Growth Factor-stimulated Cells Research Articles

Nuclear eNOS interacts with and S-nitrosates ADAR1 to modulate endothelial gene expression

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Collaborative Research Centre (CRC 1366); Chinese Scholarship Council (CSC) Introduction and Aim Nitric oxide (NO) generated by the endothelial NO synthase (eNOS) regulates vascular tone and endothelial homeostasis to counteract vascular inflammation. The effects of NO are attributable to its interaction with heme-containing proteins e.g., soluble guanylyl cyclase, or to the S-nitrosation of proteins. Most eNOS is localized to the cell membrane or the Golgi apparatus, however, it has also been detected in the nucleus. In this study, we assessed the role of nuclear eNOS in endothelial cells. Methods and Results Confocal microscopy studies confirmed the presence of eNOS in the nucleus of unstimulated human and murine endothelial cells (ex vivo and in vitro) and stimulation with vascular endothelial growth factor (VEGF) increased eNOS nuclear translocation. Co-immunoprecipitation studies coupled with proteomics revealed the VEGF-dependent association of nuclear eNOS with 81 proteins involved in RNA binding and processing. Among these, nuclear eNOS bound double-stranded RNA-specific adenosine deaminase 1 (ADAR1), an enzyme involved in RNA editing via the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). ADAR1 was S-nitrosated in human endothelial cells and the knockdown of eNOS was associated with an increase in dsRNA (immunofluorescence) and altered ADAR1-mediated A-I editing (RNA seq). The accumulation of dsRNA in human endothelial cells lacking eNOS led to the activation of the interferon α/β signalling pathway (e.g., MX2, OASL, OAS1/2/3, RNASEL) and the downregulation of cell cycle-related genes. As a result, growth factor-stimulated cell proliferation was abrogated and basal as well as tumour necrosis factor- or H2O2-induced cell death were increased. Furthermore, the increased dsRNA in endothelial cells lacking eNOS elicited the activation of interferon-induced, dsRNA-activated protein kinase, which in turn phosphorylated the eukaryotic translation initiation factor 2A. Accordingly, new protein synthesis was inhibited following eNOS knockdown (immunofluorescence and mass spectrometry). Conclusion These results demonstrated that eNOS/NO signalling regulates endothelial type I interferon signalling and affects translation, cell cycle and cell death through S-nitrosation of ADAR1.

Glutathione and Glutaredoxin in Redox Regulation and Cell Signaling of the Lens.

The ocular lens has a very high content of the antioxidant glutathione (GSH) and the enzymes that can recycle its oxidized form, glutathione disulfide (GSSG), for further use. It can be synthesized in the lens and, in part, transported from the neighboring anterior aqueous humor and posterior vitreous body. GSH is known to protect the thiols of the structural lens crystallin proteins from oxidation by reactive oxygen species (ROS) so the lens can maintain its transparency for proper visual function. Age-related lens opacity or senile cataract is the major visual impairment in the general population, and its cause is closely associated with aging and a constant exposure to environmental oxidative stress, such as ultraviolet light and the metabolic end product, H2O2. The mechanism for senile cataractogenesis has been hypothesized as the results of oxidation-induced protein-thiol mixed disulfide formation, such as protein-S-S-glutathione and protein-S-S-cysteine mixed disulfides, which if not reduced in time, can change the protein conformation to allow cascading modifications of various kinds leading to protein–protein aggregation and insolubilization. The consequence of such changes in lens structural proteins is lens opacity. Besides GSH, the lens has several antioxidation defense enzymes that can repair oxidation damage. One of the specific redox regulating enzymes that has been recently identified is thioltransferase (glutaredoxin 1), which works in concert with GSH, to reduce the oxidative stress as well as to regulate thiol/disulfide redox balance by preventing protein-thiol mixed disulfide accumulation in the lens. This oxidation-resistant and inducible enzyme has multiple physiological functions. In addition to protecting structural proteins and metabolic enzymes, it is able to regulate the redox signaling of the cells during growth factor-stimulated cell proliferation and other cellular functions. This review article focuses on describing the redox regulating functions of GSH and the thioltransferase enzyme in the ocular lens.

