Tau regulates the microtubule-dependent migration of glioblastoma cells via the Rho-ROCK signaling pathway.

The pathological significance of Tau (encoded by MAPT) in mechanisms driving cell migration in glioblastoma is unclear. By using an shRNA approach to deplete microtubule-stabilizing Tau in U87 cells, we determined its impact on cytoskeletal coordination during migration. We demonstrated here that the motility of these Tau-knockdown cells (shTau cells) was significantly (36%) lower than that of control cells. The shTau cells displayed a slightly changed motility in the presence of nocodazole, which inhibits microtubule formation. Such reduced motility of shTau cells was characterized by a 28% lower number of microtubule bundles at the non-adhesive edges of the tails. In accordance with Tau-stabilized microtubules being required for cell movement, measurements of the front, body and rear section displacements of cells showed inefficient tail retraction in shTau cells. The tail retraction was restored by treatment with Y27632, an inhibitor of Rho-ROCK signaling. Moreover, we clearly identified that shTau cells displayed relocation of the active phosphorylated form of p190-RhoGAP (also known as ARHGAP35), which inhibits Rho-ROCK signaling, and focal adhesion kinase (FAK, also known as PTK2) in cell bodies. In conclusion, our findings indicate that Tau governs the remodeling of microtubule and actin networks for the retraction of the tail of cells, which is necessary for effective migration.

Direct evidence for the interaction of stathmin along the length and the plus end of microtubules in cells.

Stathmin is a prominent destabilizer of microtubules (MTs). Extensive in vitro studies have strongly suggested that stathmin could act by sequestering tubulin and/or by binding to MT tips. In cells, the molecular mechanisms of stathmin binding to tubulin and/or MTs and its implications for the MT dynamics remain unexplored. By using immunofluorescence resonance energy transfer and fluorescence recovery after photobleaching, we analyzed the ability of stathmin and its phosphorylated forms (on Ser16, -25, -38, and -63) to interact with tubulin and MTs in A549 cells. Consistent with in vitro studies, we detected stathmin-tubulin interactions at the MT plus ends and in the cytosol. Of interest, we also observed a novel pool of stathmin bound along the MT. Expression of truncated stathmin and use of MT-stabilizing taxol further showed that the C-terminal domain of stathmin is the main contributor to this binding and that the phosphorylation state of stathmin plays a role in its binding along the MT wall. Our findings demonstrate that stathmin binds directly along the MT wall. This pool of stathmin would be readily available to participate in protofilament dissociation when the moving plus end of a depolymerizing MT reaches stathmin molecules.-Nouar, R., Breuzard, G., Bastonero, S., Gorokhova, S., Barbier, P., Devred, F., Kovacic, H., Peyrot, V. Direct evidence for the interaction of stathmin along the length and the plus end of microtubules in cells.

ROS-mediated EB1 phosphorylation through Akt/GSK3ß pathway: implication in cancer cell response to microtubule-targeting agents.

Microtubule-targeting agents (MTAs) are largely administered in adults and children cancers. Better deciphering their mechanism of action is of prime importance to develop more convenient therapy strategies. Here, we addressed the question of how reactive oxygen species (ROS) generation by mitochondria can be necessary for MTA efficacy. We showed for the first time that EB1 associates with microtubules in a phosphorylation-dependent manner, under control of ROS. By using phospho-defective mutants, we further characterized the Serine 155 residue as critical for EB1 accumulation at microtubule plus-ends, and both cancer cell migration and proliferation. Phosphorylation of EB1 on the Threonine 166 residue triggered opposite effects, and was identified as a requisite molecular switch in MTA activities. We then showed that GSK3ß activation was responsible for MTA-triggered EB1 phosphorylation, resulting from ROS-mediated inhibition of upstream Akt. We thus disclosed here a novel pathway by which generation of mitochondrial ROS modulates microtubule dynamics through phosphorylation of EB1, improving our fundamental knowledge about this oncogenic protein, and pointing out the need to re-examine the current dogma of microtubule targeting by MTAs. The present work also provides a strong mechanistic rational to the promising therapeutic strategies that currently combine MTAs with anti-Akt targeted therapies.

Saccharomyces boulardii improves intestinal epithelial cell restitution by inhibiting αvß5 integrin activation state.

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of infectious or inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the disappearance of inflammation and the repair of damaged epithelium. Saccharomyces boulardii (Sb, Biocodex) is a non-pathogenic yeast widely used as a preventive and therapeutic probiotic for the prevention and treatment of diarrhea and other gastrointestinal disorders. We recently showed that it enhances the repair of intestinal epithelium through activation of α2ß1 integrin collagen receptors. In the present study, we demonstrated that α2ß1 integrin is not the sole cell-extracellular matrix receptor involved during Sb-mediated intestinal restitution. Indeed, by using cell adhesion assays, we showed that Sb supernatant contains heat sensitive molecule(s), with a molecular weight higher than 9 kDa, which decreased αvß5 integrin-mediated adhesion to vitronectin by competing with the integrin. Moreover, Sb-mediated changes in cell adhesion to vitronectin resulted in a reduction of the αvß5signaling pathway. We used a monolayer wounding assay that mimics in vivo cell restitution to demonstrate that down-modulation of the αvß5 integrin-vitronectin interaction is related to Sb-induced cell migration. We therefore postulated that Sb supernatant contains motogenic factors that enhance cell restitution through multiple pathways, including the dynamic fine regulation of αvß5 integrin binding activity. This could be of major importance in diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.

