alteration was noted after reexpression of merlin.

FIG. 7.

Effects of silencing endogenous NF2 expression. (A) HeLa and A2780 cells were transfected with control siRNA or siRNA specific for NF2. Cells were lysed, and total cellular protein was subjected to immunoblot analysis. In both HeLa and A2780 cells, exposure to NF2 siRNA resulted in a marked reduction of merlin expression, concomitant with an increase in cyclin D1 accumulation. (B) Depletion of endogenous NF2 accelerates S-phase entry. HeLa cells were grown on coverslips and transfected with control siRNA or NF2 siRNA as described above. BrdU (20 μM) was added during the last 6 h of incubation, followed by immunofluorescence staining. BrdU incorporation was detected with an anti-BrdU-Alexa 594 antibody (red). Total nuclei were visualized with DAPI (blue). (C) Histogram depicting elevated BrdU incorporation resulting from silencing of endogenous NF2 relative to control treatment. Bars are means ± standard deviations of three independent experiments. *, P < 0.05.