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Bjorn Dahlback - One of the best experts on this subject based on the ideXlab platform.

  • plasma concentrations of GAS6 and saxl correlate with disease activity in systemic lupus erythematosus
    Rheumatology, 2011
    Co-Authors: Carl Ekman, Andreas Jonsen, Gunnar Sturfelt, Anders A Bengtsson, Bjorn Dahlback
    Abstract:

    Objectives. SLE is a systemic autoimmune disease with an annual incidence of 3.8 per 100 000. Several pathogenic mechanisms are believed to be operating in SLE, including an impaired clearance of apoptotic cells, activation of the type I IFN pathway and generation of autoimmune leucocytes. Growth arrest-specific protein 6 (GAS6) and its receptor Axl are known to regulate inflammation and may be implicated in lupus pathogenesis. We have recently developed immunological methods to quantify the vitamin-K-dependent protein GAS6 and its soluble receptor sAxl in human plasma, which we have used to investigate the role of GAS6 and soluble Axl in SLE. Methods. We have investigated the relation between the plasma concentrations of GAS6 and sAxl and disease activity and specific symptoms in 96 SLE patients. Results. GAS6 and sAxl concentrations correlated with SLEDAI (r = 0.48, P < 0.001 and r = 0.39, P < 0.001, respectively). Furthermore, concentrations of GAS6 and sAxl correlated with ESR and CRP and inversely with haemoglobin levels. GAS6 and sAxl concentrations were significantly higher in patients with anti-DNA antibodies, leucopenia and GN. Conclusion. The plasma concentrations of GAS6 and sAxl vary with disease activity in SLE, in particular GN, and may have a role in lupus pathogenesis. Furthermore, GAS6 and sAxl may be of use as biomarkers of disease activity.

  • plasma concentrations of GAS6 growth arrest specific protein 6 and its soluble tyrosine kinase receptor saxl in sepsis and systemic inflammatory response syndromes
    Critical Care, 2010
    Co-Authors: Carl Ekman, Adam Linder, Per Akesson, Bjorn Dahlback
    Abstract:

    Introduction: GAS6, the protein product of the growth arrest specific gene 6, is present in human circulation at subnanomolar concentrations. It is secreted by endothelial cells and is important for the activation of endothelium during inflammation. Axl, the tyrosine kinase receptor for GAS6, is also present in endothelium and can be cleaved and released into the circulation. The soluble of form Axl (sAxl), which is present in plasma, can bind GAS6 and inhibit Axl-mediated cell signalling. Methods: We have developed reproducible and accurate enzyme-linked immunosorbent assays for both GAS6 and sAxl and used them to investigate plasma samples from 70 patients with severe sepsis, 99 patients with sepsis, 42 patients with various infections causing fever but no systemic inflammatory response syndrome (SIRS), 20 patients with SIRS without verified infection, and 100 blood donors that served as controls. Correlations between GAS6 and sAxl concentrations and other commonly used analytes were investigated. Results: The patients with severe sepsis, sepsis, infection or SIRS had all increased concentrations of GAS6, approximately double compared to what was found in the controls. The concentrations of sAxl were also increased in the patient groups compared to the controls. GAS6 correlated with C-reactive protein, procalcitonin and interleukin 6, whereas sAxl correlated to bilirubin and procalcitonin. Conclusions: We can confirm results of earlier studies showing that circulating GAS6 is increased in sepsis and related syndromes. sAxl is increased, but less pronounced than GAS6. The concentrations of GAS6 and sAxl correlate with a number of inflammatory markers, suggesting a role in systemic inflammation.

  • GAS6 is complexed to the soluble tyrosine kinase receptor axl in human blood
    Journal of Thrombosis and Haemostasis, 2010
    Co-Authors: Carl Ekman, Jonas Stenhoff, Bjorn Dahlback
    Abstract:

    Summary Background: The vitamin K-dependent GAS6 protein (product of growth arrest specific gene 6) binds to, and activates the TAM receptor tyrosine kinases Tyro3, Axl, and Mer. GAS6 and the TAM receptors have been suggested to be important for primary platelet functions, but GAS6 cannot be found in human platelets. However, GAS6 is present in human plasma at a concentration of around 0.2 nM, which is 1,000-fold lower than that of the homologous protein S. The Axl and Mer receptors can be cleaved close to the cell membrane, yielding soluble molecules consisting of the extracellular parts of the receptors. Objective: To investigate if soluble Axl (sAxl) is present in human serum and plasma and if GAS6 circulates in complex with sAxl. Methods: We expressed recombinant sAxl, raised antibodies, developed and validated an ELISA for Axl. Serum and plasma were analyzed using ELISAs for GAS6, Axl, and sAxl-GAS6 complexes. Serum was gel filtered and fractions analyzed by the different ELISAs to determine if GAS6 in serum is free or complexed. Immunoprecipitation was used to investigate binding between GAS6 and sAxl in serum. Results: sAxl is present in serum and plasma at around 0.6 nM and all GAS6 is bound to sAxl. No complexes between GAS6 and the soluble forms of Mer and Tyro3 could be detected, indicating that sAxl is the physiological binder of GAS6 in human serum. Conclusions: GAS6 in circulation is bound to sAxl suggesting circulating GAS6 to be inhibited and incapable of stimulating the TAM receptors. (Less)

