Megakaryocytes use in vivo podosome-like structures working collectively to penetrate the endothelial barrier of bone marrow sinusoids.

BACKGROUND: Blood platelets are anucleate cell fragments that prevent bleeding and minimize blood vessel injury. They are formed from the cytoplasm of megakaryocytes located in the bone marrow. For successful platelet production, megakaryocyte fragments must pass through the sinusoid endothelial barrier by a cell biology process unique to these giant cells as compared with erythrocytes and leukocytes. Currently, the mechanisms by which megakaryocytes interact and progress through the endothelial cells are not understood, resulting in a significant gap in our knowledge of platelet production. OBJECTIVE: The aim of this study was to investigate how megakaryocytes interact and progress through the endothelial cells of mouse bone marrow sinusoids. METHODS: We used a combination of fluorescence, electron, and three-dimensional microscopy to characterize the cellular events between megakaryocytes and endothelial cells. RESULTS: We identified protrusive, F-actin-based podosome-like structures, called in vivo-MK podosomes, which initiate the formation of pores through endothelial cells. These structures present a collective and spatial organization through their interconnection via a contractile network of actomyosin, essential to regulate the endothelial openings. This ensures proper passage of megakaryocyte-derived processes into the blood circulation to promote thrombopoiesis. CONCLUSION: This study provides novel insight into the in vivo function of podosomes of megakaryocytes with critical importance to platelet production.

Profiling of phosphoinositide molecular species in human and mouse platelets identifies new species increasing following stimulation.

Phosphoinositides are bioactive lipids essential in the regulation of cell signaling as well as cytoskeleton and membrane dynamics. Their metabolism is highly active in blood platelets where they play a critical role during activation, at least through two well identified pathways involving phospholipase C and phosphoinositide 3-kinases (PI3K). Here, using a sensitive high-performance liquid chromatography-mass spectrometry method recently developed, we monitored for the first time the profiling of phosphatidylinositol (PI), PIP, PIP2 and PIP3 molecular species (fatty-acyl profiles) in human and mouse platelets during the course of stimulation by thrombin and collagen-related peptide. Furthermore, using class IA PI3K p110α or p110ß deficient mouse platelets and a pharmacological inhibitor, we show the crucial role of p110ß and the more subtle role of p110α in the production of PIP3 molecular species following stimulation. This comprehensive platelet phosphoinositides profiling provides important resources for future studies and reveals new information on phosphoinositides biology, similarities and differences in mouse and human platelets and unexpected dramatic increase in low-abundance molecular species of PIP2 during stimulation, opening new perspectives in phosphoinositide signaling in platelets.

The importance of blood platelet lipid signaling in thrombosis and in sepsis.

Blood platelets are the first line of defense against hemorrhages and are also strongly involved in the processes of arterial thrombosis, a leading cause of death worldwide. Besides their well-established roles in hemostasis, vascular wall repair and thrombosis, platelets are now recognized as important players in other processes such as inflammation, healing, lymphangiogenesis, neoangiogenesis or cancer. Evidence is accumulating they are key effector cells in immune and inflammatory responses to host infection. To perform their different functions platelets express a wide variety of membrane receptors triggering specific intracellular signaling pathways and largely use lipid signaling systems. Lipid metabolism is highly active in stimulated platelets including the phosphoinositide metabolism with the phospholipase C (PLC) and the phosphoinositide 3-kinase (PI3K) pathways but also other enzymatic systems producing phosphatidic acid, lysophosphatidic acid, platelet activating factor, sphingosine 1-phosphate and a number of eicosanoids. While several of these bioactive lipids regulate intracellular platelet signaling mechanisms others are released by activated platelets acting as autocrine and/or paracrine factors modulating neighboring cells such as endothelial and immune cells. These bioactive lipids have been shown to play important roles in hemostasis and thrombosis but also in vessel integrity and dynamics, inflammation, tissue remodeling and wound healing. In this review, we will discuss some important aspects of platelet lipid signaling in thrombosis and during sepsis that is an important cause of death in intensive care unit. We will particularly focus on the implication of the different isoforms of PI3Ks and on the generation of eicosanoids released by activated platelets.

