Akt In Response Research Articles

A red wine polyphenolic extract reduces the activation phenotype of cultured human liver myofibroblasts

To test the effect of a standardized red wine polyphenolic extract (RWPE) on the phenotype of human liver myofibroblasts in culture. Human myofibroblasts grown from liver explants were used in this study. Cell proliferation was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Signaling events were analyzed by western blot with phospho-specific antibodies. Matrix-metalloproteinase activity was measured with gel zymography. We found that cell proliferation was dose-dependently decreased by up to 90% by RWPE while cell viability was not affected. Exposure to RWPE also greatly decreased the phosphorylation of ERK1/ERK2 and Akt in response to stimulation by the mitogenic factor platelet-derived growth factor BB (PDGF-BB). Finally, RWPE affected extracellular matrix remodeling by decreasing the secretion by myofibroblasts of matrix-metalloproteinase-2 and of tissue inhibitor of matrix-metalloproteinases-1. Altogether, RWPE decreases the activation state of liver myofibroblasts. The identification of the active compounds in RWPE could offer new therapeutic strategies against liver fibrosis.

YRRL Motifs in the Cytoplasmic Domain of the Thrombopoietin Receptor Mpl Regulate Receptor Internalization and Degradation.

Thrombopoietin (TPO), acting through its receptor Mpl, promotes survival and proliferation of hematopoietic progenitor cells and also drives megakaryocyte differentiation. The pro-proliferation and survival signals activated by TPO must therefore be tightly regulated to prevent uncontrolled cell growth. Several mechanisms to down-regulate hematopoietic growth factor signaling have been identified, including increased expression of suppressors of cytokine signaling (SOCS) proteins, activation of protein phosphatases, and endocytosis and degradation of activated growth factor receptors. In this work we determined the mechanisms that control TPO-stimulated Mpl internalization and defined the processes leading to Mpl degradation using IL-3-dependent BaF3 cells engineered to express wild type (WT) and mutant forms of human Mpl. Stimulation of BaF-Mpl cells with TPO lead to rapid endocytosis of Mpl which was blocked by pre-treatment with the clathrin-mediated endocytosis inhibitor monodansylcadaverine, indicating that Mpl internalization is clathrin-mediated. Additionally, we found that inhibition of Janus kinase 2 (Jak2) and Src-family kinases greatly reduced Mpl internalization. Sites of clathrin assembly on the plasma membrane contain the adaptor protein complex AP2, which associates with transmembrane proteins enabling targeted endocytosis. Association of AP2 and transmembrane proteins relies on an interaction between AP2μ2 subunit and internalization motifs YXXΦ (where X=any amino acid and Φ=bulky hydrophobic) expressed by target proteins. Mpl contains two YXXΦ motifs, at cytoplasmic Y8 (Y8RRL) and Y78 (Y78RRL). BaF-Mpl cells transfected with siRNA targeted to AP2 displayed greatly reduced TPO-mediated internalization of Mpl, demonstrating that AP2-Mpl association is critical for Mpl endocytosis. Next, we introduced Y to F point mutations at Mpl cytoplasmic Y8 and Y78 individually, and in combination (Y8+78F). Internalization was greatly reduced in Mpl Y78F and Mpl Y8+78F after TPO stimulation, whereas Mpl Y8F internalization was only slightly attenuated. Furthermore, we found that Mpl Y78F and Y8+78F exhibited increased proliferation and increased strength and duration of activated Jak2 and AKT in response to TPO, compared to Mpl WT and Y8F. In addition to mediating endocytosis, cytoplasmic YXXΦ motifs located between 6 and 9 residues from the transmembrane domain have been shown to mediate lysosomal targeting of proteins following endocytosis. Consequently, we studied the role of Mpl Y8RRL motif in Mpl lysosome targeting. BaF-Mpl WT and Y8F cells were pretreated with cyclohexamide and stimulated with TPO for up to 2 hours. Mpl Y8F displayed greatly reduced Mpl degradation in response to TPO compared to WT. Furthermore, Mpl Y8F recycled back to the plasma membrane following TPO starvation quicker than WT Mpl, suggesting that the Mpl Y8F remains in endosomes, rather than being targeted to lysosomes. Our data shows that Mpl cytoplasmic YRRL motifs are responsible for both TPO-mediated internalization via interactions with AP2 and lysosomal targeting following endocytosis. These findings significantly advance our understanding of normal Mpl function. Further study of internalization motifs, which are present in receptors for other hematopoietic growth factors including; erythropoietin, granulocyte colony stimulating factor, leukemia inhibitory factor and interleukin, may highlight the importance of these sequences in mediating hematopoiesis and potentially aid identification of novel targets in hematological disease.