The transcriptional programme of human heart valves reveals the natural history of infective endocarditis.

Infective endocarditis (IE) is an infectious disease that is mainly caused by Staphylococcus aureus and Streptococcus sp. It usually leads to valvular destruction and vegetation formation. Its pathophysiology is badly understood and likely involves immune and coagulation systems with close interactions with the microorganism. Our objective was to evaluate host response by comparing transcriptional profiles of cardiac valves from IE patients with controls. Hierarchical clustering revealed a signature of IE consisting of 146 genes. Among the 89 up-regulated genes, we identified two genes strongly associated with IE: metalloproteinase 12 (MMP-12) and aquaporin-9, a member of the aquaglyceroporin membrane channel family. The up-regulation of MMP-12 gene is strengthened by the down-modulation of the gene encoding its inhibitor TIMP3. In addition, MMP-12 was expressed in macrophages infiltrating EI valves. We also found that aquaporin-9 was expressed in endothelial cells lining neo-vessel lumen, suggesting that aquaporin-9 might be associated with neovascularization of infected valves leading to tissue oedema secondary to the inflammatory process. The Gene Ontology annotation and the resulting functional classification showed that most up-regulated genes account for recruitment of inflammatory cells in vegetations, angiogenesis and remodelling of endocardium tissue. A network analysis confirmed the involvement of molecules related to the remodelling of endocardium tissue and angiogenesis in IE. It also evidenced the role of caspases, especially that of caspase-9 and intrinsic apoptotic pathway in IE. Based on this study we propose a scenario for the natural history of IE in humans. Some parameters identified in this work could become tools for measuring the disease activity and should be tested as biomarkers for diagnosis or prognosis assessment in future studies.

New microbicidal functions of tracheal glands: defective anti-infectious response to Pseudomonas aeruginosa in cystic fibrosis.

Tracheal glands (TG) may play a specific role in the pathogenesis of cystic fibrosis (CF), a disease due to mutations in the cftr gene and characterized by airway inflammation and Pseudomonas aeruginosa infection. We compared the gene expression of wild-type TG cells and TG cells with the cftr DeltaF508 mutation (CF-TG cells) using microarrays covering the whole human genome. In the absence of infection, CF-TG cells constitutively exhibited an inflammatory signature, including genes that encode molecules such as IL-1alpha, IL-beta, IL-32, TNFSF14, LIF, CXCL1 and PLAU. In response to P. aeruginosa, genes associated with IFN-gamma response to infection (CXCL10, IL-24, IFNgammaR2) and other mediators of anti-infectious responses (CSF2, MMP1, MMP3, TLR2, S100 calcium-binding proteins A) were markedly up-regulated in wild-type TG cells. This microbicidal signature was silent in CF-TG cells. The deficiency of genes associated with IFN-gamma response was accompanied by the defective membrane expression of IFNgammaR2 and altered response of CF-TG cells to exogenous IFN-gamma. In addition, CF-TG cells were unable to secrete CXCL10, IL-24 and S100A8/S100A9 in response to P. aeruginosa. The differences between wild-type TG and CF-TG cells were due to the cftr mutation since gene expression was similar in wild-type TG cells and CF-TG cells transfected with a plasmid containing a functional cftr gene. Finally, we reported an altered sphingolipid metabolism in CF-TG cells, which may account for their inflammatory signature. This first comprehensive analysis of gene expression in TG cells proposes a protective role of wild-type TG against airborne pathogens and reveals an original program in which anti-infectious response was deficient in TG cells with a cftr mutation. This defective response may explain why host response does not contribute to protection against P. aeruginosa in CF.

Inhibition by TNF-alpha and IL-4 of cationic lipid mediated gene transfer in cystic fibrosis tracheal gland cells.

BACKGROUND: In vivo, tracheal gland serous cells highly express the cystic fibrosis transmembrane conductance regulator (cftr) gene. This gene is mutated in the lethal monogenic disease cystic fibrosis (CF). Clinical trials in which the human CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in weak and transient gene expression. METHODS AND RESULTS: As CF is characterized by mucus inspissation, airway infection, and severe inflammation, we tested the hypothesis that inflammation and especially two cytokines involved in the Th1/Th2 inflammatory response, interleukin 4 (IL-4) and TNFalpha, could inhibit gene transfer efficiency using a model of human CF tracheal gland cells (CF-KM4) and Lipofectamine reagent as a transfection reagent. The specific secretory defects of CF-KM4 cells were corrected by Lipofectamine-mediated human CFTR gene transfer. However, this was altered when cells were pre-treated with IL-4 and TNFalpha. Inhibition of luciferase reporter gene expression by IL-4 and TNFalpha pre-treated CF-KM4 cells was measured by activity and real-time RT-PCR. Both cytokines induced similar and synergistic inhibition of transgene expression and activity. This cytokine-mediated inhibition could be prevented by anti-inflammatory agents such as glucocorticoids but not by non-steroidal (NSAI) agents. CONCLUSIONS: This data suggests that an inflammatory context generated by IL-4 and TNFalpha can inhibit human CFTR gene transfer in CF tracheal gland cells and that glucocorticoids may have a protecting action.