Tanshinone IIA modulates cancer cell morphology and movement via Rho GTPases-mediated actin cytoskeleton remodeling.

Actin filaments form unique structures with robust actin bundles and cytoskeletal networks affixed to the extracellular matrix and interact with neighboring cells, which are crucial structures for cancer cells to acquire a motile phenotype. This study aims to investigate a novel antitumor mechanism by which Tanshinone IIA (Tan IIA) modulates the morphology and migration of liver cancer cells via actin cytoskeleton regulation. 97H and Huh7 exhibited numerous tentacle-like protrusions that interacted with neighboring cells. Following treatment with Tan IIA, 97H and Huh7 showed a complete absence of cytoplasmic protrusion and adherens junctions, thereby effectively impeding their migration capability. The fluorescence staining of F-actin and microtubules indicated that these tentacle-like protrusions and cell-cell networks were actin-based structures that led to morphological changes after Tan IIA treatment by retracting and reorganizing beneath the membrane. Tan IIA can reverse the actin depolymerization and cell morphology alterations induced by latrunculin A. Tan IIA down-regulated actin and Rho GTPases expression significantly, as opposed to inducing Rho signaling activation. Preventing the activity of proteasomes and lysosomes had no discernible impact on the modifications in cellular structure and protein expression induced by Tan IIA. However, as demonstrated by the puromycin labeling technique, the newly synthesized proteins were significantly inhibited by Tan IIA. In conclusion, Tan IIA can induce dramatic actin cytoskeleton remodeling by inhibiting the protein synthesis of actin and Rho GTPases, resulting in the suppression of tumor growth and migration. Targeting the actin cytoskeleton of Tan IIA is a promising strategy for HCC treatment.

A novel anti-apoptotic role for Cdc42/ACK-1 signaling in neurons.

Neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's and Parkinson's disease are caused by a progressive and aberrant destruction of neurons in the brain and spinal cord. These disorders lack effective long-term treatments that impact the underlying mechanisms of pathogenesis and as a result, existing options focus primarily on alleviating symptomology. Dysregulated programmed cell death (i.e., apoptosis) is a significant contributor to neurodegeneration, and is controlled by a number of different factors. Rho family GTPases are molecular switches with recognized importance in proper neuronal development and migration that have more recently emerged as central regulators of apoptosis and neuronal survival. Here, we investigated a role for the Rho GTPase family member, Cdc42, and its downstream effectors, in neuronal survival and apoptosis. We initially induced apoptosis in primary cultures of rat cerebellar granule neurons (CGNs) by removing both growth factor-containing serum and depolarizing potassium from the cell medium. We then utilized both chemical inhibitors and adenoviral shRNA targeted to Cdc42 to block the function of Cdc42 or its downstream effectors under either control or apoptotic conditions. Our in vitro studies demonstrate that functional inhibition of Cdc42 or its downstream effector, activated Cdc42-associated tyrosine kinase-1 (ACK-1), had no adverse effects on CGN survival under control conditions, but significantly sensitized neurons to cell death under apoptotic conditions. In conclusion, our results suggest a key pro-survival role for Cdc42/ACK-1 signaling in neurons, particularly in regulating neuronal susceptibility to pro-apoptotic stress such as that observed in neurodegenerative disorders.

MiR-936 Targets GPR78 and Regulates Chemotherapy Resistance in Non-Small Cell Lung Cancer by Activating the Galphaq Rho GTPase Pathway.

