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Results for “Signal Transduction”

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PubMed2000

Changes in signal transduction in the platelets of patients with peripheral occlusive arterial disease.

BACKGROUND: The finding that platelets of patients with peripheral occlusive arterial disease (POAD) circulate in an activated state prompted us to study platelet signal transduction. We hypothesised that platelet hyperreactivity is caused by changes in intracellular signalling. METHODS EXPERIMENTAL DESIGN: a single blood sample was taken from the antecubital vein of each participant prior to the start of intravenous treatment with prostaglandins. SETTING: patients were recruited from our inpatient Department of Cardiology and Angiology at the University Hospital. PARTICIPANTS: 15 hospitalised patients with symptomatic POAD were randomly selected. Patients receiving antiplatelet drugs and those with diabetes were excluded. The control group consisted of 15 healthy volunteers from the medical staff. INTERVENTIONS: blood tests were performed on the day of admission before any therapeutic intervention. MEASURES: the platelet activation marker P-selectin was quantified on peripheral blood platelets before and after in vitro stimulation with platelet agonists (adenosine diphosphate, thrombin receptor activator peptide-6). The signal transduction cascade was also selectively blocked by preincubation with either: 1) forskolin, 2) phospholipase C inhibitor U-73122, or 3) bisindolylmaleimide. RESULTS: A stronger inhibitory effect on ADP-stimulated platelets was seen in patients with U-73122, as indicated by a decrease in mean fluorescence intensity of 51% versus 34% in controls (p<0.0005). CONCLUSIONS: Our findings support the assumption that changes in platelet signal transduction in POAD lead to platelet hyper-reactivity.

Adenosine Diphosphate↗

PubMed2003

Two common naturally occurring mutations in the human gonadotropin-releasing hormone (GnRH) receptor have differential effects on gonadotropin gene expression and on GnRH-mediated signal transduction.

Studies of naturally occurring human GnRH receptor (GnRHR) mutants may provide a useful approach to dissecting the signal transduction pathways involved in mediating the effects of GnRH. We have analyzed two common mutations in the GnRHR, corresponding to amino acid substitutions Gln106Arg and Arg262Gln, for their effects on the stimulation of gonadotropin subunit and GnRHR gene expression by GnRH. Despite similar impairment of GnRH-stimulated inositol phosphate production, dose-response analyses indicated that Gln106Arg and Arg262Gln both reduced the sensitivity of the FSH beta gene promoter to a greater extent than LH beta or alpha GSU, suggesting the involvement of more than one signaling pathway. Furthermore, although the sensitivities of the LH beta and FSH beta gene promoters to GnRH were similarly affected by both mutants, alpha GSU sensitivity was decreased to a greater extent by Arg262Gln than by Gln106Arg. Similarly, GnRHR gene promoter sensitivity was significantly reduced only by Arg262Gln. To further characterize the differential downstream effects of these mutant GnRHRs, we investigated their effects on additional signal transduction pathways. The mutant receptors differentially affected GnRH-mediated activation of the ERK pathway and GnRH stimulation of cAMP response element-mediated transcription. These results indicate that measurement of inositol phosphate production alone may not be adequate for assessing mutant GnRHR function and additional signal transduction pathways may better reflect physiologically relevant effects. The differential stimulation of LH beta, FSH beta, and alpha GSU gene expression may contribute to the varied phenotypes observed among patients harboring these mutations.

Animals↗

PubMed2003

Two components of a secreted cell number-counting factor bind to cells and have opposing effects on cAMP signal transduction in Dictyostelium.

