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International audience; BACKGROUND AND PURPOSE: Vascular smooth muscle cell (SMC) migration within the arterial wall is a crucial event in atherogenesis and restenosis. Monocyte chemotactic protein-1/CC-chemokine receptor 2 (MCP-1/CCR2) signalling is involved in SMC migration processes but the molecular mechanisms have not been well characterized. We investigated the role of PI3Kγ in SMC migration induced by MCP-1. EXPERIMENTAL APPROACHES: A pharmacological PI3Kγ inhibitor, adenovirus encoding inactive forms of PI3Kγ and genetic deletion of PI3Kγ were used to investigate PI3Kγ functions in the MCP-1 and platelet-derived growth factor (PDGF) signalling pathway and migration process in primary aortic SMC. KEY RESULTS: The γ isoform of PI3K was shown to be the major signalling molecule mediating PKB phosphorylation in MCP-1-stimulated SMC. Using a PI3Kγ inhibitor and an adenovirus encoding a dominant negative form of PI3Kγ, we demonstrated that PI3Kγ is essential for SMC migration triggered by MCP-1. PDGF receptor stimulation induced MCP-1 mRNA and protein accumulation in SMCs. Blockade of the MCP-1/CCR2 pathway or pharmacological inhibition of PI3Kγ reduced PDGF-stimulated aortic SMC migration by 50%. Thus PDGF promotes an autocrine loop involving MCP-1/CCR2 signalling which is required for PDGF-mediated SMC migration. Furthermore, SMCs isolated from PI3Kγ-deficient mice (PI3Kγ(-/-)), or mice expressing an inactive PI3Kγ (PI3Kγ(KD/KD)), migrated less than control cells in response to MCP-1 and PDGF. CONCLUSIONS AND IMPLICATIONS: PI3Kγ is essential for MCP-1-stimulated aortic SMC migration and amplifies cell migration induced by PDGF by an autocrine/paracrine loop involving MCP-1 secretion and CCR2 activation. PI3Kγ is a promising target for the treatment of aortic fibroproliferative pathologies.

International audience; RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.

At the end of the preimplantation period, the inner cell mass (ICM) of the mouse blastocyst is composed of two distinct cell lineages, the pluripotent epiblast (EPI) and the primitive endoderm (PrE). The current model for their formation involves initial co-expression of lineage-specific markers followed by mutual-exclusive expression resulting in a salt-and-pepper distribution of lineage precursors within the ICM. Subsequent to lineage commitment, cell rearrangements and selective apoptosis are thought to be key processes driving and refining the emergence of two spatially distinct compartments. Here, we have addressed a role for Platelet Derived Growth Factor (PDGF) signaling in the regulation of programmed cell death during early mouse embryonic development. By combining genetic and pharmacological approaches, we demonstrate that embryos lacking PDGF activity exhibited caspase-dependent selective apoptosis of PrE cells. Modulating PDGF activity did not affect lineage commitment or cell sorting, suggesting that PDGF is involved in the fine-tuning of patterning information. Our results also indicate that PDGF and fibroblast growth factor (FGF) tyrosine kinase receptors exert distinct and non-overlapping functions in PrE formation. Taken together, these data uncover an early role of PDGF signaling in PrE cell survival at the time when PrE and EPI cells are segregated.

International audience; RATIONALE: Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by pulmonary arterial muscularization due to excessive pulmonary vascular cell proliferation and migration, a phenotype dependent upon growth factors and activation of receptor tyrosine kinases (RTKs). p130(Cas) is an adaptor protein involved in several cellular signaling pathways that control cell migration, proliferation, and survival. OBJECTIVES: We hypothesized that in experimental and human PAH p130(Cas) signaling is overactivated, thereby facilitating the intracellular transmission of signal induced by fibroblast growth factor (FGF)2, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). MEASUREMENTS AND MAIN RESULTS: In patients with PAH, levels of p130(Cas) protein and/or activity are higher in the serum, in the walls of distal pulmonary arteries, in cultured smooth muscle cells (PA-SMCs), and in pulmonary endothelial cells (P-ECs) than in control subjects. These abnormalities in the p130(Cas) signaling were also found in the chronically hypoxic mice and monocrotaline-injected rats as models of human PAH. We obtained evidence for the convergence and amplification of the growth-stimulating effect of the EGF-, FGF2-, and PDGF-signaling pathways via the p130(Cas) signaling pathway. We found that daily treatment with the EGF-R inhibitor gefitinib, the FGF-R inhibitor dovitinib, and the PDGF-R inhibitor imatinib started 2 weeks after a subcutaneous monocrotaline injection substantially attenuated the abnormal increase in p130(Cas) and ERK1/2 activation and regressed established pulmonary hypertension. CONCLUSIONS: Our findings demonstrate that p130(Cas) signaling plays a critical role in experimental and idiopathic PAH by modulating pulmonary vascular cell migration and proliferation and by acting as an amplifier of RTK downstream signals.