Objective: We aimed to explore the mechanism of microRNA-936 (miR-936) targeting G protein coupled receptor 78 (GPR78) regulating chemoresistance of non-small cell lung cancer (NSCLC) by activating the Galphaq Rho GTPase pathway. Methods: We added cisplatin to DMEM medium of HCC827/cisplatin cells and adjusted the final concentration to 1 µg/mL. Cells were divided into the control group and the miR-936 transfection group. Tissue samples were divided into the normal tissue group and the NSCLC tissue group. The mRNA expression of miR-936 in tissue samples was analyzed via reverse transcription polymerase chain reaction (RT-PCR). Cell migration and invasion were detected by wound healing assay. Cell counting kit 8 (CCK-8) was used to detect the cell viability 1, 2 and 3 days after cisplatin induction. The toxicity of cisplatin was analyzed by flow cytometry. The targeting relationship between miR-936 and GPR78 was detected by luciferase reporter gene assay. The regulation of miR-936 on GPR78/Rho GTPase was analyzed by Western blot. Results: The expression of miR-936 in NSCLC was lower than in normal tissues (P < .05). The number of cell migrations and invasions in the miR-936 transfection group was lower than in the control group (P < .05). The cell viability in the miR-936 transfection group was lower than in the control group on the 1st, 2nd and 3rd day (P < .05). With the increase in cisplatin concentration, the apoptosis rate of cells increased in a dependent manner (P < .05). Compared with GPR78 Mut, overexpression of miR-936 inhibited the luciferase activity of GPR78 WT 3'- UTR (P < .05). The expression of GPR78, RhoA, Rac1 and ABCB1 protein in the miR-936 transfection group was lower than in the control group (P < .05). The expression of GPR78 protein in the inhibitor+miR-936 transfection group was lower than in the inhibitor+control group (P < .05). Conclusion: miR-936 targets GPR78 and improves the sensitivity of NSCLC cells to cisplatin via the Galphaq Rho GTPase pathway.

Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism.

Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion. Our results show that F-actin content significantly increases upon chronic cholesterol depletion, but not during acute cholesterol depletion. In addition, utilizing inhibitors targeting the cholesterol biosynthetic pathway at different steps, we show that reorganization of the actin cytoskeleton could occur due to the synergistic effect of multiple pathways, including prenylated Rho GTPases and availability of membrane phosphatidylinositol 4,5-bisphosphate. These results constitute one of the first comprehensive dissections of the mechanistic basis underlying the interplay between cellular actin levels and cholesterol biosynthesis. We envision these results will be relevant for future understating of the remodeling of the actin cytoskeleton in pathological conditions with altered cholesterol.

Claudin-1/4 as directly target gene of HIF-1α can feedback regulating HIF-1α by PI3K-AKT-mTOR and impact the proliferation of esophageal squamous cell though Rho GTPase and p-JNK pathway.

Immunohistochemical microarray comprising 80 patients with esophageal squamous cell carcinoma (ESCC) and discovered that the expression of CLDN1 and CLDN4 were significantly higher in cancer tissues compared to para-cancerous tissues. Furthermore, CLDN4 significantly affected the overall survival of cancer patients. When two ESCC cell lines (TE1, KYSE410) were exposed to hypoxia (0.1% O2), CLDN1/4 was shown to influence the occurrence and development of esophageal cancer. Compared with the control culture group, the cancer cells cultured under hypoxic conditions exhibited obvious changes in CLDN1 and CLDN4 expression at both the mRNA and protein levels. Through genetic intervention and Chip, we found that HIF-1α could directly regulate the expression of CLDN1 and CLDN4 in cancer cells. Hypoxia can affect the proliferation and apoptosis of cancer cells by regulating the PI3K-Akt-mTOR pathway. Molecular analysis further revealed that CLDN1 and CLDN4 can participate in the regulation process and had a feedback regulatory effect on HIF-1α expression in cancer cells. In vitro cellular experiments and vivo experiments in nude mice further revealed that changes in CLDN4 expression in cancer cells could affect the proliferation of cancer cells via regulation of Rho GTP and p-JNK pathway. Whether CLDN4 can be target for the treatment of ESCC needs further research.

Re-expression of DIRAS3 and p53 induces apoptosis and impaired autophagy in head and neck squamous cell carcinoma.