A secreted 450-kDa complex of proteins called counting factor (CF) is part of a negative feedback loop that regulates the size of the groups formed by developing Dictyostelium cells. Two components of CF are countin and CF50. Both recombinant countin and recombinant CF50 decrease group size in Dictyostelium. countin- cells have a decreased cAMP-stimulated cAMP pulse, whereas recombinant countin potentiates the cAMP pulse. We find that CF50 cells have an increased cAMP pulse, whereas recombinant CF50 decreases the cAMP pulse, suggesting that countin and CF50 have opposite effects on cAMP signal transduction. In addition, countin and CF50 have opposite effects on cAMP-stimulated Erk2 activation. However, like recombinant countin, recombinant CF50 increases cell motility. We previously found that cells bind recombinant countin with a Hill coefficient of approximately 2, a KH of 60 pm, and approximately 53 sites/cell. We find here that cells also bind 125I-recombinant CF50, with a Hill coefficient of approximately 2, a KH of approximately 15 ng/ml (490 pm), and approximately 56 sites/cell. Countin and CF50 require each other's presence to affect group size, but the presence of countin is not necessary for CF50 to bind to cells, and CF50 is not necessary for countin to bind to cells. Our working hypothesis is that a signal transduction pathway activated by countin binding to cells modulates a signal transduction pathway activated by CF50 binding to cells and vice versa and that these two pathways can be distinguished by their effects on cAMP signal transduction.

Animals↗

PubMed1991

Luteal enzymes of the luteinizing hormone and beta-adrenergic signal transduction pathways in hypophysectomized rabbits do not require pituitary hormone support.

Experiments were conducted to determine whether continuous pituitary hormone support is required for expression of the LH and beta-adrenergic cAMP signal transduction pathways in rabbit CL during pseudopregnancy. Parameters of the LH and catecholamine cAMP signal transduction pathways in CL of estrogen-treated hypophysectomized rabbits were compared to those of pituitary-intact rabbits. Results showed that each of the parameters of the LH and beta-adrenergic cAMP signal transduction pathways was retained in CL taken from estrogen-treated pseudopregnant rabbits that had been hypophysectomized for as long as 13 days at levels not significantly different from those of estrogen-treated pituitary-intact rabbits. These included luteal basal, and LH-, epinephrine-, and fluoride-stimulated adenylyl cyclase activities; total luteal cAMP levels; the number and affinity of cAMP-dependent protein kinase regulatory subunit cAMP binding sites; binding activity of the type I and type II regulatory subunits; and the amount of catalytic subunit protein of cAMP-dependent protein kinase. We conclude that expression of the proteins of the cAMP signal pathway for LH and beta-adrenergic hormones in CL of estrogen-treated rabbits does not require pituitary hormone support.

Animals↗

PubMed2003

SLAM-associated protein deficiency causes imbalanced early signal transduction and blocks downstream activation in T cells from X-linked lymphoproliferative disease patients.

Deficiency of SAP (SLAM (signaling lymphocyte activation molecule)-associated protein) protein is associated with a severe immunodeficiency, the X-linked lymphoproliferative disease (XLP) characterized by an inappropriate immune reaction against Epstein-Barr virus infection often resulting in a fatal clinical course. Several studies demonstrated altered NK and T cell function in XLP patients; however, the mechanisms underlying XLP disease are still largely unknown. Here, we show that non-transformed T cell lines obtained from XLP patients were defective in several activation events such as IL-2 production, CD25 expression, and homotypic cell aggregation when cells were stimulated via T cell antigen receptor (TCR).CD3 but not when early TCR-dependent events were bypassed by stimulation with phorbol 12-myristate 13-acetate/ionomycin. Analysis of proximal T cell signaling revealed imbalanced TCR.CD3-induced signaling in SAP-deficient T cells. Although phospholipase C gamma 1 phosphorylation and calcium response were both enhanced in T cells from XLP patients, phosphorylation of VAV and downstream signal transduction events such as mitogen-activated protein kinase phosphorylation and IL-2 production were diminished. Importantly, reconstitution of SAP expression by retroviral-mediated gene transfer completely restored abnormal signaling events in T cell lines derived from XLP patients. In conclusion, SAP mutation or deletion in XLP patients causes profound defects in T cell activation, resulting in immune deficiency. Moreover, these data provide evidence that SAP functions as an essential integrator in early TCR signal transduction.

