Actin-rich protrusions and nonlocalized GTPase activation in Merlin-deficient schwannomas

Schwannomas lack both alleles for the tumor suppressor Merlin, a cytoskeleton-membrane linker. Previous results showed increased cell spreading of schwannoma cells, but little is known about the underlying mechanisms. Electron microscopy reveals that schwannoma cells not only show more lamellipodia/ruffles but also multiple filopodia. We show that Cdc42, important in filopodia formation, is activated. Both Rac1 and Cdc42 are found all around the cell periphery and in colocalization with their effector phospho-p21 activated kinase in human schwannoma cells. We therefore claim that Rac1 and Cdc42 are activated in a nonlocalized manner, which explains the disperse distribution of lamellipodia/ruffles and filopodia. Using live cell imaging, we further demonstrate continuous remodeling of the many actin-rich protrusions in schwannoma cells. The underlying cytoskeleton of these structures is thin and extensively branched. The actin-related protein 2/3 complex, a major regulator of actin branching, is enriched in the many lamellipodia and ruffles of human primary schwannoma cells. We suggest that the Merlin deficiency in human primary schwannoma cells leads to a random, nonlocalized activation of Rac1 and Cdc42, inducing many actin-rich protrusion zones, not only at the leading edge but also all around the cell periphery. Their nondirectional occurrence together with the continuous and highly dynamic actin remodeling results in the dedifferentiation of these tumor cells.     

Rearrangements of the intermediate filament GFAP in primary human schwannoma cells

Loss of the tumor suppressor protein merlin causes a variety of benign tumors such as schwannomas, meningiomas, and gliomas in man. We previously reported primary human schwannoma cells to show enhanced integrin-dependent adhesion and a hyperactivation of the small RhoGTPase Rac1. Here we show that the main intermediate filament protein of Schwann cells, the glial fibrillary acidic protein, is collapsed to the perinuclear region instead of being well-spread from the nucleus to the cell periphery. This cytoskeletal reorganization is accompanied by changes in cell shape and increased cell motility. Moreover, we report tyrosine phosphorylation to be enhanced in schwannoma cells, already described earlier in intermediate filament breakdown. Thus, we believe that Rac activation via tyrosine kinase stimulation leads to GFAP collapse in human schwannoma cells, and suggest that this process plays an important role in vivo where schwannoma cells become motile, unspecifically ensheathing extracellular matrix and forming pseudomesaxons.     

Impaired intercellular adhesion and immature adherens junctions in merlin-deficient human primary schwannoma cells

Schwannomas that occur spontaneously or in patients with neurofibromatosis Type 2, lack both alleles for the tumor suppressor and plasma membrane-cytoskeleton linker merlin. We have shown that human primary schwannoma cells display activation of the RhoGTPases Rac1 and Cdc42 which results in highly dynamic and ongoing protrusive activity like ruffling. Ruffling is an initial and temporally limited step in the formation of intercellular contacts like adherens junctions that are based on the cadherin-catenin system. We tested if there is a connection between Rac1-induced ongoing ruffling and the maintenance, stabilization and functionality of adherens junctions and if this is of relevance in human, merlin-deficient schwannoma cells. We show intense ongoing ruffling is not limited to membranes of single human primary schwannoma cells, but occurs also in membranes of contacting cells, even when confluent. Live cell imaging shows that newly formed contacts are released after a short time, suggesting disturbed formation or stabilization of adherens junctions. Morphology, high phospho-tyrosine levels and cortactin staining indicate that adherens junctions are immature in human primary schwannoma cells, whereas they display characteristics of mature adherens junctions in human primary Schwann cells. When merlin is reintroduced, human primary schwannoma cells show only initial ruffling in contacting cells and adherens junctions appear more mature. We therefore propose that ongoing Rac-induced ruffling causes immature adherens junctions and leads to impaired, nonfunctional intercellular adhesion in aggregation assays in merlin-deficient schwannoma cells that could be an explanation for increased proliferation rates due to loss of contact inhibition or tumor development in general.     