BACKGROUND: p53 and DIRAS3 are tumor suppressors that are frequently silenced in tumors. In this study, we sought to determine whether the concurrent re-expression of p53 and DIRAS3 could effectively induce head and neck squamous cell carcinoma (HNSCC) cell death. METHODS: CAL-27 and SCC-25 cells were treated with Ad-DIRAS3 and rAd-p53 to induce re-expression of DIRAS3 and p53 respectively. The effects of DIRAS3 and p53 re-expression on the growth and apoptosis of HNSCC cells were examined by TUNEL assay, flow cytometric analysis and MTT. The effects of DIRAS3 and p53 re-expression on Akt phosphorylation, oncogene expression, and the interaction of 4E-BP1 with eIF4E were determined by real-time PCR, Western blotting and immunoprecipitation analysis. The ability of DIRAS3 and p53 re-expression to induce autophagy was evaluated by transmission electron microscopy, LC3 fluorescence microscopy and Western blotting. The effects of DIRAS3 and p53 re-expression on HNSCC growth were evaluated by using an orthotopic xenograft mouse model. RESULTS: TUNEL assay and flow cytometric analysis showed that the concurrent re-expression of DIRAS3 and p53 significantly induced apoptosis (P < 0.001). MTT and flow cytometric analysis revealed that DIRAS3 and p53 re-expression significantly inhibited proliferation and induced cell cycle arrest (P < 0.001). Mechanistically, the concurrent re-expression of DIRAS3 and p53 down-regulated signal transducer and activation of transcription 3 (STAT3) and up-regulated p21WAF1/CIP1 and Bax (P < 0.001). DIRAS3 and p53 re-expression also inhibited Akt phosphorylation, increased the interaction of eIF4E with 4E-BP1, and reduced the expression of c-Myc, cyclin D1, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor receptor (EGFR) and Bcl-2 (P < 0.001). Moreover, the concurrent re-expression of DIRAS3 and p53 increased the percentage of cells with GFP-LC3 puncta compared with that in cells treated with control adenovirus (50.00% ± 4.55% vs. 4.67% ± 1.25%, P < 0.001). LC3 fluorescence microscopy and Western blotting further showed that DIRAS3 and p53 re-expression significantly promoted autophagic activity but also inhibited autophagic flux, resulting in overall impaired autophagy. Finally, the concurrent re-expression of DIRAS3 and p53 significantly decreased the tumor volume compared with the control group in a HNSCC xenograft mouse model [(3.12 ± 0.75) mm3 vs. (189.02 ± 17.54) mm3, P < 0.001]. CONCLUSIONS: The concurrent re-expression of DIRAS3 and p53 is a more effective approach to HNSCC treatment than current treatment strategies.

Oncogenic DIRAS3 promotes malignant phenotypes of glioma by activating EGFR-AKT signaling.

Epidermal growth factor receptor (EGFR)-Akt signaling cascade activation plays a pivotal role in gliomas malignant phenotype, especially in Classical and Mesenchymal subtype gliomas. However, the molecules and mechanisms underlying regulate and maintain the activation of EGFR-AKT signaling remains unclear. Previously reports showed that DIRAS3 inhibits cell proliferation and induces autophagy in ovarian, breast, lung and prostate cancers, which is heterozygosity loss or down-regulated in aforementioned cancers and functionally as a tumor suppressor, whereas the role of DIRAS3 in glioma is still veiled. Here, in this study, we investigated the biological function and role of DIRAS3 in gliomas, and found that DIRAS3 is up-regulated in gliomas and is positively correlated with poor prognosis of glioma patients, meanwhile, over-expressed DIRAS3 promotes glioma cells proliferation and invasion. Further mechanistic study showed that the expression level of DIRAS3 in Classical and Mesenchymal subtype GBMs is higher, and over-expression of DIRAS3 promotes EGFR-AKT signaling activation at the downstream of EGFR and increases AKT phosphorylation, meanwhile suppression of AKT by MK-2206 reverses the tumor promoting function of DIRAS3. Taken together, these findings reveal a novel oncogenic role of DIRAS3 in the development and progression of glioma, which suggest that DIRAS3 could serve as a potential diagnostic marker and a promising therapeutic target of gliomas.

Rho GTPases control ciliary epithelium cells proliferation and progenitor profile induction in vivo.