CD3 Complex↗

PubMed1999

Response of Djun and Dfos mRNA abundance to signal transduction pathways in cultured cells of Drosophila melanogaster.

The mammalian proto-oncogenes c-jun and c-fos are situated at the end of multiple signal transduction pathways and activation of their products Jun and Fos, components of the transcription factor AP-1, are able to regulate gene transcription in response to extracellular stimuli. Djun and Dfos, the products of the Drosophila proto-oncongenes Djun and Dfos, are similar in size and sequence to their mammalian counterparts c-Jun and c-Fos and are related to their mammalian counterparts by their antigenic properties. However, very little is known about how they are regulated through signal transduction pathways. This paper has investigated the response of their mRNA abundance levels to three signal transduction pathways in Drosophila cultured cells. Various agonists and antagonists that stimulate and inhibit specific enzymes in the pathways have been tested. The results suggest that Djun and Dfos mRNA are continuously expressed and their abundance levels are transiently regulated by multiple signaling pathways, the peak response coming at 1-2 hours after perturbation. Dfos is more highly regulated than Djun which is only modulated. The receptor tyrosine kinase pathways positively regulate Dfos and Djun. The cAMP-mediated pathway positively regulates Dfos but negatively regulates Djun. The protein kinase C-activated pathway does not affect Djun whereas it negatively regulates Dfos.

Animals↗

PubMed1994

Protein tyrosine kinase activation and protein kinase C translocation are functional components of CD40 signal transduction in resting human B cells.

CD40 is a 47kD glycoprotein expressed on all B cells that plays an important role in B cell development and activation. Previous investigations have focused on signal transduction events in activated B cells and B cell lines, and little information is available regarding CD40 signal transduction in resting, normal B cells. Because CD40 plays a critical role in the regulation and activation of resting B cells, we studied the signaling mechanisms involved in functional responses to CD40 ligation in these cells. Treatment of dense tonsil B cells with either protein tyrosine kinase (PTK) or protein kinase C (PKC) inhibitors, but not with an inhibitor of cyclic nucleotide dependent kinases, resulted in abrogation of CD40-mediated cell adhesion, suggesting a role for both PTK and PKC in CD40-mediated B cell adhesion. Direct evidence for CD40-mediated PTK activation was demonstrated by increased tyrosine phosphorylation as detected by anti-phosphotyrosine Western blots of cell lysates prepared from dense resting tonsil B cells stimulated with biotinylated anti-CD40 monoclonal antibody plus avidin. CD40 engagement also resulted in PKC activation, as detected by translocation of cytosolic PKC activity to the membrane-bound compartment. CD40-mediated PKC translocation was dependent on PTK activation, whereas PTK activity induced by CD40 ligation was independent of PKC activity, suggesting that PTK activation precedes PKC activation in resting B cells stimulated with anti-CD40 mAb. The results of our experiments identify PTK and PKC activation as components of CD40 signal transduction in normal, resting B cells and establish a functional role for these events.

Antigens, CD↗

PubMed2003

Differential activation of signal transduction pathways mediating oxidative burst by chicken heterophils in response to stimulation with lipopolysaccharide and lipoteichoic acid.