IB05204, a dichloropyridodithienotriazine, inhibits angiogenesis in vitro and in vivo

In the course of a blind screening program for inhibitors of angiogenesis, IB05204 (4,8-dichloro-12-phenylpyrido[5',6':4'',5'';3',2':4,5]dithieno[3'',2''-d':3,2-d]-1,2,3-ditriazine) was selected for its ability to inhibit endothelial tubule-like network formation on Matrigel. IB05204 inhibits the in vivo angiogenesis in the chorioallantoic membrane (CAM) and the mouse Matrigel plug assays. Antiangiogenic activity seems to be highly dependent on the chloro substituents because their removal results in a complete loss of the in vitro inhibitory activity of endothelial differentiation and in vivo antiangiogenic activity in CAM assay. Although IB05204 inhibits the growth of endothelial and tumor cells in culture, its antiangiogenic activity seems to be mainly dependent on the prevention of endothelial capillary-like tube formation and inhibition of endothelial migration because these effects are recorded at lower concentrations. IB05204 treatment inhibits matrix metalloproteinase-2 (MMP-2) production in endothelial and tumor cells, down-regulates endothelial cyclooxygenase-2 expression, and represses phosphorylation of endothelial Akt in response to serum stimulation, suggesting that IB05204 interferes with molecular mechanisms of cell migration and survival. IB05204 induces apoptosis in endothelial cells through cytochrome c release and caspase activation. Data here shown altogether indicate that IB05204 is a compound that interferes with several key steps of angiogenesis, making it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.

Activation of the Mitogen-Activated Protein Kinases and Akt in Response to Pulmonary Artery Banding in the Fetal Sheep Heart Is Developmentally Regulated

Background: The postnatal heart responds to biomechanical stress with hypertrophy. The fetal heart may also undergo hyperplasia, but the percentage of mitotically active cardiomyocytes decreases throughout gestation. The signaling pathways controlling growth and proliferation in the fetal heart are poorly understood. Objective: To determine whether activation of the mitogen-activated protein kinases and Akt in the acute response to pressure loading in the fetal heart is developmentally regulated. Methods: Pulmonary artery banding (PAB) was performed in 100- or 128-day fetuses of twin gestation (n = 6 per group) for 6 h. One twin served as a control. Right ventricular (RV) and left ventricular (LV) mitogen-activated protein kinase, Akt, and cyclin D1 protein levels were determined by Western blot. Results: Within each gestational age group, hemodynamic and arterial blood gas values were similar between PAB and control fetuses. The total mitogen-activated protein kinase and Akt protein levels were unchanged by PAB at both gestational ages, as were active p38 and JNK levels. The RV levels of active ERK tended to decrease in 128-day PAB fetuses as compared with controls, and LV active ERK normalized to total ERK was significantly decreased. At gestational age (GA) 100 days, RV active ERK levels were significantly higher in PAB animals as compared with controls, with a trend towards increased active Akt levels. No differences were seen in the 100-day LV. The levels of the cell cycle promoter cyclin D1 were unchanged in all animals. Conclusions: Pressure loading of the fetal sheep heart leads to developmentally regulated cell signaling profiles. ERK and possibly Akt may be important regulators of in vivo cardiomyocyte hyperplasia.

