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PubMed2006

Mechanisms and modulation of pituitary adenylate cyclase-activating protein-induced calcium mobilization in human neutrophils.

The neuropeptide pituitary adenylate cyclase-activating protein (PACAP) acts via the G protein-coupled receptor vasoactive intestinal peptide (VIP)/PACAP receptor-1 to induce phospholipase C (PLC)/calcium and mitogen-activated protein kinase (MAPK)-dependent proinflammatory activities in human polymorphonuclear neutrophils (PMNs). In this article, we evaluate other mechanisms that regulate PACAP-evoked calcium transients, the nature of the calcium sources, and the role of calcium in proinflammatory activities. Reduction in the activity of PMNs to respond to PACAP was observed after cell exposure to inhibitors of the cAMP/protein kinase A (PKA), protein kinase C (PKC), and PI3K pathways, to pertussis toxin (PTX), genistein, and after chelation of intracellular calcium or after extracellular calcium depletion. Mobilization of intracellular calcium stores was based on the fact that PACAP-associated calcium transient was decreased after exposure to (a) thapsigargin (Tg), (b) xestospongin C (XeC), and (c) the protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; inhibition of calcium increase by calcium channel blockers, by nifedipine and verapamil, indicated that PACAP was also acting on calcium influx. Such mobilization was not dependent on a functional actin cytoskeleton. Homologous desensitization with nanomoles of PACAP concentration and heterologous receptors desensibilization by G protein-coupled receptor agonists were observed. Intracellular calcium depletion modulated PACAP-associated ERK but not p38 phosphorylation; in contrast, extracellular calcium depletion modulated PACAP-associated p38 but not ERK phosphorylation. In PACAP-treated PMNs, reactive oxygen species production and CD11b membrane upregulation in contrast to lactoferrin release were dependent on both intra- and extracellular calcium, whereas matrix metalloproteinase-9 release was unaffected by extracellular calcium depletion. These data indicate that both extracellular and intracellular calcium play key roles in PACAP proinflammatory activities.

Calcium↗
PubMed2001

GH regulation of IGF-I and suppressor of cytokine signaling gene expression in C2C12 skeletal muscle cells.

GH is required for normal postnatal growth and metabolism. GH stimulates postnatal growth through induction of IGF-I gene expression. Although the liver is the major site of GH-regulated IGF-I, recent evidence indicates that GH-regulated IGF-I expression in nonhepatic tissues is sufficient for normal postnatal growth. One potentially important nonhepatic site of GH-stimulated IGF-I expression is skeletal muscle, as injection of GH into animals leads to increased IGF-I mRNA in this tissue. Nevertheless, direct effects of GH in skeletal muscle cells in culture have not been reported. We therefore tested the C2C12 myogenic cell line for its response to GH and demonstrate that C2C12 skeletal muscle cells rapidly respond to physiological levels of GH with increased tyrosine phosphorylation of the GH receptor, Janus kinase 2, signal transducer and activator of transcription-5a and -5b, insulin receptor substrate-1, and activation of MAPKs/ERKs and protein kinase B/Akt. In these cells, GH stimulates the expression of IGF-I and two members of the suppressors of cytokine signaling family, cytokine-inducible SH2-containing protein and suppressor of cytokine signaling-2. Treatment of C2C12 myoblasts with either the MAPK kinase inhibitor PD98059 or the PI3K inhibitor wortmannin results in higher levels of GH-induced IGF-I and suppressor of cytokine signaling-2 mRNA expression, suggesting that activation of MAPK and PI3K pathways has an inhibitory role in IGF-I and suppressor of cytokine signaling-2 gene regulation. Therefore, C2C12 cells provide the first in vitro model system to study various aspects of GH action in skeletal muscle.

Animals↗
PubMed2001

PRL antiapoptotic effect in the rat decidua involves the PI3K/protein kinase B-mediated inhibition of caspase-3 activity.