Toll-like receptors (TLRs) have been previously shown to mediate oxidative burst in chicken heterophils. This study was conducted to begin to map the molecular pathways that regulate TLR-mediated oxidative burst. Peripheral blood heterophils from neonatal chicks were isolated and exposed to known inhibitors of signal transduction pathways for either 20 min (genistein, verapamil, or chelerythrine) or 120 min (pertussis toxin) at 39 degrees C. The cells were then stimulated for 30 min at 39 degrees C with Salmonella enteritidis lipopolysaccharide (LPS) or Staphylococcus aureus lipoteichoic acid (LTA). The heterophil oxidative burst was then quantitated by luminol-dependent chemiluminescence (LDCL). Genistein (a tyrosine kinase inhibitor), verapamil (a calcium channel blocker), chelerythrine (a protein kinase C inhibitor), and pertussis toxin (a G-protein inhibitor) significantly reduced LPS-stimulated oxidative burst in chicken heterophils by 34, 50, 63, and 51%, respectively. Although genistein had a statistically significant effect on reducing LPS-stimulated LDCL biologically it seems to play only a minor role within the oxidative burst pathway. Heterophils stimulated with the gram-positive TLR agonist, LTA, activated a different signal transduction pathway since chelerythrine was the only inhibitor that significantly reduced (72%) LTA-stimulated oxidative burst. These findings demonstrate that distinct signal transduction pathways differentially regulate the stimulation of oxidative burst in avian heterophils. Pertussis toxin-sensitive, protein kinase C-dependent, Ca(++)-dependent G proteins appear to regulate oxidative burst of avian heterophils stimulated with gram-negative agonist LPS; whereas, a protein kinase C-dependent signal transduction pathway plays the major role activating the oxidative burst of avian heterophils stimulated with gram-positive agonists. The distinct differences in the response of heterophils to these two agonists illustrate the specificity of TLRs to pathogen-associated molecular patterns (PAMP)s.

Animals↗

PubMed2004

Effects of acute hyperinsulinemia on insulin signal transduction and glucose transporters in ovine fetal skeletal muscle.

To test the effects of acute fetal hyperinsulinemia on the pattern and time course of insulin signaling in ovine fetal skeletal muscle, we measured selected signal transduction proteins in the mitogenic, protein synthetic, and metabolic pathways in the skeletal muscle of normally growing fetal sheep in utero. In experiment 1, 4-h hyperinsulinemic-euglycemic clamps were conducted in anesthetized twin fetuses to produce selective fetal hyperinsulinemia-euglycemia in one twin and euinsulinemia-euglycemia in the other. Serial skeletal muscle biopsies were taken from each fetus during the clamp and assayed by Western blot for selected insulin signal transduction proteins. Tyrosine phosphorylation of the insulin receptor, insulin receptor substrate-1, and the p85 subunit of phosphatidylinositol 3-kinase doubled at 30 min and gradually returned to control values by 240 min. Phosphorylation of extracellular signal-regulated kinase 1,2 was increased fivefold through 120 min of insulin infusion and decreased to control concentration by 240 min. Protein kinase B phosphorylation doubled at 30 min and remained elevated throughout the study. Phosphorylation of p70 S6K increased fourfold at 30, 60, and 120 min. In the second experiment, a separate group of nonanesthetized singleton fetuses was clamped to intermediate and high hyperinsulinemic-euglycemic conditions for 1 h. GLUT4 increased fourfold in the plasma membrane at 1 h, and hindlimb glucose uptake increased significantly at the higher insulin concentration. These data demonstrate that an acute increase in fetal plasma insulin concentration stimulates a unique pattern of insulin signal transduction proteins in intact skeletal muscle, thereby increasing pathways for mRNA translation, glucose transport, and cell growth.

Animals↗

PubMed2001

Sulfhydryl G proteins and phospholipase A(2)-associated G proteins are involved in adrenergic signal transduction in the rat pineal gland.