Sphingosine 1-phosphate S1P2 and S1P3 receptor-mediated Akt activation protects against in vivo myocardial ischemia-reperfusion injury

Sphingosine 1-phosphate (S1P) is released at sites of tissue injury and effects cellular responses through activation of G protein-coupled receptors. The role of S1P in regulating cardiomyocyte survival following in vivo myocardial ischemia-reperfusion (I/R) injury was examined by using mice in which specific S1P receptor subtypes were deleted. Mice lacking either S1P(2) or S1P(3) receptors and subjected to 1-h coronary occlusion followed by 2 h of reperfusion developed infarcts equivalent to those of wild-type (WT) mice. However, in S1P(2,3) receptor double-knockout mice, infarct size following I/R was increased by >50%. I/R leads to activation of ERK, JNK, and p38 MAP kinases; however, these responses were not diminished in S1P(2,3) receptor knockout compared with WT mice. In contrast, activation of Akt in response to I/R was markedly attenuated in S1P(2,3) receptor knockout mouse hearts. Neither S1P(2) nor S1P(3) receptor deletion alone impaired I/R-induced Akt activation, which suggests redundant signaling through these receptors and is consistent with the finding that deletion of either receptor alone did not increase I/R injury. The involvement of cardiomyocytes in S1P(2) and S1P(3) receptor mediated activation of Akt was tested by using cells from WT and S1P receptor knockout hearts. Akt was activated by S1P, and this was modestly diminished in cardiomyocytes from S1P(2) or S1P(3) receptor knockout mice and completely abolished in the S1P(2,3) receptor double-knockout myocytes. Our data demonstrate that activation of S1P(2) and S1P(3) receptors plays a significant role in protecting cardiomyocytes from I/R damage in vivo and implicate the release of S1P and receptor-mediated Akt activation in this process.

Reduction of Low Molecular Weight Protein-tyrosine Phosphatase Expression Improves Hyperglycemia and Insulin Sensitivity in Obese Mice

To investigate the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in glucose metabolism and insulin action, a specific antisense oligonucleotide (ASO) was used to reduce its expression both in vitro and in vivo. Reduction of LMW-PTP expression with the ASO in cultured mouse hepatocytes and in liver and fat tissues of diet-induced obese (DIO) mice and ob/ob mice led to increased phosphorylation and activity of key insulin signaling intermediates, including insulin receptor-beta subunit, phosphatidylinositol 3-kinase, and Akt in response to insulin stimulation. The ASO-treated DIO and ob/ob animals showed improved insulin sensitivity, which was reflected by a lowering of both plasma insulin and glucose levels and improved glucose and insulin tolerance in DIO mice. The treatment did not decrease body weight or increase metabolic rate. These data demonstrate that LMW-PTP is a key negative regulator of insulin action and a potential novel target for the treatment of insulin resistance and type 2 diabetes.

FOXO transcription factors

FOXO transcription factors

Intrahepatic insulin resistance in a murine model of steatohepatitis: effect of PPARγ agonist pioglitazone

Hepatic insulin resistance is associated with hepatic steatosis and is thought to play an important role in the pathogenesis of steatohepatitis. Using a murine model of steatohepatitis (mice fed a diet deficient in methionine and choline—MCD diet), we tested the effects of the insulin-sensitising, PPARγ agonist drug pioglitazone (PGZ) on systemic and intrahepatic insulin sensitivity and on liver pathology. Compared to controls, mice fed the MCD diet develop a significant steatohepatitis, have enhanced glucose tolerance and enhanced systemic response to insulin. PGZ did not affect the systemic insulin sensitivity in control or MCD-fed mice as assessed in vivo by intraperitoneal glucose or insulin dynamic tests. However, PGZ prevented hepatic fat accumulation and steatohepatitis induced by the MCD diet. This effect was associated with an increased mass of adipose tissue and increased expression and release of adiponectin, while hepatic acyl co-enzyme A oxidase and acyl-co-enzyme A carboxylase, regulating hepatic β-oxidation of fatty acid, remained unchanged. Steatohepatitis in MCD-diet-fed mice was associated with intrahepatic insulin resistance as shown by a reduced phosphorylation of hepatic insulin receptor, and Akt in response to an insulin stimulus. PGZ to MCD-fed mice restored the activation of the insulin receptor and of the Akt pathway in response to insulin. In conclusion, PGZ alleviates steatosis and steatohepatitis induced by the MCD diet, an effect associated with correction of intrahepatic insulin resistance.