  • genetic loss of GAS6 induces plaque stability in experimental atherosclerosis
    The Journal of Pathology, 2008
    Co-Authors: Esther Lutgens, Peter Carmeliet, Bjorn Dahlback, Marc Tjwa, Garcia P De Frutos, Erwin Wijnands, Linda Beckers, Mat J A P Daemen, Lieve Moons
    Abstract:

    The growth arrest-specific gene 6 (GAS6) plays a role in pro-atherogenic processes such as endothelial and leukocyte activation, smooth muscle cell migration and thrombosis, but its role in atherosclerosis remains uninvestigated. Here, we report that GAS6 is expressed in all stages of human and mouse atherosclerosis, in plaque endothelial cells, smooth muscle cells and macrophages. GAS6 expression is most abundant in lesions containing high amounts of macrophages, ie thin fibrous cap atheroma and ruptured plaque. Genetic loss of GAS6 does not affect the number and size of initial and advanced plaques in ApoE(-/-) mice, but alters its plaque composition. Compared to GAS6(+/+): ApoE(-/-) mice, initial and advanced plaques of GAS6(-/-): ApoE(-/-) mice contained more smooth muscle cells and more collagen and developed smaller lipid cores, while the expression of TGFbeta was increased. In addition, fewer macrophages were found in advanced plaques of GAS6(-/-): ApoE(-/-) mice. Hence, loss of GAS6 promotes the formation of more stable atherosclerotic lesions by increasing plaque fibrosis and by attenuating plaque inflammation. These findings identify a role for GAS6 in plaque composition and stability.

  • analysis of GAS6 in human platelets and plasma
    Arteriosclerosis Thrombosis and Vascular Biology, 2005
    Co-Authors: Istvan Balogh, Sassan Hafizi, Jonas Stenhoff, Karin M Hansson, Bjorn Dahlback
    Abstract:

    Objective— GAS6 is a member of the vitamin K-dependent protein family. GAS6-deficient mice were found to be resistant to thrombosis because of defective platelet function. Mouse GAS6 was demonstrated to be present in platelets and found to be involved in platelet aggregation. The aim of this study was to investigate the presence of GAS6 in human platelets and plasma and determine its role in platelet function. Methods and Results— The presence of GAS6 in human platelets and plasma was analyzed using sensitive immunologic methods. Mass spectrometry and ELISA were used to identify and quantify GAS6 in plasma. GAS6 was demonstrated to be present in human plasma, at a concentration determined to be 13 to 23 ng/mL (0.16 to 0.28 nM). Furthermore, plasma GAS6 levels were found to be lower in patients administered with warfarin. However, GAS6 was undetectable in human platelets. Conclusions— This is the first report to identify and quantify GAS6 in human plasma. However, GAS6 protein was not detected in human platelets, suggesting that any potential platelet-specific function could be because of GAS6 from the circulation. These findings open up new directions regarding the role of GAS6 in normal and pathophysiological situations such as inflammation, autoimmune disease, thrombosis and arteriosclerosis.

Toru Nakano - One of the best experts on this subject based on the ideXlab platform.

  • Growth arrest-specific protein 6 deficiency impairs liver tissue repair after acute toxic hepatitis in mice.
    Journal of Hepatology, 2009
    Co-Authors: Fouad Lafdil, Toru Nakano, Marie-noële Chobert, Vanessa Deveaux, Elie-serge Zafrani, Philippe Mavier, Yannick Laperche, Arthur Brouillet
    Abstract:

    BACKGROUND/AIMS: Resident macrophages and myofibroblasts derived from hepatic stellate cells play a key role in liver wound healing. We previously reported that these sinusoidal cells secrete the growth arrest-specific protein 6 (GAS6) and express Axl, one of its receptors. Here we address the role of GAS6 in the healing process during acute liver injury. METHODS: Toxic hepatitis was induced by a single carbon tetrachloride injection in GAS6 deficient (GAS6(-/-)) mice and liver recovery was compared with wild-type animals. RESULTS: GAS6 deficiency did not cause any change in CCl(4)-induced liver damage. At 72 h, an efficient tissue repair was observed in wild-type animals whereas in GAS6(-/-) mice, we noticed a defective wound healing accounted by reduced Kupffer cell activation revealed by a decrease in the induction of CD14, TNF-alpha, IL6 and MCP-1. GAS6-deficiency, by limiting cytokine/chemokine release, prevents hepatocyte proliferation, recruitment of circulating monocytes and accumulation of myofibroblasts in healing areas. We also report a direct chemotactic effect of GAS6 on circulating monocytes which might explain defective macrophage infiltration in liver necrotic areas of GAS6(-/-) mice. Interestingly in GAS6(-/-) mice, we observed a high and constitutive expression of Axl and an induction of the suppressor of cytokine signaling SOCS1 after CCl(4) treatment. CONCLUSIONS: The lower level of cytokines/chemokines in GAS6(-/-) mice after CCl(4) injury, is the consequence of an inhibitory signal arising from Axl receptor overexpression, leading to delayed liver repair in deficient mice.

