Independent Signal Transduction Pathways Research Articles

Mechanisms Implicated in the Response of System A to Hypertonic Stress and Amino Acid Deprivation Still Can Be Different

Alfieri et al. (2004) analyzed the response of system A to hypertonic stress by determining changes in SNAT2 mRNA levels in a panel of cell lines, including the same cell type, CHO-K1, that we routinely used in the past for this kind of studies. It is evident that a discrepancy between their results and ours regarding the effect of hypertonicity on SAT2/SNAT2 mRNA levels exists. Our CHO-K1 clone was originally obtained from E. Englesberg (University of California, Santa Barbara, CA), who developed a panel of somatic mutant cell lines with altered system A activity and regulatory properties (Moffett and Englesberg, 1984, 1986; Englesberg and Moffett, 1986; Moffett et al., 1987; Qian et al., 1991; among others). In our experiments, the SAT2/SNAT2 cDNA probe used for Northern blot analysis was a fragment cloned from that particular cell line (this is the hamster SNAT2 orthologue, accession no. of the partial sequence {type:entrez-nucleotide,attrs:{text:AF363584,term_id:19880221}}AF363584). Although we do not believe these slight differences might explain this discrepancy, the study we published in the Journal of General Physiology (Lopez-Fontanals et al., 2003) does not particularly rely upon this observation to conclude that the osmoregulatory and the amino acid–regulated responses of system A are mediated by different signal transduction pathways. In that study, we combined inhibitors of the MAP kinase pathway, as well as negative dominant cells for selected kinases in this transduction pathway, and modulators of the cell cycle machinery, to demonstrate that, at least in CHO-K1 cells, both stimuli trigger independent responses. All these experiments were performed by looking at system A functional activity, which is presumably associated with SNAT2 expression in CHO-K1 cells. This is in agreement with our previous work (Ruiz-Montasell et al., 1994) in which we used a particular somatic cell CHO-K1 mutant (CHO-K1 alar4), generated at Ellis Englesberg's laboratory (Moffett and Englesberg, 1984, 1986), that lacked the ability to respond to amino acid starvation but, interestingly, still retained the hyperosmotic response. Although we agree that the existence of the system A activating protein is still an open question, as Alfieri et al. discuss, the conclusion that both pathways must converge at some point cannot be drawn from the mere observation that the two stimuli induce an increase in SAT2/SNAT2 mRNA levels. We respectfully believe that this is a simple interpretation of these data. Most genes can be transcriptionally activated by different agents/stimuli without bearing any common step in their transduction pathways, except that they converge, obviously, at the end somewhere on the gene promoter. Nevertheless, as the authors point out, even for the response of system A to amino acid starvation, traditionally assumed to be exclusively associated with gene transcription even before system A cloning, it has been now reported that protein recruitment from intracellular stores is also responsible for this response (Ling et al., 2001). Thus, even if SAT2/SNAT2 mRNA accumulates after hypertonicity in all cell lines they have tested, it does not rule out other mechanisms, which would explain for instance why cells lacking the amino acid–regulated response still show an increase in system A activity after hypertonic shock. In fact, the authors cite, as a demonstration of the hypertonic sensitivity of the system A gene, a recent paper by Trama et al. (2002), showing that SAT2/SNAT2 gene shows dependence on NFAT5, also known as TonEBP, a transcription factor implicated in osmotic responses. However Alfieri et al. do not comment on the fact that these authors themselves conclude that “system A amino acid transporter gene ATA2 exhibited NFAT5 dependence under hypertonic conditions but not in response to amino acid deprivation.” This would argue against common pathways mediating both stimuli. We do not believe that the Northern blot shown by Alfieri et al. provides unequivocal basis for a rebuttal of the key message of our contribution, which is that both stimuli trigger system A up-regulation, measured at the functional level (system A transport activity) by independent signal transduction pathways.

CREB Overexpression In Vivo Results in Increased Proliferation, Blast Transformation, and Earlier Engraftment of Myeloid Progenitor Cells.