Caveolin-1 inhibits neurite growth by blocking Rac1/Cdc42 and p21-activated kinase 1 interactions

Growth factors such as basic fibroblast growth factors (bFGFs) could induce the differentiation of mouse neuroblastoma cells. We examine the effect of caveolin-1 on bFGF-induced differentiation of N2a cells. Caveolin-1 blocked the formation of neurites and the phosphorylation of Erk upon bFGF treatment in N2a cells. Active mutants of Rho family small GTPases (Rac1 and Cdc42) could not affect the inhibitory effect of caveolin-1, but we could restore the differentiation of N2a cells by introducing active mutants of p21-activated kinase 1 (PAK1). Over-expressed caveolin-1 could be coimmunoprecipitated with PAK1, which interrupted the steady-state Rac1/Cdc42-PAK1 interactions. From these results, we suggest that the up-regulated caveolin-1 in neuronal cells can inhibit the bFGF signaling pathway from small GTPases to PAK1 by directly binding to PAK1.     

Targeting ERK1/2 activation and proliferation in human primary schwannoma cells with MEK1/2 inhibitor AZD6244

Deficiency of the tumor suppressor merlin leads to the development of multiple tumors of the nervous system, such as schwannomas, meningiomas, and ependymomas. Due to the benign character of these tumors, classical chemotherapy is ineffective. Current therapies, surgery, and radiosurgery are local and quite invasive, thus new systemic treatments are required. We have previously described the Raf/mitogen-activated kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway activation and its role in schwannoma growth. Here, we targeted MEK1/2 known as a convergence point for multiple cascades towards ERK1/2 activation and cell proliferation, using MEK1/2 inhibitor AZD6244 (ARRY-142886; Astra Zeneca).We show that AZD6244 at low concentration completely abolished platelet-derived growth factor-DD-mediated ERK1/2 activation and cell proliferation in human primary schwannoma cells. Moreover, this drug was not toxic for either schwannoma or Schwann cells and has been reported to be safe with tolerable side effects. Thus, AZD6244 can be considered as a drug candidate for schwannoma treatment.     

The atypical Guanine-nucleotide exchange factor, dock7, negatively regulates schwann cell differentiation and myelination

In development of the peripheral nervous system, Schwann cells proliferate, migrate, and ultimately differentiate to form myelin sheath. In all of the myelination stages, Schwann cells continuously undergo morphological changes; however, little is known about their underlying molecular mechanisms. We previously cloned the dock7 gene encoding the atypical Rho family guanine-nucleotide exchange factor (GEF) and reported the positive role of Dock7, the target Rho GTPases Rac/Cdc42, and the downstream c-Jun N-terminal kinase in Schwann cell migration (Yamauchi et al., 2008). We investigated the role of Dock7 in Schwann cell differentiation and myelination. Knockdown of Dock7 by the specific small interfering (si)RNA in primary Schwann cells promotes dibutyryl cAMP-induced morphological differentiation, indicating the negative role of Dock7 in Schwann cell differentiation. It also results in a shorter duration of activation of Rac/Cdc42 and JNK, which is the negative regulator of myelination, and the earlier activation of Rho and Rho-kinase, which is the positive regulator of myelination. To obtain the in vivo evidence, we generated Dock7 short hairpin (sh)RNA transgenic mice. They exhibited a decreased expression of Dock7 in the sciatic nerves and enhanced myelin thickness, consistent with in vitro observation. The effects of the in vivo knockdown on the signals to Rho GTPases are similar to those of the in vitro knockdown. Collectively, the signaling through Dock7 negatively regulates Schwann cell differentiation and the onset of myelination, demonstrating the unexpected role of Dock7 in the interplay between Schwann cell migration and myelination.     