  • GAS6 axl pi3k akt pathway plays a central role in the effect of statins on inorganic phosphate induced calcification of vascular smooth muscle cells
    European Journal of Pharmacology, 2007
    Co-Authors: Koichi Kozaki, Toru Nakano, Katsuya Iijima, Masahiro Akishita, Yasuyoshi Ouchi
    Abstract:

    Apoptosis is essential for the initiation and progression of vascular calcification. Recently, we showed that 3-hydroxy-3-methylglutaryl (HMG) CoA reductase inhibitors (statins) have a protective effect against vascular smooth muscle cell calcification by inhibiting apoptosis, where growth arrest-specific gene 6 (GAS6) plays a pivotal role. In the present study, we clarified the downstream targets of GAS6-mediated survival signaling in inorganic phosphate (Pi)-induced apoptosis and examined the effect of statins. We found that fluvastatin and pravastatin significantly inhibited Pi-induced apoptosis and calcification in a concentration-dependent manner in human aortic smooth muscle cells (HASMC), as was found with atorvastatin previously. GAS6 and its receptor, Axl, expression were downregulated in the presence of Pi, and recombinant human GAS6 (rhGAS6) significantly inhibited apoptosis and calcification in a concentration-dependent manner. During apoptosis, Pi suppressed Akt phosphorylation, which was reversed by rhGAS6. Wortmannin, a specific phosphatidylinositol 3-OH kinase (PI3K) inhibitor, abolished the increase in Akt phosphorylation by rhGAS6 and eliminated the inhibitory effect of rhGAS6 on both Pi-induced apoptosis and calcification, suggesting that PI3K-Akt is a downstream signal of the GAS6-mediated survival pathway. Pi reduced phosphorylation of Bcl2 and Bad, and activated caspase 3, all of which were reversed by rhGAS6. The inhibitory effect of statins on Pi-induced apoptosis was accompanied by restoration of the GAS6-mediated survival signal pathway: upregulation of GAS6 and Axl expression, increased phosphorylation of Akt and Bcl2, and inhibition of Bad and caspase 3 activation. These findings indicate that the GAS6-mediated survival pathway is the target of statins' effect to prevent vascular calcification.

  • GAS6 induces akt mtor mediated mesangial hypertrophy in diabetic nephropathy
    Kidney International, 2005
    Co-Authors: Kojiro Nagai, Toru Nakano, Motoko Yanagita, Atsushi Fukatsu, Takeshi Matsubara, Akira Mima, Eriko Sumi, Hiroshi Kanamori, Noriyuki Iehara, Yoshikazu Ishimoto
    Abstract:

    GAS6 induces Akt/mTOR-mediated mesangial hypertrophy in diabetic nephropathy. Background. We have already reported GAS6 is involved in glomerular hypertrophy observed in diabetic nephropathy. However, the molecular mechanisms involved in glomerular hypertrophy are still unknown, especially in vivo. Methods. In vivo, diabetes was induced in rats and mice by streptozotocin (STZ) and the activation of the Akt/mTOR path- way in glomeruli was examined. In vitro, mesangial hypertrophy was assessed by ( 3 H)leucine incorporation and measuring cell areas. Results. Akt, p70 S6 kinase, and 4E-BP-1 were induced and phosphorylated in rat glomerular lysates after 12 weeks of STZ injection when mesangial and glomerular hypertrophy was ob- served. We then examined the role of GAS6 by treating STZ- rats with warfarin, and found that warfarin treatment inhibited the phosphorylation of these molecules as well as the hyper- trophy. We next examined whether high glucose stimulation can induce the expression of GAS6/Axl in mesangial cells. Stim- ulation of the cells with 25 mmol/L of glucose increased the expression of GAS6/Axl and mesangial cell size compared with that with 5.6 mmol/L of glucose. This hypertrophic effect was abolished in mesangial cells derived from GAS6 knockout mice. We also found that LY294002 and rapamycin blocked GAS6- induced activation of the Akt/mTOR pathway and mesangial hypertrophy. Furthermore, less phosphorylated Akt-positive or 4E-BP-1-positive areas were found in STZ-treated GAS6 knock- out mice than in STZ-treated wild-type mice. Conclusion. Our study indicates that the Akt/mTOR path- way is a key signaling cascade in GAS6-mediated mesangial and glomerular hypertrophy and revealed a crucial role of GAS6/Axl and the Akt/mTOR pathway in the development of diabetic nephropathy.

  • essential role of GAS6 for glomerular injury in nephrotoxic nephritis
    Journal of Clinical Investigation, 2002
    Co-Authors: Motoko Yanagita, Toru Nakano, Atsushi Fukatsu, Hidenori Arai, Yoshikazu Ishimoto, Kojiro Nagai, Tsuyoshi Ito, David J Salant, Toshio Doi, Toru Kita
    Abstract:

    Growth-arrest specific gene 6 (GAS6) is a vitamin K-dependent growth factor for mesangial and epithelial cells. To investigate whether GAS6 is essential for progressive glomerular injury, we constructed GAS6(-/-) mice and examined the role of GAS6 in accelerated nephrotoxic nephritis (NTN), a model of progressive glomerulonephritis. We found less mortality and proteinuria in GAS6(-/-) mice than in wild-type mice following injection of nephrotoxic serum. Glomerular cell proliferation, glomerular sclerosis, crescent formation, and deposition of fibrin/fibrinogen in glomeruli were also reduced in GAS6(-/-) mice. Furthermore, administering GAS6(-/-) mice recombinant wild-type GAS6, but not GAS6 lacking a previously characterized N-terminal gamma-carboxyl group, induced massive proteinuria, glomerular cell proliferation, and glomerulosclerosis, comparable to responses seen in wild-type mice. These data indicate that GAS6 induces glomerular cell proliferation in NTN and suggest that this factor contributes to glomerular injury and the progression of chronic nephritis.