Enrichment of Tyrosine Phosphorylated Peptides for Quantitative Mass Spectrometry Analysis of RTK Signaling Dynamics.

Cells sense and respond to mitogens by activating a cascade of signaling events, primarily mediated by tyrosine phosphorylation (pY). Because of its key roles in cellular homeostasis, deregulation of this signaling is often linked to oncogenesis. To understand the mechanisms underlying these signaling pathway aberrations, it is necessary to quantify tyrosine phosphorylation on a global scale in cancer cell models. However, the majority of the protein phosphorylation events occur on serine (86%) and threonine (12%) residues, whereas only 2% of phosphorylation events occur on tyrosine residues ( Olsen et al., 2006 ). The low stoichiometry of tyrosine phosphorylation renders it difficult to quantify cellular pY events comprehensively with high mass accuracy and reproducibility. Here, we describe a detailed protocol for isolating and quantifying tyrosine phosphorylated peptides from drug-perturbed, growth factor-stimulated cancer cells, using immunoaffinity purification and tandem mass tags (TMT) coupled with mass spectrometry.

Preparation and Characterization of Liposomal Everolimus by Thin-Film Hydration Technique

In 10% to 40% of the cases of coronary stent implantation, patients face in-stent restenosis due to an inflammatory response, which induces artery thickening. Everolimus, a drug that inhibits growth factor-stimulated cell proliferation of endothelial cells, represents a promising alternative to prevent in-stent restenosis. In this study, everolimus was encapsulated by a film hydration technique in liposomes by using phosphatidylcholine and cholesterol at different ratios. As the ratio of cholesterol increases, it modulates the rigidity of the structure which can affect the encapsulation efficiency of the drug due to steric hindrance. Moreover, various lipid : drug ratios were tested, and it was found that as the lipid : drug ratio increases, the encapsulation efficiency also increases. This behavior is observed because everolimus is a hydrophobic drug; therefore, if the lipidic region increases, more drug can be entrapped into the liposomes. In addition, stability of the encapsulated drug was tested for 4 weeks at 4°C. Our results demonstrate that it is possible to prepare liposomal everolimus by film hydration technique followed by extrusion with high entrapment efficiency as a viable drug delivery system.

SAT-133 Breast Tumor Kinase (Brk/PTK6) Mediates Triple Negative Breast Cancer Cell Migration and Taxol Resistance via SH2 Domain-Dependent Activation of RhoA and AhR

Triple negative breast cancer (TNBC) patients have higher recurrence rates and a worse prognosis relative to patients diagnosed with other breast cancer subtypes. Protein tyrosine kinase 6 (PTK6; also called Brk), a soluble tyrosine kinase, is overexpressed in 86% of breast cancer patients, however its precise function in the context of TNBC is poorly defined. PTK6 expression is elevated in TNBC models in response to both cellular and endocrine stress, coordinated transcriptionally by the Hypoxia-Inducible Factors (HIFs) and glucocorticoid receptor (GR). We showed previously that PTK6 expression, but not its intrinsic kinase activity, is required for breast cancer cell motility. To further delineate the mechanisms of PTK6 signaling, we created kinase-intact domain structure mutants of PTK6 via in frame deletions of the N-terminal SH3 or SH2 domains. MDA-MB-231 cells expressing a PTK6 variant lacking the SH2 domain (SH2-del PTK6) were less responsive to growth factor-stimulated cell motility relative to wild type or kinase dead (KM) controls. To identify signal transduction pathways activated in TNBC cells harboring PTK6 domain mutants, we used a reverse phase protein array (RPPA), which revealed that the SH2 domain of PTK6 mediates TNBC cell motility via activation of the RhoA and/or AhR signaling pathways. Moreover, in TNBC cells, including a taxane-refractory TNBC model, addition of AhR or Rho inhibitors to paclitaxel (Taxol) enhanced cytotoxicity. Together, these studies reveal that the SH2-domain of PTK6 is an effector of advanced cancer phenotypes in GR+ TNBC cells and identify RhoA and AhR as novel therapeutic targets in PTK6+ tumors.