The netrin-1 receptor DCC promotes filopodia formation and cell spreading by activating Cdc42 and Rac1

Netrins are a family of secreted proteins that function as tropic cues directing cell and axon migration during neural development. We show that the netrin-1 receptor, deleted in colorectal cancer (DCC), is present at filopodia tips in growth cones of embryonic rat spinal commissural neurons. To further investigate DCC function, we characterized the expression of netrins and netrin receptors in HEK293T cells and NG108-15 cells and found that they express netrin-1 but do not express DCC. Ectopic expression of DCC produced a netrin-1-dependent increase in the number of filopodia and in cell surface area. Coexpression of DCC and dominant negative Cdc42 or dominant negative Rac1 blocked the increase in filopodia number and cell surface area, respectively. Furthermore, addition of netrin-1 to cells expressing DCC caused a persistent activation of Cdc42 and Rac1. These findings suggest that netrin-1, via DCC, influences cellular motility by regulating actin-based membrane extension through the activation of Cdc42 and Rac1.     

The role of insulin-like growth factors signaling in merlin-deficient human schwannomas

Loss of the tumor suppressor merlin causes development of the tumors of the nervous system, such as schwannomas, meningiomas, and ependymomas occurring spontaneously or as part of a hereditary disease Neurofibromatosis Type 2 (NF2). Current therapies, (radio) surgery, are not always effective. Therefore, there is a need for drug treatments for these tumors. Schwannomas are the most frequent of merlin‐deficient tumors and are hallmark for NF2. Using our in vitro human schwannoma model, we demonstrated that merlin‐deficiency leads to increased proliferation, cell–matrix adhesion, and survival. Increased proliferation due to strong activation of extracellular‐signal‐regulated kinase 1/2 (ERK1/2) is caused by overexpression/activation of platelet‐derived growth factor receptor‐β (PDGFR‐β) and ErbB2/3 which we successfully blocked with AZD6244, sorafenib, or lapatinib. Schwannoma basal proliferation is, however, only partly dependent on PDGFR‐β and is completely independent of ErbB2/3. Moreover, the mechanisms underlying pathological cell–matrix adhesion and survival of schwannoma cells are still not fully understood. Here, we demonstrate that insulin‐like growth factor‐I receptor (IGF‐IR) is strongly overexpressed and activated in human primary schwannoma cells. IGF‐I and ‐II are overexpressed and released from schwannoma cells. We show that ERK1/2 is relevant for IGF‐I‐mediated increase in proliferation and cell–matrix adhesion, c‐Jun N‐terminal kinases for increased proliferation and AKT for survival. We demonstrate new mechanisms involved in increased basal proliferation, cell–matrix adhesion, and survival of schwannoma cells. We identified therapeutic targets IGF‐IR and downstream PI3K for treatment of schwannoma and other merlin‐deficient tumors and show usefulness of small molecule inhibitors in our model. PI3K is relevant for both IGF‐IR and previously described PDGFR‐β signaling in schwannoma. © 2012 Wiley Periodicals, Inc.     

Distinct functions of Rac1 and Cdc42 during axon guidance and growth cone morphogenesis in Drosophila

Rho family small GTPases are thought to be key molecules in the regulation of cytoskeletal organization, especially for actin filaments. In order to examine the functions of Rac1 and Cdc42 in axon guidance at the midline of the central nervous system in Drosophila embryos, we either activated or inactivated Rac1 and Cdc42 in all postmitotic neurons. We found that the phenotypes of Cdc42 activation and Rac1 inactivation were similar to those of roundabout mutants, in that many extra axons crossed the midline. We also found that Rac1 inactivation is dominant over Roundabout receptor activation. Our observations indicate that Rac1 and Cdc42 have distinct functions in downstream signalling events triggered by Roundabout receptors. In order to further examine the functional difference between Rac1 and Cdc42 in the growth cone morphogenesis, we used primary embryonic cultures to closely observe neurite formation. We showed that activation of Rac1 and Cdc42 has distinct effects on neurite formation, particularly on growth cone morphology and the actin filaments within. Both Rac1 and Cdc42 activation induced large growth cones and long filopodia, but Cdc42 did so more efficiently than Rac1. Only Rac1 activation, however, induced thick actin bundles in the filopodia. We also found a clear difference between Rac1 and Cdc42 in terms of the response to an inhibitor of actin polymerization. Our results suggest that Cdc42 is specifically involved in the regulation of actin filaments in growth cones, whereas Rac1 is involved in additional functions.     