  • GAS6 induces mesangial cell proliferation via latent transcription factor stat3
    Journal of Biological Chemistry, 2001
    Co-Authors: Motoko Yanagita, Toru Nakano, Kazumasa Ohashi, Atsushi Fukatsu, Hidenori Arai, Kensaku Mizuno, Toru Kita
    Abstract:

    Abstract Mesangial cell proliferation is essential for the pathogenesis and progression of glomerular disease. Previously, we showed that GAS6 plays a pivotal role in mesangial cell proliferationin vitro and in vivo. In the present study, we identified downstream targets of GAS6 signaling to examine the role in mesangial cell proliferation in vitro and in vivo. We found that GAS6 tyrosine phosphorylates STAT3 (signal transducers and activators of transcription) with concomitant translocation to the nucleus and induces STAT3-dependent transcriptional activation in cultured mesangial cells. Expressing dominant negative STAT3 inhibited GAS6-mediated transcriptional activation of STAT3 and abolished GAS6-induced mesangial cell proliferation. In a model of mesangial proliferative glomerulonephritis, STAT3 is phosphorylated in mesangial cells, and its phosphorylation peaks at day 8 after the injection of anti-Thy1.1 antibody. Inhibition of GAS6 by warfarin and the extracellular domain of its receptor, Axl, abolished phosphorylation of STAT3 in vivo. Thus, our in vitro and in vivo findings indicate that autocrine growth factor GAS6 induces mesangial cell proliferation via latent transcription factor STAT3. Therefore, STAT3 might be a new therapeutic target for kidney disease induced by mesangial proliferation.

Mark Blostein - One of the best experts on this subject based on the ideXlab platform.

  • Prostaglandin E synthase is upregulated by GAS6 during cancer-induced venous thrombosis.
    Blood, 2015
    Co-Authors: Meghedi N. Aghourian, Catherine A. Lemarié, Francois-rene Bertin, Mark Blostein
    Abstract:

    Venous thromboembolism is a common complication of cancer. Based on recent evidence that (1) growth arrest-specific 6 (GAS6) regulates the expression of tissue factor during venous thrombosis, and (2) cancer promotes a procoagulant milieu, we hypothesize that GAS6 may be involved in cancer-induced coagulopathy. Venous thrombi were induced in both wild-type (WT) and GAS6-deficient ((-/-)) mice with cancer. WT mice with cancer developed larger thrombi than their healthy counterparts; these larger thrombi induced by cancer were not seen in GAS6(-/-) mice. Whole genome microarray analysis of differential gene expression in WT and GAS6(-/-) endothelial cells exposed to M27 murine lung carcinoma cells reveal that GAS6 increases prostaglandin E synthase (Ptges) expression in endothelial cells. This was confirmed using real-time polymerase chain reaction and immunofluorescence staining. Culture of WT endothelial cells with M27 increases the secretion of prostaglandin E2 (PGE2), the enzymatic product of Ptges, in WT but not in GAS6(-/-) endothelial cells. In WT endothelial cells, Ptges expression was regulated through extracellular signal-regulated kinase 1/2 phosphorylation (ERK1/2). In vitro, PGE2 activates platelets after binding to its receptor, EP3. In vivo, EP3 receptor antagonism reversed the effect of cancer-induced thrombosis in WT mice. These results show that GAS6, through upregulation of PGE2, contributes to cancer-induced venous thrombosis.

  • vascular GAS6 contributes to thrombogenesis and promotes tissue factor up regulation after vessel injury in mice
    Blood, 2013
    Co-Authors: Richard S Robins, Catherine A. Lemarié, Meghedi N. Aghourian, Sandrine Laurance, Mark Blostein
    Abstract:

    GAS6 (growth-arrest specific gene 6) plays a role in thrombus stabilization. GAS6 null (-/-) mice are protected from lethal venous and arterial thromboembolism through platelet signaling defects induced only by 5 μM ADP and 10 μM of the thromboxane analog, U46619. This subtle platelet defect, despite a dramatic clinical phenotype, raises the possibility that GAS6 from a source other than platelets contributes to thrombus formation. Thus, we hypothesize that GAS6 derived from the vascular wall plays a role in venous thrombus formation. Bone marrow transplantation and platelet depletion/reconstitution experiments generating mice with selective ablations of GAS6 from either the hematopoietic or nonhematopoietic compartments demonstrate an approximately equal contribution by GAS6 from both compartments to thrombus formation. Tissue factor expression was significantly reduced in the vascular wall of GAS6(-/-) mice compared with WT. In vitro, thrombin-induced tissue factor expression was reduced in GAS6(-/-) endothelial cells compared with wild-type endothelium. Taken together, these results demonstrate that vascular GAS6 contributes to thrombus formation in vivo and can be explained by the ability of GAS6 to promote tissue factor expression and activity. These findings support the notion that vascular wall-derived GAS6 may play a pathophysiologic role in venous thromboembolism.