During gestation, the uterus undergoes severe changes to accommodate and protect the developing conceptus. In particular, stromal endometrial cells proliferate and differentiate to form the decidual tissue, which produces PRL. Once the conceptus begins to grow, extensive regression by apoptosis take place in the decidua coincident with the loss of the PRL receptor in this tissue. In this report we have established for the first time that PRL, acting through the long form of the PRL receptor and the PI3K pathway, exerts an antiapoptotic effect in rat decidua. We have also shown that protein kinase B phosphorylation on serine 473 as well as its nuclear translocation are stimulated by PRL in decidual cells. Moreover, we have found that caspase-3, a well known effector of apoptosis, becomes expressed and active in the rat decidua just at a time when this tissue undergoes extensive apoptosis. PRL was able to down-regulate both caspase-3 mRNA levels as well as activity. Furthermore, using a protein kinase B dominant-negative expression vector, we provide evidence that PRL inhibition of caspase-3 requires an intact protein kinase B pathway. Finally, we have also found that rat placental lactogen I and II dose-dependently inhibit caspase-3 mRNA, suggesting multiple sources of PRL in the hormonal control of rat decidual regression. In summary, the results of this study have defined an important role for decidual PRL in the normal progress of pregnancy, specifically in the regression and reorganization of the decidua.

Animals↗
PubMed2006

Context-specific requirements for Fgfr1 signaling through Frs2 and Frs3 during mouse development.

Fibroblast growth factor receptor 1 (Fgfr1) plays pleiotropic roles during embryonic development, but the mechanisms by which this receptor signals in vivo have not previously been elucidated. Biochemical studies have implicated Fgf receptor-specific substrates (Frs2, Frs3) as the principal mediators of Fgfr1 signal transduction to the MAPK and PI3K pathways. To determine the developmental requirements for Fgfr1-Frs signaling, we generated mice (Fgfr1(Delta)Frs/DeltaFrs) in which the Frs2/3-binding site on Fgfr1 is deleted. Fgfr1(Delta)Frs/DeltaFrs embryos die during late embryogenesis, and exhibit defects in neural tube closure and in the development of the tail bud and pharyngeal arches. However, the mutant receptor is able to drive Fgfr1 functions during gastrulation and somitogenesis, and drives normal MAPK responses to Fgf. These findings indicate that Fgfr1 uses distinct signal transduction mechanisms in different developmental contexts, and that some essential functions of this receptor are mediated by Frs-independent signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Fundamentally different roles for LFA-1, Mac-1 and alpha4-integrin in neutrophil chemotaxis.

Although the LFA-1, Mac-1 and alpha(4) integrins are required for chemotaxis, it is unknown how they are regulated or what specific role they play. Previously we demonstrated that fMLP and IL-8 induce chemotaxis via the p38 MAPK and phosphoinositide 3-kinase (PI3K) pathways, respectively. Here we show that these chemoattractants also activate and use Mac-1 and LFA-1 in a differential manner during chemotaxis. Using integrin-specific substrata, we demonstrate that cell movement in response to IL-8 is mediated by Mac-1, whereas LFA-1 is required for directional migration. By contrast, chemotaxis to fMLP requires Mac-1 for cell movement, whereas LFA-1 and alpha(4)-integrin are required for directional migration. On serum protein, which contains ligands for LFA-1, Mac-1 and alpha(4)-integrin, chemotaxis to fMLP is dependent on Mac-1, whereas chemotaxis to IL-8 is dependent on LFA-1. These results suggest that Mac-1 is the dominant integrin involved in chemotaxis to fMLP, and LFA-1 is the dominant integrin involved in chemotaxis to IL-8. Consistent with these observations, higher quantities of high-affinity Mac-1 are found on cells chemotaxing to fMLP then on cells chemotaxing to IL-8. Moreover, a much larger quantity of clustered LFA-1 was found on cells migrating to IL-8 compared to cells moving towards fMLP. When cells are presented with competing gradients of fMLP and IL-8, they preferentially migrate towards fMLP and activate/utilize integrins in a manner identical to fMLP alone. Under the same conditions, p38 MAPK inhibition abolishes the preferential migration to fMLP; instead, the cells migrate preferentially towards IL-8. The activation and utilization of integrins under these conditions are consistent with patterns observed with IL-8 alone. Together, these data suggest that fMLP and IL-8 differentially activate integrins for use during chemotaxis, that p38 MAPK is a major mediator in the activation and utilization of integrins, and selective integrin activation occurs during chemotaxis between opposing gradients.

