Cytoplasmic RNA modulators of an inside-out signal-transduction cascade

A vaccinia virus-based RNA expression system enabled high-level cytoplasmic expression of RNA aptamers directed against the intracellular domain of the β2 integrin LFA-1, a transmembrane protein that mediates cell adhesion to intercellular adhesion molecule-1 (ICAM-1). In two different cell types, cytoplasmic expression of integrin-binding aptamers reduced inducible cell adhesion to ICAM-1. The aptamers specifically target, and thereby define, a functional cytoplasmic subdomain important for the regulation of cell adhesion in leukocytes. Our approach of aptamer-controlled blocking of signaling pathways in vivo could potentially be applied wherever targeted modulation of a signal-transduction cascade is desired.

The TOR (target of rapamycin) signal transduction pathway regulates the stability of translation initiation factor eIF4G in the yeast Saccharomyces cerevisiae

Initiation factor eIF4G is an essential protein required for initiation of mRNA translation via the 5′ cap-dependent pathway. It interacts with eIF4E (the mRNA 5′ cap-binding protein) and serves as an anchor for the assembly of further initiation factors. With treatment of Saccharomyces cerevisiae with rapamycin or with entry of cells into the diauxic phase, eIF4G is rapidly degraded, whereas initiation factors eIF4E and eIF4A remain stable. We propose that nutritional deprivation or interruption of the TOR signal transduction pathway induces eIF4G degradation.

ErbB2 expression increases the spectrum and potency of ligand-mediated signal transduction through ErbB4

Interleukin 3-dependent murine 32D cells do not detectably express members of the ErbB receptor family and do not proliferate in response to known ligands for these receptors. 32D transfectants were generated expressing human ErbB4 alone (32D.E4) or with ErbB2 (32D.E2/E4). Epidermal growth factor (EGF), neuregulin 1-β (NRG1-β), betacellulin (BTC), transforming growth factor-α (TGF-α), heparin binding-EGF (HB-EGF), and amphiregulin were analyzed for their ability to mediate mitogenesis in these transfectants. 32D.E4 responded mitogenically to NRG1-β and BTC. Surprisingly, EGF also induced significant DNA synthesis and TGF-α was negligibly mitogenic on 32D.E4 cells, whereas HB-EGF and amphiregulin were inactive. Although coexpression of ErbB2 with ErbB4 in 32D.E2/E4 cells did not significantly alter DNA synthesis in response to NRG1-β or BTC, it greatly enhanced mitogenesis elicited by EGF and TGF-α and unmasked the ability of HB-EGF to induce proliferation. EGF-related ligands that exhibited potent mitogenic activity on 32D.E2/E4 cells at low concentrations induced adherence, morphological alterations, and up-regulation of the Mac-1 integrin and FcγRII/III at higher concentrations. While 125I-EGF could be specifically crosslinked to both 32D.E4 and 32D.E2/E4 cells, its crosslinking capacity was greatly enhanced in the cotransfected cells. The ability of the various ligands to mediate proliferation and/or adhesion in the two transfectants correlated with their capacity to induce substrate tyrosine phosphorylation and to initiate and sustain activation of mitogen-activated protein kinase. We conclude that the ability of ErbB4 to mediate signal transduction through EGF-like ligands is broader than previously assumed and can be profoundly altered by the concomitant expression of ErbB2.

Interleukin-2 induces β2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB)

β2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of β2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4+ T cell lines obtained from healthy donors and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in β2 integrin (CD18)-positive but not in β2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation of the 125-kDa protein but not other proteins in β2-integrin-positive T cells. Likewise, a β2 integrin (CD18) antibody selectively inhibits induction of the 125-kDa phosphotyrosine protein, whereas cytokine-mediated tyrosine phosphorylation of other proteins is largely unaffected. Immunoprecipitation experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in β2-integrin-positive but not in β2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fakB-tyrosine phosphorylation in β2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125FAK. In conclusion, our data indicate that IL-2 induces β2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB.

Platelet signal transduction defect with Gα subunit dysfunction and diminished Gαq in a patient with abnormal platelet responses

G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5′-[γ-thio]triphosphate (GTP[γS]) was diminished in the patient’s platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of α-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[γS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by Gαs) and its inhibition (mediated by Gαi) by thrombin in the patient’s platelet membranes were normal. Immunoblot analysis of Gα subunits in the patient’s platelet membranes showed a decrease in Gαq (<50%) but not Gαi, Gαz, Gα12, and Gα13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein α-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Gαq in thrombin-induced responses.