International audience; RATIONALE: Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by pulmonary arterial muscularization due to excessive pulmonary vascular cell proliferation and migration, a phenotype dependent upon growth factors and activation of receptor tyrosine kinases (RTKs). p130(Cas) is an adaptor protein involved in several cellular signaling pathways that control cell migration, proliferation, and survival. OBJECTIVES: We hypothesized that in experimental and human PAH p130(Cas) signaling is overactivated, thereby facilitating the intracellular transmission of signal induced by fibroblast growth factor (FGF)2, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). MEASUREMENTS AND MAIN RESULTS: In patients with PAH, levels of p130(Cas) protein and/or activity are higher in the serum, in the walls of distal pulmonary arteries, in cultured smooth muscle cells (PA-SMCs), and in pulmonary endothelial cells (P-ECs) than in control subjects. These abnormalities in the p130(Cas) signaling were also found in the chronically hypoxic mice and monocrotaline-injected rats as models of human PAH. We obtained evidence for the convergence and amplification of the growth-stimulating effect of the EGF-, FGF2-, and PDGF-signaling pathways via the p130(Cas) signaling pathway. We found that daily treatment with the EGF-R inhibitor gefitinib, the FGF-R inhibitor dovitinib, and the PDGF-R inhibitor imatinib started 2 weeks after a subcutaneous monocrotaline injection substantially attenuated the abnormal increase in p130(Cas) and ERK1/2 activation and regressed established pulmonary hypertension. CONCLUSIONS: Our findings demonstrate that p130(Cas) signaling plays a critical role in experimental and idiopathic PAH by modulating pulmonary vascular cell migration and proliferation and by acting as an amplifier of RTK downstream signals.

International audience; INTRODUCTION: Soft-tissue sarcomas (STS) represent a heterogeneous group of malignant tumors originating from connective tissues. Over recent years, this heterogeneity has led to a molecular breakdown of STS and subsequent use of targeted agents in several molecularly defined subgroups. After the initial success of imatinib in gastrointestinal stromal tumors, several other compounds have shown promising activity in some but not all subgroups of sarcoma. AREAS COVERED: This review discusses the rational and clinical results, when available, that support this subtype-directed approach. In the vast majority of cases, these agents have been tested only in patients with advanced disease; as chemotherapeutic agents are developed as non-histotype-specific therapies, they are not discussed here. The PubMed literature was searched using the terms 'sarcoma', 'angiogenesis', 'mTOR' and 'targeted agents'. Proceedings of the annual meeting of the American Society of Clinical Oncology as well as those of the Connective Tissue Oncology Society were also searched for relevant information. EXPERT OPINION: Many agents are currently developed in a subtype-specific manner in STS and this represents a significant leap forward. However, much remains to be done to improve our understanding of the molecular biology of this heterogeneous group of diseases.

International audience; BACKGROUND: Persistence of myofibroblasts is believed to contribute to the development of fibrosis in idiopathic pulmonary fibrosis (IPF). Transforming growth factor beta1 (TGFbeta1) irreversibly converts fibroblasts into pathological myofibroblasts, which express smooth muscle alpha-actin (alpha-SMA) and produce extracellular matrix proteins, such as procollagen I (alpha1). Reactive oxygen species produced by NADPH oxidases (NOXs) have been shown to regulate cell differentiation. It was hypothesised that NOX could be expressed in parenchymal pulmonary fibroblasts and could mediate TGFbeta1-stimulated conversion of fibroblasts into myofibroblasts. METHODS: Fibroblasts were cultured from the lung of nine controls and eight patients with IPF. NOX4, alpha-SMA and procollagen I (alpha1) mRNA and protein expression, reactive oxygen species production and Smad2/3 phosphorylation were quantified, in the absence and in the presence of incubation with TGFbeta1. Migration of platelet-derived growth factor (PDGF)-induced fibroblasts was also assessed. RESULTS: It was found that (1) NOX4 mRNA and protein expression was upregulated in pulmonary fibroblasts from patients with IPF and correlated with mRNA expression of alpha-SMA and procollagen I (alpha1) mRNA; (2) TGFbeta1 upregulated NOX4, alpha-SMA and procollagen I (alpha1) expression in control and IPF fibroblasts; (3) the change in alpha-SMA and procollagen I (alpha1) expression in response to TGFbeta1 was inhibited by antioxidants and by a NOX4 small interfering RNA (siRNA); (4) NOX4 modulated alpha-SMA and procollagen I (alpha1) expression by controlling activation of Smad2/3; and (5) NOX4 modulated PDGF-induced fibroblast migration. CONCLUSION: NOX4 is critical for modulation of the pulmonary myofibroblast phenotype in IPF, probably by modulating the response to TGFbeta1 and PDGF.