Chemotaxis, Leukocyte↗
PubMed2006

[Functions of Y-box binding protein and its role in tumorigenicity].

Y-box binding proteins, belonging to a family of multifunctional proteins conserved from bacteria to human, are involved in transcriptional and translational regulation of various genes, mRNA alternative splicing, DNA replication and repair, as well as cell proliferation. A typical Y-box binding protein contains three structure domains, namely the N-terminal domain, the hydrophilic C-terminal domain and the conserved cold shock domain (CSD) which binds strongly to inverted CCAAT box found in different promoters and determines protein function.Y-box binding proteins may play an important physiological role in cell proliferation.For example, the human Y-box protein 1 (YB-1) may be repressed in the oncogenic phosphoinositide 3-kinase (PI3K) pathway. In addition, it may also act as a negative regulator of p53. It has been demonstrated that YB-1 represses transcription of the p53 promoter in a sequence-specific manner using specifically reporter assays. This implies that YB-1 may, in some situations, protect cells from p53-mediated apoptosis, indicating that YB-1 may be a good target for the development of a new therapeutics.The function of Y-box binding protein and its effect on carcinogenesis are summarized in the paper. We hope to further explore the functional roles of Y-box binding protein, and provide some helpful lines and suggestions for tumor control.

Animals↗
PubMed2006

HIV protease inhibitors decrease VEGF/HIF-1alpha expression and angiogenesis in glioblastoma cells.

Glioblastomas are malignant brain tumors that are rarely curable, even with aggressive therapy (surgery, chemotherapy, and radiation). Glioblastomas frequently display loss of PTEN and/or epidermal growth factor receptor activation, both of which activate the PI3K pathway. This pathway can increase vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1alpha expression. We examined the effects of two human immunodeficiency virus protease inhibitors, nelfinavir and amprenavir, which inhibit Akt signaling, on VEGF and HIF-1alpha expression and on angiogenesis. Nelfinavir decreased VEGF mRNA expression and VEGF secretion under normoxia. Downregulation of P-Akt decreased VEGF secretion in a manner similar to that of nelfinavir, but the combination of the two had no greater effect, consistent with the idea that nelfinavir decreases VEGF through the PI3K/Akt pathway. Nelfinavir also decreased the hypoxic induction of VEGF and the hypoxic induction of HIF-1alpha, which regulates VEGF promoter. The effect of nelfinavir on HIF-1alpha was most likely mediated by decreased protein translation. Nelfinavir's effect on VEGF expression had the functional consequence of decreasing angiogenesis in in vivo Matrigel plug assays. Similar effects on VEGF and HIF-1alpha expression were seen with a different protease inhibitor, amprenavir. Our results support further research into these protease inhibitors for use in future clinical trials for patients with glioblastoma multiformes.

Brain Neoplasms↗
PubMed2005

Gonadotropins upregulate the epidermal growth factor receptor through activation of mitogen-activated protein kinases and phosphatidyl-inositol-3-kinase in human ovarian surface epithelial cells.

Gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have been implicated as probable risk factors in epithelial ovarian carcinomas, most of which are derived from ovarian surface epithelium (OSE). Since epidermal growth factor (EGF) increases the growth of ovarian surface epithelial cells, we determined the effect of gonadotropins on the expression of epidermal growth factor receptor (EGFR). We investigated the basal levels of EGFR mRNA and protein, and the mechanisms involved in the regulation of EGFR at the transcriptional and translational levels by FSH and LH. The immortalized OSE cell lines (IOSE) derived from normal OSE cells by transfecting SV40 T-antigen (IOSE-80 and IOSE-80PC, a post-crisis line) and ovarian cancer cell lines were employed. A significantly lower level of EGFR was observed in both IOSE-80 and IOSE-80PC cells when compared with the ovarian cancer cell lines, OVCAR-3 and SKOV-3. Treatment of IOSE-80PC cells with FSH and LH (10(-7) and 10(-6) g/ml) resulted in a significant increase in EGFR mRNA at 24 h and EGFR protein at 48 h, whereas the treatment with gonadotropins for 24-48 h induced a mild increase in EGFR in OVCAR-3, but not in SKOV-3 cells. In addition, IOSE-80PC cells treated with gonadotropins and EGF (10 nM) exhibited an additive stimulation of mitogenesis. Further, FSH and LH significantly increased activities of various kinases at 5-10 min, and pre-treatments with LY294002 (an inhibitor of PI3K) or PD98059 (an inhibitor of ERK1/2) partially blocked the gonadotropin-induced up-regulation of EGFR in IOSE-80PC cells. We investigated whether the effect of gonadotropins on EGFR mRNA levels is induced by increased transcription and/or by altered mRNA stability. Treatment of IOSE-80PC cells with FSH (10(-7) and 10(-6) g/ml) significantly enhanced the activity of the EGFR promoter (120 and 140% increase, respectively) at 24 h, and treatment with LH (10(-7) g/ml) for 24 h induced an increase in the activity of EGFR promoter (30%) in these cells. On the other hand, LH resulted in a significant increase in EGFR mRNA stability in the decay curves. Taken together, these results suggest that the effect of gonadotropins on the expression of EGFR may affect cell growth via ERK-1/-2 and PI3K pathways in pre-neoplastic ovarian surface epithelial cells, and that FSH and LH increase EGFR mRNA by different mechanisms. The former increased EGFR gene transcription essentially, whereas the latter mainly enhanced EGFR mRNA stability.

Epithelial Cells↗
PubMed2005

Homocysteine thiolactone inhibits insulin-stimulated DNA and protein synthesis: possible role of mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3) and p70 S6K phosphorylation.

Hyperhomocysteinemia and insulin resistance are independent factors for cardiovascular disease. Most of the angiotoxic effects of homocysteine are related to the formation of homocysteine thiolactone and the consequent increase in oxidative stress. We have recently found that homocysteine thiolactone inhibits insulin receptor tyrosine kinase activity, which results in decreased phosphatidylinositol 3-kinase (PI3K) activity and inhibition of glycogen synthesis. Oxidative stress seemed to be the mechanism underlying these effects, since glutathione was able to restore the insulin signaling as well as the insulin-mediated glycogen synthesis. In the present work we have further investigated insulin receptor signaling studying mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3) and p70 S6K phosphorylation. Again, homocysteine thiolactone (50 microM) prevented insulin-mediated MAPK, GSK-3 and p70 S6K phosphorylation and these effects were blocked by glutathione (250 microM). Since MAPK and PI3K pathways, including GSK3 and S6K, seem to mediate insulin-mediated growth and proliferation, we measured DNA and protein synthesis. We have found that homocysteine thiolactone (50 microM) inhibits insulin-mediated growth and proliferation, as previously shown for glycogen synthesis. Again, these effects seem to be mediated by oxidative stress, since 250 microM glutathione completely abolished the effects of homocysteine thiolactone on insulin-stimulated DNA and protein synthesis. In conclusion, these data suggest that homocysteine thiolactone impairs insulin signaling by a mechanism involving oxidative stress, leading to a defect in the action of insulin on growth and proliferation.

Animals↗
PubMed2006

Lessons from the drug discovery of lapatinib, a dual ErbB1/2 tyrosine kinase inhibitor.

Protein Kinases offer many opportunities for drug intervention points since phosphorylation is the most common post-translational modification. Phosphorylation regulates activity, location, degradation, conformation and the aberrant activity is implicated in many diseases, including cancer, inflammation, cardiovascular and central nervous system diseases. The focus of this review will be on the generation of highly effective signaling inhibitors targeting members of the erbB family of receptor tyrosine kinases, EGFR and ErbB-2, also known as transmembrane Type 1 receptor tyrosine kinases of the HER family of receptors. Ligand binding to the receptor causes a conformational change which activates the tyrosine kinase domain leading to autophosphorylation. This autophosphorylation activates the RAS/mitogen activated protein (MAP) kinase and phosphoinositol-3-kinase (PI3K) pathways leading to a myriad of signaling and cellular activities. Type 1 receptors are over-expressed in a variety of cancers and generally correlate with poor prognosis. For this reason, scientists at GlaxoSmithKline and many others in the scientific community, target the ATP binding site of the intracellular portion of the protein to block the aberrant signaling event. This review intends to cover the lessons learned in the discovery of lapatinib (GW572016, GW2016) by pulling together the various different publications that have been generated in distinct disciplines on aspects of the drug discovery program. Data analyses and correlation of assay data to help with the design of drug like molecules will be included and will demonstrate a break from the traditional focus on absolute potency as a guiding factor in lead compound selection.