Critical role for lipid raft-associated Src kinases in activation of PI3K-Akt signalling

Lipid rafts are membrane microdomains distinct from caveolae, whose functions in polypeptide growth factor signalling remain unclear. Here we show that in small cell lung cancer (SCLC) cells, specific growth factor receptors such as c-Kit associate with lipid rafts and that these domains play a critical role in the activation of phosphoinositide 3-kinase (PI3K) signalling. The class IA p85/p110α associated with Src in lipid rafts and was activated by Src in vitro. Lipid raft integrity was essential for Src activation in response to stem cell factor (SCF) and raft disruption selectively inhibited activation of protein kinase B (PKB)/Akt in response to SCF stimulation. Moreover, inhibition of Src kinases blocked PKB/Akt activation and SCLC cell growth. The use of fibroblasts with targeted deletion of the Src family kinase genes confirmed the role of Src kinases in PKB/Akt activation by growth factor receptors. Moreover a constitutively activated mutant of Src also stimulated PI3K/Akt in lipid rafts, indicating that these microdomains play a role in oncogenic signalling.Together our data demonstrate that lipid rafts play a key role in the activation of PI3K signalling by facilitating the interaction of Src with specific PI3K isoforms.

Lysophospholipids Control Integrin-dependent Adhesion in Splenic B Cells through Gi and G12/G13 Family G-proteins but Not through Gq/G11

Integrin-mediated adhesion is a crucial step in lymphocyte extravasation and homing. We show here that not only the chemokines CXCL12 and CXCL13 but also the lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) enhance adhesion of murine follicular and marginal zone B cells to ICAM-1 in vitro. This process involves clustering of integrin LFA-1 and is blocked by pertussis toxin, suggesting that G(i) family G-proteins are involved. In addition, lysophospholipid-induced adhesion on ICAM-1 depends on Rho and Rhokinase, indicative of an involvement of G(12)/G(13), possibly also G(q)/G(11) family G-proteins. We used G(12)/G(13)- or G(q)/G(11)-deficient B cells to study the role of these G-protein families in lysophospholipid-induced adhesion and found that the pro-adhesive effects of LPA and S1P are completely abrogated in G(12)/G(13)-deficient marginal zone B cells, reduced in G(12)/G(13)-deficient follicular B cells, and normal in G(q)/G(11)-deficient B cells. We also show that loss of lysophospholipid-induced adhesion results in disinhibition of migration in response to the follicular chemokine CXCL13, which might contribute to the abnormal localization of splenic B cell populations observed in B cell-specific G(12)/G(13)-deficient mice in vivo. Taken together, this study shows that lysophospholipids regulate integrin-mediated adhesion of splenic B cells to ICAM-1 through G(i) and G(12)/G(13) family G-proteins but not through G(q)/G(11).

Akt-mediated GSK-3beta inhibition prevents migration of polyamine-depleted intestinal epithelial cells via Rac1.