Secretase-independent and RhoGTPase/PAK/ERK-dependent regulation of cytoskeleton dynamics in astrocytes by NSAIDs and derivatives

Profens like ibuprofen, R-flurbiprofen, or CHF5074 are being considered for the treatment of Alzheimer's disease because epidemiological data indicates that non-steroidal anti-inflammatory drugs are protective against neurodegeneration. Rho-GTPases are small G proteins, including RhoA, Cdc42, and Rac1, which control cytoskeleton dynamics. Because ibuprofen promotes axon growth via RhoA in neurons, we examined whether profens modulate astrocyte plasticity via Rho-GTPases. We report that ibuprofen (100-500 μM), R-flurbiprofen (100-500 μM), and CHF5074 (10-30 μM) caused a concentration-dependent stellation of astrocytes in primary cultures, associated with the reorganization of GFAP and actin filaments. The stellation was independent of COX2, α-, β- or γ-secretase as judged by the lack of effect of inhibitors of these enzymes. RhoA, PAK, and Cdc42, but not Rac1, accounted for the profen-mediated stellation, as concluded from the joint analyses of activities and reversal experiments with adenoviral or pharmacological manipulations. Ibuprofen accelerated migration in a scratch-wound assay, while R-flurbiprofen had no effect and CHF5074 caused deceleration. Cell polarity regulation by Cdc42 and ERK1/2 may underlie the paradoxical effects of profens on migration. We conclude that profens regulate cytoskeleton dynamics in astrocytes via Rho-GTPases, PAK, and ERK1/2. Since migration is a hallmark of astrocyte response during inflammation we propose that, in addition to (or instead of) lowering amyloid-β42 via secretases, ibuprofen and its derivatives may prevent Alzheimer's disease instead of AD by modulating astrocyte reactivity through Rho-GTPase/PAK/ERK-dependent signaling.     

SAG: a Schwann cell membrane glycoprotein.

We report on characterization of a 170,000 Da glycoprotein found exclusively in the PNS. We refer to this protein as the Schwann cell membrane glycoprotein (SAG). SAG contains the HNK-1 carbohydrate, which is considered by some to be a marker of adhesion molecules. Its N-terminal sequence is not similar to previously known polypeptide sequences. SAG is found exclusively in the PNS, is present in rat sciatic nerve prior to myelination, and is in both myelinating and nonmyelinating Schwann cells. Tumors of Schwann cell lineage express SAG where axons are present (neurofibromas) but do not in the absence of axons (schwannomas). Schwannoma cells in culture do not express SAG even when exposed to forskolin, an activator of adenylate cyclase. However, schwannoma cells grown in the presence of a neuronal cell line (PC12) express SAG.     

Regulation of endothelial cell integrin function and angiogenesis by COX-2, cAMP and Protein Kinase A

Angiogenesis, the development of new blood vessels from preexisting vessels, is a key step in tumor growth, invasion and metastasis formation. Inhibition of tumor angiogenesis is considered as an attractive approach to suppress cancer progression and spreading. Adhesion receptors of the integrin family promote tumor angiogenesis by mediating cell migration, proliferation and survival of angiogenic endothelial cells. Integrins up regulated and highly expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, have been considered as relevant targets for anti-angiogenic therapies. Small molecular integrin antagonists or blocking antibodies suppress angiogenesis and tumor progression in many animal models, and some of them are currently being tested in cancer clinical trials as anti-angiogenic agents. COX-2 inhibitors exert anti-cancer effects, at least in part, by inhibiting tumor angiogenesis. We have recently shown that COX-2 inhibitors suppress endothelial cell migration and angiogenesis by preventing alphaVbeta3-mediated and cAMP/PKA-dependent activation of the small GTPases Rac and Cdc42. Here we will review the evidence for the involvement of vascular integrins in mediating angiogenesis and the role of COX-2 metabolites in modulating the cAMP/Protein Kinase A pathway and alphaVbeta3-dependent Rac activation in endothelial cells.     