Animals↗
PubMed2006

Ca2+-dependent cytoprotective effects of ursodeoxycholic and tauroursodeoxycholic acid on the biliary epithelium in a rat model of cholestasis and loss of bile ducts.

Chronic cholestatic liver diseases are characterized by impaired balance between proliferation and death of cholangiocytes, as well as vanishing of bile ducts and liver failure. Ursodeoxycholic acid (UDCA) is a bile acid widely used for the therapy of cholangiopathies. However, little is known of the cytoprotective effects of UDCA on cholangiocytes. Therefore, UDCA and its taurine conjugate tauroursodeoxycholic acid (TUDCA) were administered in vivo to rats simultaneously subjected to bile duct ligation and vagotomy, a model that induces cholestasis and loss of bile ducts by apoptosis of cholangiocytes. Because these two bile acids act through Ca2+ signaling, animals were also treated with BAPTA/AM (an intracellular Ca2+ chelator) or Gö6976 (a Ca2+-dependent protein kinase C-alpha inhibitor). The administration of UDCA or TUDCA prevented the induction of apoptosis and the loss of proliferative and functional responses observed in the bile duct ligation-vagotomized rats. These effects were neutralized by the simultaneous administration of BAPTA/AM or Gö6976. UDCA and TUDCA enhanced intracellular Ca2+ and IP3 levels, together with increased phosphorylation of protein kinase C-alpha. Parallel changes were observed regarding the activation of the MAPK and PI3K pathways, changes that were abolished by addition of BAPTA/AM or Gö6976. These studies provide information that may improve the response of cholangiopathies to medical therapy.

Animals↗
PubMed2001

Ceramide mediates tumor-induced dendritic cell apoptosis.

Induction of apoptosis in dendritic cells (DC) is one of the escape mechanisms of tumor cells from the immune surveillance system. This study aimed to clarify the underlying mechanisms of tumor-induced DC apoptosis. The supernatants (SN) of murine tumor cell lines B16 (melanoma), MCA207, and MCA102 (fibrosarcoma) increased C16 and C24 ceramide as determined by electrospray mass spectrometry and induced apoptosis in bone marrow-derived DC. N-oleoylethanolamine or D-L-threo 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which inhibits acid ceramidase or glucosylceramide synthase and then increases endogenous ceramide, enhanced DC apoptosis and ceramide levels in the presence of tumor SN. Pretreatment with L-cycloserine, an inhibitor of de novo ceramide synthesis, or phorbol ester, 12-O-tetradecanoylphorbol-13-acetate reduced endogenous ceramide levels and protected DC from tumor-induced apoptosis. However, other DC survival factors, including LPS and TNF-alpha, failed to do so. The protective activity of 12-O-tetradecanoylphorbol-13-acetate is abrogated by pretreatment with phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. Therefore, down-regulation of PI3K is the major facet of tumor-induced DC apoptosis. Tumor SN, N-oleoylethanolamine, or PDMP suppressed Akt, NF-kappaB, and bcl-x(L) in DC, suggesting that the accumulation of ceramide impedes PI3K-mediated survival signals. Taken together, ceramide mediates tumor-induced DC apoptosis by down-regulation of the PI3K pathway.

Animals↗
PubMed2004

Neuromedin U elicits cytokine release in murine Th2-type T cell clone D10.G4.1.