International audience; BACKGROUND: Persistence of myofibroblasts is believed to contribute to the development of fibrosis in idiopathic pulmonary fibrosis (IPF). Transforming growth factor beta1 (TGFbeta1) irreversibly converts fibroblasts into pathological myofibroblasts, which express smooth muscle alpha-actin (alpha-SMA) and produce extracellular matrix proteins, such as procollagen I (alpha1). Reactive oxygen species produced by NADPH oxidases (NOXs) have been shown to regulate cell differentiation. It was hypothesised that NOX could be expressed in parenchymal pulmonary fibroblasts and could mediate TGFbeta1-stimulated conversion of fibroblasts into myofibroblasts. METHODS: Fibroblasts were cultured from the lung of nine controls and eight patients with IPF. NOX4, alpha-SMA and procollagen I (alpha1) mRNA and protein expression, reactive oxygen species production and Smad2/3 phosphorylation were quantified, in the absence and in the presence of incubation with TGFbeta1. Migration of platelet-derived growth factor (PDGF)-induced fibroblasts was also assessed. RESULTS: It was found that (1) NOX4 mRNA and protein expression was upregulated in pulmonary fibroblasts from patients with IPF and correlated with mRNA expression of alpha-SMA and procollagen I (alpha1) mRNA; (2) TGFbeta1 upregulated NOX4, alpha-SMA and procollagen I (alpha1) expression in control and IPF fibroblasts; (3) the change in alpha-SMA and procollagen I (alpha1) expression in response to TGFbeta1 was inhibited by antioxidants and by a NOX4 small interfering RNA (siRNA); (4) NOX4 modulated alpha-SMA and procollagen I (alpha1) expression by controlling activation of Smad2/3; and (5) NOX4 modulated PDGF-induced fibroblast migration. CONCLUSION: NOX4 is critical for modulation of the pulmonary myofibroblast phenotype in IPF, probably by modulating the response to TGFbeta1 and PDGF.

International audience; In response to insulin, glucose transporter GLUT4 translocates from intracellular compartments towards the plasma membrane where it enhances cellular glucose uptake. Here, we show that sera from various species contain a factor that dose-dependently induces GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes, human adipocytes, myoblasts and myotubes. Notably, the effect of this factor on GLUT4 is fully maintained in insulin-resistant cells. Our studies demonstrate that the serum-induced increase in cell surface GLUT4 levels is not due to inhibition of its internalization and is not mediated by insulin, PDGF, IGF-1, or HGF. Similarly to insulin, serum also augments cell surface levels of GLUT1 and TfR. Remarkably, the acute effect of serum on GLUT4 is largely additive to that of insulin, while it also sensitizes the cells to insulin. In accordance with these findings, serum does not appear to activate the same repertoire of downstream signaling molecules that are implicated in insulin-induced GLUT4 translocation. We conclude that in addition to insulin, at least one other biological proteinaceous factor exists that contributes to GLUT4 regulation and still functions in insulin resistance. The challenge now is to identify this factor.

International audience; Motile cilia have long been known to play a role in processes such as cell locomotion and fluid movement whereas the functions of primary cilia have remained obscure until recent years. To date, ciliary dysfunction has been shown to be causally linked to a number of clinical manifestations that characterize the group of human disorders known as ciliopathies. This classification reflects a common or shared cellular basis and implies that it is possible to associate a series of different human conditions with ciliary dysfunction, which allows gaining insight into the cellular defect in disorders of unknown etiology solely based on phenotypic observations. Furthermore, to date we know that the cilium participates in a number of biological processes ranging from chemo- and mechanosensation to the transduction of a growing list of paracrine signaling cascades that are critical for the development and maintenance of different tissues and organs. Consequently, the primary cilium has been identified as a key structure necessary to regulate and maintain cellular and tissue homeostasis and thus its study is providing significant information to understand the pathogenesis of the different phenotypes that characterize these human conditions. Finally, the similarities between different ciliopathies at the phenotypic level are proving to be due to their shared cellular defect and also their common genetic basis. To this end, recent studies are showing that mutations in a given ciliary gene often appear involved in the pathogenesis of more than one clinical entity, complicating their genetic dissection, and hindering our ability to generate accurate genotype-phenotype correlations.