Local production of tenascin-C acts as a trigger for monocyte/macrophage recruitment that provokes cardiac dysfunction.

Aims: Tenascin-C (TNC) is an endogenous danger signal molecule strongly associated with inflammatory diseases and with poor outcome in patients with cardiomyopathies. Its function within pathological cardiac tissue during pressure overload remains poorly understood. Methods and results: We showed that TNC accumulates after 1 week of transverse aortic constriction (TAC) in the heart of 12-week-old male mice. By cross bone marrow transplantation experiments, we determined that TNC deposition relied on cardiac cells and not on haematopoietic cells. The expression of TNC induced by TAC, or by administration of a recombinant lentivector coding for TNC, triggered a pro-inflammatory cardiac microenvironment, monocyte/macrophage (MO/MΦ) accumulation, and systolic dysfunction. TNC modified macrophage polarization towards the pro-inflammatory phenotype and stimulated RhoA/Rho-associated protein kinase (ROCK) pathways to promote mesenchymal to amoeboid transition that enhanced macrophage migration into fibrillar collagen matrices. The amplification of inflammation and MO/MΦ recruitment by TNC was abrogated by genetic invalidation of TNC in knockout mice. These mice showed less ventricular remodelling and an improved cardiac function after TAC as compared with wild-type mice. Conclusions: By promoting a pro-inflammatory microenvironment and macrophage migration, TNC appears to be a key factor to enable the MO/MΦ accumulation within fibrotic hearts leading to cardiac dysfunction. As TNC is highly expressed during inflammation and sparsely during the steady state, its inhibition could be a promising therapeutic strategy to control inflammation and immune cell infiltration in heart disease.

A dual role for the class III PI3K, Vps34, in platelet production and thrombus growth.

To uncover the role of Vps34, the sole class III phosphoinositide 3-kinase (PI3K), in megakaryocytes (MKs) and platelets, we created a mouse model with Vps34 deletion in the MK/platelet lineage (Pf4-Cre/Vps34lox/lox). Deletion of Vps34 in MKs led to the loss of its regulator protein, Vps15, and was associated with microthrombocytopenia and platelet granule abnormalities. Although Vps34 deficiency did not affect MK polyploidisation or proplatelet formation, it dampened MK granule biogenesis and directional migration toward an SDF1α gradient, leading to ectopic platelet release within the bone marrow. In MKs, the level of phosphatidylinositol 3-monophosphate (PI3P) was significantly reduced by Vps34 deletion, resulting in endocytic/trafficking defects. In platelets, the basal level of PI3P was only slightly affected by Vps34 loss, whereas the stimulation-dependent pool of PI3P was significantly decreased. Accordingly, a significant increase in the specific activity of Vps34 lipid kinase was observed after acute platelet stimulation. Similar to Vps34-deficient platelets, ex vivo treatment of wild-type mouse or human platelets with the Vps34-specific inhibitors, SAR405 and VPS34-IN1, induced abnormal secretion and affected thrombus growth at arterial shear rate, indicating a role for Vps34 kinase activity in platelet activation, independent from its role in MKs. In vivo, Vps34 deficiency had no impact on tail bleeding time, but significantly reduced platelet prothrombotic capacity after carotid injury. This study uncovers a dual role for Vps34 as a regulator of platelet production by MKs and as an unexpected regulator of platelet activation and arterial thrombus formation dynamics.

Internalized Receptor for Glucose-dependent Insulinotropic Peptide stimulates adenylyl cyclase on early endosomes.

Until very recently, G-protein dependent signal of GPCRs was thought to originate exclusively from the plasma membrane and internalized GPCRs were considered silent. Here, we demonstrated that, once internalized and located in the membrane of early endosomes, glucose-dependent Insulinotropic receptor (GIPR) continues to trigger production of cAMP and PKA activation. Direct evidence is based on identification of the active form of Gαs in early endosomes containing GIPR using a genetically encoded GFP tagged nanobody, and on detection of a distinct FRET signal accounting for cAMP production at the surface of endosomes containing GIP, compared to endosomes without GIP. Furthermore, decrease of the sustained phase of cAMP production and PKA activation kinetics as well as reversibility of cAMP production and PKA activity following GIP washout in cells treated with a pharmacological inhibitor of GIPR internalization, and continuous increase of cAMP level over time in the presence of dominant-negative Rab7, which causes accumulation of early endosomes in cells, were noticed. Hence the GIPR joins the few GPCRs which signal through G-proteins both at plasma membrane and on endosomes.