  • growth arrest specific gene 6 GAS6 and vascular hemostasis
    Advances in Nutrition, 2012
    Co-Authors: Sandrine Laurance, Catherine A. Lemarié, Mark Blostein
    Abstract:

    GAS6 (growth arrest-specific 6) belongs structurally to the family of plasma vitamin K-dependent proteins. GAS6 has a high structural homology with the natural anticoagulant protein S, sharing the same modular composition. Interestingly, despite the presence of a γ-carboxyglutamic acid domain in its structure, no role in the coagulation cascade has been identified for GAS6. GAS6 has been shown to be involved in vascular homeostasis and more precisely is involved in proliferation, apoptosis, efferocytosis, leukocyte migration, and sequestration and platelet aggregation. It is also involved in the activation of different cell types, from platelets to endothelial and vascular smooth muscle cells. Thus, it has been shown to play a role in several pathophysiological processes such as atherosclerosis, cancer, and thrombosis. Interestingly, studies using GAS6 null mice highlighted that GAS6 may represent a novel potential target for anticoagulant therapy, because these animals are protected from lethal venous thromboembolism without excessive bleeding. However, the mechanism in thrombus occurrence remains to be further explored. In the present review, we will focus on the role of GAS6 in innate immunity, atherosclerosis, thrombosis, and cancer-related events.

  • GAS6-mediated signaling is dependent on the engagement of its gamma-carboxyglutamic acid domain with phosphatidylserine.
    Biochemical and Biophysical Research Communications, 2008
    Co-Authors: Isabelle Rajotte, Ines Hasanbasic, Mark Blostein
    Abstract:

    Abstract GAS6 is a vitamin K-dependent protein containing gamma-carboxyglutamic acid (Gla) at its N-terminus and a receptor binding domain at its C-terminus. GAS6–Axl binding is necessary but not sufficient to support endothelial cell survival as decarboxylated GAS6 inhibits the pro-survival function of GAS6 by binding and inhibiting Axl, even though decarboxylated GAS6 cannot support endothelial cell survival itself. It is hypothesized that interactions between the Gla domain of GAS6 and phosphatidylserine (PS), though not required for GAS6 binding to Axl, are necessary for GAS6–Axl function. In support of this hypothesis are results showing that (1) two specific inhibitors of Gla–PS interactions, namely soluble PS and Annexin V, abrogate GAS6-mediated endothelial cell survival and (2) Soluble PS inhibits Akt activation, a downstream intracellular event triggered by GAS6–Axl binding. In conclusion, we propose a heretofore unknown function of Gla, where Gla–PS binding on the N-terminus of GAS6 is necessary for a GAS6 function mediated through its binding to Axl via its C-terminus.

  • the role of gamma carboxylation in the anti apoptotic function of GAS6
    Journal of Thrombosis and Haemostasis, 2005
    Co-Authors: Ines Hasanbasic, Isabelle Rajotte, Mark Blostein
    Abstract:

    GAS6 is a novel member of the vitamin K-dependent family of gamma-carboxylated proteins and is a ligand for the receptor tyrosine kinase Axl. GAS6-Axl interactions have been shown to mediate cell survival in vascular endothelium. Although the receptor-binding portion of GAS6 lies in the C-terminus, the significance of the N-terminal gamma-carboxylated residues (Gla domain) is not clear. To address this question, this study examines the role of the Gla domain in phospholipid binding as well as in the promotion of cell survival, especially in endothelial cells. The results show that carboxylated GAS6 binds to phosphatidylserine-containing phospholipid membranes in an analogous manner to other gamma-carboxylated proteins whereas decarboxylated GAS6 does not. The gamma-carboxylation inhibitor warfarin abrogates GAS6-mediated protection of NIH3T3 fibroblasts from serum starvation-induced apoptosis. Furthermore, the role of gamma-carboxylation in GAS6's survival effect on endothelium is demonstrated directly in that only carboxylated, but not decarboxylated, GAS6 protects endothelial cells from serum starvation-induced apoptosis. gamma-carboxylation is also required for both Axl phosphorylation and PI3 kinase activation. Taken together, these findings demonstrate that gamma-carboxylation is necessary not only for GAS6 binding to phospholipid membranes, but also for GAS6-mediated endothelial cell survival.

Claudio Schneider - One of the best experts on this subject based on the ideXlab platform.