The rapid migration of intestinal epithelial cells (IEC) is important for the healing of mucosal wounds. We have previously shown that polyamine depletion inhibits migration of IEC-6 cells. Akt activation and its downstream target GSK-3beta have been implicated in the regulation of migration. Here we investigated the significance of elevated phosphatidylinositol 3-kinase (PI3K)/Akt signaling on migration of polyamine-depleted cells. Polyamine-depleted cells had high Akt (Ser473) and GSK-3beta (Ser9) phosphorylation. Pretreatment with 20 microM LY294002 (PI3K inhibitor) for 30 min inhibited phosphorylation of Akt, increased migration by activating Rac1 in polyamine-depleted IEC-6 cells, and restored the actin structure similar to that in cells grown in control medium. Treatment of cells with a GSK-3beta inhibitor (AR-A014418) altered the actin cytoskeleton and inhibited migration, mimicking the effects of polyamine depletion. Thus, our results indicate that sustained activation of Akt in response to polyamine depletion inhibits migration through GSK-3beta and Rac1.

From Ras to PI3K

The catalytic p110 subunits of class 1 phosphoinositide 3-kinases (PI3Ks) have a conserved Ras binding domain (RBD), and an interaction has been noted between Ras guanosine triphosphatase (GTPase) and p110 PI3K in vitro and in overexpression systems. However, the biological relevance of this putative regulatory input has been unclear. Two groups now report a physiological role for Ras interactions with PI3K in flies and mouse neutrophils. Orme et al . created transgenic flies carrying a Dp110 (the only Drosophila p110) with mutations in the RBD that did not interact with Ras (either Ras1 or Ras2) but was otherwise biochemically normal. This Dp110 RBD rescued flies from lethal deletion of Dp110 ; however, the rescued flies were smaller and females exhibited reduced fecundity, laying 60% fewer eggs. Dp110 RBD flies also had decreased activation of Akt in response to insulin in the brain and imaginal discs and decreased basal Akt activation in the ovaries. Although the interaction with Ras does not appear essential for PI3K signaling, it does appear to maximize the response. The authors suggest that different developmental contexts may require different levels of signaling. In this case, egg production, which requires massive growth, is one process with the highest dependency on the intensity of the PI3K signal. In mammals there are multiple isoforms of PI3K, and Suire et al . showed that the p110γ isoform, which is stimulated by G protein-coupled receptors through the action of Gβγ and an adaptor (in neutrophils the adaptor is p101), also appeared to be activated by Ras. They compared the neutrophil responsiveness to chemotactic agents fMLP or C5a, which are G protein-coupled receptor ligands, in cells isolated from mice in which p101 was knocked out to prevent Gβγ activation of p110γ or in which the RBD of p110γ was mutated (p110γ DASAA ) to prevent Ras activation. For cells carrying either mutation, production of phosphoinositide (3,4) diphosphate or phosphoinositide (3,4,5) triphosphate, activation of Akt, and chemotaxis were decreased compared with wild-type cells or cells from mice heterozygous for p110γ. In vivo, recruitment of neutrophils in response to peritonitis was diminished in the mutant mice. The production of reactive oxygen species in response to agonist stimulation showed selectivity in that only the cells from the p110γ DASAA mice exhibited a reduction in this arm of the PI3K pathway, suggesting that one role of Ras may be to activate a separate pool of PI3K from that activated by Gβγ. These two articles provide examples in which Ras may control the intensity of the PI3K signal in one context and provide selectivity to the signaling process in another. M. H. Orme, S. Alrubaie, G. L. Bradley, C. D. Walker, S. J. Leevers, Input from Ras is required for maximal PI(3)K signalling in Drosophila . Nat. Cell Biol. 8 , 1298-1302 (2006). [PubMed] S. Suire, A. M. Condliffe, G. J. Ferguson, C. D. Ellson, H. Guillou, K. Davidson, H. Welch, J. Coadwell, M. Turner, E. R. Chilvers, P. T. Hawkins, L. Stephens, Gβγs and the Ras binding domain of p110γ are both important regulators of PI(3)Kγ signalling in neutrophils. Nat. Cell. Biol. 8 , 1303-1309 (2006). [PubMed]

Rapid CD40‐mediated rescue from CD95‐induced apoptosis requires TNFR‐associated factor‐6 and PI3K