PLIF: A rapid, accurate method to detect and quantitatively assess protein-lipid interactions.

Phosphoinositides are a type of cellular phospholipid that regulate signaling in a wide range of cellular and physiological processes through the interaction between their phosphorylated inositol head group and specific domains in various cytosolic proteins. These lipids also influence the activity of transmembrane proteins. Aberrant phosphoinositide signaling is associated with numerous diseases, including cancer, obesity, and diabetes. Thus, identifying phosphoinositide-binding partners and the aspects that define their specificity can direct drug development. However, current methods are costly, time-consuming, or technically challenging and inaccessible to many laboratories. We developed a method called PLIF (for "protein-lipid interaction by fluorescence") that uses fluorescently labeled liposomes and tethered, tagged proteins or peptides to enable fast and reliable determination of protein domain specificity for given phosphoinositides in a membrane environment. We validated PLIF against previously known phosphoinositide-binding partners for various proteins and obtained relative affinity profiles. Moreover, PLIF analysis of the sorting nexin (SNX) family revealed not only that SNXs bound most strongly to phosphatidylinositol 3-phosphate (PtdIns3P or PI3P), which is known from analysis with other methods, but also that they interacted with other phosphoinositides, which had not previously been detected using other techniques. Different phosphoinositide partners, even those with relatively weak binding affinity, could account for the diverse functions of SNXs in vesicular trafficking and protein sorting. Because PLIF is sensitive, semiquantitative, and performed in a high-throughput manner, it may be used to screen for highly specific protein-lipid interaction inhibitors.

Phosphoinositides: Important lipids in the coordination of cell dynamics.

By interacting specifically with proteins, phosphoinositides organize the spatiotemporal formation of protein complexes involved in the control of intracellular signaling, vesicular trafficking and cytoskeleton dynamics. A set of specific kinases and phosphatases ensures the production, degradation and inter-conversion of phosphoinositides to achieve a high level of precision in the regulation of cellular dynamics coordinated by these lipids. The direct involvement of these enzymes in cancer, genetic or infectious diseases, and the recent arrival of inhibitors targeting specific phosphoinositide kinases in clinic, emphasize the importance of these lipids and their metabolism in the biomedical field.

The role of class I, II and III PI 3-kinases in platelet production and activation and their implication in thrombosis.

Blood platelets play a pivotal role in haemostasis and are strongly involved in arterial thrombosis, a leading cause of death worldwide. Besides their critical role in pathophysiology, platelets represent a valuable model to investigate, both in vitro and in vivo, the biological roles of different branches of the phosphoinositide metabolism, which is highly active in platelets. While the phospholipase C (PLC) pathway has a crucial role in platelet activation, it is now well established that at least one class I phosphoinositide 3-kinase (PI3K) is also mandatory for proper platelet functions. Except class II PI3Kγ, all other isoforms of PI3Ks (class I α, ß, γ, δ; class II α, ß and class III) are expressed in platelets. Class I PI3Ks have been extensively studied in different models over the past few decades and several isoforms are promising drug targets to treat cancer and immune diseases. In platelet activation, it has been shown that while class I PI3Kδ plays a minor role, class I PI3Kß has an important function particularly in thrombus growth and stability under high shear stress conditions found in stenotic arteries. This class I PI3K is a potentially interesting target for antithrombotic strategies. The role of class I PI3Kα remains ill defined in platelets. Herein, we will discuss our recent data showing the potential impact of inhibitors of this kinase on thrombus formation. The role of class II PI3Kα and ß as well as class III PI3K (Vps34) in platelet production and function is just emerging. Based on our data and those very recently published in the literature, we will discuss the impact of these three PI3K isoforms in platelet production and functions and in thrombosis.

