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Merlin Regulates Transmembrane Receptor Accumulation and Signaling at the Plasma Membrane in Primary Mouse Schwann Cells and in Human Schwannomas

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Merlin Regulates Transmembrane Receptor Accumulation and Signaling at the Plasma Membrane in Primary Mouse Schwann Cells and in Human Schwannomas Oncogene (2009) 28, 854–865 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Merlin regulates transmembrane receptor accumulation and signaling at the plasma membrane in primary mouse Schwann cells and in human schwannomas D Lallemand1,2, J Manent1,2, A Couvelard3, A Watilliaux1,2, M Siena1,2, F Chareyre1,2, A Lampin1,2, M Niwa-Kawakita1,2, M Kalamarides1,2,4 and M Giovannini1,2 1INSERM, U674, Paris, France; 2Universite´ Paris 7, Denis Diderot, Institut Universitaire d 0He´matologie, Paris, France; 3APHP, Hoˆpital Beaujon, Service d’Anatomie et de Cytologie Pathologique, Clichy, France and 4APHP, Hoˆpital Beaujon, Service de Neurochirurgie, Clichy, France The NF2 gene product,merlin/schwannomin,is a membrane proteins (Bretscher et al., 2002). Merlin cytoskeleton organizer with unique growth-inhibiting inhibits the proliferation of many different cell types, activity in specific cell types. A narrow spectrum of although the proposed mechanisms vary considerably. tumors is associated with NF2 deficiency,mainly schwan- Merlin was shown to downregulate a wide range of nomas and meningiomas,suggesting cell-specific mecha- mitogenic signaling pathways, such as Ras, Rac or PI3K nisms of growth control. We have investigated merlin (Fraenzer et al., 2003; Kissil et al., 2003; Lim et al., 2003; function in mouse Schwann cells (SCs). We found that Hirokawa et al., 2004; McClatchey and Giovannini, merlin regulates contact inhibition of proliferation by 2005; Morrison et al., 2007). Moreover, Nf2 inactivation limiting the delivery of several growth factor receptors at results in destabilization of adherens junctions (AJs) the plasma membrane of primary SCs. Notably,upon cell- that are essential for contact inhibition of proliferation to-cell contact,merlin downregulates the membrane in mouse embryonic fibroblasts (MEFs) (Lallemand levels of ErbB2 and ErbB3,thus inhibiting the activity et al., 2003). In addition, merlin can block ligand- of the downstream mitogenic signaling pathways protein induced epidermal growth factor receptor internaliza- kinase B and mitogen-activated protein kinase. Conse- tion and signaling in various cell types (Curto et al., quently,loss of merlin activity is associated with elevated 2007). In a rat schwannoma cells, merlin also inhibits levels of ErbB receptors in primary SCs. We also observed growth by promoting platelet-derived growth factor accumulation of growth factor receptors such as ErbB2 receptor (PDGFR) degradation (Fraenzer et al., 2003). and 3,insulin-like growth factor 1 receptor and platelet- Finally, in Drosophila, D-Merlin and Expanded coop- derived growth factor receptor in peripheral nerves of Nf2- erate to stimulate endocytosis of membrane proteins, mutant mice and in human NF2 schwannomas,suggesting such as Notch and epidermal growth factor receptor, that this mechanism could play an important role in thereby reducing their levels at the cell surface and tumorigenesis. inhibiting downstream signaling (Maitra et al., 2006). Oncogene (2009) 28, 854–865; doi:10.1038/onc.2008.427; Despite these pieces of evidence, there is no consensus as published online 24 November 2008 to whether these mechanisms are central to specific NF2-related tumors. One striking feature of NF2 is the Keywords: neurofibromatosis type 2; growth factor narrow tumor spectrum composed essentially of benign receptors; trafficking; Schwann cells; proliferation; schwannomas and meningiomas. Therefore, we decided schwannoma to study merlin function in mouse Schwann cells (SCs) that are most likely to provide pertinent information about the molecular mechanisms of NF2 tumor suppression. Introduction Large cultures of primary SC can be derived from mice harboring a conditional Nf2 allele (Giovannini Merlin is the product of the NF2 tumor suppressor gene et al., 2000; Manent et al., 2003). In these cells, Nf2 is that shares strong sequence and structural homology inactivated following adenovirus-mediated Cre expres- with the ERM proteins (Ezrin, Radixin and Moesin), as sion, allowing the study of the early consequences of well as the capacity to bind to F-actin and plasma merlin loss on SC proliferation and tumor initiation. We found that merlin controls contact-dependent inhibition Correspondence: Dr M Giovannini, House Ear Institute, Center for of SC proliferation by inhibiting the delivery of Neural Tumor Research, 2100W Third Street, Los Angeles, CA 90057, receptors to the plasma membrane. Notably, merlin USA. E-mail: [email protected] downregulates the membrane levels of ErbB2 and Received 19 March 2008; revised 10 September 2008; accepted 15 ErbB3, thus inhibiting downstream mitogenic signaling September 2008; published online 24 November 2008 pathways. Moreover, we show that loss of merlin Merlin regulates transmembrane receptor accumulation D Lallemand et al 855 activity is associated with elevated levels of ErbB