Interferon β transduction of peripheral blood lymphocytes from HIV-infected donors increases Th1-type cytokine production and improves the proliferative response to recall antigens

We are developing a gene therapy method of HIV infection based on the constitutive low production of interferon (IFN) β. Peripheral blood lymphocytes (PBL) from HIV-infected patients at different clinical stages of infection were efficiently transduced with the HMB-HbHuIFNβ retroviral vector. The constitutive low production of IFN-β in cultured PBL from HIV-infected patients resulted in a decreased viral production and an enhanced survival of CD4+ cells, and this protective effect was observed only in the PBL derived from donors having a CD4+ cell count above 200 per mm3. In IFN-β-transduced PBL from healthy and from HIV-infected donors, the production of the Th1-type cytokines IFN-γ and interleukin (IL)-12 was enhanced. In IFN-β-transduced PBL from HIV-infected donors, the production of IL-4, IL-6, IL-10, and tumor necrosis factor α was maintained at normal levels, contrary to the increased levels produced by the untransduced PBL. The proliferative response to recall antigens was partially restored in IFN-β-transduced PBL from donors with an impaired antigen response. Thus, in addition to inhibiting HIV replication, IFN-β transduction of PBL from HIV-infected donors improves several parameters of immune function.

The selectivity of the hair cell’s mechanoelectrical-transduction channel promotes Ca2+ flux at low Ca2+ concentrations

The mechanoelectrical-transduction channel of the hair cell is permeable to both monovalent and divalent cations. Because Ca2+ entering through the transduction channel serves as a feedback signal in the adaptation process that sets the channel’s open probability, an understanding of adaptation requires estimation of the magnitude of Ca2+ influx. To determine the Ca2+ current through the transduction channel, we measured extracellular receptor currents with transepithelial voltage-clamp recordings while the apical surface of a saccular macula was bathed with solutions containing various concentrations of K+, Na+, or Ca2+. For modest concentrations of a single permeant cation, Ca2+ carried much more receptor current than did either K+ or Na+. For higher cation concentrations, however, the flux of Na+ or K+ through the transduction channel exceeded that of Ca2+. For mixtures of Ca2+ and monovalent cations, the receptor current displayed an anomalous mole-fraction effect, which indicates that ions interact while traversing the channel’s pore. These results demonstrate not only that the hair cell’s transduction channel is selective for Ca2+ over monovalent cations but also that Ca2+ carries substantial current even at low Ca2+ concentrations. At physiological cation concentrations, Ca2+ flux through transduction channels can change the local Ca2+ concentration in stereocilia in a range relevant for the control of adaptation.

Molecular switch in signal transduction: Reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the γ-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD–Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of α2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of α2 and facilitates the unwinding of a helical turn of α2 (residues 66–69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.

Reduction in levels of the cyclin-dependent kinase inhibitor p27kip-1 coupled with transforming growth factor β neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells

Successful gene therapy depends on stable transduction of hematopoietic stem cells. Target cells must cycle to allow integration of Moloney-based retroviral vectors, yet hematopoietic stem cells are quiescent. Cells can be held in quiescence by intracellular cyclin-dependent kinase inhibitors. The cyclin-dependent kinase inhibitor p15INK4B blocks association of cyclin-dependent kinase (CDK)4/cyclin D and p27kip-1 blocks activity of CDK2/cyclin A and CDK2/cyclin E, complexes that are mandatory for cell-cycle progression. Antibody neutralization of β transforming growth factor (TGFβ) in serum-free medium decreased levels of p15INK4B and increased colony formation and retroviral-mediated transduction of primary human CD34+ cells. Although TGFβ neutralization increased colony formation from more primitive, noncycling hematopoietic progenitors, no increase in M-phase-dependent, retroviral-mediated transduction was observed. Transduction of the primitive cells was augmented by culture in the presence of antisense oligonucleotides to p27kip-1 coupled with TGFβ-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGFβ, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations.

Mechanical stretch induces vascular permeability factor in human mesangial cells: Mechanisms of signal transduction

Hemodynamic abnormalities have been implicated in the pathogenesis of the increased glomerular permeability to protein of diabetic and other glomerulopathies. Vascular permeability factor (VPF) is one of the most powerful promoters of vascular permeability. We studied the effect of stretch on VPF production by human mesangial cells and the intracellular signaling pathways involved. The application of mechanical stretch (elongation 10%) for 6 h induced a 2.4-fold increase over control in the VPF mRNA level (P < 0.05). There was a corresponding 3-fold increase in VPF protein level by 12 h (P < 0.001), returning to the baseline by 24 h. Stretch-induced VPF secretion was partially prevented both by the protein kinase C (PKC) inhibitor H7 (50 μM: 72% inhibition, P < 0.05) and by pretreatment with phorbol ester (phorbol-12-myristate-13 acetate 10−7 M: 77% inhibition, P < 0.05). A variety of protein tyrosine kinase (PTK) inhibitors, genistein (20 μg/ml), herbimycin A (3.4 μM), and a specific pp60src peptide inhibitor (21 μM) also significantly reduced, but did not entirely prevent, stretch-induced VPF protein secretion (respectively 63%, 80%, and 75% inhibition; P < 0.05 for all). The combination of both PKC and PTK inhibition completely abolished the VPF response to mechanical stretch (100% inhibition, P < 0.05). Stretch induces VPF gene expression and protein secretion in human mesangial cells via PKC- and PTK-dependent mechanisms.