PURPOSE: Rho GTPases play a central role in actin-based cytoskeleton reorganization and regulate multiple signaling pathways that control gene transcription, cell survival, and proliferation. We investigated the effect of Rho GTPases on cell cycle regulation and progenitor genes expression on mouse ciliary epithelium (CE), a potential source of progenitor/stem cells in the adult retina. METHODS: Rho GTPases were activated by intraocular injection of lysophosphatidic acid and inactivated by Clostridium difficile Toxin A (general Rho GTPase inhibitor), NSC23766 (Rac1 activation inhibitor), or Y27632 (Rho-associated protein kinase [ROCK] inhibitor). Thereafter, we assayed for RhoA, RhoB, and Rac1 protein localization in CE cells. Proliferation was examined by the expression levels of cell cycle regulators p27(kip), p16(INK4a), and Ki67 and the effects on progenitors by determining the changes in Pax6 and Chx10 progenitor markers expression. RESULTS: All GTPases investigated were expressed in mouse CE cells. Activation increased the coexpression of Pax6 and Chx10, but had no significant effect on the proliferation of CE cells. In contrast, Rho GTPases inactivation increased cell proliferation and potentiated the proliferative effect of growth factors. Specific inactivation of Rac1 or ROCK increased the levels of Ki67 and decreased the expression of the cell cycle inhibitors p27(kip) and p16(INK4a). CONCLUSIONS: This study reports that Rho GTPase modulation (activation and inactivation) controls the expression of retinal progenitor genes and proliferation, respectively, in the adult ciliary epithelial progenitor/stem cells of rodent eyes. The modulation of these two different mechanisms (proliferation and reprogramming) may provide a potential new approach in retinal repair.

In vitro reconstitution of activation of PLCepsilon by Ras and Rho GTPases.

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phophatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to diacylglycerol (DAG) and inositol 1,4,5-triphosphate [Ins(1,4,5)P3]. PLCepsilon is a recently discovered isoform that has been shown to be activated by members of the Ras and Rho families of guanosine trisphosphatases (GTPases) as well as subunits of heterotrimeric G-proteins. We describe a method for expressing a truncated PLCepsilon variant as an MBP fusion protein in E. coli. Subsequently, we describe the methodology necessary to reconstitute this protein with K-Ras-4B and RhoA GTPases and measure its activation.

RhoA and RhoC proteins promote both cell proliferation and cell invasion of human oesophageal squamous cell carcinoma cell lines in vitro and in vivo.

There is growing evidence that Rho proteins are deregulated by overexpression in tumours; and according to some reports, this correlates with disease progression. Our previous clinical study had demonstrated a correlation between RhoA expression and tumour progression in oesophageal squamous cell carcinoma (ESCC). These findings prompted us to study, using nude mice, pathological roles of Rho proteins in human ESCC cells. Western blot analysis in ESCC cell lines, in addition to cell proliferation and in vitro migration assays, were performed to observe the malignant potential of RhoA and RhoC in untransfected and transfected cells. Constitutively active RhoA, RhoC and dominant negative RhoA (dnRhoA) proteins were transfected to ESCC (TE-1 and TE-2) cells. The stably transfected cells were injected into nude mice, and the growth and metastasis of these cells to the lungs were analysed. Tumour tissues were then examined using immunohistochemical methods for proteins Ki-67 (MIB-1), FAK, MMP-1, MMP-9 and TIMP-3. Protein levels of RhoA and RhoC in ESCC cell lines were visualised by Western blotting, and showed highest expression in TE-2 cells. Results from the migration assay illustrated that both RhoA and RhoC play a role in migration of ESCC cells. In TE-2 transfected cells, RhoC showed greater migration compared to RhoA. By using an experimental metastasis model in nude mice, RhoA was found to promote more tumour growth than RhoC, whereas RhoC induced lung metastasis in comparison to RhoA. Ki-67 labelling index was used to evaluate the proliferation potential of tumour tissue inoculated from nude mice. In TE-2 cells RhoA gave a proliferation capacity of 24.8+/-0.5, which was significantly higher than those of TE-2 RhoC 10+/-0.4 (P<0.01). Strong immunoreactivity for FAK, MMP-1 and MMP-9 proteins was present in all tumour cells. By contrast, loss of TIMP-3 expression was observed in all tumour cells. In conclusion, our results indicate that pro-oncogenic Rho proteins are involved in promoting tumour growth, cell migration and metastasis in human ESCC cells in nude mice. The results from this study suggest that active Rho proteins may induce a transforming effect that leads to a malignant phenotype.