The activation molecule CD40 and the death receptor CD95/Fas play important roles in regulating B cells so that effective antimicrobial immunity occurs without autoimmunity. CD40 signaling increases CD95 expression, sensitizing cells to apoptosis, but sustained CD40 signals rescue B cells from CD95 killing. Here we describe a mechanism of early CD40-mediated rescue from CD95-induced apoptosis in B cells. Maximal rescue was achieved when CD40 signals were given within 1-2 h of initiating CD95 apoptosis. CD40 signaling did not block association of Fas-associated death domain-containing protein with CD95, but decreased CD95-induced activation of caspases 3 and 8. Rapid CD40 rescue did not require NF-kappaB activation and was independent of de novo protein synthesis, but was dependent upon active PI3 K. Signaling via a CD40 mutant that does not bind TNFR-associated factor (TRAF)1, TRAF2, and TRAF3 rescued B cells from CD95-induced apoptosis. TRAF1/2/3-independent rescue was confirmed in B cell lines made deficient in these TRAF molecules by gene targeting. In contrast, CD40 rescue was completely abrogated in TRAF6-deficient B cells, which showed reduced activation of Akt in response to CD40 engagement. These results reveal a new rapid mechanism to balance B cell activation and apoptosis.

Nicotine activates cell-signaling pathways through muscle-type and neuronal nicotinic acetylcholine receptors in non-small cell lung cancer cells

Nicotinic acetylcholine receptors (nAChR) are expressed on non-neuronal cell types, including normal bronchial epithelial cells, and nicotine has been reported to cause Akt activation in cultured normal airway cells. This study documents mRNA and protein expression of subunits known to form a muscle-type nAChR in non-small cell lung cancer (NSCLC) cell lines. In one NSCLC examined, mRNA and protein for a heteropentamer neuronal-type α3 β2 nAChR was detected in addition to a muscle-type receptor. Protein for the α5 nAChR was also detected in NSCLC cells. Although, mRNA for the α7 nAChR subunit was observed in all cell lines, α7 protein was not detectable by immunoblot in NSCLC cell extracts. Immunohistochemistry (IHC) of NSCLC primary tissues from 18 patients demonstrated protein expression of nAChR α1 and β1 subunits, but not α7 subunit, in lung tumors, indicating preferential expression of the muscle-type receptor. In addition, the β1 subunit showed significantly increased expression in lung tumors as compared to non-tumor bronchial tissue. The α1 subunit also showed evidence of high expression in lung tumors. Nicotine at a concentration of 10 μM caused phosphorylation of mitogen-activated protein kinase (MAPK) (p44/42) that could be inhibited using nAChR antagonists. Inhibition was observed at 100 nM α-bungarotoxin ( α-BTX) or 10 μM hexamethonium (HEX); maximal inhibition was achieved using a combination of α-BTX and HEX. Akt was also phosphorylated in NSCLC cells after exposure to nicotine; this effect was inhibited by the PI3K inhibitor LY294002 and antagonists to the neuronal-type nAChR, but not to the muscle-type receptor. Nicotine triggered influx of calcium in the 273T NSCLC cell line, suggesting that L-type calcium channels were activated. 273T cells also showed greater activation of p44/42 MAPK than of Akt in response to nicotine. Cultures treated with nicotine and the EGFR tyrosine kinase inhibitor gefitinib showed a significant increase in the number of surviving cells compared to gefitinib alone. These data indicate that the muscle-type nAChR, rather than the α7 type, is highly expressed in NSCLC and leads to downstream activation of the p44/42 MAPK pathway. Neuronal-type receptors are also present and functional, as evidenced by antagonist studies, although, the expression levels are lower than muscle-type nAChR. They also lead to downstream activation of MAPK and Akt. Nicotine may play a role in regulating survival of NSCLC cells and endogenous acetylcholine released locally in the lung and/or chronic nicotine exposure might play a role in NSCLC development. In addition, exposure of NSCLC patients to nicotine through use of nicotine replacement products or use of tobacco products may alter the efficacy of therapy with EGFR inhibitors.