The rat pineal gland with its circadian noradrenaline-regulated melatonin rhythm is an excellent model for studying adrenergic signal transduction with respect to cAMP and cGMP formation. The stimulatory G(s) proteins play a well-established role in this process. In contrast, the potential roles of the inhibitory G(i) proteins, the functionally unclear other G(o) proteins, and a number of G protein subtypes are not known. The present study examines the effects on beta(1)- and beta(1)-plus-alpha(1)-stimulated cAMP and cGMP formation of a number of G protein modulators in rat pinealocyte suspension cultures. The effects of the nitric oxide donor sodium nitroprusside on cGMP were also examined. The results showed that drugs that activate G proteins of the G(i)/G(o) family, i.e., pertussis toxin, mastoparan, and compound 48/80, had no effect on unstimulated, isoproterenol (beta(1))-stimulated, or combined isoproterenol/phenylephrine (beta(1)-plus()-alpha(1))-stimulated cAMP and cGMP accumulation. However, in this experimental paradigm, the inhibitors of sulfhydryl G proteins (N-ethylmaleimide) and those of phospholipase A2-related G proteins (isotetrandrine) exerted a clear inhibitory effect. Sodium-nitroprusside-stimulated cGMP accumulation was also inhibited. These results confirm a previous report that members of the G(i)/G(o) family, which are present in the rat pineal gland, do not play a major role in adrenergic signal transduction. The new finding that sulfhydryl G proteins and phospholipase A2-associated G proteins exert a clear stimulatory effect on adrenergic signal transduction suggests that they are subtypes of G(s) proteins.

Adrenergic alpha-Agonists↗

PubMed1996

Elucidation of a MgATP signal transduction pathway in the nitrogenase iron protein: formation of a conformation resembling the MgATP-bound state by protein engineering.

The present work defines one MgATP signal transduction pathway in the nitrogenase iron (Fe) protein. Deletion of an amino acid (Leu 127) by site-directed mutagenesis in the protein chain between Asp 125, located in the ATP binding site, and Cys 132, a ligand to the [4Fe-4S] cluster, resulted in protein conformational changes resembling the MgATP-bound state in the absence of any bound nucleotides. Specifically, 1H nuclear magnetic resonance, electron paramagnetic resonance, and circular dichroism spectroscopic properties, along with Fe chelation assays, suggested that deletion of Leu 127 in the Fe protein resulted in changes in the electronic properties of the [4Fe-4S] cluster similar to those normally observed upon MgATP binding to the wild-type Fe protein. Deletion of Leu 127 of the Fe protein lowered the redox potential of of the [4FE-4S] cluster by 112 mV compared to the wild-typeFe protein (-412mV compared to -294 mV). A nearly identical lowering of the redox potential by 120 mV occurs in the wild-type Fe protein upon binding MgATP (-294 mV compared to 420 mV). The L127delta Fe protein did not contain bound nucleotides which could account for the observed conformational changes. The present results support a model in which the protein chain from ASP 125 to Cys 132 acts as one pathway for MgATP signal transduction and suggests a mechanism for this transduction to the [4Fe-4S] cluster. The L127delta Fe protein was found to still bind 2 MgATP or 2 MgADP molecules/Fe protein. Unlike the wild-type Fe protein, the L127delta Fe protein bound 2 ADP molecules/Fe protein in the absence of Mg2+. Finally, the L127delta protein was found to bind to the MoFe protein, although the complex did not catalyze MgATP hydrolysis or substrate reduction. In concurrence with previous models, homologies between the Asp 125 to Cys 132 transduction pathway in Fe protein and the switch II region of the broad class of GTPase signal transduction proteins (G-proteins) are discussed.

Adenosine Triphosphate↗

PubMed1995

Agonist-independent, muscle-type-specific signal transduction pathways in cat esophageal and lower esophageal sphincter circular smooth muscle.

