Ichiko Saotome

The Nf2 Tumor Suppressor, Merlin, Functions in Rac-Dependent Signaling

Mutations in the neurofibromatosis type II (NF2) tumor suppressor predispose humans and mice to tumor development. The study of Nf2+/- mice has demonstrated an additional effect of Nf2 loss on tumor metastasis. The NF2-encoded protein, merlin, belongs to the ERM (ezrin, radixin, and moesin) family of cytoskeleton:membrane linkers. However, the molecular basis for the tumor- and metastasis- suppressing activity of merlin is unknown. We have now placed merlin in a signaling pathway downstream of the small GTPase Rac. Expression of activated Rac induces phosphorylation and decreased association of merlin with the cytoskeleton. Furthermore, merlin overexpression inhibits Rac-induced signaling in a phosphorylation-dependent manner. Finally, Nf2-/- cells exhibit characteristics of cells expressing activated alleles of Rac. These studies provide insight into the normal cellular function of merlin and how Nf2 mutation contributes to tumor initiation and progression.

Nf2/Merlin controls progenitor homeostasis and tumorigenesis in the liver

The molecular signals that control the maintenance and activation of liver stem/progenitor cells are poorly understood, and the role of liver progenitor cells in hepatic tumorigenesis is unclear. We report here that liver-specific deletion of the neurofibromatosis type 2 (Nf2) tumor suppressor gene in the developing or adult mouse specifically yields a dramatic, progressive expansion of progenitor cells throughout the liver without affecting differentiated hepatocytes. All surviving mice eventually developed both cholangiocellular and hepatocellular carcinoma, suggesting that Nf2(-/-) progenitors can be a cell of origin for these tumors. Despite the suggested link between Nf2 and the Hpo/Wts/Yki signaling pathway in Drosophila, and recent studies linking the corresponding Mst/Lats/Yap pathway to mammalian liver tumorigenesis, our molecular studies suggest that Merlin is not a major regulator of YAP in liver progenitors, and that the overproliferation of Nf2(-/-) liver progenitors is instead driven by aberrant epidermal growth factor receptor (EGFR) activity. Indeed, pharmacologic inhibition of EGFR blocks the proliferation of Nf2(-/-) liver progenitors in vitro and in vivo, consistent with recent studies indicating that the Nf2-encoded protein Merlin can control the abundance and signaling of membrane receptors such as EGFR. Together, our findings uncover a critical role for Nf2/Merlin in controlling homeostasis of the liver stem cell niche.

Ezrin and Moesin Function Together to Promote T Cell Activation

The highly homologous proteins ezrin, radixin, and moesin link proteins to the actin cytoskeleton. The two family members expressed in T cells, ezrin and moesin, are implicated in promoting T cell activation and polarity. To elucidate the contributions of ezrin and moesin, we conducted a systematic analysis of their function during T cell activation. In response to TCR engagement, ezrin and moesin were phosphorylated in parallel at the regulatory threonine, and both proteins ultimately localized to the distal pole complex (DPC). However, ezrin exhibited unique behaviors, including tyrosine phosphorylation and transient localization to the immunological synapse before movement to the DPC. To ask whether these differences reflect unique requirements for ezrin vs moesin in T cell signaling, we generated mice with conditional deletion of ezrin in mature T cells. Ezrin−/− T cells exhibited normal immunological synapse organization based upon localization of protein kinase C-θ, talin, and phospho-ZAP70. DPC localization of CD43 and RhoGDP dissociation inhibitor, as well as the novel DPC protein Src homology region 2 domain-containing phosphatase-1, was also unaffected. However, recruitment of three novel DPC proteins, ezrin binding protein of 50 kDa, Csk binding protein, and the p85 subunit of PI3K was partially perturbed. Biochemical analysis of ezrin−/− T cells or T cells suppressed for moesin using small interfering RNA showed intact early TCR signaling, but diminished levels of IL-2. The defects in IL-2 production were more pronounced in T cells deficient for both ezrin and moesin. These cells also exhibited diminished phospholipase C-γ1 phosphorylation and calcium flux. We conclude that despite their unique movement and phosphorylation patterns, ezrin and moesin function together to promote T cell activation.

Ezrin-mediated apical integrity is required for intestinal homeostasis.