Simvastatin decreases IL-6 and IL-8 production in epithelial cells.

Many cardiovascular studies have suggested that 3-hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors (statins) have anti-inflammatory effects independent of cholesterol lowering. As a chronic inflammatory disease, periodontitis shares some mechanisms with atherosclerosis. Since oral epithelial cells participate importantly in periodontal inflammation, we measured simvastatin effects on interleukin-6 and interleukin-8 production by cultured human epithelial cell line (KB cells) in response to interleukin-1alpha. Simvastatin decreased production, an effect reversed by adding mevalonate or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate. Simvastatin was found to reduce NF-kappaB and AP-1 promoter activity in KB cells. Dominant-negative Rac1 severely inhibited interleukin-1alpha-induced NF-kappaB and AP-1 promoter activity. Our results may indicate an anti-inflammatory effect of simvastatin on human oral epithelial cells, apparently involving Rac1 GTPase inhibition.

Regulation of microtubule dynamics in 3T3 fibroblasts by Rho family GTPases.

To get insight into the action of Rho GTPases on the microtubule system we investigated the effects of Cdc42, Rac1, and RhoA on the dynamics of microtubules in Swiss 3T3 fibroblasts. In control cells microtubule ends were dynamic: plus ends frequently switched between growth, shortening and pauses; the growth phase predominated over shortening. Free minus ends of microtubules depolymerized rapidly and never grew. Free microtubules were short-lived, and the microtubule network was organized into a radial array. In serum-starved cells microtubule ends became more stable: although plus ends still transited between growth and shortening, polymerization and depolymerization excursions became shorter and balanced each other. Microtubule minus ends were also stabilized. Consequently lifespan of free microtubules increased and microtubule array changed its radial pattern into a random one. Activation of Cdc42 and Rac1 in serum-starved cells promoted dynamic behavior of microtubule plus and minus ends, while inhibition of these GTPases in serum-grown cells suppressed microtubule dynamics and mimicked all effects of serum starvation. Activation of RhoA in serum-grown cells had effects similar to Cdc42 /Rac1 inactivation: it suppressed the dynamics of plus and minus ends, reduced the length of growth and shrinking episodes, and disrupted the radial organization of microtubules. However, in contrast to Cdc42 and Rac1 inactivation, active RhoA had no effect on the balance between microtubule growth and shortening. We conclude that Cdc42 and Rac1 have similar stimulating effects on microtubule dynamics while RhoA acts in an opposite way.

A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts.

Osteoclast maturation is accompanied by changes in podosome patterning, resulting in the formation of a peripheral belt, which requires an intact microtubule network. Here, we report that by inhibiting Rho, the podosome belt is maintained at the cell periphery despite depolymerisation of microtubules by nocodazole. Rho inhibition was correlated to the increase in microtubule stabilisation and microtubule acetylation. By microinjecting activated Rho or its activated effector mDia2 in osteoclasts, we found that the podosome belt was disrupted and the level of microtubule acetylation dramatically decreased. We further characterised the molecular mechanism responsible for microtubule deacetylation by co-immunoprecipitation experiments. We found that not only was mDia2 coprecipitating with the recently identified microtubule deacetylase HDAC6 but that it also activated the microtubule deacetylase activity of HDAC6 in an in vitro deacetylase assay. Finally, we found that during osteoclastogenesis, there is a correlation between the increase in microtubule acetylation and the podosome belt stabilisation and that if Rho is inhibited in the early stages of osteoclast differentiation, it accelerates both microtubule acetylation and podosome belt stabilisation. Altogether, our data reveal a pathway in which Rho interferes with the osteoclast maturation process by controlling the level of microtubule acetylation and actin organisation through mDIA2 and HDAC6.

Modulation of Rho GTPase activity alleviates chondroitin sulfate proteoglycan-dependent inhibition of neurite extension.