Neuromedin U (NmU), originally isolated from porcine spinal cord and later from other species, is a novel peptide that potently contracts smooth muscle. NmU interacts with two G protein-coupled receptors designated as NmU-1R and NmU-2R. This study demonstrates a potential proinflammatory role for NmU. In a mouse Th2 cell line (D10.G4.1), a single class of high affinity saturable binding sites for (125)I-labeled NmU (K(D) 364 pM and B(max) 1114 fmol/mg protein) was identified, and mRNA encoding NmU-1R, but not NmU-2R, was present. Competition binding analysis revealed equipotent, high affinity binding of NmU isopeptides to membranes prepared from D10.G4.1 cells. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular Ca(2+) concentration (EC(50) 4.8 nM for human NmU). In addition, NmU also significantly increased the synthesis and release of cytokines including IL-4, IL-5, IL-6, IL-10, and IL-13. Studies using pharmacological inhibitors indicated that maximal NmU-evoked cytokine release required functional phospholipase C, calcineurin, MEK, and PI3K pathways. These data suggest a role for NmU in inflammation by stimulating cytokine production by T cells.

Animals↗
PubMed2005

Differential calcium regulation of proinflammatory activities in human neutrophils exposed to the neuropeptide pituitary adenylate cyclase-activating protein.

The neuropeptide pituitary adenylate cyclase-activating protein (PACAP) acts via the G protein-coupled receptor vasoactive intestinal peptide/PACAP receptor-1 to induce phospholipase C/calcium and MAPK-dependent proinflammatory activities in human polymorphonuclear neutrophils (PMNs). In this study, we evaluate other mechanisms that regulate PACAP-evoked calcium transients, the nature of the calcium sources, and the role of calcium in proinflammatory activities. Reduction in the activity of PMNs to respond to PACAP was observed after cell exposure to inhibitors of the cAMP/protein kinase A, protein kinase C, and PI3K pathways, to pertussis toxin, genistein, and after chelation of intracellular calcium or after extracellular calcium depletion. Mobilization of intracellular calcium stores was based on the fact that PACAP-associated calcium transient was decreased after exposure to 1) thapsigargin, 2) Xestospongin C, and 3) the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenyl hydrazone; inhibition of calcium increase by calcium channel blockers, by nifedipine and verapamil, indicated that PACAP was also acting on calcium influx. Such mobilization was not dependent on a functional actin cytoskeleton. Homologous desensitization with nanomoles of PACAP concentration and heterologous receptors desensibilization by G protein-coupled receptor agonists were observed. Intracellular calcium depletion modulated PACAP-associated ERK but not p38 phosphorylation; in contrast, extracellular calcium depletion modulated PACAP-associated p38 but not ERK phosphorylation. In PACAP-treated PMNs, reactive oxygen species production and CD11b membrane up-regulation in contrast to lactoferrin release were dependent on both intra- and extracellular calcium, whereas matrix metalloproteinase-9 release was unaffected by extracellular calcium depletion. These data indicate that both extracellular and intracellular calcium play key roles in PACAP proinflammatory activities.

CD11b Antigen↗
PubMed2006

The chemokine CX3CL1 reduces migration and increases adhesion of neurons with mechanisms dependent on the beta1 integrin subunit.

Fractalkine/CX3CL1 and its specific receptor CX3CR1 are constitutively expressed in several regions of the CNS and are reported to mediate neuron-microglial interaction, synaptic transmission, and neuronal protection from toxic insults. CX3CL1 is released both by neuronal and astrocytic cells, whereas CX3CR1 is mainly expressed by microglial cells and neurons. Microglial cells efficiently migrate in response to CX3CL1, whereas no evidence is reported to date on CX3CL1-induced neuronal migration. For this reason, we have investigated in vitro the effects of CX3CL1 on basal migration of neurons and of the microglial and astrocytic populations, all these cells being obtained from the hippocampus and the cerebellum of newborn rats. We report that CX3CL1 stimulates microglial cell migration but efficiently reduces basal neuronal movement, regardless of the brain source. The effect of CX3CL1 is pertussis toxin (PTX) sensitive and PI3K dependent on hippocampal neurons, while it is PTX sensitive, PI3K dependent, and ERK dependent on cerebellar granules. Interestingly, CX3CL1 also increases neuron adhesion to the extracellular matrix component laminin, with mechanisms dependent on PTX-sensitive G proteins, and on the ERK and PI3K pathways. Both the reduction of migration and the increase of neuron adhesion require the activation of the beta(1) and alpha(6) integrin subunits with the exception of cerebellar neuron migration, which is only dependent on the beta(1) subunit. More importantly, in neurons, CX3CL1/CXCL12 cotreatment abolished the effect mediated by a single chemokine on chemotaxis and adhesion. In conclusion, our findings indicate that CX3CL1 reduces neuronal migration by increasing cell adhesion through integrin-dependent mechanisms in hippocampal and cerebellar neurons.