Essential role of class II PI3K-C2α in platelet membrane morphology.

The physiologic roles of the class II phosphoinositide 3-kinases (PI3Ks) and their contributions to phosphatidylinositol 3-monophosphate (PI3P) and PI(3,4)P2 production remain elusive. Here we report that mice heterozygous for a constitutively kinase-dead PI3K-C2α display aberrant platelet morphology with an elevated number of barbell-shaped proplatelets, a recently discovered intermediate stage in the final process of platelet production. Platelets with heterozygous PI3K-C2α inactivation have critical defects in α-granules and membrane structure that are associated with modifications in megakaryocytes. These platelets are more rigid and unable to form filopodia after stimulation. Heterozygous PI3K-C2α inactivation in platelets led to a significant reduction in the basal pool of PI3P and a mislocalization of several membrane skeleton proteins known to control the interactions between the plasma membrane and cytoskeleton. These alterations had repercussions on the performance of platelet responses with delay in the time of arterial occlusion in an in vivo model of thrombosis and defect in thrombus formation in an ex vivo blood flow system. These data uncover a key role for PI3K-C2α activity in the generation of a basal housekeeping PI3P pool and in the control of membrane remodeling, critical for megakaryocytopoiesis and normal platelet production and function.

BIN1/M-Amphiphysin2 induces clustering of phosphoinositides to recruit its downstream partner dynamin.

Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

Phosphatidylinositol 5-phosphate regulates invasion through binding and activation of Tiam1.

PtdIns5P is a lipid messenger acting as a stress-response mediator in the nucleus, and known to maintain cell activation through traffic alterations upon bacterial infection. Here, we show that PtdIns5P regulates actin dynamics and invasion via recruitment and activation of the exchange factor Tiam1 and Rac1. Restricted Rac1 activation results from the binding of Tiam1 DH-PH domains to PtdIns5P. Using an assay that mimics Rac1 membrane anchoring by using Rac1-His and liposomes containing Ni(2+)-NTA modified lipids, we demonstrate that intrinsic Tiam1 DH-PH activity increases when Rac1 is anchored in a PtdIns5P-enriched environment. This pathway appears to be general since it is valid in different pathophysiological models: receptor tyrosine kinase activation, bacterial phosphatase IpgD expression and the invasive NPM-ALK(+) lymphomas. The discovery that PtdIns5P could be a keystone of GTPases and cytoskeleton spatiotemporal regulation opens important research avenues towards unravelling new strategies counteracting cell invasion.

Phosphatidylinositol 5-phosphate: a nuclear stress lipid and a tuner of membranes and cytoskeleton dynamics.

Phosphatidylinositol 5-phosphate (PtdIns5P), the least characterized among the three phosphatidylinositol monophosphates, is emerging as a bioactive lipid involved in the control of several cellular functions. Similar to PtdIns3P, it is present in low amounts in mammalian cells, and can be detected at the plasma membrane and endomembranes as well as in the nucleus. Changes in PtdIns5P levels are observed in mammalian cells following specific stimuli or stresses, and in human diseases. Recently, the contribution of several enzymes such as PIKfyve, myotubularins, and type II PtdInsP-kinases to PtdIns5P metabolism has gained a strong experimental support. Here, we provide a picture emerging from recent studies showing how this lipid can be generated and act as a regulator of membrane and cytoskeleton dynamics, and as a modulator of gene expression. We briefly summarize the current methods and tools for studying PtdIns5P, and discuss how PtdIns5P can integrate and coordinate different functions in a spatiotemporal manner.

CIP4 controls CCL19-driven cell steering and chemotaxis in chronic lymphocytic leukemia.