CREB or cAMP responsive element binding protein is a 43-kDa-basic/leucine zipper (bZip) transcription factor that regulates gene expression through the activation of cAMP-dependent or -independent signal transduction pathways. CREB promotes growth and survival in a variety of cell types and is overexpressed in the bone marrow of greater than 60% of AML patients. To understand the role of CREB in myelopoiesis, we characterized the effects of CREB overexpression in transgenic mice. We created mice in which CREB expression was targeted to the myeloid lineage using the hMRP8 promoter. CREB transgenic mice showed evidence of monocytosis, compared to age-matched littermate controls. We performed colony assays with methylcellulose containing SCF, IL-6, and IL-3. Bone marrow cells from CREB transgenic mice formed robust colonies earlier and had increased numbers of colony forming units (CFU-GM) when compared to control mice. Cytospin analysis of these cells showed the presence of more immature myeloid cells compared to controls. At day 12, cells from colonies were 50% c-Kit positive, 83% Gr-1 positive, and 67% Mac-1 positive by FACS analysis. To assess self-renewal of progenitors from CREB transgenic mice, serial replating experiments were performed. Bone marrow cells from transgenic mice were highly successful in repopulating the methylcellulose containing SCF, IL-6, and IL-3, in contrast to the control cells, which were unable to grow after serial replating. Following tertiary replating of the CREB transgenic mouse bone marrow, we observed that the colonies (96+3.5) appeared more homogeneous with immature cells that were >99% c-Kit positive and <1% GR-1, Mac-1 positive. These results suggest that persistent expression of CREB leads to a blast-like phenotype in the absence of differentiation. To determine whether increased CREB expression confers growth factor-independence, we cultured bone marrow cells in methylcellulose that did not contain cytokines. We observed a 10-fold increase in the numbers of cells from CREB transgenic mice (two different founder lines) compared to normal bone marrow. When cultured in methylcellulose containing M-CSF, the bone marrow cells from CREB transgenic mice formed larger and significantly greater numbers of colonies. However, these cells did not grow in the presence of G-CSF or EPO alone. To determine if the myeloproliferative (monocytosis) phenotype was transplantable into wild type recipient mice we injected 4x106 bone marrow cells from CREB transgenic mice into wild type C57/BL6 recipient mice. Serial analysis of the peripheral blood counts and cell surface markers by FACS analysis showed earlier myeloid engraftment at 6 weeks following transplantation compared to normal control mice. The transgenic recipients showed increased monocytes and neutrophils in the peripheral blood with a corresponding increase in Mac-1 positive, Gr-1 positive cell populations at 8 weeks after transplantation. At the same time, the lymphocyte count was significantly lower in CREB transgenic recipient mice than controls. Our results suggest that CREB plays a critical role in the regulation of normal hematopoiesis and stem cell self-renewal.

The ARE-dependent mRNA-destabilizing activity of BRF1 is regulated by protein kinase B

Butyrate response factor (BRF1) belongs to the Tis11 family of CCCH zinc-finger proteins, which bind to mRNAs containing an AU-rich element (ARE) in their 3' untranslated region and promote their deadenylation and rapid degradation. Independent signal transduction pathways have been reported to stabilize ARE-containing transcripts by a process thought to involve phosphorylation of ARE-binding proteins. Here we report that protein kinase B (PKB/Akt) stabilizes ARE transcripts by phosphorylating BRF1 at serine 92 (S92). Recombinant BRF1 promoted in vitro decay of ARE-containing mRNA (ARE-mRNA), yet phosphorylation by PKB impaired this activity. S92 phosphorylation of BRF1 did not impair ARE binding, but induced complex formation with the scaffold protein 14-3-3. In vivo and in vitro data support a model where PKB causes ARE-mRNA stabilization by inactivating BRF1 through binding to 14-3-3.

Transcription repression in oncogenic transformation: common targets of epigenetic repression in cells transformed by Fos, Ras or Dnmt1.

Fos and Ras function in both dependent and independent signal transduction pathways, and sustained activity of either oncogene is sufficient to induce cell transformation and tumorigenesis. Increased DNA (cytosine-5) methyltransferse (Dnmt1) activity is involved in the mechanism of transformation by both oncogenes, suggesting that inappropriate epigenetic transcription regulation may be a common route of oncogenesis, and that cell transformation may model aspects of the epigenetic deregulation that often occurs in tumors. Here, we have taken a microarray-based gene expression approach to identify differentially expressed genes in cells transformed by c-fos, v-fos, ras or Dnmt1. The cohort of genes differentially expressed in all four transformation systems includes an over-representation of repressed genes, many of which have been functionally implicated in the suppression of transformation or tumorigenesis. Furthermore, we identified four potential tumor suppressor genes subject to epigenetic transcriptional repression in transformed cells. The results emphasize the role of transcription repression in oncogenesis, and they provide insights into the potential common epigenetic mechanisms impacting cell transformation.

