Jean-Pierre Salles

Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization

Exosomes are small vesicles secreted from multivesicular bodies, which are able to stimulate the immune system leading to tumour cell eradication. We have analysed lipids of exosomes secreted either upon stimulation from rat mast cells (RBL-2H3 cells), or constitutively from human dendritic cells. As compared with parent cells, exosomes displayed an enrichment in sphingomyelin, but not in cholesterol. Phosphatidylcholine content was decreased, but an enrichment was noted in disaturated molecular species as in phosphatidylethanolamines. Lyso(bis)phosphatidic acid was not enriched in exosomes as compared with cells. Fluorescence anisotropy demonstrated an increase in exosome-membrane rigidity from pH 5 to 7, suggesting their membrane reorganization between the acidic multivesicular body compartment and the neutral outer cell medium. NMR analysis established a bilayer organization of exosome membrane, and ESR studies using 16-doxyl stearic acid demonstrated a higher flip-flop of lipids between the two leaflets as compared with plasma membrane. In addition, the exosome membrane exhibited no asymmetrical distribution of phosphatidylethanolamines. Therefore exosome membrane displays a similar content of the major phospholipids and cholesterol, and is organized as a lipid bilayer with a random distribution of phosphatidylethanolamines. In addition, we observed tight lipid packing at neutral pH and a rapid flip-flop between the two leaflets of exosome membranes. These parameters could be used as a hallmark of exosomes.

Hyperghrelinemia precedes obesity in Prader-Willi syndrome.

BACKGROUND High plasma ghrelin levels have been reported in Prader-Willi syndrome (PWS). However, little is known about plasma ghrelin in these children during the first years of life characterized by a failure to thrive. OBJECTIVE The objective of the study was to investigate total plasma ghrelin levels in children with PWS and controls from 2 months to 17 years. SUBJECTS AND METHODS Forty children with PWS [24 boys, 16 girls, median age 3.6 yr, median body mass index (BMI) Z-score 0.3] were compared with 84 controls (57 boys, 27 girls, median age 4.2 yr median BMI Z-score 0.1). Children were then divided into two groups according to age and GH treatment. RESULTS Median plasma ghrelin levels were significantly higher in children with PWS, compared with controls at any age (568 vs. 173, P < 0.0001) and decreased with age in both groups (P < 0.0001). In the whole group of PWS, we found an inverse relationship between ghrelin and BMI Z-score, insulin, homeostasis model assessment insulin resistance index, leptin, and lean mass. Plasma ghrelin levels were higher in children with PWS than controls, both in the youngest children below 3 yr who were not receiving GH (771 vs. 233, P < 0.0001) and in the children older than 3 yr, all of whom were treated with GH (428 vs. 159, P < 0.0001). CONCLUSIONS Plasma ghrelin levels in children with PWS are elevated at any age, including during the first years of life, thus preceding the development of obesity.

Genetic and pharmacological targeting of phosphoinositide 3-kinase-gamma reduces atherosclerosis and favors plaque stability by modulating inflammatory processes.

Background— The role of inflammation at all stages of the atherosclerotic process has become an active area of investigation, and there is a notable quest for novel and innovative drugs for the treatment of atherosclerosis. The lipid kinase phosphoinositide 3-kinase-&ggr; (PI3K&ggr;) is thought to be a key player in various inflammatory, autoimmune, and allergic processes. These properties and the expression of PI3K&ggr; in the cardiovascular system suggest that PI3K&ggr; plays a role in atherosclerosis. Methods and Results— Here, we demonstrate that a specific PI3K&ggr; inhibitor (AS605240) is effective in murine models of established atherosclerosis. Intraperitoneal administration of AS605240 (10 mg/kg daily) significantly decreased early atherosclerotic lesions in apolipoprotein E–deficient mice and attenuated advanced atherosclerosis in low-density lipoprotein receptor–deficient mice. Furthermore, PI3K&ggr; levels were elevated in both human and murine atherosclerotic lesions. Comparison of low-density lipoprotein receptor–deficient mice transplanted with wild-type or PI3K&ggr;-deficient bone marrow demonstrated that functional PI3K&ggr; in the hematopoietic lineage is required for atherosclerotic progression. Alleviation of atherosclerosis by targeting of PI3K&ggr; activity was accompanied by decreased macrophage and T-cell infiltration, as well as increased plaque stabilization. Conclusions— These data identify PI3K&ggr; as a new target in atherosclerosis with the potential to modulate multiple stages of atherosclerotic lesion formation, such as fatty streak constitution, cellular composition, and final fibrous cap establishment.

PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes

Exosomes are small vesicles secreted by different immune cells and which display anti‐tumoral properties. Stimulation of RBL‐2H3 cells with ionomycin triggered phospholipase D2 (PLD2) translocation from plasma membrane to intracellular compartments and the release of exosomes. Although exosomes carry the two isoforms of PLD, PLD2 was enriched and specifically sorted on exosomes when overexpressed in cells. PLD activity present on exosomes was clearly increased following PLD2 overexpression. PLD2 activity in cells was correlated to the amount of exosome released, as measured by FACS. Therefore, the present work indicates that exosomes can vehicle signaling enzymes.