Solid tumor dissemination relies on the reprogramming of molecular pathways controlling chemotaxis. Whether the motility of nonsolid tumors such as leukemia depends on the deregulated expression of molecules decoding chemotactic signals remains an open question. We identify here the membrane remodeling F-BAR adapter protein Cdc42-interacting protein 4 (CIP4) as a key regulator of chemotaxis in chronic lymphocytic leukemia (CLL). CIP4 is expressed at abnormally high levels in CLL cells, where it is required for CCL19-induced chemotaxis. Upon CCL19 stimulation of CLL cells, CIP4 associates with GTP-bound Cdc42 and is recruited to the rear of the lamellipodium and along microspikes radiating through the lamellipodium. Consistent with its cellular distribution, CIP4 removal impairs both the assembly of the polarized lamellipodium and directional migration along a diffusible CCL19 gradient. Furthermore, CIP4 depletion results in decreased activation of WASP, but increased activation of PAK1 and p38 mitogen-activated protein kinase (MAPK). Notably, p38 MAPK inhibition results in impaired lamellipodium assembly and loss of directional migration. This suggests that CIP4 modulates both the WASP and p38 MAPK pathways to promote lamellipodium assembly and chemotaxis. Overall, our study reveals a critical role of CIP4 in mediating chemotaxis of CLL cells by controlling the dynamics of microspike-containing protrusions and cell steering.

A novel mass assay to quantify the bioactive lipid PtdIns3P in various biological samples.

PtdIns3P is recognized as an important player in the control of the endocytotic pathway and in autophagy. Recent data also suggest that PtdIns3P contributes to molecular mechanisms taking place at the plasma membrane and at the midbody during cytokinesis. This lipid is present in low amounts in mammalian cells and remains difficult to quantify either by traditional techniques based on radiolabelling followed by HPLC to separate the different phosphatidylinositol monophosphates, or by high-sensitive liquid chromatography coupled to MS, which is still under development. In the present study, we describe a mass assay to quantify this lipid from various biological samples using the recombinant PtdIns3P 5-kinase, PIKfyve. Using this assay, we show an increase in the mass level of PtdIns3P in mouse and human platelets following stimulation, loss of this lipid in Vps34-deficient yeasts and its relative enrichment in early endosomes isolated from BHK cells.

Regulation of the DH-PH tandem of guanine nucleotide exchange factor for Rho GTPases by phosphoinositides.

Rho GTPases act as molecular switches central in cellular processes such as cytoskeleton dynamics, migration, cell proliferation, growth or survival. Their activation is tightly regulated downstream of cell surface receptors by Guanine nucleotide Exchange Factors (GEFs), that are responsible for the specificity, the accuracy, and the spatial restriction of Rho GTPases response to extracellular cues. Because there is about four time more RhoGEFs that Rho GTPases, and GEFs do not always show a strict specificity for GTPases, it is clear that their regulation depends on specific interactions with the subcellular environment. RhoGEFs bear a peculiar structure, highly conserved though evolution, consisting of a DH-PH tandem, the DH (Dbl homology) domain being responsible for the exchange activity. The function of the PH (Pleckstrin homology) domain known to bind phosphoinositides, however, remains elusive, and reports are in many cases rather confusing. This review summarizes data on the regulation of RhoGEFs activity through interaction of the PH-associated DH domain with phosphoinositides which are considered as critical players in the spatial organization of major signaling pathways.

Phosphoinositides and cellular pathogens.

Phosphoinositides are considered as highly dynamic players in the spatiotemporal organization of key signaling pathways, actin cytoskeleton rearrangements, establishment of cell polarity and intracellular vesicle trafficking. Their metabolism is accurately controlled and mutations in several phosphoinositide metabolizing enzymes take part in the development of human pathologies. Interestingly, evidence is accumulating that modulation of the phosphoinositide metabolism is critical for pathogenicity and virulence of many human pathogens. Given the importance of phosphoinositides, which link membrane and cytoskeleton dynamics to cell responses, it is not surprising that many invasive pathogens hijack their metabolism as part of their strategies to establish infection. In fact, according to their lifestyle, cellular pathogens use the phosphoinositide metabolism in order to trigger their uptake in nonphagocytic cells and/or modulate the maturation of the pathogen-containing vacuole to establish their replicative niche or escape in the cytosol and promote host cell survival. The last two decades have been marked by the discovery of different tactics used by cellular pathogens to modulate the phosphoinositide metabolism as part of their strategies to survive, proliferate and disseminate in a hostile environment.