Patch-clamp and amperometric recordings from norepinephrine transporters: Channel activity and voltage-dependent uptake

Transporters for the biogenic amines dopamine, norepinephrine, epinephrine and serotonin are largely responsible for transmitter inactivation after release. They also serve as high-affinity targets for a number of clinically relevant psychoactive agents, including antidepressants, cocaine, and amphetamines. Despite their prominent role in neurotransmitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single transporter level. The resolution of radiotracer-based flux techniques limits the opportunities to dissect these problems. Here we combine patch-clamp recording techniques with microamperometry to record the transporter-mediated flux of norepinephrine across isolated membrane patches. These data reveal voltage-dependent norepinephrine flux that correlates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked with bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmitter across the membrane in discrete shots containing hundreds of molecules. Amperometry is used widely to study neurotransmitter distribution and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present application is the use of amperometry on inside-out patches with synchronous recording of flux and current. Thus, our results further demonstrate a powerful method to assess transporter function and regulation.

Induction of host signal transduction pathways by Helicobacter pylori

Adherence of Helicobacter pylori to cultured gastric epithelial cells is associated with several cellular events, including the tyrosine phosphorylation of a 145-kDa host protein; the reorganization of the host cell actin and associated cellular proteins, like vasodilator-stimulated phosphoprotein, adjacent to the attached bacterial cell; and the subsequent release of the cytokine, interleukin 8 (IL-8). H. pylori isolated from patients with ulcer disease and gastric cancer contain a DNA insertion, the cag pathogenicity island (PAI), that is not present in bacteria isolated from individuals with asymptomatic infection. Mutations in a number of PAI genes abolish tyrosine phosphorylation and IL-8 synthesis but not the cytoskeletal rearrangements. Kinase inhibition studies suggest there are two distinct pathways operative in stimulating IL-8 release from host cells and one of these H. pylori pathways is independent of the tyrosine phosphorylation step.

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans

The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid “Spider” media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7, encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.

The protein kinases of Caenorhabditis elegans: A model for signal transduction in multicellular organisms

Caenorhabditis elegans should soon be the first multicellular organism whose complete genomic sequence has been determined. This achievement provides a unique opportunity for a comprehensive assessment of the signal transduction molecules required for the existence of a multicellular animal. Although the worm C. elegans may not much resemble humans, the molecules that regulate signal transduction in these two organisms prove to be quite similar. We focus here on the content and diversity of protein kinases present in worms, together with an assessment of other classes of proteins that regulate protein phosphorylation. By systematic analysis of the 19,099 predicted C. elegans proteins, and thorough analysis of the finished and unfinished genomic sequences, we have identified 411 full length protein kinases and 21 partial kinase fragments. We also describe 82 additional proteins that are predicted to be structurally similar to conventional protein kinases even though they share minimal primary sequence identity. Finally, the richness of phosphorylation-dependent signaling pathways in worms is further supported with the identification of 185 protein phosphatases and 128 phosphoprotein-binding domains (SH2, PTB, STYX, SBF, 14-3-3, FHA, and WW) in the worm genome.

Prolonged activation of the N-methyl-d-aspartate receptor–Ca2+ transduction pathway causes spontaneous recurrent epileptiform discharges in hippocampal neurons in culture

The molecular basis for developing symptomatic epilepsy (epileptogenesis) remains ill defined. We show here in a well characterized hippocampal culture model of epilepsy that the induction of epileptogenesis is Ca2+-dependent. The concentration of intracellular free Ca2+ ([Ca2+]i) was monitored during the induction of epileptogenesis by prolonged electrographic seizure activity induced through low-Mg2+ treatment by confocal laser-scanning fluorescent microscopy to directly correlate changes in [Ca2+]i with alterations in membrane excitability measured by intracellular recording using whole-cell current–clamp techniques. The induction of long-lasting spontaneous recurrent epileptiform discharges, but not the Mg2+-induced spike discharges, was prevented in low-Ca2+ solutions and was dependent on activation of the N-methyl-d-aspartate (NMDA) receptor. The results provide direct evidence that prolonged activation of the NMDA–Ca2+ transduction pathway causes a long-lasting plasticity change in hippocampal neurons causing increased excitability leading to the occurrence of spontaneous, recurrent epileptiform discharges.