In Vivo Restoration of Physiological Levels of Truncated TrkB.T1 Receptor Rescues Neuronal Cell Death in a Trisomic Mouse Model

Imbalances in neurotrophins or their high-affinity Trk receptors have long been reported in neurodegenerative diseases. However, a molecular link between these gene products and neuronal cell death has not been established. In the trisomy 16 (Ts16) mouse there is increased apoptosis in the cortex, and hippocampal neurons undergo accelerated cell death that cannot be rescued by administration of brain-derived neurotrophic factor (BDNF). Ts16 neurons have normal levels of the TrkB tyrosine kinase receptor but an upregulation of the TrkB.T1 truncated receptor isoform. Here we show that restoration of the physiological level of the TrkB.T1 receptor by gene targeting rescues Ts16 cortical cell and hippocampal neuronal death. Moreover, it corrects resting Ca2+ levels and restores BDNF-induced intracellular signaling mediated by full-length TrkB in Ts16 hippocampal neurons. These data provide a direct link between neuronal cell death and abnormalities in Trk neurotrophin receptor levels.

Expression of Vascular Endothelial Growth Factor Receptor 1 in Bone Marrow-derived Mesenchymal Cells Is Dependent on Hypoxia-inducible Factor 1

Bone marrow-derived cells are recruited to sites of ischemia, where they promote tissue vascularization. This response is dependent upon the expression of vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1), which mediates cell migration in response to VEGF or placental growth factor (PLGF). In this study, we found that exposure of cultured mouse bone marrow-derived mesenchymal stromal cells (MSC) to hypoxia or an adenovirus encoding a constitutively active form of hypoxia-inducible factor 1 (HIF-1) induced VEGFR1 mRNA and protein expression and promoted ex vivo migration in response to VEGF or PLGF. MSC in which HIF-1 activity was inhibited by a dominant negative or RNA interference approach expressed markedly reduced levels of VEGFR1 and failed to migrate or activate AKT in response to VEGF or PLGF. Thus, loss-of-function and gain-of-function approaches demonstrated that HIF-1 activity is necessary and sufficient for basal and hypoxia-induced VEGFR1 expression in bone marrow-derived MSC.

A Bex‐cycle built for two

Neurotrophins promote neuronal survival, but it has been recognized that they also stimulate differentiation. Indeed, in early studies by Hamburger, Levi‐Montelcini and Cohen, nerve growth factor (NGF) not only promoted survival, but also caused marked neurite outgrowth when added to explants of sensory ganglia (Cohen et al , 1954). The ability of NGF to promote differentiation was particularly well illustrated when Greene & Tischler (1976) showed that a pheochromocytoma cell line (PC12) treated with NGF exited the cell cycle and differentiated into a sympathetic neuron‐like phenotype. Since then, it has been established that the survival and differentiation effects of the neurotrophins are primarily mediated by binding to members of the Trk family of tyrosine kinase receptors (Huang & Reichardt, 2003); however, the first receptor identified for NGF was a 75‐kDa protein referred to as p75 (Chao et al , 1986; Radeke et al , 1987). In the past few years, the p75 receptor has received a lot of attention because of its multifunctional signalling ability. This receptor can promote cell survival or induce apoptosis, regulate neurite outgrowth and promote peripheral myelination, depending on the specific cellular context, the expression of co‐receptors and the activating ligand (Barker, 2004). The role of p75 in promoting differentiation and cell‐cycle arrest has been largely overlooked in recent years, even though this was one of the first cellular responses to be shown to result from the binding of NGF to the receptor. Long considered to be simply a co‐receptor for the Trks, p75 came to be recognized as a bona fide signalling molecule when it was shown to activate sphingomyelinase. This resulted in the production of ceramide, which led to the differentiation of T9 glioma cells as indicated by their cell‐cycle arrest and process outgrowth (Dobrowsky et al , 1994). Although the focus of most subsequent research …