ALK+ALCLs induce cutaneous, HMGB-1-dependent IL-8/CXCL8 production by keratinocytes through NF-κB activation.

Anaplastic large-cell lymphomas (ALCLs) bearing the t(2;5) translocation (ALK(+)ALCLs) are frequently characterized by skin colonization and associated with a poor prognosis. Using conditional transgenic models of anaplastic lymphoma kinase-positive (ALK(+)) lymphomas and human ALK(+)ALCL cell lines, in the present study, we show that high-mobility-group box-1 (HMGB-1), a proinflammatory cytokine, is released by ALK(+) cells, and demonstrate extracellular HMGB-1-stimulated secretion of the IL-8 chemokine by HaCaT keratinocytes through the involvement of MMP-9, PAR-2, and the NF-κB pathway. Furthermore, we demonstrate that, in vitro, IL-8 is able to induce the invasiveness of ALK(+) cells, which express the IL-8 receptors CXCR1 and CXCR2. In vitro and in vivo, HMGB-1 inhibition achieved by glycyrrhizin treatment led to a drastic reduction in ALK(+) cell invasiveness. The pathophysiological relevance of our observations was confirmed by demonstrating that the HMGB-1 and IL-8 receptors are expressed in ALK(+)ALCL biopsies. We have also shown that IL-8 secretion is correlated with leukemic dissemination of ALK(+) cells in a significant number of patients. The results of the present study demonstrate for the first time a relationship among the pro-inflammatory mediators HMGB-1, MMP-9, PAR-2, and IL-8. We propose that these mediators create a premetastatic niche within the skin, thereby participating in ALK(+) lymphoma epidermotropism.

The p.Arg63Trp polymorphism controls Vav1 functions and Foxp3 regulatory T cell development.

CD4(+) regulatory T cells (T(reg) cells) expressing the transcription factor Foxp3 play a pivotal role in maintaining peripheral tolerance by inhibiting the expansion and function of pathogenic conventional T cells (T(conv) cells). In this study, we show that a locus on rat chromosome 9 controls the size of the natural T(reg) cell compartment. Fine mapping of this locus with interval-specific congenic lines and association experiments using single nucleotide polymorphisms (SNPs) identified a nonsynonymous SNP in the Vav1 gene that leads to the substitution of an arginine by a tryptophan (p.Arg63Trp). This p.Arg63Trp polymorphism is associated with increased proportion and absolute numbers of T(reg) cells in the thymus and peripheral lymphoid organs, without impacting the size of the T(conv) cell compartment. This polymorphism is also responsible for Vav1 constitutive activation, revealed by its tyrosine 174 hyperphosphorylation and increased guanine nucleotide exchange factor activity. Moreover, it induces a marked reduction in Vav1 cellular contents and a reduction of Ca(2+) flux after TCR engagement. Together, our data reveal a key role for Vav1-dependent T cell antigen receptor signaling in natural T(reg) cell development.

Shigella flexneri infection generates the lipid PI5P to alter endocytosis and prevent termination of EGFR signaling.

The phosphoinositide metabolic pathway, which regulates cellular processes implicated in survival, motility, and trafficking, is often subverted by bacterial pathogens. Shigella flexneri, a bacterium that causes dysentery, injects IpgD, a phosphoinositide phosphatase that generates the lipid phosphatidylinositol 5-phosphate (PI5P), into host cells, thereby activating the phosphoinositide 3-kinase-Akt survival pathway. We show that epidermal growth factor receptor (EGFR) is required for PI5P-dependent activation of Akt in infected HeLa cells or cells ectopically expressing IpgD. Cells treated with PI5P had increased numbers of early endosomes with activated EGFR, no detectable EGFR in the late endosomal or lysosomal compartments, and prolonged EGFR signaling. Endosomal recycling and retrograde pathways were spared, indicating that the effect of PI5P on the degradative route to the late endocytic compartments was specific. Thus, we identified PI5P, which was enriched in endosomes, as a regulator of vesicular trafficking that alters growth factor receptor signaling by impairing lysosomal degradation, a property used by S. flexneri to favor survival of host cells.