Rôle de la GTPase ARF6 dans la prolifération cellulaire

Bien qu'elle soit vitale en conditions physiologiques, la prolifération cellulaire pourrait contribuer à la genèse et la progression de maladies graves telles que l'athérosclérose et le cancer. Dans cette étude, nous démontrons que la GTPase ARF6, connue principalement pour son rôle dans l'endocytose, le trafic membrane plasmique-endosomes et le remodelage de l'actine, contrôle la prolifération cellulaire à deux niveaux différents du cycle cellulaire soit le début de la phase G1 et la mitose. D'abord, dans un contexte cardiovasculaire, nous rapportons qu'ARF6 contrôle la production des ROS induite par l'AngII en régulant positivement l'activation de la GTPase Rac1 et l'expression de la NADPH oxydase NOX1. Les ROS agissent comme médiateurs pour favoriser la transactivation de l'EGFR et l'activation des MAP Kinases ERK1/2, p38MAPK et JNK1/2 induisant l'expression des gènes nécessaires à la transition G1/S et la prolifération. Ensuite, nous montrons qu'ARF6 joue un rôle important dans la dernière phase du cycle cellulaire, la mitose. En effet, ARF6 est nécessaire pour la stabilté des fibres-K et le bon positionnement des chromosomes au niveau de la plaque de métaphase, ce qui permet la satisfaction du SAC, l'ubiquitination de la cycline B, sa dégradation et la ségrégation chromosomique (l'anaphase). Finalement, en étudiant la voie mTOR dans les cellules déplétées en ARF6, nous avons identifié une nouvelle régulation de cette voie durant la mitose. Nous démontrons que les kinases p85S6K1 et S6K2 s'activent d'une manière dépendante de CDK1 pour phosphoryler la protéine ribosomale S6 durant la mitose.Although it is vital in normal physiological conditions, cell proliferation, when poorly controlled, could contribute to the pathogenesis and progression of serious diseases such as atherosclerosis and cancer. In this study, we demonstrate that the GTPase ARF6, classically known for its role in endocytosis, plasma membrane-endosomal trafficking and actin remodeling, controls cell proliferation at two different levels of the cell cycle, G1 phase and mitosis. First, in a cardiovascular context, we show that ARF6 controls AngII-induced ROS production by positively regulating Rac1 activation and NOX1 expression. ROS act as mediators to promote the transactivation of EGFR and the activation of the three MAP Kinases ERK1/2, p38MAPK and JNK1/2 inducing the expression of genes required for G1/S transition and proliferation. Second, we show that ARF6 plays an important role in the last phase of the cell cycle, mitosis. Indeed, ARF6 is required for the stability of microtubules (K-fibres) and the proper positioning of chromosomes at the metaphase plate allowing SAC satisfaction, triggering of cyclin B ubiquitination, its degradation and chromosomal segregation (anaphase). Finally, by studying the mTOR pathway in ARF6 depleted cells, we characterized a new regulation of this pathway during mitosis. We demonstrate that the kinases p85S6K1 and S6K2 are activated in a CDK1-dependent manner to phosphorylate the ribosomal protein S6 during mitosis

C9ORF72 interaction with cofilin modulates actin dynamics in motor neurons.

Intronic hexanucleotide expansions in C9ORF72 are common in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non-ATG-initiated translation are responsible for the pathophysiology. We determined the interactome of C9ORF72 in motor neurons and found that C9ORF72 was present in a complex with cofilin and other actin binding proteins. Phosphorylation of cofilin was enhanced in C9ORF72-depleted motor neurons, in patient-derived lymphoblastoid cells, induced pluripotent stem cell-derived motor neurons and post-mortem brain samples from ALS patients. C9ORF72 modulates the activity of the small GTPases Arf6 and Rac1, resulting in enhanced activity of LIM-kinases 1 and 2 (LIMK1/2). This results in reduced axonal actin dynamics in C9ORF72-depleted motor neurons. Dominant negative Arf6 rescues this defect, suggesting that C9ORF72 acts as a modulator of small GTPases in a pathway that regulates axonal actin dynamics

The GTPase ARF6 Controls ROS Production to Mediate Angiotensin II-Induced Vascular Smooth Muscle Cell Proliferation.

High reactive oxygen species (ROS) levels and enhanced vascular smooth muscle cells (VSMC) proliferation are observed in numerous cardiovascular diseases. The mechanisms by which hormones such as angiotensin II (Ang II) acts to promote these cellular responses remain poorly understood. We have previously shown that the ADP-ribosylation factor 6 (ARF6), a molecular switch that coordinates intracellular signaling events can be activated by the Ang II receptor (AT1R). Whether this small GTP-binding protein controls the signaling events leading to ROS production and therefore Ang II-dependent VSMC proliferation, remains however unknown. Here, we demonstrate that in rat aortic VSMC, Ang II stimulation led to the subsequent activation of ARF6 and Rac1, a key regulator of NADPH oxidase activity. Using RNA interference, we showed that ARF6 is essential for ROS generation since in conditions where this GTPase was knocked down, Ang II could no longer promote superoxide anion production. In addition to regulating Rac1 activity, ARF6 also controlled expression of the NADPH oxidase 1 (Nox 1) as well as the ability of the EGFR to become transactivated. Finally, ARF6 also controlled MAPK (Erk1/2, p38 and Jnk) activation, a key pathway of VSMC proliferation. Altogether, our findings demonstrate that Ang II promotes activation of ARF6 to controls ROS production by regulating Rac1 activation and Nox1 expression. In turn, increased ROS acts to activate the MAPK pathway. These signaling events represent a new molecular mechanism by which Ang II can promote proliferation of VSMC

Schematic representation of the molecular mechanism by which ARF6 mediates Ang II promoted ROS generation and proliferation of VSMC.

<p>Stimulation of the AT₁R by Ang II leads to the activation of ARF6, which is essential for the activation of Rac1. This Rho GTPase acts to control NADPH oxidase and formation of ROS. These signaling intermediates play numerous roles in VSMC. They are essential for EGFR transactivation and MAPK phosphorylation. In addition, ARF6 can regulate Nox1 expression to further support ROS production. Altogether, our findings show that ARF6 is a molecular switch regulating cellular proliferation.</p

ARF6 regulates Nox1 expression.

<p>(A) Nox1 and Nox4 protein expression was examined in control and ARF6 depleted VSMC using Western blot analysis. Levels of ARF6 and actin were also determined. Graph represents quantification of all data (n = 3, ***P< 0.001). (B) mRNA levels of Nox1 and Nox4 were also assessed in cells infected with the control and ARF6 shRNA. Data were normalized to two control mRNA (GADPH and 4-HPRT) and presented as fold change over one control experiment (n = 3, *P < 0.05). (C) Noxa1 and Noxo1 protein levels were measured in control and ARF6 depleted VSMC using Western blot analysis. Graph represents quantification of three independent experiments (n = 3). (D) Nox1 and Nox4 protein expression was also examined in control and Rac1 depleted VSMC as in (A) (n = 3). (E) VSMC were transiently transfected with empty vector, HA-ARF6, HA-ARF6 T¹⁵⁷A, HA-ARF6 T²⁷N, myc-Rac1, myc-Rac1 Q⁶¹L or myc-Rac1 T¹⁷N and Nox1, actin, HA-tag and myc-tag levels were detected using Western blot analysis (n = 3).</p

ARF6 mediates Ang II induced cellular proliferation through ROS.

<p>(A) Control and ARF6 depleted cells were stimulated or not with Ang II (100 nM) for the indicated times. Manual cell count was performed for each experimental condition (n = 3, ***P < 0.001). (B) Proliferation of control and ARF6 depleted cells stimulated or not with Ang II was assessed using the MTT assay (n = 3, ***P < 0.001). (C) Control and Rac1 T¹⁷N expressing cells were left untreated or stimulated with Ang II (100 nM) for 72h. Proliferation was assessed using the MTT assay as in (B) (n = 3, **<i>P</i> < 0.01, ***<i>P</i> < 0.001). (D) Cells incubated with DMSO, AG1478 (100 nM), ML171 (5 μM) or DPI (1 μM) were stimulated or not with Ang II for 72h and proliferation was assessed using MTT assay (n = 3, ***P < 0.001). (E). Cellular viability of control, ARF6 depleted and DPI treated cells stimulated or not with Ang II was determined using trypan blue (n = 3)</p

Ang II-induced superoxide anion production is ARF6 dependent.

<p>(A) Control and ARF6 depleted VSMC were stimulated with Ang II (100 nM) for the indicated time and incubated with DHE. Images are representative of each condition. Cells were examined to confirm ARF6 knock down by Western blot. (B) Graph represents quantitative analysis of fluorescence intensity mean per cell presented as fold change over basal (scrambled shRNA, t = 0). 3–4 images representative of 1000–1500 cells per time point of Ang II stimulation and per condition were analyzed using ImageJ software. DHE fluorescence were quantified and normalized to cells number following DAPI staining (n = 3, *P < 0.05, **P < 0.01). (C) Quiescent control and ARF6 depleted VSMC were stimulated or not with AngII (100 nM, 60 min) and superoxide anion levels were measured using cytochrome C reduction assay as described in materials and methods (n = 3, *P < 0.05). (D) Control and Rac1 T¹⁷N overexpressing VSMC were stimulated or not with Ang II. Superoxide anion levels were then evaluated by DHE staining as in (A). Rac1 T¹⁷N overexpression was confirmed by Western blot analysis. (E) Results obtained in (D) were quantified by analysis of fluorescence intensity average per cell as in (B) (n = 3, **<i>P</i> < 0.01).</p

ARF6 controls Ang II-induced Rac1 activation.

<p>VSMC were stimulated for the indicated times with Ang II (100 nM) then lysed. (A). Endogenous levels of activated ARF6 (ARF6-GTP) captured by GST pulldown assay and total ARF6 (input) were assessed by Western blot analysis (n = 4, *P < 0.05). (B). Activated and total Rac1 levels were also determined by Western blot analysis (n = 3, **P < 0.01, ***P< 0.001). (C). VSMC were infected with scrambled or ARF6 shRNA lentiviruses. At the third day of infection, cells were serum starved for 48h and stimulated or not with Ang II for 5 min. Activated Rac1 was assessed as in (B). Quantifications are presented as fold-change over basal (cnt shRNA, t = 0) and are normalized to total protein content (n = 3, **<i>P</i> < 0.01).</p

ARF6 is required for the activation of the ROS sensitive Erk1/2, p38 and Jnk.

<p>(A) Control and ARF6 depleted VSMC were stimulated with Ang II (100 nM) for the indicated times. Phosphorylation and total levels of Erk1/2, p38, and Jnk were examined (n = 3, **P < 0.01). (B) Cells were treated with vehicle or DPI (10 μM) and stimulated with Ang II (100 nM) for the indicated times. Phosphorylation levels of Erk1/2, p38 and Jnk were assessed as in (A). Results are representative of three independent experiments and quantifications are the mean ± SEM (n = 3, *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001). (C) Cells were treated with vehicle, AG1478 (100 nM) or ML171 (5 μM) for 30 min then stimulated with Ang II for the indicated times. Phosphorylation and total levels of Erk1/2, p38, and Jnk were assessed by Western blot analysis (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001).</p

β-catenin mediates growth defects induced by centrosome loss in a subset of APC mutant colorectal cancer independently of p53.

Colorectal cancer is the third most common cancer and the second leading cause of cancer-related deaths worldwide. The centrosome is the main microtubule-organizing center in animal cells and centrosome amplification is a hallmark of cancer cells. To investigate the importance of centrosomes in colorectal cancer, we induced centrosome loss in normal and cancer human-derived colorectal organoids using centrinone B, a Polo-like kinase 4 (Plk4) inhibitor. We show that centrosome loss represses human normal colorectal organoid growth in a p53-dependent manner in accordance with previous studies in cell models. However, cancer colorectal organoid lines exhibited different sensitivities to centrosome loss independently of p53. Centrinone-induced cancer organoid growth defect/death positively correlated with a loss of function mutation in the APC gene, suggesting a causal role of the hyperactive WNT pathway. Consistent with this notion, β-catenin inhibition using XAV939 or ICG-001 partially prevented centrinone-induced death and rescued the growth two APC-mutant organoid lines tested. Our study reveals a novel role for canonical WNT signaling in regulating centrosome loss-induced growth defect/death in a subset of APC-mutant colorectal cancer independently of the classical p53 pathway