Serotonin potentiation of glycine-activated whole-cell currents in the superficial laminae neurons of the rat spinal dorsal horn is mediated by protein kinase C

The modulatory effects of serotonin (5-HT) on glycine (Gly)-activated whole-cell currents were investigated in neurons acutely dissociated from the superficial laminae (I and II) of the rat spinal dorsal horn using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Our results demonstrate that (1) Gly acted on strychnine (STR)-sensitive Gly receptors and elicited inward Cl − currents ( I Gly) at a holding potential of −40 mV; (2) 5-HT potentiated I Gly without affecting the reversal potential of I Gly; (3) the agonist ( α-methyl-5-HT) and antagonist (ketanserine) of 5-HT 2 receptor mimicked and blocked the potentiating effect of 5-HT on I Gly, respectively; (4) bisindolylmaleimide I (BIM), a selective inhibitor of protein kinase C (PKC), reduced the potentiating effect of 5-HT on I Gly; and (5) 5-HT-induced enhancement of I Gly was not affected by pretreatment with 1,2-bis-(2-aminophenoxy) ethane- N, N, N′, N′-tetraacetic acid tetrakis (acetoxy-methyl) ester (BAPTA AM), a Ca 2+ chelator. These results indicate that (1) the potentiation of 5-HT on I Gly is mediated by 5-HT 2 receptor and through Ca 2+-independent PKC intracellular signal transduction pathway; and (2) the interactions between 5-HT and Gly might modulate the transmission of nociceptive information through the spinal cord.

Regulation of alternative oxidase gene expression in soybean.

Soybean (Glycine max cv. Stevens) suspension cells were used to investigate the expression of the alternative oxidase (Aox) multigene family. Suspension cells displayed very high rates of cyanide-insensitive respiration, but Aox3 was the only isoform detected in untreated cells. Incubation with antimycin A, citrate, salicylic acid or at low temperature (10 degrees C) specifically induced the accumulation of the Aox1 isoform. Aox2 was not observed under any conditions in the cells. Increases in Aox1 protein correlated with increases in Aox1 mRNA. Treatment of soybean cotyledons with norflurazon also induced expression of Aox1. Reactive oxygen species (ROS) were detected upon incubation of cells with antimycin, salicylic acid or at low temperature, but not during incubation with citrate. Aox1 induction by citrate, but not by antimycin, was prevented by including the protein kinase inhibitor staurosporine in the medium. The results suggest that multiple pathways exist in soybean to regulate expression of Aox genes and that Aox1 specifically is induced by a variety of stress and metabolic conditions via at least two independent signal transduction pathways.

Endothelial NOS-dependent activation of c-Jun NH(2)- terminal kinase by oxidized low-density lipoprotein.

Oxidized low-density lipoprotein (oxLDL) is known to activate a number of signal transduction pathways in endothelial cells. Among these are the c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase, and extracellular signal-regulated kinase (ERK). These mitogen-activated protein kinases (MAP kinase) determine cell survival in response to environmental stress. Interestingly, JNK signaling involves redox-sensitive mechanisms and is activated by reactive oxygen and nitrogen species derived from both NADPH oxidases, nitric oxide synthases (NOS), peroxides, and oxidized low-density lipoprotein (oxLDL). The role of endothelial NOS (eNOS) in the activation of JNK in response to oxLDL has not been examined. Herein, we show that on exposure of endothelial cells to oxLDL, both ERK and JNK are activated through independent signal transduction pathways. A key role of eNOS activation through a phosphatidylinositol-3-kinase-dependent mechanism leading to phosphorylation of eNOS is demonstrated for oxLDL-dependent activation of JNK. Moreover, we show that activation of ERK by oxLDL is critical in protection against the cytotoxicity of oxLDL.

ELF3 encodes a circadian clock-regulated nuclear protein that functions in an Arabidopsis PHYB signal transduction pathway.