Decoding Dopamine Signaling

Dopamine is a key neurotransmitter that is important for many physiological functions including motor control, mood, and the reward pathway. In this issue of Cell, the laboratories of Marc Caron and Li-Huei Tsai identify two very different molecules--beta-arrestin 2 and Par-4, respectively--that unexpectedly are involved in dopamine signaling via the D2 receptor. These two new signaling pathways mediate the actions of dopamine on behavior and facilitate crosstalk between different signaling pathways that are activated by binding of dopamine to the D2 receptor.

Roles of Tyrosine Phosphorylation and Cleavage of Protein Kinase Cδ in Its Protective Effect Against Tumor Necrosis Factor-related Apoptosis Inducing Ligand-induced Apoptosis

Protein kinase Cdelta (PKCdelta) regulates cell apoptosis in a cell- and stimulus-specific manner. Here, we studied the role of PKCdelta in the apoptotic effect of TRAIL in glioma cells. We found that transfection of the cells with a PKCdelta kinase-dead mutant (K376R) or with a small interfering RNA targeting the PKCdelta mRNA increased the apoptotic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), whereas overexpression of PKCdelta decreased it. PKCdelta acted downstream of caspase 8 and upstream of cytochrome c release from the mitochondria. TRAIL induced cleavage of PKCdelta within 2-3 h of treatment, which was abolished by caspase 3, 8, and 9 inhibitors. The cleavage of PKCdelta was essential for its protective effect because overexpression of a caspase-resistant mutant (PKCdeltaD327A) did not protect glioma cells from TRAIL-induced apoptosis but rather increased it. TRAIL induced translocation of PKCdelta to the perinuclear region and the endoplasmic reticulum and phosphorylation of PKCdelta on tyrosine 155. Using a PKCdeltaY155F mutant, we found that the phosphorylation of PKCdelta on tyrosine 155 was essential for the cleavage of PKCdelta in response to TRAIL and for its translocation to the endoplasmic reticulum. In addition, phosphorylation of PKCdelta on tyrosine 155 was necessary for the activation of AKT in response to TRAIL. Our results indicate that PKCdelta protects glioma cells from the apoptosis induced by TRAIL and implicate the phosphorylation of PKCdelta on tyrosine 155 and its cleavage as essential factors in the anti-apoptotic effect of PKCdelta.

The Lsc RhoGEF mediates signaling from thromboxane A2 to actin polymerization and apoptosis in thymocytes

The Lsc RhoGEF (also known as p115-RhoGEF) is a GTP exchange factor (GEF), an activator of GTPases of the Rho family. Lsc has a RhoGEF domain specific for Rho GTPase and a regulator of G protein signaling (RGS) domain specific for Galpha(12/13) subunits. One G protein receptor that can couple to Galpha(12/13) subunits is the receptor for thromboxane A(2 )(TXA(2)), thromboxane-prostanoid (called TP), which is highly expressed in immature thymocytes. TXA(2) has been implicated in thymocyte apoptosis. We found that Lsc(-/-) mice on a BALB/c background show thymic hyperplasia due to increased numbers of thymocytes and that these numbers further increase with the age of the mice. To investigate a role for Lsc in TXA(2) signaling, we analyzed activation of primary thymocytes by TXA(2) in vitro. TXA(2)-induced apoptosis of double-positive thymocytes and Rho activation required Lsc, and TXA(2) stimulation of actin polymerization and cofilin phosphorylation required both Lsc and Rho kinase (ROCK). Additionally, in the absence of Lsc, phosphorylation of the survival kinase Akt in response to TXA(2) was greatly enhanced. Together, these data demonstrate that Lsc is essential for mediating TXA(2 )signaling involved in apoptosis and actin organization and suggest that TXA(2) regulates thymic cellularity via Lsc.