SUN-007 Discovery of Novel Signaling Pathways Downstream of PTK6 in TNBC Models

Triple negative breast cancer (TNBC) patients have a worse prognosis relative to other breast cancer subtypes. The glucocorticoid receptor (GR) is a ubiquitous steroid receptor that mediates homeostatic responses to life-induced stressors via glucocorticoids synthesized in the adrenal cortex. High GR expression predicts poor outcome in TNBC. The molecular mechanisms for how stress and GR contribute to TNBC progression are largely unknown. We previously described overexpression of protein tyrosine kinase 6 (PTK6) in response to both cellular and endocrine stress, coordinated by GR/HIF complexes in TNBC models. PTK6-induced signaling further amplified phosphorylation of GR on Ser134. PTK6 knock-down impaired TNBC motility, and blocked outgrowth of cancer stem-like cells (CSC) as measured by tumorsphere assays. Furthermore, PTK6 expression was integral for cancer cell survival upon chemotherapy treatment (Taxol, 5-FU). Interestingly, kinase activity of PTK6 was not required for TNBC cell motility or CSC outgrowth. This finding is of critical importance for drug design, given that previous efforts have primarily focused on targeting the PTK6 kinase domain. To probe the mechanisms of PTK6 signaling, we have created kinase-intact domain structure mutants of PTK6 via in frame deletions of the N-terminal SH3 or SH2 domain. MDA-MB-231 cells expressing PTK6 which lacks an SH2 domain (SH2-del PTK6) exhibited increased formation of tumorspheres relative to wt or kinase-dead (KM) full-length PTK6. Surprisingly, SH2-del PTK6+ cells were strikingly less responsive to both serum- and growth factor-stimulated cell motility relative to wt or KM controls. To track signal transduction pathways activated in TNBC cells harboring PTK6 domain mutants (wt, KM, SH2-del PTK6, PTK6 null), we used Reverse Phase Protein Array (RPPA). Our data suggests that the SH2 domain of PTK6 mediates TNBC motility via activation of the Rho and/or AhR signaling pathways, while CSC outgrowth primarily occurs via activation of the p38 and/or canonical Wnt signaling pathways. Together, these studies nominate PTK6 as an effector of advanced cancer phenotypes in GR+ TNBC cells and identify novel therapeutic targets (Rho, AhR) for treatment of TNBC tumors. Additionally, our work defines PTK6 domain-specific functions and reveals new opportunities for pharmacologically targeting the PTK6 SH2-domain in TNBC.

Endogenous Selenoprotein P, a Liver-Derived Secretory Protein, Mediates Myocardial Ischemia/Reperfusion Injury in Mice.

Selenoprotein P (SeP), a liver-derived secretory protein, functions as a selenium supply protein in the body. SeP has been reported to be associated with insulin resistance in humans through serial analysis of gene expression. Recently, SeP has been found to inhibit vascular endothelial growth factor-stimulated cell proliferation in human umbilical vein endothelial cells, and impair angiogenesis in a mouse hind limb model. In this study, the role of SeP in ischemia/reperfusion (I/R) injury has been investigated. SeP knockout (KO) and littermate wild-type (WT) mice were subjected to 30 min of myocardial ischemia followed by 24 h of reperfusion. The myocardial infarct area/area at risk (IA/AAR), evaluated using Evans blue (EB) and 2,3,5-triphenyltetrazolium chloride (TTC) staining, was significantly smaller in SeP KO mice than in WT mice. The number of terminal de-oxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei was significantly lower in SeP KO mice than in WT mice. In addition, caspase-3 activation was reduced in SeP KO mice compared to that in WT mice. Furthermore, phosphoinositide 3-kinase/Akt and Erk levels were examined for the reperfusion injury salvage kinase (RISK) pathway. Interestingly, SeP KO significantly increased the phosphorylation of IGF-1, Akt, and Erk compared to that in WT mice after I/R. Finally, I/R-induced myocardial IA/AAR was significantly increased in SeP KO mice overexpressing SeP in the liver compared to other SeP KO mice. These results, together, suggest that inhibition of SeP protects the heart from I/R injury through upregulation of the RISK pathway.