Smooth muscle cells isolated from the circular muscle layer of cat esophagus and lower esophageal sphincter (LES) exhibit distinct contractile intracellular signal transduction pathways in response to acetylcholine. To determine whether these contractile pathways are muscle type dependent, the authors examined the signal transduction pathways utilized by substance P and bombesin, which in other tissues, use different signal transduction pathways, and by the GTP analog, guanosine 5'-O-3-thiotriphosphate (GTP gamma S), which activates all available G proteins. Western blot analysis of esophageal and LES circular muscle revealed the presence of Gq-G11 (42 kD), Gi1-Gi2 (40 kD) and Go-Gi3 (40 kD) types of G proteins. The responses of esophageal cells to bombesin and substance P were blocked by 1) a Gi3 protein antibody, 2) the inhibitor of specific phosphatidylcholine-phospholipase C (PLC) D609 potassium tricyclo-[5.2.1.0(2.6)]-decyl-(9[8])-xanthogenate, 3) inhibition of phosphatidic acid phosphohydrolase by propranolol, 4) the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and 5) incubation in Ca(++)-free medium. Conversely, the responses of LES muscle cells to bombesin and substance P were blocked by 1) a Gq-G11 antibody, 2) a phosphatidylinositol-specific PLC antagonist U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione), 3) the calmodulin inhibitor CGS9343B (1,3-Dihydro-1-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin++ +-4 - yl)methyl-4-piperindinyl]-2H-benzimidazol-2-one maleate) and 4) incubation in Sr++. After permeabilization by saponin, inositol 1,4,5-trisphosphate contracted LES but not esophageal cells. The inositol 1,4,5-trisphosphate receptor antagonist heparin and depletion of intracellular Ca++ stores by thapsigargin or A23187 4-Benzoxazolecarboxylic acid, 5-(methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol- 2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-, [6s-[6.alpha. (2S*,3S*),8.beta. (R*), 9.beta., 11. alpha.]]-(9Cl), blocked bombesin- and substance P-induced contraction of LES but not of esophageal muscle. In addition, contraction in response to GTP gamma S, which activates all G proteins, was blocked in esophageal cells by a Gi3-protein antibody, propranolol, D609 and H7. In LES muscle cells, the response to GTP gamma S was blocked by a Gq protein antibody, U-73122 and CGS934B. These data demonstrate that, in esophageal muscle, different agonists activate the same Gi3 protein, phosphatidylcholine-specific phospholipases and protein kinase C-dependent pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Type I collagen substrate increases calcitonin and parathyroid hormone receptor-mediated signal transduction in UMR 106-06 osteoblast-like cells.

Components of the extracellular matrices (ECM) exert pleiotropic effects in many cell systems, but little is known of the effect of ECM on hormone signal transduction. We have investigated the effect of ECM substrates on cell growth and signal transduction by calcitonin (CT) and parathyroid hormone (PTH) using the rat osteosarcoma cell line, UMR 106-06. Type I collagen (collagen[I]) and Matrigel changed the morphology of the cells and significantly inhibited cell growth by 37% or 23%, respectively, compared with control. None of laminin, fibronectin, or type IV collagen affected cell shape or proliferation. Cells cultured on collagen (I)-coated plates showed increased specific binding of labeled CT compared with cells on plastic plates. The effect was apparent by 24 h and persisted for at least 72 h. None of the other ECM affected CT binding. Scatchard analysis revealed that collagen(I) increased CT receptor numbers but not receptor affinity. Consistent with increased binding capacity, cells plated on collagen(I) had increased responses to each of CT and PTH in terms of cyclic adenosine monophosphate (cAMP) production compared to control cells. In addition, cAMP production by prostaglandin E2, cholera toxin, and forskolin was increased by 30-70% compared to control. These data suggest that collagen(I) had effects not only on membrane receptors but on guanosine triphosphate (GTP) binding proteins (G proteins). The effect of collagen(I) on CT binding was no longer present when the cells were freed from the plates by enzymatic dispersion and binding measured in cell suspensions. In UMR 106-01 cells transiently transfected with the porcine CT receptor cDNA, binding was similarly induced by collagen(I). These data are the first demonstration that collagen(I) may play an important role in signal transduction, affecting both receptors and G proteins in UMR 106-06 cells. These results draw attention to the potential role of the ECM of bone in hormone-induced responses.

Animals↗

PubMed1998

Kinetic analysis of a signal-transduction pathway by time-resolved somatic complementation of mutants.