Many aspects of plant development are regulated by photoreceptor function and the circadian clock. Loss-of-function mutations in the Arabidopsis EARLY FLOWERING 3 (ELF3) and PHYTOCHROME B (PHYB) genes cause early flowering and influence the activity of circadian clock-regulated processes. We demonstrate here that the relative abundance of the ELF3 protein, which is a novel nucleus-localized protein, displays circadian regulation that follows the pattern of circadian accumulation of ELF3 transcript. Furthermore, the ELF3 protein interacts with PHYB in the yeast two-hybrid assay and in vitro. Genetic analyses show that ELF3 requires PHYB function in early morphogenesis but not for the regulation of flowering time. This suggests that ELF3 is a component of a PHYB signaling complex that controls early events in plant development but that ELF3 and PHYB control flowering via independent signal transduction pathways.

Wound signalling in plants.

Plants undergoing the onslaught of wound-causing agents activate mechanisms directed to healing and further defence. Responses to mechanical damage are either local or systemic or both and hence involve the generation, translocation, perception, and transduction of wound signals to activate the expression of wound-inducible genes. Although the central role for jasmonic acid in plant responses to wounding is well established, other compounds, including the oligopeptide systemin, oligosaccharides, and other phytohormones such as abscisic acid and ethylene, as well as physical factors such as hydraulic pressure or electrical pulses, have also been proposed to play a role in wound signalling. Different jasmonic acid-dependent and -independent wound signal transduction pathways have been identified recently and partially characterized. Components of these signalling pathways are mostly similar to those implicated in other signalling cascades in eukaryotes, and include reversible protein phosphorylation steps, calcium/calmodulin-regulated events, and production of active oxygen species. Indeed, some of these components involved in transducing wound signals also function in signalling other plant defence responses, suggesting that cross-talk events may regulate temporal and spatial activation of different defences.

Targeting G Proteins with Membrane-Permeable Peptides

Many receptors can activate multiple independent signal transduction pathways. Chang et al. have developed a method for creating membrane-permeable peptides to specifically disrupt individual signal transduction pathways to elucidate the role of each pathway in the generating the cellular responses. The peptides were directed to block the interactions of the G protein with the specific target effectors. Three systems were used to demonstrate the principle: (i) the selective disruption of signaling through G q to phospholipase-Cβ by the serotonin receptor 5-HT 2c , (ii) the selective disruption of signaling through G βγ to mitogen-activated protein kinase by α 2A adrenergic receptors, and (iii) the selective disruption of signaling through G q to phospholipase-Cβ by the thrombin receptor. Additionally, their methods allow for modular synthesis of the membrane-permeable peptides, providing versatility, increased yield, and lower costs compared with other methods of peptide synthesis. Chang, M., Zhang, L., Tam, J.P., and Sanders-Bush, E. (2000) Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides. J. Biol. Chem. 275 : 7021-7029. [Abstract] [Full Text]

The Trimeric GTP-binding Protein (Gq/G11) α Subunit Is Required for Insulin-stimulated GLUT4 Translocation in 3T3L1 Adipocytes

To investigate the potential role of trimeric GTP-binding proteins regulating GLUT4 translocation in adipocytes, wild type and constitutively active G(q) (G(q)/Q209L), G(i) (G(i)/Q205L), and G(s) (G(s)/Q227L) alpha subunit mutants were expressed in 3T3L1 adipocytes. Although expression of neither the wild type nor G(i)/Q205L and G(s)/Q227L alpha subunit mutants had any effect on the basal or insulin-stimulated translocation of a co-expressed GLUT4-enhanced green fluorescent protein (EGFP) fusion protein, expression of G(q)/Q209L resulted in GLUT4-EGFP translocation in the absence of insulin. In contrast, microinjection of an inhibitory G(q)/G(11) alpha subunit-specific antibody but not a G(i) or G(s) alpha subunit antibody prevented insulin-stimulated endogenous GLUT4 translocation. Consistent with a required role for GTP-bound G(q)/G(11), expression of the regulators of G protein signaling (RGS4 and RGS16) also attenuated insulin-stimulated GLUT4-EGFP translocation. To assess the relationship between G(q)/G(11) function with the phosphatidylinositol 3-kinase dependent pathway, expression of a dominant-interfering p85 regulatory subunit, as well as wortmannin treatment inhibited insulin-stimulated but not G(q)/Q209L-stimulated GLUT4-EGFP translocation. Furthermore, G(q)/Q209L did not induce the in vivo accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), whereas expression of the RGS proteins did not prevent the insulin-stimulated accumulation of PIP(3). Together, these data demonstrate that insulin stimulation of GLUT4 translocation requires at least two independent signal transduction pathways, one mediated through the phosphatidylinositol 3-kinase and another through the trimeric GTP-binding proteins G(q) and/or G(11).

