The neurofibromatosis type 2 gene-encoded protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane-cytoskeleton-associated proteins. Recent studies suggest that the loss of neurofibromatosis type 2 function contributes to tumor development and metastasis. Although the cellular functions of merlin as a tumor suppressor are relatively well characterized, the cellular mechanism whereby merlin controls cell proliferation from membrane locations is still poorly understood. During our efforts to find potential merlin modulators through protein-protein interactions, we identified transactivation-responsive RNAbinding protein (TRBP) as a merlin-binding protein in a yeast two-hybrid screen. The interaction between TRBP and merlin was confirmed by glutathione S-transferase pull-down assays, co-immunoprecipitation, and co-localization experiments. The carboxyl-terminal regions of each protein were responsible for their interaction. Cells overexpressing TRBP showed enhanced cell growth in cell proliferation assays and also exhibited transformed phenotypes, such as anchorage-independent cell growth and tumor development in mouse xenografts. Merlin efficiently inhibited these oncogenic activities of TRBP in our experiments. These results provide the first clue to the functional interaction between TRBP and merlin and suggest a novel mechanism for the tumor suppressor function of merlin both in vitro and in vivo.

BackgroundMesenchymal stem cells (MSCs) are an attractive source of adult stem cells for therapeutic application in clinical study. Genetic modification of MSCs with beneficial genes makes them more effective for therapeutic use. However, it is difficult to transduce genes into MSCs by common transfection methods, especially nonviral methods. In this study, we applied microporation technology as a novel electroporation technique to introduce enhanced green fluorescent protein (EGFP) and brain-derived neurotropfic factor (BDNF) plasmid DNA into human umbilical cord blood-derived MSCs (hUCB-MSCs) with significant efficiency, and investigated the stem cell potentiality of engineered MSCs through their phenotypes, proliferative capacity, ability to differentiate into multiple lineages, and migration ability towards malignant glioma cells.ResultsUsing microporation with EGFP as a reporter gene, hUCB-MSCs were transfected with higher efficiency (83%) and only minimal cell damage than when conventional liposome-based reagent (<20%) or established electroporation methods were used (30-40%). More importantly, microporation did not affect the immunophenotype of hUCB-MSCs, their proliferation activity, ability to differentiate into mesodermal and ectodermal lineages, or migration ability towards cancer cells. In addition, the BDNF gene could be successfully transfected into hUCB-MSCs, and BDNF expression remained fairly constant for the first 2 weeks in vitro and in vivo. Moreover, microporation of BDNF gene into hUCB-MSCs promoted their in vitro differentiation into neural cells.ConclusionTaken together, the present data demonstrates the value of microporation as an efficient means of transfection of MSCs without changing their multiple properties. Gene delivery by microporation may enhance the feasibility of transgenic stem cell therapy.

The ability of mesenchymal stem cells (MSCs) to differentiate into neural cells makes them potential replacement therapeutic candidates in neurological diseases. Presently, overexpression of brain-derived neurotrophic factor (BDNF), which is crucial in the regulation of neural progenitor cell differentiation and maturation during development, was sufficient to convert the mesodermal cell fate of human umbilical cord bloodderived MSCs (hUCB-MSCs) into a neuronal fate in culture, in the absence of specialized induction chemicals. BDNF overexpressing hUCB-MSCs (MSCs-BDNF) yielded an increased number of neuron-like cells and, surprisingly, increased the expression of neuronal phenotype markers in a time-dependent manner compared with control hUCB-MSCs. In addition, MSCs-BDNF exhibited a decreased labeling for MSCs-related antigens such as CD44, CD73, and CD90, and decreased potential to differentiate into mesodermal lineages. Phosphorylation of the receptor tyrosine kinase B (TrkB), which is a receptor of BDNF, was increased significantly in MSC-BDNF. BDNF overexpression also increased the phosphorylation of β-catenin and extracellular signal-regulated kinases (ERKs). Inhibition of TrkB availability by treatment with the TrkBspecific inhibitor K252a blocked the BDNF-stimulated phosphorylation of β-catenin and ERKs, indicating the involvement of both the β-catenin and ERKs signals in the BDNF-stimulated and TrkB-mediated neural differentiation of hUCB-MSCs. Reduction of β-catenin availability using small interfering RNA-mediated gene silencing inhibited ERKs phosphorylation. However, β-catenin activation was maintained. In addition, inhibition of β-catenin and ERKs expression levels abrogated the BDNF-stimulated upregulation of neuronal phenotype markers. Furthermore, MSC-BDNF survived and migrated more extensively when grafted into the lateral ventricles of neonatal mouse brain, and differentiated significantly into neurons in the olfactory bulb and periventricular astrocytes. These results indicate that BDNF induces the neural differentiation of hUCB-MSCs in culture via the TrkB-mediated phosphorylation of ERKs and β-catenin and following transplantation into the developing brain.

Mounting evidence suggests that lipoxygenase (LO)-catalyzed products may play a key role in the development and progression of human cancers. In this study, we analyzed the effects of a 5-LO inhibitor, which inhibits the conversion of arachidonic acid to leukotrienes, on cell proliferation and apoptosis in human malignant glioma cells, including 5-LO-expressing cells U-87MG, A172 and 5-LO non-expressing cell U373. Growth of U-87MG and A172 cells, but not that of U373 cells, was inhibited in a dose-dependent manner by treatment with MK886. Similarly, specific 5-LO silencing by small interfering RNA reduced the growth of U-87MG and A172 cells. MK886 treatment reduced 5-LO activity independently of 5-LO-activating protein (FLAP) in human malignant glioma cells. MK886 treatment also induced cell apoptosis, measured by DNA fragmentation and nuclear condensation, in U-87MG and A172 cells but there were no signs in U373 cells. Moreover, this treatment reduced ERKs phosphorylation and anti-apoptotic molecule Bcl-2 expression, and increased Bax expression in U-87MG and A172 cells. In summary, our results show there is a link between the 5-LO expression status and the extent of MK886-inhibited cell proliferation and apoptosis. Taken together, this study suggest that 5-LO is a possible target for treating patients with gliomas, and 5-LO inhibition might be potent therapy for patients with 5-LO-expressing malignant gliomas.

Neurofibromatosis type 2 (NF2) is the most commonly mutated gene in benign tumors of the human nervous system such as schwannomas and meningiomas. The NF2 gene encodes a protein called schwannomin or merlin, which is involved in regulating cell growth and proliferation through protein-protein interactions with various cellular proteins. In order to better understand the mechanism by which merlin exerts its function, yeast two-hybrid screening was performed and Ral guanine nucleotide dissociation stimulator (RalGDS), a downstream molecule of Ras, was identified as a merlin-binding protein. The direct interaction between merlin and RalGDS was confirmed both in vitro and in the NIH3T3 cells. The domain analyses revealed that the broad Cterminal region of merlin (aa 141-595) is necessary for the interaction with the C-terminal Ras-binding domain (RBD) of RalGDS. Functional studies showed that merlin inhibits the RalGDS-induced RalA activation, the colony formation and the cell migration in mammalian cells. These results suggest that merlin can function as a tumor suppressor by inhibiting the RalGDS-mediated oncogenic signals.