Individual cell types are defined by architecturally and functionally specialized cortical domains. The Ezrin, Radixin, and Moesin (ERM) proteins play a major role in organizing cortical domains by assembling membrane protein complexes and linking them to the cortical actin cytoskeleton. Many studies have focused on the individual roles of the ERM proteins in stabilizing the membrane–cytoskeleton interface, controlling the distribution and function of apical membrane complexes, regulating the small GTPase Rho, or establishing cell–cell junctions. We previously found that deletion of the mouse Ezrin gene yields severe defects in apical integrity throughout the developing intestinal epithelium, resulting in incomplete villus morphogenesis and neonatal death. However, the molecular function of Ezrin in building the apical surface of the intestinal epithelium was not clear. By deleting Ezrin in the adult mouse intestinal epithelium, we provide evidence that Ezrin performs multiple molecular functions that collaborate to build the functional apical surface of the intestinal epithelium in vivo. The loss of Ezrin-mediated apical integrity in the adult intestine yields severe morphological consequences during intestinal homeostasis, including defects in cell geometry, extrusion, junctional remodeling, and spindle orientation. Surprisingly, deletion of Ezrin either before or after villus morphogenesis yields villus fusion, revealing a previously unrecognized step in intestinal homeostasis. Our studies indicate that the function of Ezrin in building and maintaining the apical domain is essential not only for intestinal morphogenesis but also for homeostasis in the mature intestine.

Ezrin is key regulator of Src-induced malignant phenotype in three-dimensional environment.

The oncogenic tyrosine kinase Src has a role in cancer development, especially by promoting invasive and metastatic behavior. It is, however, unclear which of the Src-regulated signaling cascades induce malignant phenotype in three-dimensional environment. One of Src substrates is ezrin, a cytoskeletal organiser and regulator of signal transduction. Ezrin expression correlates with poor outcome of diverse cancers and is essential in experimental metastatic osteosarcoma. We reconstituted genetically ezrin-deficient cells with wild-type (WT) or phosphorylation-deficient Y477F ezrin together with constitutively active Src. In two-dimensional cultures, Src induced malignant features regardless of the presence or absence of ezrin. In contrast, only WT ezrin-expressing cells grew efficiently in soft agar or in suspension. In Matrigel, only WT ezrin significantly promoted growth and invasion, and was targeted to specific regions on the plasma membrane. WT and Y477F ezrin-expressing cells showed marked differences only when growing or scattering in three-dimensional matrix. Additional experiments showed that Y477-phosphorylated ezrin is also needed for the growth of Src-transformed epithelial cells in three-dimensional matrix. Cells lacking functional ezrin had reduced cyclin D levels and fewer cells in G2+S phase, possibly as a consequence of abnormal mTOR signaling, as ezrin Y477F cells showed lower expression of phosphorylated intermediates downstream of mTOR than WT cells. We conclude that the pathways activated by Src depend on the type of environment and that ezrin is a crucial element of Src-induced malignant features in cells growing inside three-dimensional environment.

Ezrin Tunes the Magnitude of Humoral Immunity

Ezrin is a member of the ezrin–radixin–moesin family of membrane-actin cytoskeleton cross-linkers that participate in a variety of cellular processes. In B cells, phosphorylation of ezrin at different sites regulates multiple processes, such as lipid raft coalescence, BCR diffusion, microclustering, and endosomal JNK activation. In this study, we generated mice with conditional deletion of ezrin in the B cell lineage to investigate the physiological significance of ezrin’s function in Ag receptor–mediated B cell activation and humoral immunity. B cell development, as well as the proportion and numbers of major B cell subsets in peripheral lymphoid organs, was unaffected by the loss of ezrin. Using superresolution imaging methods, we show that, in the absence of ezrin, BCRs respond to Ag binding by accumulating into larger and more stable signaling microclusters. Loss of ezrin led to delayed BCR capping and accelerated lipid raft coalescence. Although proximal signaling proteins showed stronger activation in the absence of ezrin, components of the distal BCR signaling pathways displayed distinct effects. Ezrin deficiency resulted in increased B cell proliferation and differentiation into Ab-secreting cells ex vivo and stronger T cell–independent and -dependent responses to Ag in vivo. Overall, our data demonstrate that ezrin regulates amplification of BCR signals and tunes the strength of B cell activation and humoral immunity.

Ezrin Is Highly Expressed in Early Thymocytes, but Dispensable for T Cell Development in Mice

Background Ezrin/radixin/moesin (ERM) proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored. Methodology/Principal Findings We characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin−/− mice likely arise as a consequence of nutritional stress. Conclusions/Significance We conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin.