Serotonin 5-HT2A receptor-mediated hypertrophy is negatively regulated by caveolin-3 in cardiomyoblasts and neonatal cardiomyocytes.

The serotonin 5-HT(2A) receptor belongs to the G-protein-coupled receptors (GPCRs) superfamily and mediates the hypertrophic response to serotonin (5-HT) in cardiac myocytes. At present the regulatory mechanisms of 5-HT(2A) receptor-induced myocyte hypertrophy are not fully understood. The localization and the compartmentation of GPCRs within specialized membrane microdomains are known to modulate their signalling pathway. Therefore, we hypothesized that caveolae microdomains and caveolin-3, the predominant isoform of cardiac caveolae, might be regulators of 5-HT(2A) receptor signalling. We demonstrate that 5-HT(2A) receptors interact with caveolin-3 upon 5-HT stimulation and traffic into caveolae membrane microdomains. We provide evidence that caveolin-3 knockdown abolishes the redistribution of 5-HT(2A) receptors into caveolae and enhances 5-HT(2A) receptor-induced myocyte hypertrophic markers such as cell size, protein synthesis and ANF gene expression. Importantly, we demonstrate that caveolin-3 and caveolae structures are negative regulators of 5-HT(2A) receptor-induced nuclear factor of activated T cells (NFAT) transcriptional activation. Taken together, our data demonstrate that caveolin-3 and caveolae microdomains are important regulators of the hypertrophic response induced by 5-HT(2A) receptors. These findings thus open new insights to target heart hypertrophy under the enhanced serotonin system. This article is part of a Special Issue entitled "Local Signaling in Myocytes".

Essential role of TRPC1 channels in cardiomyoblasts hypertrophy mediated by 5-HT2A serotonin receptors.

Serotonin (5-HT) participates in the development of cardiac hypertrophy through 5-HT(2A) serotonin receptors. The hypertrophic growth of cardiomyoblasts induced by 5-HT(2A) receptors involves the activation of the Ca(2+) responsive calcineurin/NFAT pathway. However, the mechanism whereby NFAT is activated by 5-HT(2A) receptors remains indeterminate. In this study, we examined whether transient receptor potential canonical (TRPC) channels participate in NFAT activation and hypertrophic response triggered by 5-HT. We demonstrate that TRPC1 expression is upregulated in 5-HT-treated rat cardiomyoblasts whereas TRPC6 is induced in a mouse model of heart hypertrophy. Moreover, TRPC1 knockdown by small interfering RNA inhibits NFAT activation and hypertrophic response mediated by 5-HT(2A) receptors. These findings provide new insights about a mechanistic basis for the activation of the calcineurin/NFAT pathway by 5-HT(2A) receptors and highlight the critical role of TRPC1 in the development of cardiac hypertrophy.

Interaction of ezrin with the novel guanine nucleotide exchange factor PLEKHG6 promotes RhoG-dependent apical cytoskeleton rearrangements in epithelial cells.

The mechanisms underlying functional interactions between ERM (ezrin, radixin, moesin) proteins and Rho GTPases are not well understood. Here we characterized the interaction between ezrin and a novel Rho guanine nucleotide exchange factor, PLEKHG6. We show that ezrin recruits PLEKHG6 to the apical pole of epithelial cells where PLEKHG6 induces the formation of microvilli and membrane ruffles. These morphological changes are inhibited by dominant negative forms of RhoG. Indeed, we found that PLEKHG6 activates RhoG and to a much lesser extent Rac1. In addition we show that ezrin forms a complex with PLEKHG6 and RhoG. Furthermore, we detected a ternary complex between ezrin, PLEKHG6, and the RhoG effector ELMO. We demonstrate that PLEKHG6 and ezrin are both required in macropinocytosis. After down-regulation of either PLEKHG6 or ezrin expression, we observed an inhibition of dextran uptake in EGF-stimulated A431 cells. Altogether, our data indicate that ezrin allows the local activation of RhoG at the apical pole of epithelial cells by recruiting upstream and downstream regulators of RhoG and that both PLEKHG6 and ezrin are required for efficient macropinocytosis.

Mitophagy acts as a safeguard mechanism against human vascular smooth muscle cell apoptosis induced by atherogenic lipids.

Mitophagy is a critical cellular process that selectively targets damaged mitochondria for autophagosomal degradation both under baseline conditions and in response to stress preventing oxidative damage and cell death. Recent studies have linked alterations in mitochondria function and reduced autophagy with the development of age-related pathologies. However, the significance of mitochondrial autophagy in vessel wall in response to atherogenic lipid stressors is not known. In the present study, we investigated the role of mitophagy on human vascular smooth muscle cells (VSMC) apoptosis induced by oxidized low-density lipoproteins (LDL). We reported for the first time that the engulfment of defective mitochondria by autophagosomes occurred in human VSMC in response to oxidized LDL. The molecular mechanism mediating mitophagy in human VSMC involved dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, accumulation of PTEN-induced putative kinase 1 (PINK1) and the recruitment of the E3 ubiquitin ligase Parkin to mitochondria. Likewise, we found increased voltage-dependent anion channel 1 (VDAC1) and mitofusin 2 (Mnf2) mitochondrial proteins ubiquitination and LC3 association to mitochondria. Using flow cytometry in the presence of lysosomal inhibitors, we showed that PINK1 and Parkin silencing impaired mitophagy flux and enhanced oxidized LDL-induced VSMC apoptosis. In addition, overexpression of PINK1 and Parkin were protective by limiting cell death. Moreover, reduced Bax levels found in VSMC-overexpressing Parkin indicated cross talk among mitophagy and mitochondrial apoptotic signalling pathways. Altogether these data demonstrate that mitophagy is a safeguard mechanism against human VSMC apoptosis induced by atherogenic stressors and highlight mitophagy as a potential target to stabilize atherosclerotic plaque.

Internalization and desensitization of the human glucose-dependent-insulinotropic receptor is affected by N-terminal acetylation of the agonist.

How incretins regulate presence of their receptors at the cell surface and their activity is of paramount importance for the development of therapeutic strategies targeting these receptors. We have studied internalization of the human Glucose-Insulinotropic Polypeptide receptor (GIPR). GIP stimulated rapid robust internalization of the GIPR, the major part being directed to lysosomes. GIPR internalization involved mainly clathrin-coated pits, AP-2 and dynamin. However, neither GIPR C-terminal region nor ß-arrestin1/2 was required. Finally, N-acetyl-GIP recognized as a dipeptidyl-IV resistant analogue, fully stimulated cAMP production with a ∼15-fold lower potency than GIP and weakly stimulated GIPR internalization and desensitization of cAMP response. Furthermore, docking N-acetyl-GIP in the binding site of modeled GIPR showed slighter interactions with residues of helices 6 and 7 of GIPR compared to GIP. Therefore, incomplete or partial activity of N-acetyl-GIP on signaling involved in GIPR desensitization and internalization contributes to the enhanced incretin activity of this peptide.

Annexin II-dependent actin remodelling evoked by hydrogen peroxide requires the metalloproteinase/sphingolipid pathway.

Actin remodeling is a dynamic process associated with cell shape modification occurring during cell cycle and proliferation. Oxidative stress plays a role in actin reorganization via various systems including p38MAPK. Beside, the mitogenic response evoked by hydrogen peroxide (H2O2) in fibroblasts and smooth muscle cells (SMC) involves the metalloproteinase (MMPs)/sphingomyelinase 2 (nSMase2) signaling pathway. The aim of this work was to investigate whether this system plays a role in actin remodeling induced by H2O2. Low H2O2 dose (5µM) rapidly triggered a signaling cascade leading to nSMase2 activation, src and annexin 2 (AnxA2) phosphorylation, and actin remodeling, in fibroblasts and SMC. These events were blocked by pharmacological inhibitors of MMPs (Ro28-2653) and p38MAPK (SB203580), and were lacking in MMP2(-/-) and in nSMase2-mutant (fro) fibroblasts. Likewise, H2O2 was unable to induce actin remodeling in fro and MMP2(-/-) fibroblasts or in cells pretreated with p38MAPK, or MMP inhibitors. Finally we show that nSMase2 activation by H2O2, depends on MMP2 and p38MAPK, and is required for the src-dependent phosphorylation of AnxA2, and actin remodeling. Taken together, these findings indicate for the first time that AnxA2 phosphorylation and actin remodeling evoked by oxidative stress depend on the sphingolipid pathway, via MMP2 and p38MAPK.

Surgical mask to prevent influenza transmission in households: a cluster randomized trial.

BACKGROUND: Facemasks and respirators have been stockpiled during pandemic preparedness. However, data on their effectiveness for limiting transmission are scarce. We evaluated the effectiveness of facemask use by index cases for limiting influenza transmission by large droplets produced during coughing in households. METHODOLOGY AND PRINCIPAL FINDINGS: A cluster randomized intervention trial was conducted in France during the 2008-2009 influenza season. Households were recruited during a medical visit of a household member with a positive rapid influenza A test and symptoms lasting less than 48 hours. Households were randomized either to the mask or control group for 7 days. In the intervention arm, the index case had to wear a surgical mask from the medical visit and for a period of 5 days. The trial was initially intended to include 372 households but was prematurely interrupted after the inclusion of 105 households (306 contacts) following the advice of an independent steering committee. We used generalized estimating equations to test the association between the intervention and the proportion of household contacts who developed an influenza-like illness during the 7 days following the inclusion. Influenza-like illness was reported in 24/148 (16.2%) of the contacts in the intervention arm and in 25/158 (15.8%) of the contacts in the control arm and the difference between arms was 0.40% (95%CI: -10% to 11%, P = 1.00). We observed a good adherence to the intervention. In various sensitivity analyses, we did not identify any trend in the results suggesting effectiveness of facemasks. CONCLUSION: This study should be interpreted with caution since the lack of statistical power prevents us to draw formal conclusion regarding effectiveness of facemasks in the context of a seasonal epidemic. TRIAL REGISTRATION: clinicaltrials.gov NCT00774774.