Sensory control of sporulation in Physarum polycephalum plasmodia is mediated by a branched signal-transduction pathway that integrates blue light, far-red light, heat shock and the starvation state. Mutants defective in the pathway were isolated and three phenotypes obtained: blue-blind, general-blind and light-independent sporulating. When plasmodia of the blue-blind mutant Blu1 were exposed to a pulse of blue light and subsequently fused to non-induced wild-type plasmodia, the resulting heterokaryons sporulated, indicating a functional blue- light photoreceptor in the mutant. When the general-blind mutant Nos1 was fused to a wild-type plasmodium which had been induced by light, sporulation of the heterokaryon was blocked. However, the dominant inhibition of sporulation by Nos1 was gradually lost with increasing time between induction by light and time of fusion, suggesting that Nos1 can be bypassed by the time-dependent formation of a downstream signal-transduction intermediate. Phenotype expression in constitutively sporulating (Cos) mutants depended on starvation. The Cos2 product was titrated by fusing mutant plasmodia of different sizes to wild-type plasmodia of constant size and analysing the sporulation probability of the resulting heterokaryon. The titration curve indicates that a small change in the amount of Cos2 product can cause sporulation. We conclude that somatic complementation analysis allows the time-resolved evaluation of the regulatory function of mutations in a signal-transduction pathway without prior cloning of the gene. This shortcut allows us to characterize many mutants quickly and to select those for molecular analysis that display a well-defined regulatory function.

Animals↗

PubMed1995

BDNF-activated signal transduction in rat cortical glial cells.

Cortical glial cells in culture were found to be responsive to the neurotrophin brain-derived neurotrophic factor (BDNF), as evidenced by activation of multiple signal transduction processes. BDNF produced an increase in mitogen-activated protein (MAP) kinase tyrosine phosphorylation, MAP kinase activity, intracellular calcium concentration and c-fos expression in the glial cells. Only a subset of the glial cells responded to BDNF, as reflected in single-cell analysis of calcium transients and c-fos expression. BDNF had no detectable effect on glial mitotic activity, as measured by DNA synthesis. In parallel studies, nerve growth factor and neurotrophin-3 had no effect on signalling in these cultures. BDNF has previously been demonstrated to act via trkB receptors with a cytoplasmic tyrosine kinase domain (gp145trkB). Pretreatment of glial cultures with K252a, which at low concentrations specifically inhibits the trk tyrosine kinases, abolished BDNF effects on MAP kinase stimulation, suggesting that BDNF was acting through gp145trkB. However, subsequent studies showed that gp145trkB was expressed at extremely low levels in the cultures: gp145trkB mRNA transcripts could only be detected using the reverse transcription-polymerase chain reaction, and gp145trkB protein was not detected by either immunoblotting or immunocytochemistry. On the other hand, the glia expressed significantly higher levels of gp95trkB mRNA and protein, which represent truncated forms of trkB receptors lacking the tyrosine kinase domain. The results of these studies demonstrate that a subset of cultured CNS glia respond to BDNF with the activation of conventional signal transduction processes. The mechanism of BDNF-initiated signal transduction in glial cells most likely involves a relatively small number of gp145trkB receptors, but involvement of the more abundant truncated gp95trkB receptors cannot be excluded.

Animals↗

PubMed1999

Human cytomegalovirus inhibits IFN-alpha-stimulated antiviral and immunoregulatory responses by blocking multiple levels of IFN-alpha signal transduction.

The type I IFNs represent a primordial, tightly regulated defense system against acute viral infection. IFN-alpha confers resistance to viral infection by activating a conserved signal transduction pathway that up-regulates direct antiviral effectors and induces immunomodulatory activities. Given the critical role of IFN-alpha in anti-human cytomegalovirus (HCMV) immunity and the profound ability of HCMV to escape the host immune response, we hypothesized that HCMV blocks IFN-alpha-stimulated responses by disrupting multiple levels of the IFN-alpha signal transduction pathway. We demonstrate that HCMV inhibits IFN-alpha-stimulated MHC class I, IFN regulatory factor-1, MxA and 2',5-oligoadenylate synthetase gene expression, transcription factor activation, and signaling in infected fibroblasts and endothelial cells by decreasing the expression of Janus kinase 1 and p48, two essential components of the IFN-alpha signal transduction pathway. This investigation is the first to report inhibition of type I IFN signaling by a herpesvirus. We propose that this novel immune escape mechanism is a major means by which HCMV is capable of escaping host immunity and establishing persistence.