  • GAS6 anti apoptotic signaling requires nf κb activation
    Journal of Biological Chemistry, 2001
    Co-Authors: Francesca Demarchi, Claudio Schneider, Brian Varnum, Roberto Verardo, Claudio Brancolini
    Abstract:

    The growth arrest-specific 6 gene product GAS6 is a growth and survival factor related to protein S. GAS6 is the ligand of Axl receptor tyrosine kinase; upon binding to its receptor GAS6 activates the phosphatidylinositol 3-OH kinase (PI3K) and its downstream targets S6K and Akt. GAS6 anti-apoptotic signaling was previously shown to require functional PI3K and Akt and to involve Bad phosphorylation in serum-starved NIH 3T3 cells. Here we demonstrate that GAS6 induces a rapid and transient increase in nuclear NF-kappa B binding activity coupled to transcription activation from NF-kappa B-responsive promoters and increase in Bcl-x(L) protein level. GAS6 survival function is impaired in cells lacking p65/RelA and in NIH 3T3 cells transfected with a dominant negative I kappa B, indicating that NF-kappa B activation plays a central role in promoting survival in this system. Moreover, NF-kappa B activation can be blocked by a dominant negative Akt and by wortmannin, an inhibitor of PI3K, thus suggesting that NF-kappa B activation is a downstream event with respect to PI3K and Akt, as already described for other growth factors. In addition, we show that glycogen synthase kinase 3, which is phosphorylated in response to GAS6, can physically associate with NFKB1/p105 in living cells and can phosphorylate it in vitro. Furthermore, GAS6 treatment is coupled to a decrease in p105 protein level. Altogether these data suggest the involvement of NF-kappa B and glycogen synthase kinase 3 in GAS6 anti-apoptotic signaling and unveil a possible link between these survival pathways.

  • the growth arrest specific gene GAS6 protein is expressed in abnormal embryos sired by male golden hamsters with accessory sex glands removed
    Anatomy and Embryology, 2001
    Co-Authors: H Y Jiang, Claudio Schneider, K H Lee, P L Tang, Pak Ham Chow
    Abstract:

    Expression of growth arrest specific gene (GAS6) and its receptors in embryonic and uterine tissues in normal pregnancy and pregnancy that produces abnormal embryos sired by hamsters with partial or total deletion of male accessory sex glands was studied by in situ hybridization, immunohistochemistry, reverse-transcription polymerization reaction and enzyme-linked immunoabsorbant assay. At oestrus, very strong GAS6 mRNA and GAS6 expression were seen only in the uterine epithelium and endometrial glands. Upon implantation, both of them could be demonstrated in the decidualizing stroma. From day 4 to day 7 p.c, GAS6 mRNA was present in the embryo, but GAS6 immunoreactivity was only found in those showing features of degeneration. The GAS6:β-actin mRNA ratio was low in oestrus and at day 4 of pregnancy but rose as the embryo grew. As for the receptors, Rse was detected in embryonic cells during days 5–7 p.c., and decidual cell from days 4 to 7 p.c., but Mer could be found in decidual cells and trophoblasts. It was concluded that GAS6 had a role in endometrial transformation during decidualization and trophoblastic invasion. In the embryo, GAS6 was transcribed, but the protein was only produced in response to need, such as when normal progression of development was threatened.

  • GAS6 mediated survival in nih3t3 cells activates stress signalling cascade and is independent of ras
    Oncogene, 1999
    Co-Authors: Sandro Goruppi, Elisabetta Ruaro, Brian Varnum, Claudio Schneider
    Abstract:

    GAS6 is a growth factor membrane of the vitamin K-dependent family of proteins which is preferentially expressed in quiescent cells. GAS6 was identified as the ligand for Axl tyrosine kinase receptor family. Consistent with this, GAS6 was previously reported to induce cell cycle re-entry of serum-starved NIH3T3 cells and to prevent cell death after complete growth factor withdrawal, the survival effect being uncoupled from GAS6-induced mitogenesis. We have previously demonstrated that both GAS6 mitogenic and survival effects are mediated by Src and the phosphatidylinositol3-OH kinase (PI3K). Here we report that Ras is required for GAS6 mitogenesis but is dispensable for its survival effect. GAS6-induced survival requires the activity of the small GTPases of the Rho family, Rac and Rho, together with the downstream kinase Pak. Overexpression of the respective dominant negative constructs abrogates GAS6-mediated survival functions. Addition of GAS6 to serum starved cells results in the activation of AKT/PKB and in the phosphorylation of the Bcl-2 family member, Bad. By ectopic expression of a catalytically inactive form of AKT/PKB, we demonstrate that AKT/PKB is necessary for GAS6-mediated survival functions. We further show evidence that GAS6 stimulation of serum starved NIH3T3 cells results in a transient ERK, JNK/SAPK and p38 MAPK activation. Blocking ERK activation did not influence GAS6-induced survival, suggesting that such pathway is not involved in GAS6 protection from cell death. On the contrary we found that the late constitutive increase of p38 MAPK activity associated with cell death was downregulated in GAS6-treated NIH3T3 cells thus suggesting that GAS6 might promote survival by interfering with this pathway. Taken together the evidence here provided identity elements involved in GAS6 signalling more specifically elucidating the pathway responsible for GAS6-induced cell survival under conditions that do not allow cell proliferation.