Animals↗
PubMed2006

Cutting edge: T cell requirement for CD28 costimulation is due to negative regulation of TCR signals by PTEN.

Recent studies suggest that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) plays a critical role in the maintenance of self-tolerance. Using T cell-specific PTEN knockout mice (PTENDeltaT), we have identified a novel mechanism by which PTEN regulates T cell tolerance. We found that TCR stimulation alone, without CD28 costimulation, is sufficient to induce hyperactivation of the PI3K pathway, which leads to enhanced IL-2 production by naive PTENDeltaT T cells. Importantly, as a result of this increased response to TCR stimulation, PTENDeltaT CD4(+) T cells no longer require CD28 costimulation for in vitro or in vivo expansion. In fact, unlike wild-type T cells, PTENDeltaT CD4(+) T cells are not anergized by delivery of TCR stimulation alone. These data suggest that by negatively regulating TCR signals, PTEN imposes a requirement for CD28 costimulation, thus defining a novel mechanism for its role in self-tolerance.

Animals↗
PubMed2000

Bcr/Abl activates transcription of the Bcl-X gene through STAT5.

Several tyrosine kinase oncogenes have been associated with myeloproliferative diseases, including Bcr/Abl, Tel/Abl, Tel/Jak2, and Tel/PDGFR. One target molecule shared by these oncogenes is known to be STAT5. We generated sublines of Ba/F3 cells in which either wild-type STAT5 or a constitutively active mutant of STAT5 (STAT5-1*6) were expressed under the control of a tetracycline-inducible promoter. These cell lines were compared with a Ba/F3 cell line in which the expression of p210(Bcr/Abl) was made inducible by a similar promoter. Before induction, all cells were dependent on interleukin 3 (IL-3) for growth and survival. Both STAT5-1*6 and Bcr/Abl enhanced viability and induced proliferation in the absence of IL-3. We found that the proviability protein Bcl-X(L), but not Bcl-2, was induced by both p210(Bcr/Abl) and STAT5-1*6. Using a Bcl-X gene promoter construct fused to a luciferase complementary DNA (cDNA), both p210(Bcr/Abl) and STAT5-1*6 were shown to induce transcription of Bcl-X. The increase in transcription of the Bcl-X promoter and the increase in Bcl-X protein, due to p210(Bcr/Abl), were blocked by expression of a dominant negative STAT5 mutant. Interestingly, however, STAT5-1*6 required the continued presence of IL-3 to cause a significant increase in Bcl-X(L) protein, whereas p210(Bcr/Abl) did not need IL-3. Studies with enzyme inhibitors suggest that the extra signal supplied by IL-3 may be supplied by the PI3K pathway. Overall, these data suggest that constitutively activated STAT5 can increase viability and proliferation of Ba/F3 cells. This may contribute to, but is not likely sufficient for, the enhanced viability associated with Bcr/Abl transformation.

Animals↗
PubMed2001

[Current advance in insulin signal transduction].

Evidence has suggested that the insulin receptor substarate(IRS)-phosphoinositide 3-kinase(PI3K) pathway plays a central role in metabolic actions of insulin. The downstream effectors that mediate PI 3-kinase-dependent actions, such as Akt or atypical PKC, have been identified. Lipid and protein phosphatases that modulate the IRS-PI3 K pathway are implicated in the development of insulin resistance. The abundance of IRS, regulated at the level of both mRNA and protein, may also contribute to the sensitivity of cells to insulin. Tissue-specific insulin receptor knockout mice involved in insulin signaling are disrupted have reveled the impact of the defects of insulin signaling in a specific tissue.

Animals↗