Antiviral Agents↗

PubMed2004

beta3-Adrenergic-dependent and -independent mechanisms participate in cold-induced modulation of insulin signal transduction in brown adipose tissue of rats.

During cold exposure, homeothermic animals mobilize glucose with higher efficiency than at thermoneutrality. An interaction between the insulin signal transduction machinery and high sympathetic tonus is thought to play an important role in this phenomenon. In the present study, rats were exposed to cold during 8 days and treated, or not, with a beta3-adrenergic agonist, BRL37344 sodium 4-2-2-(3-chlorophenyl)-2-hydroxyethyl amino propyl phenoxy-acetic acid sodium (BRL37344), or antagonist, SR59230A 3-(2-ethylphenoxy)-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate (SR59230A), to evaluate the cross-talk between insulin and beta3-adrenergic intracellular signaling in brown adipose tissue. The drugs did not modify food ingestion, body temperature, and body weight in control and cold-exposed rats. Treatment of control rats with BRL37344 led to higher insulin-induced tyrosine phosphorylation of the insulin receptors, insulin receptor substrate (IRS)-1 and ERK, higher insulin-induced IRS-1/PI3-kinase association, and higher [Ser(473)] phosphorylation of Akt. Cold exposure alone promoted higher insulin-induced tyrosine phosphorylation of the insulin receptors, IRS-1, IRS-2, and ERK, and higher insulin-induced IRS-1 and IRS-2/PI3-kinase association. Except for the regulation of ERK, SR59230A abolished all the cold-induced effects upon the insulin signal transduction pathway. However, this antagonist only partially inhibited the cold-induced increase of glucose uptake. Thus, the sympathetic tonus generated during cold-exposure acts, in brown adipose tissue, through the beta3-adrenergic receptor and modulates insulin signal transduction, with the exception of ERK. However, insulin-independent mechanisms other than beta3-adrenergic activation participate in cold-induced glucose uptake in brown adipose tissue of rats.

Adaptation, Physiological↗

PubMed2005

Intracellular signal transduction pathway proteins as targets for cancer therapy.

Circulating cytokines, hormones, and growth factors control all aspects of cell proliferation, differentiation, angiogenesis, apoptosis, and senescence. These chemical signals are propagated from the cell surface to intracellular processes via sequential kinase signaling, arranged in modules that exhibit redundancy and cross talk. This signal transduction system comprising growth factors, transmembrane receptor proteins, and cytoplasmic secondary messengers is often exploited to optimize tumor growth and metastasis in malignancies. Thus, it represents an attractive target for cancer therapy. This review will summarize current knowledge of selected intracellular signaling networks and their role in cancer therapy. The focus will be on pathways for which inhibitory agents are currently undergoing clinical testing. Original data for inclusion in this review were identified through a MEDLINE search of the literature. All papers from 1966 through March 2005 were identified by the following search terms: "signal transduction," "intracellular signaling," "kinases," "proliferation," "growth factors," and "cancer therapy." All original research and review papers related to the role of intracellular signaling in oncogenesis and therapeutic interventions relating to abnormal cell signaling were identified. This search was supplemented by a manual search of the Proceedings of the Annual Meetings of the American Association for Cancer Research, American Society of Clinical Oncology, and the American Association for Cancer Research (AARC)--European Organisation for Research and Treatment of Cancer (EORTC)--National Cancer Institute (NCI) Symposium on New Anticancer Drugs.

Antineoplastic Agents↗