Nuclear and cytosolic events of light-induced, phytochrome-regulated signaling in higher plants.

Plants as sessile organisms cannot walk away but have to adapt to changes in their natural environments. Light is obviously the most important external factor, being the energy source. To regulate their fitness, the photosynthetic capacity of plants must be adapted to changes in the ambient light environment. Therefore, plants have evolved a large set of photoreceptors to monitor light quality and quantity, ranging from the UV to the infrared part of the spectrum. At least three different photoreceptor classes have been identified and analyzed: (i) UV‐B receptors, primarily characterized by action spectroscopy (Wellmann, 1983); (ii) the blue UV‐A photoreceptors, cry1 and cry2 and phototropine (Ahmad and Cashmore, 1993; Christie et al ., 1998; Lin et al ., 1998); and (iii) the red/far‐red reversible phytochromes. These photoreceptors control different aspects of plant growth and development, and in this review we focus on the physiological and molecular events regulated by the most characterized plant photoreceptors, the phytochromes. In higher plants, phytochromes are encoded by a small multigene family; five genes ( PHYA–PHYE , Sharrock and Quail, 1989; Clack et al ., 1994) have been identified in Arabidopsis . Phytochromes exist as dimers composed of two 125 kDa polypeptides, each carrying a covalently linked tetrapyrrol chromophore in the N‐terminal domain and dimerization domains in the C‐terminal domain. The photosensory function of the molecule is based on its capacity for reversible interconversion between the red light‐absorbing Pr form and the far‐red light absorbing Pfr form following sequential absorption of red and far‐red light. Photosignal perception by the receptor is followed by conformational changes, and activates, through an as yet poorly understood mechanism, signaling pathways leading to changes in the expression of genes that underlie developmental responses to light (Quail et al ., 1995). In order to finely tune their …

Alpha(2)-adrenoceptor and NPY receptor-mediated contractions of porcine isolated blood vessels: evidence for involvement of the vascular endothelium.

1. Enhanced contractions to the alpha(2)-adrenoceptor agonist UK14304 and neuropeptide Y (NPY) in the porcine ear artery can be uncovered by pharmacological manipulation. The aim of this study was to determine whether similar pharmacological manipulation can uncover enhanced contractions in the porcine splenic artery, and to determine whether the endothelium modulates these responses. 2. UK14304 (0.3 microM) and NPY (0.1 microM) produced small contractions of the porcine splenic artery. After pre-contraction of the tissue with U46619, followed by relaxation with forskolin, the responses to both UK14304 and NPY were enhanced. Enhanced contractions to both UK14304 and NPY were also obtained after relaxation with SNP. These results demonstrate that, as in the porcine ear artery, alpha(2)-adrenoceptors and NPY receptors are able to produce enhanced contractile responses through both adenylyl cyclase-dependent and -independent signal transduction pathways. 3. Removal of the endothelium had no significant effect on responses to UK14304 either alone or in the presence of U46619 and forskolin in the porcine splenic artery. On the other hand, responses to UK14304 after relaxation with SNP were reduced after endothelium-denudation in both the porcine splenic artery and ear artery. Similar results were obtained with NPY in the porcine ear artery. 4. In conclusion, enhanced contractile responses to UK14304 and NPY in the porcine splenic artery can be uncovered using methods similar to those employed in the porcine ear artery. Under certain conditions the responses to both agents are modulated by the endothelium. These data highlight further the similarities in the signal transduction pathways used by both alpha(2)-adrenoceptors and NPY receptors to induce vasoconstriction.