  • The product of a GAS6 splice variant allows the release of the domain responsible for Axl tyrosine kinase receptor activation
    FEBS letters, 1997
    Co-Authors: Sandro Goruppi, Brian Varnum, Harvey Yamane, Paolo Marcandalli, Andy Garcia, Cris Clogston, Monica Gostissa, Claudio Schneider
    Abstract:

    The product of GAS6 (GAS6) is a growth factor with high level of similarity to protein S and was identified as the ligand for Axl family of tyrosine kinase receptors. GAS6 contains an N-terminal γ-carboxylated domain (Gla), four epidermal growth factor like domains and a large C-terminal D region. An alternative GAS6 spliced form (GAS6SV) having an additional 43 amino acids between fourth EFG like and D domain was characterised. Here we show data indicating that GAS6SV is specifically cleaved within the inserted sequence, thereby splitting the D domain from the remaining part of the protein. The resulting two proteolytic products of 36 kDa and 50 kDa were separated and the 50 kDa fragment corresponding to region D was shown to be responsible for Axl receptor activation. Furthermore a deletion mutant of GAS6 containing only the D domain was shown to similarly activate Axl receptor phosphorylation unequivocally demonstrating that D domain can act as a signalling molecule. The possible roles of the proteolytic processing of GAS6SV in the regulation of growth factor availability are discussed.

  • requirement of phosphatidylinositol 3 kinase dependent pathway and src for GAS6 axl mitogenic and survival activities in nih 3t3 fibroblasts
    Molecular and Cellular Biology, 1997
    Co-Authors: Sandro Goruppi, Elisabetta Ruaro, Brian Varnum, Claudio Schneider
    Abstract:

    GAS6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that GAS6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from GAS6-induced mitogenesis. Here we report that the mitogenic effect of GAS6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, GAS6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks GAS6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for GAS6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited GAS6-stimulated entry into the S phase. Accordingly, GAS6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of GAS6. Taken together, the evidence presented here identifies elements involved in the GAS6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival.

Kensaku Mizuno - One of the best experts on this subject based on the ideXlab platform.

  • requirement of gamma carboxyglutamic acid modification and phosphatidylserine binding for the activation of tyro3 axl and mertk receptors by growth arrest specific 6
    Frontiers in Immunology, 2017
    Co-Authors: Ke Geng, Kensaku Mizuno, Stanley G Kimani, Sushil Kumar, Vladyslav Kholodovych, Sergei V Kotenko, Canan Kasikara, Oleta A Sandiford, Pranela Rameshwar, Raymond B Birge
    Abstract:

    The Tyro3, Axl, and Mertk (TAM) receptors are homologous type I receptor tyrosine kinases that have critical functions in the clearance of apoptotic cells in multicellular organisms. TAMs are activated by their endogenous ligands, growth arrest-specific 6 (GAS6), and protein S (Pros1), that function as bridging molecules between externalized phosphatidylserine (PS) on apoptotic cells and the TAM ectodomains. However, the molecular mechanisms by which GAS6/Pros1 promote TAM activation remains elusive. Using TAM/IFNγR1 reporter cell lines to monitor functional TAM activity, we found that GAS6 activity was exquisitely dependent on vitamin K-mediated γ-carboxylation, whereby replacing vitamin K with anticoagulant warfarin, or by substituting glutamic acid residues involved in PS binding, completely abrogated GAS6 activity as a TAM ligand. Furthermore, using domain and point mutagenesis, GAS6 activity also required both an intact Gla domain and intact EGF-like domains, suggesting these domains function cooperatively in order to achieve TAM activation. Despite the requirement of γ-carboxylation and the functional Gla domain, non-γ-carboxylated GAS6 and Gla deletion/EGF-like domain deletion mutants still retained their ability to bind TAMs and acted as blocking decoy ligands. Finally, we found that distinct sources of PS-positive cells/vesicles (including apoptotic cells, calcium-induced stressed cells, and exosomes) bound GAS6 and acted as cell-derived or exosome-derived ligands to activate TAMs. Taken together, our findings indicate that PS is indispensable for TAM activation by GAS6, and by inference, provides new perspectives on how PS, regulates TAM receptors and efferocytosis.

  • GAS6 induces mesangial cell proliferation via latent transcription factor stat3
    Journal of Biological Chemistry, 2001
    Co-Authors: Motoko Yanagita, Toru Nakano, Kazumasa Ohashi, Atsushi Fukatsu, Hidenori Arai, Kensaku Mizuno, Toru Kita
    Abstract:

    Abstract Mesangial cell proliferation is essential for the pathogenesis and progression of glomerular disease. Previously, we showed that GAS6 plays a pivotal role in mesangial cell proliferationin vitro and in vivo. In the present study, we identified downstream targets of GAS6 signaling to examine the role in mesangial cell proliferation in vitro and in vivo. We found that GAS6 tyrosine phosphorylates STAT3 (signal transducers and activators of transcription) with concomitant translocation to the nucleus and induces STAT3-dependent transcriptional activation in cultured mesangial cells. Expressing dominant negative STAT3 inhibited GAS6-mediated transcriptional activation of STAT3 and abolished GAS6-induced mesangial cell proliferation. In a model of mesangial proliferative glomerulonephritis, STAT3 is phosphorylated in mesangial cells, and its phosphorylation peaks at day 8 after the injection of anti-Thy1.1 antibody. Inhibition of GAS6 by warfarin and the extracellular domain of its receptor, Axl, abolished phosphorylation of STAT3 in vivo. Thus, our in vitro and in vivo findings indicate that autocrine growth factor GAS6 induces mesangial cell proliferation via latent transcription factor STAT3. Therefore, STAT3 might be a new therapeutic target for kidney disease induced by mesangial proliferation.

  • promotion of the uptake of ps liposomes and apoptotic cells by a product of growth arrest specific gene GAS6
    Journal of Biochemistry, 2000
    Co-Authors: Yoshikazu Ishimoto, Kensaku Mizuno, Kazumasa Ohashi, Toru Nakano
    Abstract:

    GAS6, a ligand of receptor tyrosine kinases Axl, Sky, and Mer, potentiates cell proliferation and prevents cell death. It also contains g-carboxylglutamic acid residues that mediate the interaction of some blood coagulation factors with negatively charged phospholipids. In our previous study, we demonstrated that GAS6 specifically binds to phosphatidylserine (PS) and links Axl-expressing cells to the PS-coated surface. In this study, to further understand the biological role of the interaction of GAS6 with PS, we examined the effect of GAS6 on the uptake of PS liposomes by macrophages. In vitro phagocytosis studies showed that GAS6 enhanced the uptake of PS liposomes approximately threefold and that the interaction of GAS6 with the surface of macrophages was essential for this enhancement. Analyses of the mechanism of the uptake of PS liposome suggested that GAS6 interacts with PS liposome via its N-terminal Gla domain and with macrophages via its C-terminal domain. Like that of PS liposomes, the uptake of apoptotic cells by macrophages was also enhanced, approximately twofold, in the presence of GAS6. These findings suggest that GAS6 may help phagocytic cells recognize cells with PS exposed on their surfaces, which is considered to be one of the mechanisms for clearing away dying cells. Thus, GAS6 may play a critical role in homeostasis by facilitating the clearance of PS-expressing cells.

  • mechanism of inhibitory effect of warfarin on mesangial cell proliferation
    Journal of The American Society of Nephrology, 1999
    Co-Authors: Motoko Yanagita, Toru Nakano, Kazumasa Ohashi, Harunobu Ozaki, Kenji Ishii, Hidenori Arai, Kensaku Mizuno, Toru Kita
    Abstract:

    Abstract . Because proliferation of mesangial cells is a hallmark of glomerular diseases, understanding the regulatory mechanism of mesangial proliferation is important for the treatment. Warfarin has long been used to treat glomerular diseases, although its mechanism of effect on mesangial proliferation has remained unknown. Therefore, this study was conducted to examine whether warfarin can inhibit mouse mesangial cell proliferation by focusing on GAS6, which has been shown to be activated by vitamin K-dependent γ-carboxylation. In mesangial cells, GAS6 and its receptor Axl were expressed. In addition, exogenous GAS6 phosphorylated Axl, activated extracellular signal-regulated kinase, and stimulated [ 3 H]-thymidine incorporation in mouse mesangial cells. This study also examined whether endogenous GAS6 stimulates mesangial proliferation. Conditioned medium (CM) from serum-starved mesangial cells could stimulate [ 3 H]-thymidine incorporation and phosphorylate extracellular signal-regulated kinase, whereas CM in the presence of warfarin could not. Simultaneous administration of vitamin K could cancel the inhibitory effect of warfarin. These results suggest that vitamin K-dependent growth factors in the CM are critical for mesangial proliferation. Addition of the extracellular domain of Axl to the CM inhibited its mitogenic effect on mesangial cells, suggesting that this vitamin K-dependent growth factor is GAS6. It is concluded that GAS6 is an endogenous mitogen in mesangial cells, and warfarin inhibits mesangial proliferation possibly by inhibiting γ-carboxylation of GAS6. This study sheds light on the regulation of mesangial proliferation and may lead to a new therapeutic strategy for glomerular diseases.

  • identification of the product of growth arrest specific gene 6 as a common ligand for axl sky and mer receptor tyrosine kinases
    Journal of Biological Chemistry, 1996
    Co-Authors: Kyoko Nagata, Toru Nakano, Kazumasa Ohashi, Hitoshi Arita, Chen Zong, Hidesaburo Hanafusa, Kensaku Mizuno
    Abstract:

    Abstract Axl, Sky, and Mer, members of an Axl/Sky receptor tyrosine kinase subfamily, are typified by the cell adhesion molecule-related extracellular domain. The product of growth arrest-specific gene 6 (GAS6), structurally homologous to the anticoagulant protein S, was recently identified as the ligand for Axl and Sky, but the ligand for Mer remained unknown. We have now obtained evidence that GAS6 can also function as a ligand for Mer. Co-precipitation analysis, using soluble receptors of Axl, Sky, and Mer (Axl-Fc, Sky-Fc, and Mer-Fc) composed of the extracellular domain of receptors fused to the Fc domain of immunoglobulin G1, clearly showed that GAS6, but not protein S, specifically bound to Axl-Fc, Sky-Fc, and Mer-Fc fusion proteins. Quantitative kinetic analyses using a BIAcore biosensor instrument revealed dissociation constants (Kd) of the binding of rat GAS6 to Axl-Fc, Sky-Fc, and Mer-Fc are 0.4, 2.7, and 29 nM, respectively. We also found that GAS6 stimulated tyrosine phosphorylation of Axl, Sky, and Mer receptors ectopically expressed in Chinese hamster ovary cells. Taken together, these findings suggest that GAS6 is a common ligand for Axl, Sky, and Mer, all known members of an Axl/Sky receptor subfamily.