Interaction of p190A RhoGAP with eIF3A and Other Translation Preinitiation Factors Suggests a Role in Protein Biosynthesis

The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr308, but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined.

Kinetic Modeling and Analysis of the Akt/Mechanistic Target of Rapamycin Complex 1 (mTORC1) Signaling Axis Reveals Cooperative, Feedforward Regulation

Mechanistic target of rapamycin complex 1 (mTORC1) controls biosynthesis and has been implicated in uncontrolled cell growth in cancer. Although many details of mTORC1 regulation are well understood, a systems-level, predictive framework synthesizing those details is currently lacking. We constructed various mathematical models of mTORC1 activation mediated by Akt and aligned the model outputs to kinetic data acquired for growth factor-stimulated cells. A model based on a putative feedforward loop orchestrated by Akt consistently predicted how the pathway was altered by depletion of key regulatory proteins. Analysis of the successful model also elucidates two dynamical motifs: neutralization of a negative regulator, which characterizes how Akt indirectly activates mTORC1, and seesaw enzyme regulation, which describes how activated and inhibited states of mTORC1 are controlled in concert to produce a nonlinear, ultrasensitive response. Such insights lend quantitative understanding of signaling networks and their precise manipulation in various contexts.

Real-Time Single Molecule Visualization of SH2 Domain Membrane Recruitment in Growth Factor Stimulated Cells.

In the last decade, single molecule tracking (SMT) techniques have emerged as a versatile tool for molecular cell biology research. This approach allows researchers to monitor the real-time behavior of individual molecules in living cells with nanometer and millisecond resolution. As a result, it is possible to visualize biological processes as they occur at a molecular level in real time. Here we describe a method for the real-time visualization of SH2 domain membrane recruitment from the cytoplasm to epidermal growth factor (EGF) induced phosphotyrosine sites on the EGF receptor. Further, we describe methods that utilize SMT data to define SH2 domain membrane dynamics parameters such as binding (τ), dissociation (k d), and diffusion (D) rates. Together these methods may allow us to gain greater understanding of signal transduction dynamics and the molecular basis of disease-related aberrant pathways.

AKT-dependent phosphorylation of the SAM domain induces oligomerization and activation of the scaffold protein CNK1

Scaffold proteins are hubs for the coordination of intracellular signaling networks. The scaffold protein CNK1 promotes several signal transduction pathway. Here we demonstrate that sterile motif alpha (SAM) domain-dependent oligomerization of CNK1 stimulates CNK1-mediated signaling in growth factor-stimulated cells. We identified Ser22 located within the SAM domain as AKT-dependent phosphorylation site triggering CNK1 oligomerization. Oligomeric CNK1 increased the affinity for active AKT indicating a positive AKT feedback mechanism. A CNK1 mutant lacking the SAM domain and the phosphorylation-defective mutant CNK1S22A antagonizes oligomerization and prevents CNK1-driven cell proliferation and matrix metalloproteinase 14 promoter activation. The phosphomimetic mutant CNK1S22D constitutively oligomerizes and stimulates CNK1 downstream signaling. Searching the COSMIC database revealed Ser22 as putative target for oncogenic activation of CNK1. Like the phosphomimetic mutant CNK1S22D, the oncogenic mutant CNK1S22F forms clusters in serum-starved cells comparable to clusters of CNK1 in growth factor-stimulated cells. CNK1 clusters induced by activating Ser22 mutants correlate with enhanced cell invasion and binding to and activation of ADP ribosylation factor 1 associated with tumor formation. Mutational analysis indicate that EGF-triggered phosphorylation of Thr8 within the SAM domain prevents AKT binding and antagonizes CNK1-mediated AKT signaling. Our findings reveal SAM domain-dependent oligomerization by AKT as switch for CNK1 activation.