The central nervous system (CNS) fails to regenerate after injury. A glial scar forms at the injury site, contributing to regenerative failure partly resulting from the chondroitin sulfate proteoglycans (CSPGs) in the glial scar. The family of Rho GTPases, which includes Cdc42, Rac1, and RhoA, is involved in growth cone dynamics. Although the response of neural cells to the inactivation of Rho when contacting myelin-related substrates, or CSPG, has been investigated, Rac1's and Cdc42's abilities to modulate CSPG-dependent inhibition have yet to be explored. In this study, a stripe assay was utilized to examine the effects of modulating all three Rho GTPases on neurite extension across inhibitory CSPG lanes. Alternating laminin (LN) and CSPG lanes were created and NG108-15 cells and E9 chick dorsal root ganglia (DRGs) were cultured on the lanes. By using the protein delivery agent Chariot, the neuronal response to exposure of constitutively active (CA) and dominant negative (DN) mutants of the Rho GTPases, along with the bacterial toxin C3, was determined by quantifying the percentage ratio of neurites crossing the CSPG lanes. CA-Cdc42, CA-Rac1, and C3 transferase significantly increased the number of neurites crossing into the CSPG lanes compared with the negative controls for both the NG108-15 cells and the E9 chick DRGs. We also show that these mutant proteins require the delivery vehicle, Chariot, to enter the neurons and affect neurite extension. Therefore, activation of Cdc42 and Rac, as well as inhibition of Rho, helps overcome the CSPG-dependent inhibition of neurite extension.

Effects of microinjected small GTPases on the actin cytoskeleton of human neutrophils.

This paper describes a method for microinjection of proteins (Rho GTPases) into neutrophils and observations on the responses of the cells to these injections. Neutrophils are extremely difficult to inject because of their small size, complex morphology and fragility. To allow microinjections they must be cultured on a substrate that enables them to settle, adhere and spread. We determined that fibronectin- and/or collagen-coated coverslips are the best substrates and we used very fine needles and short microinjection times to minimize cell damage. These methods permitted us to inject up to 100 cells in a single preparation over a period of 30 min. Effects of microinjection were assessed by using tetramethylrhodamine isothiocyanate (TRITC)-phalloidin to label F-actin filaments, and observation by fluorescence and confocal scanning microscopy. Microinjection alone resulted in cell rounding and some changes in the F-actin cytoskeleton but injected cells remained adherent at the substrate, were able to respond to microinjected GTPases (V12Rac, V14RhoA, V12Cdc42) and continued to be responsive to activation by exposure to fMet-Leu-Phe (fMLP) or O-tetradecanoylphorbal 13-acetate (TPA). V12Rac caused an increase in neutrophil membrane ruffling and short protrusions from the cell membrane, whereas V14RhoA induced a large increase in punctate F-actin structures. V12Cdc42 produced focal condensation of F-actin and induced the formation of small microspikes. The differences between these responses of neutrophils and those of other similarly treated cell types are discussed. Our findings demonstrate that microinjection is a valuable technique for studying the role of individual proteins in neutrophils.

Cytotoxic necrotizing factor-2 of Escherichia coli alters the morphology of cultured hippocampal neurons.

Certain pathogenic strains of E. coli produce the cytotoxic necrotizing factors-1 or -2. Cytotoxic necrotizing factor-1 irreversibly activates the small GTPases of the Rho family Rho, Rac and Cdc42. Cytotoxic necrotizing factor-2 may have similar effects. Since the Rho proteins play an important role in the organization of the actin cytoskeleton and neuronal differentiation, we have investigated whether cytotoxic necrotizing factor-2 affects the morphology of cultured hippocampal neurons. The toxin indeed caused dendrite retraction and axon shortening. Within 4 h of application, cytotoxic necrotizing factor-2 induced a transient formation of short finger-like extensions. To study the role of the Rho proteins in the morphological changes caused by cytotoxic necrotizing factor-2, we transfected neurons with recombinant Rho proteins. Dominant-negative forms of Rac or Rho but not of Cdc42 prevented the formation of short extensions induced by cytotoxic necrotizing factor-2, indicating synergistic effects of Rac and Rho. In contrast, the retraction of dendrites induced by cytotoxic necrotizing factor-2 was only prevented by dominant-negative Rho. Analysis with pull-down assays showed that cytotoxic necrotizing factor-2 strongly activated Rac and Rho, whereas an effect on Cdc42 was not observed. Cytotoxic necrotizing factor-2 also diminished the total amount of Rac and Rho. The degradation of Rac was so pronounced that the increase in Rac activity was only transient. In organotypic cultures of the hippocampus, cytotoxic necrotizing factor-2 reduced the number of neurites per neuron, suggesting that neurons in the tissue context were also vulnerable. We conclude that cytotoxic necrotizing factor-2 has pronounced effects on neuronal morphology, which are due to activation of the GTPases Rho and Rac.