Tollip is a mediator of protein sumoylation.

Tollip is an interactor of the interleukin-1 receptor involved in its activation. The endosomal turnover of ubiquitylated IL-1RI is also controlled by Tollip. Furthermore, together with Tom1, Tollip has a general role in endosomal protein traffic. This work shows that Tollip is involved in the sumoylation process. Using the yeast two-hybrid technique, we have isolated new Tollip partners including two sumoylation enzymes, SUMO-1 and the transcriptional repressor Daxx. The interactions were confirmed by GST-pull down experiments and immunoprecipitation of the co-expressed recombinants. More specifically, we show that the TIR domain of the cytoplasmic region of IL-1RI is a sumoylation target of Tollip. The sumoylated and unsumoylated RanGAP-1 protein also interacts with Tollip, suggesting a possible role in RanGAP-1 modification and nuclear-cytoplasmic protein translocation. In fact, Tollip is found in the nuclear bodies of SAOS-2/IL-1RI cells where it colocalizes with SUMO-1 and the Daxx repressor. We conclude that Tollip is involved in the control of both nuclear and cytoplasmic protein traffic, through two different and often contrasting processes: ubiquitylation and sumoylation.

Mechanosensing role of caveolae and caveolar constituents in human endothelial cells.

A variety of evidence suggests that endothelial cell functions are impaired in altered gravity conditions. Nevertheless, the effects of hypergravity on endothelial cell physiology remain unclear. In this study we cultured primary human endothelial cells under mild hypergravity conditions for 24-48 h, then we evaluated the changes in cell cycle progression, caveolin1 gene expression and in the caveolae status by confocal microscopy. Moreover, we analyzed the activity of enzymes known to be resident in caveolae such as endothelial nitric oxide synthase (eNOS), cycloxygenase 2 (COX-2), and prostacyclin synthase (PGIS). Finally, we performed a three-dimensional in vitro collagen gel test to evaluate the modification of the angiogenic responses. Results indicate that hypergravity shifts endothelial cells to G(0)/G(1) phase of cell cycle, reducing S phase, increasing caveolin1 gene expression and causing an increased distribution of caveolae in the cell interior. Hypergravity also increases COX-2 expression, nitric oxide (NO) and prostacyclin (PGI2) production, and inhibits angiogenesis as evaluated by 3-D collagen gel test, through a pathway not involving apoptosis. Thus, endothelial cell caveolae may be responsible for adaptation of endothelium to hypergravity and the mechanism of adaptation involves an increased caveolin1 gene expression coupled to upregulation of vasodilators as NO and PGI2.

Stage-specific gene expression in early differentiating oligodendrocytes.

The screening of a differential library from precursor and differentiated oligodendrocytes, obtained through the representational difference analysis (RDA) technique, has generated a number of cDNA recombinants corresponding to mRNA coding for known and unknown proteins: (1) mRNA coding for proteins involved in protein synthesis, (2) mRNA coding for proteins involved in the organization of the cytoskeleton, and (3) mRNA coding for proteins of unknown function. The expression profile of the mRNA was studied by Northern blot hybridization to the poly-A(+) mRNA from primary rat progenitor and differentiated oligodendrocytes. In most cases, hybridization to the precursor was higher than hybridization to the differentiated mRNA, supporting the validity of the differential screening. Hybridization of the cDNA to rat cerebral hemisphere and brain stem poly-A(+) mRNA, isolated from 1- to 90-day-old rats, confirms the results obtained with the mRNA from differentiating oligodendrocytes. The intensity of the hybridization bands decreases as differentiation proceeds. The pattern of expression observed in oligodendrocytes is different from that found in the brain only in the case of the nexin-1 mRNA, the level of which remains essentially constant throughout differentiation both in the brain stem and in the cerebral hemispheres, in agreement with the published data. In contrast, the intensity of hybridization to the oligodendrocyte mRNA is dramatically lower in the differentiated cells compared with the progenitor oligodendrocyte cells. Some of the recombinant cDNA represent mRNA sequences present at high frequency distribution in the cells, while others belong to the rare sequences group. Six recombinants code for proteins of the ribosomal family, suggesting that of approximately 70 known ribosomal proteins, only a few are upregulated during oligodendrocyte differentiation. The third category of open reading frame (ORF) is represented by rare messengers coding for proteins of unknown functions and includes six clones: RDA 279, 11, 95, 96, 254, and 288.