Testing a cell signaling circuit

Signal Transduction It remains a challenge to understand how a limited number of signaling mechanisms encode a broad range of cellular responses. Ryu et al. explored signaling by the mitogen-activated protein kinase pathway activated by various growth factors in single PC-12 rat pheochromocytoma cells. As Bluthgen explains in a commentary, the authors tested the circuit much as an electrical engineer might test an electrical circuit. They compared effects of pulses or sustained doses of growth factor. Sustained growth factor stimulus caused a heterogeneous response of single cells—some transiently and some sustaining pathway activation. Mathematical modeling and evaluation of pulsed growth factor stimulation in cells showed that different frequencies of stimulation enabled the same growth factor to cause different cell fate decisions. Mol. Syst. Biol. 10.15252/msb.20156458 (2015). Mol. Syst. Biol. 10.15252/msb.20156642 (2015).

Posttranslational Modifications of Steroid Receptors: Phosphorylation.

The detection of phosphorylation status of proteins has become a critical component of the analysis of activity, localization, and turnover studies of most proteins, particularly for those involved in signaling. The androgen receptor is no exception to this rule with its localization, transcriptional activity, and interactions determined by a series of key phosphorylations on serine residues. Here we have presented a series of techniques for the investigation of the phosphorylation status and intracellular localization of the androgen receptor after hormone and growth factor stimulation of cells in culture (in vitro) and in prostate cancer tissue (in vivo). Modified methods for immunohistochemistry, immunoblotting and immunofluorescence detection with high efficacy for the measurement and monitoring of androgen receptor are presented here alongside examples of their use.

Sulfated glycosaminoglycan derivatives affect the bioactivity of angiogenic growth factors

Event Abstract Back to Event Sulfated glycosaminoglycan derivatives affect the bioactivity of angiogenic growth factors Linda Koehler1, Sandra Rother1, Stephanie Moeller2, Matthias Schnabelrauch2, Vera Hintze1 and Dieter Scharnweber1 1 TU Dresden, Max Bergmann Center of Biomaterials, Germany 2 INNOVENT e.V., Biomaterials Department, Germany Introduction: Sulfated glycosaminoglycans (GAGs) are promising candidates for functional bio­ma­terials since their sulfate groups modulate the binding of growth factors, thereby influencing their biological activity profile. The interaction of different GAGs with several growth factors has previously been reported. These stu­dies demonstrated a sulfation- and carbohydrate backbone-dependent binding of GAGs resulting in an influence on the interaction of these mediators with recep­tors and therefore their bioactivity[1],[2]. Angiogenic growth factors, like vascular endothelial (VEGF) and basic fibroblast growth factor (bFGF) play an important role during wound healing[3],[4]. Heparin is known to distinctly regulate VEGF and bFGF signaling by altering their interaction with the respective receptors[4],[5]. Hence we hypothesize that VEGF and bFGF interaction with different GAGs influences their bioactivity depending on their degree of sul­fa­tion (D.S.) and carbohydrate backbone. This might lead to important implications for the use of GAGs as part of biomaterials for controlling angiogenesis during wound healing in health com­pro­mised patients. Materials and Methods: The interaction of VEGF and bFGF with different sulfated hyaluronan (sHA) and chondroitin sulfate (CS) derivatives was determined via surface plasmon resonance (SPR). VEGF or bFGF were im­mobilized onto a sensor chip surface and dif­fe­rent concentrations of solute GAGs were injected to reveal their binding strength. Addi­tionally, ELISA experiments with immobilized GAGs were performed to validate the SPR results. The influence of GAGs on the capability of growth factors to bind to their receptors was again investigated by SPR, while the consequences of the GAG/growth factor interaction on bioactivity were studied in cell-based bioassays. Here pre-incubated GAG/growth factor complexes were added to the cells and growth factor-stimulated cell proliferation was de­termined. Results and Discussion: In case of bFGF the binding strength of sHA and CS derivatives reveals a strong dependence on the D.S. but not on the carbohydrate backbone of the GAG. For VEGF, however, low-sHA and high-sulfated CS showed a comparable bin­ding strength. These results imply that the different molecular geometries in the carbohydrate backbone of the GAGs might render the respec­tive sulfate groups to interact differently. This is further emphasized in investigations with TGF-β1 whe­re naturally sulfated heparin demonstrated a comparable binding strength to high sulfated CS, even though it has a lower D.S[2]. The binding of growth factors to their respective recep­tors was modulated in the presence of GAGs which implies an altered biological profile. This could be verified in cell-based bioassays, since the presence of GAGs affected the growth factor-stimulated proliferation of the cells. Conclusion: The results imply that sulfated GAGs are promising candidates to be included in biomaterial coatings for controlling angiogenesis and therefore wound hea­ling processes, which would in particular be beneficial for health com­pro­mised patients. We acknowledge financial support by the DFG [SFB Transregio 67, projects A2, A3 and Z3].