Phosphorylation of the NF2 tumor suppressor in Schwann cells is mediated by Cdc42-Pak and requires paxillin binding

Mutations in the Neurofibromatosis type 2 tumor suppressor gene that encodes Schwannomin causes formation of benign schwannomas. Schwannoma cells lose their characteristic bipolar shape and become rounded with excessive ruffling membranes. Schwannomin is phosphorylated at serine 518 (S518) by p21 activated kinase (Pak). Unphosphorylated schwannomin is associated with growth inhibition but little is known about the function of the phosphorylated form, or the molecular events leading to its phosphorylation. Here, we report in SCs that schwannomin S518 phosphorylation requires binding to paxillin and targeting to the plasma membrane. Phospho-S518-schwannomin is enriched in the peripheral-most aspects of membrane specializations where paxillin, activated Pak, Cdc42 but not Rac are highly expressed. Schwannomin and Pak phosphorylation levels are not reduced in response to lowering Rac-GTP levels with NSC23766. Expression of schwannomin S518A/D-GFP variants each distinctively altered Schwann cell shape and polarity. These results are consistent with tight spatial regulation of S518 phosphorylation at the plasma membrane in a paxillin and Cdc42-Pak dependent manner that leads to local reorganization of the SC cytoskeleton.     

N-WASP regulates extension of filopodia and processes by oligodendrocyte progenitors, oligodendrocytes, and Schwann cells-implications for axon ensheathment at myelination

The molecular mechanisms used by oligodendrocyte precursor cells (OPCs), oligodendrocytes (OLs), and Schwann cells (SCs) to advance processes for motility in the developing nervous system and to ensheath axons at myelination are currently not well defined. Here we demonstrate that OPCs, OLs, and SCs express the major proteins involved in actin polymerization-driven protrusion; these key proteins including F-actin, the Arp2/3 complex, neural-Wiskott Aldrich Syndrome protein (N-WASP) and WAVE proteins, and the RhoGTPases Rac and Cdc42 are present at the leading edges of processes being extended by OPCs, OLs, and SCs. We reveal by real-time PCR that OLs and SCs have different dominant WAVE isoforms. Inhibition of the WASP/WAVE protein, N-WASP, with wiskostatin that prevents activation of the Arp2/3 complex, blocks process extension by OPCs and SCs. Inhibition of N-WASP also causes OPC and SC process retraction, which is preceded by retraction of filopodia. This implicates filopodia in OPC and SC process stability and also of N-WASP in OPC and SC process dynamics. We also demonstrate that p34 (a component of the Arp2/3 complex), WASP/WAVE proteins, actin, alpha-tubulin, Rac, Cdc42, vinculin, and focal adhesion kinase are detected in water-shocked myelin purified from brain. Inhibition of N-WASP with wiskostatin decreases the number of axons undergoing initial ensheathment in intact optic nerve samples and reduces the Po content of dorsal root ganglia:SC co-cultures. Our findings indicate that OPCs, OLs, and SCs extend processes using actin polymerization-driven protrusion dependent on N-WASP. We hypothesize that inner mesaxons of OLs and SCs use the same mechanism to ensheath axons at myelination.     