Cholesterol and tau protein--findings in Alzheimer's and Niemann Pick C's disease.

Niemann Pick C (NPC), a fatal autosomal-recessive neurovisceral lipid storage disorder, is a juvenile dementia with massive nerve-cell loss and cytoskeletal abnormalities in cerebral neurons. These abnormalities consist of tangles of tau protein, which is otherwise highly soluble and usually stabilizes the microtubules. Immunologically and ultrastructurally similar tangles are seen some decades later in patients with Alzheimer's disease (AD). There is evidence that tangle-bearing cells in both diseases show higher levels of free (i. e. filipin-positive) cholesterol than adjacent tangle-free nerve cells. The cholesterol accumulates either in a more diffuse way (mainly in AD) or in granule-like accumulations (mainly in NPC). In NPC, neuron cholesterol may originate from sources other than the alimentary tract. Experiments with a NPC mouse model revealed that even in pure neuron cultures, the NPC -/- neurons accumulate free cholesterol in contrast to NPC-wt littermates, suggesting that the cholesterol is either synthesized by the neurons or liberated from degenerated ones before being taken up by the endosomal/lysosomal pathway. The accumulation of free cholesterol in the somata of NPC neurons is associated with a decrease of cholesterol levels in myelin sheaths. In terms of tau protein, NPC -/- mice exhibit higher levels of AT8-positive tau, suggesting that the phosphorylation-dependent mAb AT8 has detected a tau-epitope in a state considered to represent early stages of tangle formation. Concomitantly to the increase in free intracellular cholesterol, the rate-limiting enzyme in cholesterol and isoprenoid biosynthesis, HMG-CoA reductase, was found to be significantly reduced. Experimental blockade of the enzyme's activity by application of the lipid-lowering drug lovastatin showed subcellular shifts in tau phosphorylation as monitored with mAbs AT8, 12E8 and others. In summary, the data showed interesting similarities between NPC and AD suggesting some pathological metabolic pathway in common.

[Lymphocytes: what is "Vav"]. / Les lymphocytes: comment ça "Vav"?

Guanine exchange factors (GEF) of the Vav family are critical activators of Rho GTPases, which control actin cytoskeletal reorganization and gene transcription. Among all GEFs identified, Vav proteins are the only GEFs regulated by tyrosine phosphorylation. Moreover, their structure contains several protein-protein or protein-lipid interaction domains. These domains are involved in the formation of multimolecular signalling complexes, highlighting the adaptor role of Vav proteins. The unique combination of these properties makes Vav proteins privileged integrators of multiple signalling pathways in a broad range of tissues and cells. Lymphocyte function during inflammatory and immune responses requires a dynamic remodeling of cellular architecture. Thus, it is not surprising that Vav proteins have been found to play a central role in the regulation of physiologic and pathologic lymphocyte responses.

[Rho proteins: their function in neurons]. / Les protéines Rho: leur rôle dans les neurones.

Neurons possess a polarized morphology. In general, each neuron has several dendrites but only one axon. Such morphology is the basis for directionalized rapid signaling, information flowing from the short dendrites to the long axon. The mechanisms involved in the establishment of the neuronal polarity remain largely unknown. However, recently, members of Rho family proteins have been implicated in the regulation of neuronal morphology especially development of neuronal polarity, axon outgrowth and guidance, dendritic tree elaboration and synapse formation. Moreover, the Rho GTPases have been reported to be directly or indirectly involved in some neurological conditions such as X-linked mental retardation as well as Alzheimer's and Parkinson's diseases. These findings demonstrate the importance of Rho GTPases in the development, maintenance and function of the nervous system.