Directed random walks and constraint programming reveal active pathways in hepatocyte growth factor signaling.

An effective means to analyze mRNA expression data is to take advantage of established knowledge from pathway databases, using methods such as pathway-enrichment analyses. However, pathway databases are not case-specific and expression data could be used to infer gene-regulation patterns in the context of specific pathways. In addition, canonical pathways may not always describe the signaling mechanisms properly, because interactions can frequently occur between genes in different pathways. Relatively few methods have been proposed to date for generating and analyzing such networks, preserving the causality between gene interactions and reasoning over the qualitative logic of regulatory effects. We present an algorithm (MCWalk) integrated with a logic programming approach, to discover subgraphs in large-scale signaling networks by random walks in a fully automated pipeline. As an exemplary application, we uncover the signal transduction mechanisms in a gene interaction network describing hepatocyte growth factor-stimulated cell migration and proliferation from gene-expression measured with microarray and RT-qPCR using in-house perturbation experiments in a keratinocyte-fibroblast co-culture. The resulting subgraphs illustrate possible associations of hepatocyte growth factor receptor c-Met nodes, differentially expressed genes and cellular states. Using perturbation experiments and Answer Set programming, we are able to select those which are more consistent with the experimental data. We discover key regulator nodes by measuring the frequency with which they are traversed when connecting signaling between receptors and significantly regulated genes and predict their expression-shift consistently with the measured data. The Java implementation of MCWalk is publicly available under the MIT license at: https://bitbucket.org/akittas/biosubg.

Heparin responses in vascular smooth muscle cells involve cGMP-dependent protein kinase (PKG).

Published data provide strong evidence that heparin treatment of proliferating vascular smooth muscle cells results in decreased signaling through the ERK pathway and decreases in cell proliferation. In addition, these changes have been shown to be mimicked by antibodies that block heparin binding to the cell surface. Here, we provide evidence that the activity of protein kinase G is required for these heparin effects. Specifically, a chemical inhibitor of protein kinase G, Rp-8-pCPT-cGMS, eliminates heparin and anti-heparin receptor antibody effects on bromodeoxyuridine incorporation into growth factor-stimulated cells. In addition, protein kinase G inhibitors decrease heparin effects on ERK activity, phosphorylation of the transcription factor Elk-1, and heparin-induced MKP-1 synthesis. Although transient, the levels of cGMP increase in heparin treated cells. Finally, knock down of protein kinase G also significantly decreases heparin effects in growth factor-activated vascular smooth muscle cells. Together, these data indicate that heparin effects on vascular smooth muscle cell proliferation depend, at least in part, on signaling through protein kinase G.

Modulation of Epidermal Growth Factor Stimulated ERK Phosphorylation and Cell Motility by Inositol Trisphosphate Kinase.

Epidermal growth factor [EGF] mediated stimulation of its receptor in endothelial cell [EC] is accompanied by phosphorylation of the EGF-receptor [EGFR] and activation of phospholipase C-γ, resulting in the breakdown of phosphatidylinositol(4,5)-bisphosphate and generating inositol (1,4,5)-trisphosphate [IP3] and diacylglycerol. IP3 thus formed can be further converted to inositol (1,3,4,5)-tetrakisphosphate [IP4] by an enzyme called IP3-kinase [IP3K]. In this study we have investigated the effect of modulation of intracellular IP3K activity by the use of an inhibitor, 2-trifluoromethyl [6-(4-nitrobenzyl)-purine] [IP3KI] and siRNA against IP3KB on EGF-induced ERK-phosphorylation and cell motility. EGF stimulated ERK-phosphorylation that has been implicated in EGF-stimulated cell migration was inhibited by both IP3KI and siRNA against IP3KB. Inhibition of ERK-phosphorylation was accompanied by decreased cell migration in the presence of IP3KI.

