The pSUPER.retro.neo-YAP-siRNA construct was also generated in pSUPER.retro.neo using the strategy described above and a previously described YAP target sequence (CCAGAGAATCAGTCAGAGA) [30]. to increased S-phase entry. In addition, merlin loss in both meningioma cell lines and main tumors resulted in increased YAP expression and nuclear localization. Finally, siRNA-mediated reduction of YAP inNF2-deficient meningioma cells rescued the effects of merlin loss on cell proliferation and S-phase access. Collectively, these Angiotensin 1/2 (1-9) results represent the first demonstration that merlin regulates cell growth in human malignancy cells by suppressing YAP. == Introduction == Neurofibromatosis type 2 (NF2) is usually a malignancy predisposition syndrome phenotypically characterized by the occurrence of multiple nervous system tumors. The two most common tumors in this inherited syndrome are schwannomas and meningiomas [1]. Whereas meningiomas from individuals with NF2 exhibit biallelic inactivation of theNF2gene, loss ofNF2expression is also detected in as many as 60% of sporadic meningiomas [2]. Similarly, genetically designed mice with leptomeningealNF2inactivation also develop meningiomas [3,4]. These findings strongly implicate theNF2gene in the pathogenesis of meningiomas; however, the molecular mechanism by whichNF2regulates cell growth relevant to meningioma tumorigenesis remains unsolved. Merlin (or schwannomin), the product of theNF2gene, is usually a member of the protein 4.1 family that links the actin cytoskeleton to plasma membrane proteins [5]. Although few studies have examined merlin loss in meningioma cells, loss of merlin in fibroblasts and Schwann cells results in loss of contact-dependent inhibition of proliferation, enhanced growth in soft agar and tumor formation in mice [6,7]. In these cell types, merlin has been implicated in epidermal growth factor receptor [8], 1-integrin [9], and CD44 [7] function as well as Ras [10], Rac1 [11,12], phosphatidylinositol 3-kinase [13], mitogen-activated protein kinase [14], and transmission transducer and activator of transcription [15] intracellular signaling. It is not known whether any of these growth control pathways are deregulated inNF2-deficient meningioma tumors. Unfavorable regulation of growth by merlin is usually conserved inDrosophila, where it functions upstream of the Hippo signaling pathway to coordinately regulate cell proliferation and apoptosis [16,17]. Mutations in merlin or other components of the Hippo pathway such as the serine/threonine kinases, Hippo and Warts, the adaptor molecule, Salvador, or Mats, results in activation of the transcriptional coactivator, Yorkie. Yorkie regulates expression of downstream target genes includingcyclin EandDIAP1(Drosophilainhibitor of apoptosis protein 1) causing increased growth, delayed cell cycle exit, inhibition of apoptosis, and enhanced cell survival [16,17]. Individual components of the Hippo pathway are highly conserved in mammals, where they also regulate cell proliferation and apoptosis (Physique 1) [18,19]. Mice and humans have two Warts orthologs, Lats1 and Lats2. Mice deficient for Lats1 develop soft-tissue sarcomas and ovarian tumors [20]. The human ortholog of Salvador, hWW45, is usually mutated in malignancy cell lines [21]. The two mammalian Hippo homologues, Mst1 and Mst2, promote apoptosis Angiotensin 1/2 (1-9) and regulate cell cycle exit [22]. Vertebrate Mst2 can rescue the lethality and overgrowth phenotypes of Hippo mutants inDrosophila[23]. Much like theirDrosophilacounterparts, human Mst2 phosphorylates and activates both Lats1 and Lats2 [24]. The Yes-associated protein (YAP), the mammalian ortholog of Yorkie, is the main effector of the mammalian Hippo pathway. Similar to the function of Yorkie inDrosophila, YAP causes aberrant tissue growth in mice and induces epithelial transformation in mammary cells [25,26]. == Physique 1. == Schematic of the mammalian Hippo signaling pathway. The Hippo pathway is an evolutionary conserved cellular pathway that coordinately regulates cell proliferation and apoptosis. Merlin has been proposed to interact with unknown membrane proteins and transduce a signal that stimulates the phosphorylation of LATS1/2 by the serine/threonine kinases MST1/2 that interact with hWW45. LATS1/2 inhibits the transcriptional coactivator YAP resulting in suppressed expression of downstream target genes such as cyclins that are involved in cell growth and proliferation. Given the conservation of components and mechanisms that operate downstream of merlin betweenDrosophilaand mammals, we tested the functional relationship among merlin, Hippo pathway regulation, and growth suppression in human meningioma tumors. We developed nonneoplastic and neoplastic meningeal cell lines that mimic gain or loss ofNF2expression and used these matched lines to examine merlin regulation of YAP. We found that absence of merlin results in loss of contact-dependent inhibition of growth and promotes anchorage-independent growth. Merlin loss enhances cell proliferation by increasing access into the S-phase and cyclin E1 expression. Inactivation of merlin Cited2 Angiotensin 1/2 (1-9) results in increased YAP expression and nuclear accumulation of YAP in these meningioma cell lines and in main human meningioma tumors. Finally, we show that YAP suppression reverses the proliferation effects associated with merlin loss in meningiomas. Collectively,.