Salmon gonadotropin IIβ subunit promoter contains multiple DNA elements responsible for stimulation by gonadotropin-releasing hormone through protein kinase C-dependent and -independent pathways

Gonadotropin-releasing hormone (GnRH) stimulates gonadotropin (GTH) production by activating GTH subunit gene transcription. In salmonid fish, the expression of the β subunit gene of GTH II (sGTH IIβ) is stimulated by GnRH at the final stages of reproduction. DNA elements required for the GnRH stimulation were examined by analyzing sGTH IIβ promoter activity by transfection studies in a gonadotrope-derived cell line, αT3-1. A GnRH analog (GnRHa) specifically stimulated the sGTH IIβ promoter (3358 bp) expression 3.6-fold, while phorbol myristate acid (PMA) stimulated it 6.2–9-fold. Analysis of a series of 5′-deletion mutants has revealed that a proximal region (−258 to −199) was important in GnRHa stimulation through protein kinase C (PKC)-independent signal transduction pathways, because an internal deletion mutant (Δ(246−217)/3358) showed a significant decrease in the level of GnRHa stimulation, but showed no change in stimulation by PMA. A large upstream region (−3358 to −1260) showed an enhancing activity of the GnRHa stimulation, and a far upstream 530 bp segment in this region (−3358 to −2829) may be responsible for this activity. The present results suggest that sGTH IIβ gene may be controlled by GnRH through multiple DNA elements including those responsive to PKC-dependent and -independent signal transduction pathways.

Independent Signaling Pathways Regulate Cellular Turgor during Hyperosmotic Stress and Appressorium-Mediated Plant Infection by Magnaporthe grisea

The phytopathogenic fungus Magnaporthe grisea elaborates a specialized infection cell called an appressorium with which it mechanically ruptures the plant cuticle. To generate mechanical force, appressoria produce enormous hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control of cellular turgor, we analyzed the response of M. grisea to hyperosmotic stress. During acute and chronic hyperosmotic stress adaptation, M. grisea accumulates arabitol as its major compatible solute in addition to smaller quantities of glycerol. A mitogen-activated protein kinase-encoding gene OSM1 was isolated from M. grisea and shown to encode a functional homolog of HIGH-OSMOLARITY GLYCEROL1 (HOG1), which encodes a mitogen-activated protein kinase that regulates cellular turgor in yeast. A null mutation of OSM1 was generated in M. grisea by targeted gene replacement, and the resulting mutants were sensitive to osmotic stress and showed morphological defects when grown under hyperosmotic conditions. M. grisea deltaosm1 mutants showed a dramatically reduced ability to accumulate arabitol in the mycelium. Surprisingly, glycerol accumulation and turgor generation in appressoria were unaltered by the Deltaosm1 null mutation, and the mutants were fully pathogenic. This result indicates that independent signal transduction pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection. Consistent with this, exposure of M. grisea appressoria to external hyperosmotic stress induced OSM1-dependent production of arabitol.

NO and cardiac diastolic function.

Time for primary review 34 days. Since the first report of a role for nitric oxide (NO) in modulating myocardial contractile function in 1991 [1], an enormous number of studies have been published in this field (recently reviewed in Refs. [2–4]). It is now recognised that, under physiological conditions, the major sources of NO that are important for contractile regulation are (1) endothelial-type nitric oxide synthase (eNOS) expressed in cardiac endothelial cells, and (2) eNOS expressed in cardiac myocytes themselves. In a variety of pathological situations, contractile function may also be influenced by inducible NOS (iNOS) expressed in several cell types, including infiltrating inflammatory cells, coronary microvascular and endocardial endothelial cells, coronary vascular smooth muscle, and cardiac myocytes. In contrast to the relatively well-defined, essentially species-independent primary action of NO in the vasculature (i.e., mediation of endothelium-dependent vasodilatation), in the heart multiple and sometimes contradictory actions of NO have been reported (Table 1). Some of these actions may be species-specific while others have not always been reproduced by independent laboratories despite the use of apparently similar experimental preparations and protocols. Apart from species and methodological differences, additional relevant factors that are likely to influence the response to NO include: the cellular source of NO, the amount released (or experimentally studied), prevailing redox balance and antioxidant status, the target tissue (e.g., atrial or ventricular tissue), interactions with neurohumoral and other stimuli, the presence of immune activation or disease, and the activation of distinct cGMP-dependent and -independent subcellular signal transduction pathways. View this table: Table 1 Reported actions of NO on the heart. Due to constraints of space, only a limited number of original references have been cited. Many other original references may be found in the bibliography of Refs. [2–4] Most studies that have investigated the effects of NO on contractile function have focused on … * Corresponding author. Tel.: +32-53-724-433

Two distinct interleukin-3-mediated signal pathways, Ras-NFIL3 (E4BP4) and Bcl-xL, regulate the survival of murine pro-B lymphocytes.

