The Neurofibromatosis 2 Protein,Merlin, Regulates Glial Cell Growth in an ErbB2- and Src-Dependent Manner

Individuals with the inherited cancer predisposition syndrome neurofibromatosis 2 (NF2) develop several central nervous system (CNS) malignancies, including glial cell neoplasms (ependymomas). Recent studies have suggested that the NF2 protein, merlin (or schwannomin), may regulate receptor tyrosine kinase signaling, intracellular mitogenic growth control pathways, or adherens junction organization in non-nervous-system cell types. For this report, we used glial fibrillary acidic protein conditional knockout mice and derivative glia to determine how merlin regulates CNS glial cell proliferation. We show that the loss of merlin in glial cells results in increased proliferation in vitro and in vivo. Merlin regulation of glial cell growth reflects deregulated Src activity, such that pharmacologic or genetic inhibition of Src activation reduces Nf2^−/− glial cell growth to wild-type levels. We further show that Src regulates Nf2^−/− glial cell growth by sequentially regulating FAK and paxillin phosphorylation/activity. Next, we demonstrate that Src activation results from merlin regulation of ErbB2 activation and that genetic or pharmacologic ErbB2 inhibition reduces Nf2^−/− glial cell Src/Src effector activation and proliferation to wild-type levels. Lastly, we show that merlin competes with Src for direct binding to ErbB2 and present a novel molecular mechanism for merlin regulation of ErbB2-dependent Src signaling and growth control.Neurofibromatosis type 2 (NF2) is an autosomal dominant inherited cancer syndrome in which affected individuals develop nervous system tumors, including peripheral nerve tumors (schwannomas), leptomeningeal tumors (meningiomas), and glial fibrillary acidic protein (GFAP)-immunoreactive glial cell tumors (spinal ependymomas). NF2 results from a germ line mutation in the NF2 tumor suppressor gene, located on chromosome 22q (46, 60). Tumors in this disorder arise following somatic inactivation of the one remaining wild-type (WT) NF2 allele in specific cell types. In this regard, NF2-associated schwannomas, meningiomas, and ependymomas all exhibit biallelic NF2 gene inactivation (33, 47, 61). In addition, NF2 gene inactivation is also observed in 50 to 78% of sporadic schwannomas, 32 to 84% of sporadic meningiomas, and 37% of sporadic ependymomas (21, 29), suggesting that this gene is also a key growth regulator in nonhereditary nervous system cancers.The NF2 gene was identified in 1993 and found to code for a 595-amino-acid protein, termed merlin or schwannomin (46, 60). Analysis of the predicted protein sequence revealed striking sequence similarity between merlin and a family of protein 4.1 family members that link the actin cytoskeleton to cell surface glycoproteins (55). In particular, merlin most closely resembles the ezrin/radixin/moesin (ERM) subfamily and has been shown to bind actin as well as to associate with several cell surface glycoproteins, including CD44 and β1-integrin (5, 32, 48). However, unlike the ERM proteins, merlin is unique in its capacity to function as a nervous system tumor suppressor gene.In order to identify the key signaling pathways regulated by the merlin tumor suppressor protein, previous studies have focused on merlin growth regulation in fibroblasts, primary Schwann cell and human schwannoma cell cultures, meningioma and schwannoma tumor cell lines, and other non-central nervous system (non-CNS) cell types. These investigations have resulted in the identification of a large number of nonintersecting growth control pathways regulated by merlin in different cell types. In this regard, merlin has been implicated in epidermal growth factor receptor (EGFR) (9), β1-integrin (15), and CD44 (1, 35, 48) function as well as in Ras (25, 59), Rac1 (34, 52), phosphatidylinositol 3-kinase (44), mitogen-activated protein kinase (MAPK) (7, 30), and STAT (51) intracellular signaling. While each of these pathways is involved in growth control in the brain, it is not known which of these intracellular signaling pathways are deregulated in NF2-deficient CNS cell types.To gain insights into the role of the NF2 gene in glial cell growth control relevant to the development of targeted therapies for NF2-associated glial cell malignancies, we studied the consequence of merlin loss on the growth of primary brain glial cells (astrocytes) in vitro and in vivo, using Nf2 conditional knockout genetically engineered mice (GEM). We demonstrate for the first time that merlin regulates brain glial cell growth by controlling the phosphorylation/activity of Src and its downstream effectors, FAK and paxillin. Furthermore, we show that merlin regulation of Src phosphorylation/activation is modulated by ErbB2 phosphorylation/activation and ErbB2-Src binding. Finally, we show that merlin competitively inhibits Src binding to ErbB2 and, in this manner, prevents ErbB2-mediated Src phosphorylation and downstream mitogenic signaling. Based on these findings, we propose a novel mechanism for merlin growth regulation in CNS glia.