Cell cycle regulation by the nutrient-sensing mammalian target of rapamycin (mTOR) pathway.

Cell division involves a series of ordered and controlled events that lead to cell proliferation. Cell cycle progression implies not only demanding amounts of cell mass, protein, lipid, and nucleic acid content but also a favorable energy state. The mammalian target of rapamycin (mTOR), in response to the energy state, nutrient status, and growth factor stimulation of cells, plays a pivotal role in the coordination of cell growth and the cell cycle. Here, we review how the nutrient-sensing mTOR-signaling cascade molecularly integrates nutritional and mitogenic/anti-apoptotic cues to accurately coordinate cell growth and cell cycle. First, we briefly outline the structure, functions, and regulation of the mTOR complexes (mTORC1 and mTORC2). Second, we concisely evaluate the best known ability of mTOR to control G1-phase progression. Third, we discuss in detail the recent evidence that indicates a new genome stability caretaker function of mTOR based on the specific ability of phosphorylated forms of several mTOR-signaling components (AMPK, raptor, TSC, mTOR, and S6K1), which spatially and temporally associate with essential mitotic regulators at the mitotic spindle and at the cytokinetic cleavage furrow.

Antibodies Directed to Neisseria gonorrhoeae Impair Nerve Growth Factor-Dependent Neurite Outgrowth in Rat PC12 Cells

In children born from mothers with prenatal infections with the Gram-negative bacterium Neisseria gonorrhoeae, schizophrenia risk is increased in later life. Since cortical neuropil formation is frequently impaired during this disease, actions of a rabbit polyclonal antiserum directed to N. gonorrhoeae on neurite outgrowth in nerve growth factor-stimulated PC12 cells were investigated here. It turned out that 10μg/ml of the antiserum leads indeed to a significant reduction in neurite outgrowth, whereas an antiserum directed to Neisseria meningitidis had no such effect. Furthermore, reduction in neurite outgrowth could be reversed by the neuroleptic drugs haloperidol, clozapine, risperidone, and olanzapine. On the molecular level, the observed effects seem to include the known neuritogenic transcription factors FoxO3a and Stat3, since reduced neurite outgrowth caused by the antiserum was accompanied by a reduced phosphorylation of both factors. In contrast, restitution of neurite outgrowth by neuroleptic drugs revealed no correlation to the phosphorylation state of these factors. The present report gives a first hint that bacterial infections could indeed lead to impaired neuropil formation in vitro; however, the in vivo relevance of this finding for schizophrenia pathogenesis remains to be clarified in the future.

MAPK uncouples cell cycle progression from cell spreading and cytoskeletal organization in cycling cells

Integrin-mediated cytoskeletal tension supports growth-factor-induced proliferation, and disruption of the actin cytoskeleton in growth factor-stimulated cells prevents the re-expression of cyclin D and cell cycle re-entry from quiescence. In contrast to cells that enter the cell cycle from G0, cycling cells continuously express cyclin D, and are subject to major cell shape changes during the cell cycle. Here, we investigated the cell cycle requirements for cytoskeletal tension and cell spreading in cycling mammalian cells that enter G1-phase from mitosis. Disruption of the actin cytoskeleton at progressive time-points in G1-phase induced cell rounding, FA disassembly, and attenuated both integrin signaling and growth factor-induced p44/p42 mitogen-activated protein kinase activation. Although cyclin D expression was reduced, the expression of cyclin A and entry into S-phase were not affected. Moreover, expression of cyclin B1, progression through G2- and M-phase, and commitment to a new cell cycle occurred normally. In contrast, cell cycle progression was strongly prevented by inhibition of MAPK activity in G1-phase, whereas cell spreading, cytoskeletal organization, and integrin signaling were not impaired. MAPK inhibition also prevented cytoskeleton-independent cell cycle progression. Thus, these results uncouple the requirements for cell spreading and cytoskeletal organization from MAPK signaling, and show that cycling mammalian cells can proliferate independently of actin stress fibers, focal adhesions, or cell spreading, as long as a threshold level of MAPK activity is sustained.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-012-1130-2) contains supplementary material, which is available to authorized users.