Adsorption and release of BMP-2 on nanocrystalline apatite-coated and uncoated hydroxyapatite/β-tricalcium phosphate porous ceramics

The association of bone morphogenetic proteins (BMPs) with calcium phosphate bioceramics is known to confer them osteoinductive properties. The aim of this study was to evaluate the surface properties, especially regarding recombinant human BMP-2 (rhBMP-2) adsorption and release, of commercial sintered biphasic calcium phosphate ceramics after coating with biomimetic nanocrystalline apatite. The raw and coated ceramics exhibited similar macroporous structures but different nanometer-sized pores contents. Both types of ceramics showed Langmuir-type adsorption isotherms of rhBMP-2. The coating noticeably increased the rate of adsorption and the total amount of growth factor taken up, but the maximum coverage per surface area unit as well as the affinity constant appeared lower for coated ceramics compared with raw ceramic surfaces. The limited advantage gained by coating the ceramics can be assigned to a lower accessibility of the surface adsorption sites compared with the raw ceramics. The quantity of rhBMP-2 spontaneously released in cell culture medium during the first weeks was lower for coated samples than for uncoated ceramics and represented a minor fraction of the total adsorbed amount. In conclusion, the nanocrystalline apatite coating was found to favor the adsorption of rhBMP-2 while providing a mean to fine tune the release of the growth factor.

SHIP-2 and PTEN Are Expressed and Active in Vascular Smooth Muscle Cell Nuclei, but Only SHIP-2 Is Associated with Nuclear Speckles

Recently, the control of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3)-dependant signaling by phosphatases has emerged, but there is a shortage of information on intranuclear PtdIns(3,4,5)P3 phosphatases. Therefore, we investigated the dephosphorylation of [32P]PtdIns(3,4,5)P3 specifically labeled on the D-3 position of the inositol ring in membrane-free nuclei isolated from pig aorta vascular smooth muscle cells (VSMCs). In vitro PtdIns(3,4,5)P3 phosphatase assays revealed the production of both [32P]PtdIns(3,4)P2 and inorganic phosphate, demonstrating the presence of PtdIns(3,4,5)P3 5- and 3-phosphatase activities inside the VSMC nucleus, respectively. Both activities presented the same potency in cellular lysates, whereas the nuclear PtdIns(3,4,5)P3 5-phosphatase activity appeared to be the most efficient. Immunoblot experiments showed for the first time the expression of the 5-phosphatase SHIP-2 (src homology 2 domain-containing inositol phosphatase) as well as the 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) in VSMC nuclei. In addition, immunoprecipitations from nuclear fractions indicated a [32P]PtdIns(3,4,5)P3 dephosphorylation by both SHIP-2 and PTEN. Moreover, confocal microscopy analyses demonstrated that SHIP-2 but not PTEN colocalized with a speckle-specific component, the SC35 splicing factor. These results suggest that SHIP-2 may be the primary enzyme for metabolizing PtdIns(3,4,5)P3 into PtdIns(3,4)P2 within the nucleus, thus producing another second messenger, whereas PTEN could down-regulate nuclear phosphoinositide 3-kinase signaling. Finally, intranuclear PtdIns(3,4,5)P3 phosphatases might be involved in the control of VSMC proliferation and the pathogenesis of vascular proliferative disorders.

Functional Effects of PTPN11 (SHP2) Mutations Causing LEOPARD Syndrome on Epidermal Growth Factor-Induced Phosphoinositide 3-Kinase/AKT/Glycogen Synthase Kinase 3β Signaling

ABSTRACT LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11). How these alterations cause the disease remains unknown. We report here that fibroblasts isolated from LS patients displayed stronger epidermal growth factor (EGF)-induced phosphorylation of both AKT and glycogen synthase kinase 3β (GSK-3β) than fibroblasts from control patients. Similar results were obtained in HEK293 cells expressing LS mutants of SHP2. We found that the GAB1/phosphoinositide 3-kinase (PI3K) complex was more abundant in fibroblasts from LS than control subjects and that both AKT and GSK-3β hyperphosphorylation were prevented by reducing GAB1 expression or by overexpressing a GAB1 mutant unable to bind to PI3K. Consistently, purified recombinant LS mutants failed to dephosphorylate GAB1 PI3K-binding sites. These mutants induced PI3K-dependent increase in cell size in a model of chicken embryo cardiac explants and in transcriptional activity of the atrial natriuretic factor (ANF) gene in neonate rat cardiomyocytes. In conclusion, SHP2 mutations causing LS facilitate EGF-induced PI3K/AKT/GSK-3β stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology.