growth factor and adhesion receptors. We found that receptors in peripheral nerves of Nf2-mutant mice the amounts of several growth factor receptors (ErbB2, as well as in human NF2 schwannomas. Taken toge- ErbB3, insulin-like growth factor 1 receptor (IGF1R), ther, our data suggest that the altered delivery of PDGFR-b) and adhesion molecules (N-and E-cadher- ErbB receptors plays a major role in schwannoma in) were elevated in total membrane extracts from development. confluent Nf2À/À SCs compared with Nf2 þ / þ SCs, whereas b-catenin levels were not (Figure 2b). These observations were confirmed by surface protein bioti- nylation (Figure 2d). In contrast, receptor levels were Results nearly identical in subconfluent Nf2 þ / þ SCs and Nf2À/À SCs (Supplementary Figure 2). Quantification of the Proliferative advantage of Nf2À/À SCs signals showed that the higher levels of receptors were We did not observe any difference in the growth rates associated with an increase of phosphorylated receptors. Nf2 þ / þ Nf2À/À between and SCs as long as they These molecular phenotypes were strictly regulated by remained subconfluent (Supplementary Figure 1). Once merlin because its reintroduction into Nf2À/À SCs Nf2 þ / þ SC cultures reached confluence, we saw pro- markedly reduced the levels of ErbB2, ErbB3, IGF1R, gressive growth arrest between days 5 and 13 of culture PDGFR-b and E-and N-cadherin at the plasma (Figure 1a). In contrast, Nf2À/À SCs kept growing and membrane (Figures 2c and d). Quantification of specific reached saturation densities 2–3 times higher than markers EEA1, Lamp1, p115 or Rab11 in membrane Nf2 þ / þ SCs (Figures 1a–c). Adenovirus-mediated re- extracts by western blot (Figure 2e) and by immuno- expression of merlin was sufficient to restore saturation fluorescence staining (Supplementary Figure 3) sug- densities similar to those observed in Nf2 þ / þ SCs, gested that the accumulation of membrane proteins in illustrating that loss of merlin was responsible for the Nf2À/À SCs was not the result of an expansion of phenotype (Figures 1b and c). intracellular compartments, such as the Golgi apparatus Primary cells in culture undergo progressive and or the endocytic vesicles. In addition, saturation of the irreversible growth arrest after several passages, a plasma membrane with the lipophilic fluorescent dye process referred to as replicative senescence. Primary FM1-43X demonstrated that this compartment was not mouse cells can escape this fate by losing expression of enlarged in Nf2À/À SCs (Figure 2f). Finally, quantitative tumor suppressor genes, such as p21Cip, p53 or pRb reverse transcription–PCR (RT–PCR) analysis showed (Bringold and Serrano, 2000; Itahana et al., 2004). We no increase of ErbB2, ErbB3 and IGF1R mRNA observed that Nf2 þ / þ SCs became senescent after five to expression levels in Nf2À/À SCs and a slight increase of seven passages in culture when they stopped proliferat- Pdgfrb (1.3-fold). Only N-cadherin mRNA levels were ing, lost their bipolar shape and became very large and increased following loss of merlin expression flat, the typical morphological features of senescent cells (Figure 2g). In conclusion, Nf2 inactivation in SCs (Figures 1d and e). In contrast, Nf2À/À SCs never leads to the accumulation of growth factor receptors at stopped proliferating (Figure 1d) and showed none of the plasma membrane, which is not due to increased the morphological features of senescence (Figure 1e). protein synthesis. Although levels of p107, p130, p53, p19Arf and p21Cip increased in late-passage Nf2À/À SCs, their expression þ / þ levels remained always lower than in Nf2 SC Merlin regulates the delivery of growth factor receptors (Figure 1f). p27Kip levels were not affected by Nf2 À/À to the plasma membrane in SCs inactivation. Beyond passage 8, Nf2 SCs never Several reports have proposed a role for merlin in became senescent and spontaneously transformed. protein transport (Maitra et al., 2006; Curto et al., 2007) and the involvement of the ERM proteins in vesicular Transmembrane receptors accumulate at the plasma trafficking is well established (Pujuguet et al., 2003; membrane of Nf2-deficient SCs Tamma et al., 2005; Stanasila et al., 2006). We Following Nf2 inactivation in SCs, we observed a clear compared growth factor internalization in Nf2 þ / þ and morphological change (Figure 2a, upper panels) and Nf2À/À SC cultures using a surface protein biotin pulse- profound alterations of the actin cytoskeleton chase approach and found no differences (data not (Figure 2a, lower panels), suggesting that, as in MEFs shown). As the phenotype might be too subtle to be (Lallemand et al., 2003), Nf2 inactivation results in the detected using this method, we decided to overexpress loss of cell–cell contact. AJs were visualized by staining merlin in Nf2À/À SCs to accentuate a putative trafficking of b-catenin, a component of these structures. In defect. We observed no difference in the rate of contrast to Nf2 þ / þ SCs, staining of b-catenin in internalization of the receptors 20 min after the biotin Nf2-deficient SCs was not clearly localized in regions pulse (or at later times) when comparing Nf2À/À SCs of cell–cell contact (Figure 2a, middle panels).
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