Rho family GTPases,Rac and Cdc42, control the localization of neonatal dentate granule cells during brain development

The hippocampus is generally considered as a brain center for learning and memory. We have recently established an electroporation‐mediated gene transfer method to investigate the development of neonatal dentate granule cells in vivo. Using this new technique, we introduced knockdown vectors against Rac1 small GTPase into precursors for dentate granule cells at postnatal day 0. After 21 days, Rac1‐deficient cells were frequently mispositioned between the granule cell layer (GCL) and hilus. About 60% of these mislocalized cells expressed a dentate granule cell marker, Prox1. Both the dendritic spine density and the ratio of mature spine were reduced when Rac1 was silenced. Notably, the deficient cells have immature thin processes during migrating in the early neonatal period. Knockdown of another Rac isoform, Rac3, also resulted in mislocalization of neonatally born dentate granule cells. In addition, knockdown of Cdc42, another Rho family protein, also caused mislocalization of the cell, although the effects were moderate compared to Rac1 and 3. Despite the ectopic localization, Rac3‐ or Cdc42‐disrupted mispositioned cells expressed Prox1. These results indicate that Rho signaling pathways differentially regulate the proper localization and differentiation of dentate granule cells.     

Activation of oncogenic pathways in degenerating neurons in Alzheimer disease

A number of recent findings have highlighted the similarities between neurogenesis during development and neurodegeneration during Alzheimer disease. In fact, neuronal populations that are known to degenerate in Alzheimer disease exhibit phenotypic changes characteristic of cells re-entering the cell division cycle. In this study, we extended these findings by investigating components of the cell cycle, known to trigger progression through G1 through activation of signal transduction cascades. Specifically, we found that proteins implicated in G1 transition, namely Cdc42/Rac, are upregulated in select neuronal populations in cases of Alzheimer disease in comparison to age-matched controls. Importantly, Cdc42/Rac shows considerable overlap with early cytoskeletal abnormalities suggesting that these changes are an extremely proximal event in the pathogenesis of the disease. Given the functional role of Cdc42/Rac in various cellular processes known to be perturbed in Alzheimer disease, namely cytoskeletal organization, oxidative balance, and oncogenic signaling, it is likely that increased neuronal Cdc42/Rac is highly significant in relation to the pathogenic process and contributes to neuronal degeneration. In fact, these findings suggest that Alzheimer disease is an oncogenic process.     

Receptor for advanced glycation end products (RAGE) mediates neuronal differentiation and neurite outgrowth

The receptor for advanced glycation end products (RAGE) plays a crucial role in several disease processes, such as diabetes, inflammation, and neurodegeneration. In this article we report multiple roles of RAGE in neuronal differentiation and neurite outgrowth. In retinoic-induced P19 embryonic carcinoma stem cells, silencing the expression of RAGE by RNA interference (RNAi) blocked differentiation of the P19 cells into neuronal cells and enhanced the formation of vimentin-positive fibroblast-like cells. RAGE knockdown inhibited retinoic acid-induced activation and blocked nuclear translocation of NF-kappaB, suggesting RAGE regulates activation of NF-kappaB. RAGE was also shown to be involved in survival of P19 cells during retinoic acid differentiation. Additionally, knockdown of RAGE strongly inhibited neurite outgrowth in retinoic acid-differentiated P19 cells, indicating that RAGE is required for neurite outgrowth of differentiated P19 cells. Retinoic acid-treated P19 cells activated GTPases, Rac1, and Cdc42. This activation of the GTPases was inhibited in RAGE-knockdown cells. In primary cerebellar granule neurons, the knockdown of RAGE also inhibited neurite outgrowth. In these cells, overexpression of dominant-negative forms of Rac1 and Cdc42 inhibited neurite outgrowth, whereas overexpression of constitutively active forms of Rac1 and Cdc42 in RAGE-deficient neurons restored neurite outgrowth, indicating that RAGE mediated neurite outgrowth through the Rac1/Cdc42 pathway. This is the first report on the role of RAGE in cell lines and primary neurons, as determined by RNAi knockdown.