International audience; Spatial regulation is an important feature of signal specificity elicited by cytoplasmic tyrosine kinases of the Src family (SRC family protein tyrosine kinases [SFK]). Cholesterol-enriched membrane domains, such as caveolae, regulate association of SFK with the platelet-derived growth factor receptor (PDGFR), which is needed for kinase activation and mitogenic signaling. PAG, a ubiquitously expressed member of the transmembrane adaptor protein family, is known to negatively regulate SFK signaling though binding to Csk. We report that PAG modulates PDGFR levels in caveolae and SFK mitogenic signaling through a Csk-independent mechanism. Regulation of SFK mitogenic activity by PAG requires the first N-terminal 97 aa (PAG-N), which include the extracellular and transmembrane domains, palmitoylation sites, and a short cytoplasmic sequence. We also show that PAG-N increases ganglioside GM1 levels at the cell surface and, thus, displaces PDGFR from caveolae, a process that requires the ganglioside-specific sialidase Neu-3. In conclusion, PAG regulates PDGFR membrane partitioning and SFK mitogenic signaling by modulating GM1 levels within caveolae independently from Csk.

The Src-like adaptor protein (SLAP) belongs to the subfamily of adapter proteins that negatively regulate cellular signalling initiated by tyrosine kinases. SLAP has a unique, myristylated N-terminus, followed by SH3 and SH2 domains with high homology to Src family tyrosine kinases (SFK) and a unique C-terminal tail, which is important for c-Cbl binding. We have previously shown that SLAP negatively regulates platelet-derived growth factor (PDGF)-induced mitogenesis in fibroblasts and we now report that it regulates F-actin assembly for dorsal ruffles formation. c-Cbl mediated SLAP inhibition towards actin remodelling. Moreover, SLAP enhanced PDGF-induced c-Cbl phosphorylation by SFK. In contrast, SLAP mitogenic inhibition was not mediated by c-Cbl, but it rather involved a competitive mechanism with SFK for PDGF-receptor (PDGFR) association and mitogenic signalling. Accordingly, phosphorylation of the Src mitogenic substrates Stat3 and Shc were reduced by SLAP. Thus, we concluded that SLAP regulates PDGFR signalling by two independent mechanisms: a competitive mechanism for PDGF-induced Src mitogenic signalling and a non-competitive mechanism for dorsal ruffles formation mediated by c-Cbl.

International audience; PURPOSE: Low molecular weight carbonyl compounds, such as the alpha-ketoaldehydes methylglyoxal (MGO) and glyoxal (GO), are formed under hyperglycemic conditions and behave as advanced glycation end product (AGE) precursors. They form adducts on proteins, thereby inducing cellular dysfunctions involved in chronic complications of diabetes. METHODS AND MAIN FINDINGS: Nontoxic concentrations of GO or MGO altered the PDGF-induced PDGFRbeta-phosphorylation, ERK1/2-activation, and nuclear translocation, and the subsequent proliferation of mesenchymal cells (smooth muscle cells and skin fibroblasts). This resulted mainly from inhibition of the intrinsic tyrosine kinase of PDGFRbeta and in part from altered PDGF-BB binding to PDGFRbeta. Concomitantly, the formation of AGE adducts (N(epsilon)carboxymethyl-lysine and N(epsilon)carboxyethyl-lysine) was observed on immunoprecipitated PDGFRbeta. Arginine and aminoguanidine, used as carbonyl scavengers, reversed the inhibitory effect and the formation of AGE adducts on PDGFRbeta. AGE-PDGFRbeta adducts were also detected by anti-AGE antibodies in PDGFRbeta immunopurified from aortas of diabetic (streptozotocin-treated) compared to nondiabetic apolipoprotein E-null mice. Mass spectrometry analysis of aortas demonstrated increased AGE formation in diabetic specimens. CONCLUSIONS: These data indicate that MGO and GO induce desensitization of PDGFRbeta that helps to reduce mesenchymal cell proliferation.

International audience; Focal vascular injury and impaired endothelial function are features of pulmonary hypertension (PH) that lead to enhanced platelet endothelial cell interactions. Vascular endothelial growth factor (VEGF) is contained in platelets and released at sites of vascular injury to promote endothelial repair and wound healing in combination with platelet-derived nonspecific mitogens such as platelet-derived growth factor (PDGF). The overall balance between platelet VEGF and PDGF was investigated in 21 patients with primary PH, 8 with secondary PH, and 27 with chronic hypoxemic lung disease (CHLD), as well as in 29 control subjects. Platelet VEGF content was increased in patients with primary and secondary PH as compared with control subjects (518 +/- 89, 675 +/- 156, and 166 +/- 29 fg/10(5) platelets, respectively; p < 0.01), whereas platelet PDGF content was similar in the three groups (31 +/- 2, 36 +/- 4, and 33 +/- 3 pg/10(5) platelets, respectively; NS). Patients treated with a continuous prostacyclin infusion had a higher platelet VEGF but a similar platelet PDGF content as compared with untreated patients. Moderate increases in platelet VEGF and PDGF contents were observed in the CHLD patients. We conclude that patients with primary or secondary PH have an increase in platelet VEGF content, but not in platelet PDGF content, and that their platelet VEGF content increases further in response to prostacyclin infusion. We suggest that imbalance between platelet VEGF and PDGF is beneficial to patients with PH.