The adaptor protein Gab1 couples the stimulation of vascular endothelial growth factor receptor-2 to the activation of phosphoinositide 3-kinase.

Phosphoinositide 3-kinase (PI3K) mediates essential functions of vascular endothelial growth factor (VEGF), including the stimulation of endothelial cell proliferation and migration. Nevertheless, the mechanisms coupling the receptor VEGFR-2 to PI3K remain obscure. We observed that the Grb2-bound adapter Gab1 is tyrosine-phosphorylated and relocated to membrane fractions upon VEGF stimulation of endothelial cells. We could detect the PI3K regulatory subunit p85 in immunoprecipitates of endogenous Gab1, and vice versa, and measure a Gab1-associated lipid kinase activity upon VEGF stimulation. Furthermore, transfection of the Gab1-YF3 mutant lacking all p85-binding sites strongly repressed PI3K activation measured in vitro. Moreover, Gab1-YF3 severely decreased the cellular amount of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generated in response to VEGF. Furthermore, adenoviral expression of Gab1-YF3 suppressed both Akt phosphorylation and recovery of wounded human umbilical vein endothelial cell monolayers, a VEGF-dependent process involving cell migration and proliferation under PI3K control. Transfection of other Gab1 mutants, lacking Grb2-binding sites or the pleckstrin homology (PH) domain, also prevented Akt activation, further demonstrating Gab1 involvement in PI3K activation. These mutants were also used to show that interactions with both Grb2 and PtdIns(3,4,5)P3 mediate Gab1 recruitment by VEGFR-2. Importantly, Gab1 mobilization was impaired by (i) PI3K inhibitors, (ii) deletion of Gab1 PH domain, (iii) PTEN (phosphatase and tensin homolog deleted on chromosome 10) overexpression to repress PtdIns(3,4,5)P3 production, and (iv) overexpression of a competitor PH domain for PtdIns(3,4,5)P3 binding, which altogether demonstrated that PI3K is also an upstream regulator of Gab1. Gab1 thus appears as a primary actor in coupling VEGFR-2 to PI3K/Akt, recruited through an amplification loop involving PtdIns(3,4,5)P3 and its PH domain.

Modulation of phosphoinositide 3‐kinase activation by cholesterol level suggests a novel positive role for lipid rafts in lysophosphatidic acid signalling

Methyl‐β‐cyclodextrin (MβCD) was used to explore a role for cholesterol‐enriched plasma membrane microdomains in coupling lysophosphatidic acid (LPA) stimulation to phosphoinositide 3‐kinase (PI3K) activation. Cholesterol depletion strongly inhibited the production of phosphatidylinositol 3,4‐bisphosphate and phosphatidylinositol 3,4,5‐trisphosphate in Vero cells stimulated with LPA. In agreement, the phosphorylation of Akt/protein kinase B, but not of Erk kinases, was suppressed by MβCD. MβCD did not interfere with the overall phospholipid metabolism, and its effects were reversed in cholesterol add‐back experiments. Finally, PI3K was detected in lipid rafts prepared from control but not MβCD‐treated cells, suggesting that these microdomains contribute to LPA signalling by compartmentalising component(s) of the PI3K pathway.

Noonan syndrome-causing SHP2 mutants inhibit insulin-like growth factor 1 release via growth hormone-induced ERK hyperactivation, which contributes to short stature

Noonan syndrome (NS), a genetic disease caused in half of cases by activating mutations of the tyrosine phosphatase SHP2 (PTPN11), is characterized by congenital cardiopathies, facial dysmorphic features, and short stature. How mutated SHP2 induces growth retardation remains poorly understood. We report here that early postnatal growth delay is associated with low levels of insulin-like growth factor 1 (IGF-1) in a mouse model of NS expressing the D61G mutant of SHP2. Conversely, inhibition of SHP2 expression in growth hormone (GH)-responsive cell lines results in increased IGF-1 release upon GH stimulation. SHP2-deficient cells display decreased ERK1/2 phosphorylation and rat sarcoma (RAS) activation in response to GH, whereas expression of NS-associated SHP2 mutants results in ERK1/2 hyperactivation in vitro and in vivo. RAS/ERK1/2 inhibition in SHP2-deficient cells correlates with impaired dephosphorylation of the adaptor Grb2-associated binder-1 (GAB1) on its RAS GTPase-activating protein (RASGAP) binding sites and is rescued by interfering with RASGAP recruitment or function. We demonstrate that inhibition of ERK1/2 activation results in an increase of IGF-1 levels in vitro and in vivo, which is associated with significant growth improvement in NS mice. In conclusion, NS-causing SHP2 mutants inhibit GH-induced IGF-1 release through RAS/ERK1/2 hyperactivation, a mechanism that could contribute to growth retardation. This finding suggests that, in addition to its previously shown beneficial effect on NS-linked cardiac and craniofacial defects, RAS/ERK1/2 modulation could also alleviate the short stature phenotype in NS caused by PTPN11 mutations.