Hematopoietic cells require cytokine-initiated signals for survival as well as proliferation. The pathways that transduce these signals, ensuring timely regulation of cell fate genes, remain largely undefined. The NFIL3 (E4BP4) transcription factor, Bcl-xL, and constitutively active mutants of components in Ras signal transduction pathways have been identified as key regulation proteins affecting murine interleukin-3 (IL-3)-dependent cell survival. Here we show that expression of NFIL3 is regulated by oncogenic Ras mutants through both the Raf-mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. NFIL3 inhibits apoptosis without affecting Bcl-xL expression. By contrast, Bcl-xL levels are regulated through the membrane proximal portion in the cytoplasmic domain of the receptor (betac chain), which is shared by IL-3 and granulocyte-macrophage colony-stimulating factor. Activation of either pathway alone is insufficient to ensure cell survival, indicating that multiple independent signal transduction pathways mediate the survival of developing B-lymphoid cells.

Independent signal-transduction pathways for vanadate and for insulin in the activation of glycogen synthase and glycogenesis in rat adipocytes.

The activating effect of vanadate on glycogenesis and on glycogen synthase (uridine diphosphate-glucose-glycogen glucosyl transferase) activity was studied in rat adipocytes and compared with that of insulin. Using several approaches and specific blockers, we found that vanadate and insulin resemble each other, in the activation of glycogen synthase, in several aspects: both require nonarrested protein phosphatase 1 activity; they are equally suppressed by conditions that elevate cAMP-levels; and both depend on the activation of phosphatidylinositol-3 kinase. The basic differences between them are as follows: 1) vanadate promotes glycogenesis through the activation of a cytosolic protein tyrosine kinase, in an insulin-receptor-independent manner; 2) vanadate elevates glucose-6-phosphate (G-6-P) to a higher level than insulin; 3) vanadate-activated glycogenesis is accompanied by an increase in the cellular content of immunoreactive glycogen synthase, an effect less noticeable with insulin; 4) adipose glucose-6-phosphatase is inhibited by vanadate (dose for 50% inhibition, IC50 = 7 +/- 0.7 microM) but not by insulin. We have concluded that insulin and vanadate activate glycogenesis through a phosphatidylinositol-3 kinase and dephosphorylation-dependent mechanism. Vanadate, however, uses a receptor-independent pathway and is superior to insulin in elevating the level of G-6-P, a key metabolite for activating glycogen synthase. This is attributed to the combined effect of vanadate in enhancing glucose entry and in inhibiting dephosphorylation of endogenously formed G-6-P. The latter effect is not exerted by insulin.

Independent Signal-Transduction Pathways for Vanadate and for Insulin in the Activation of Glycogen Synthase and Glycogenesis in Rat Adipocytes

Independent Signal-Transduction Pathways for Vanadate and for Insulin in the Activation of Glycogen Synthase and Glycogenesis in Rat Adipocytes

Two Distinct Signaling Pathways Mediate DIF Induction of Prestalk Gene Expression inDictyostelium

DuringDictyosteliumdevelopment, the differentiation inducing factor (DIF) triggers expression of the prestalk geneecmBand induces stalk cell differentiation, a form of programmed cell death. The effects of DIF are mediated by a sustained increase in cytosolic Ca2+levels. The Ca2+ATPase inhibitor BHQ causes a similar rise in Ca2+levels and also induces prestalk gene expression. We show here that Ca2+is a specific intermediate for prestalk gene induction, since BHQ represses transcription of the cAMP-inducible aggregative genePDE,the postaggregative geneCP2,and the prespore geneD19.The prestalk geneecmAis also induced by DIF, but induction appears to occur in two steps, which occur within 1 h and after 2 h, respectively. The slow step shows the same kinetics asecmBinduction and similar toecmBinduction, this step is BHQ inducible and requires an initial round of protein synthesis. The fast step does not require protein synthesis and cannot be induced by BHQ. This indicates that in addition to the slow Ca2+-mediated pathway, there is probably a second fast Ca2+-independent signal transduction pathway for DIF.