Independent Signal Transduction Pathways Research Articles

AtC3H3, an Arabidopsis Non-TZF Gene, Enhances Salt Tolerance by Increasing the Expression of Both ABA-Dependent and -Independent Stress-Responsive Genes.

Salinity causes widespread crop loss and prompts plants to adapt through changes in gene expression. In this study, we aimed to investigate the function of the non-tandem CCCH zinc-finger (non-TZF) protein gene AtC3H3 in response to salt stress in Arabidopsis. AtC3H3, a gene from the non-TZF gene family known for its RNA-binding and RNase activities, was up-regulated under osmotic stress, such as high salt and drought. When overexpressed in Arabidopsis, AtC3H3 improved tolerance to salt stress, but not drought stress. The expression of well-known abscisic acid (ABA)-dependent salt stress-responsive genes, namely Responsive to Desiccation 29B (RD29B), RD22, and Responsive to ABA 18 (RAB18), and representative ABA-independent salt stress-responsive genes, namely Dehydration-Responsive Element Binding protein 2A (DREB2A) and DREB2B, was significantly higher in AtC3H3-overexpressing transgenic plants (AtC3H3 OXs) than in wild-type plants (WT) under NaCl treatment, indicating its significance in both ABA-dependent and -independent signal transduction pathways. mRNA-sequencing (mRNA-Seq) analysis using NaCl-treated WT and AtC3H3 OXs revealed no potential target mRNAs for the RNase function of AtC3H3, suggesting that the potential targets of AtC3H3 might be noncoding RNAs and not mRNAs. Through this study, we conclusively demonstrated that AtC3H3 plays a crucial role in salt stress tolerance by influencing the expression of salt stress-responsive genes. These findings offer new insights into plant stress response mechanisms and suggest potential strategies for improving crop resilience to salinity stress.

Dual-Mode Paper Biosensing Platform Based on Oxidase-Like CoFeMn Nanozymes for Point-of-Care Detection of Glutathione

A paper-based biosensing platform is a point-of-care (POC) analysis toolkit for detection of various biomarkers. Nonetheless, its lack of high sensitivity, selectivity, and multi-signal readout poses a high degree of challenge. Herein, we introduce an interesting colorimetric-temperature dual-signal readout biosensor based on multifunctional CoFeMn biomimetic oxidase. Due to the cost-effectiveness, no additional detector requirement, dual-signal readout capacity, and darker signal change, CoFeMn hydroxide is innovatively fabricated by a facile hydrothermal method using Mn as an active center to construct a multifunctional signal tag for mimicking the structure of oxidase. This strategy allows CoFeMn hydroxide to exhibit an unusual oxidase-like activity, transforming O2 into ·OH in the absence of H2O2 and resulting in the generation of a colorimetric temperature dual signal by oxidizing 3,3′,5,5′-tetramethylbenzidine (TMB). We used glutathione (GSH) as the analyst and successfully applied this biosensor to determine GSH, which showed excellent analytical performance of rapidity, high specificity, sensitivity, and accuracy. Employing the colorimetric and temperature test strips, these toolkits are designed to visualize GSH. Benefiting from a relatively independent signal transduction pathway, the constructed test strip POC devices display satisfactory coupling efficiency and are further employed to monitor GSH in tumor cells. This research will be conducive to the rational design of a mimetic oxidase-based dual-response nanoprobe in paper-based POC devices, the exploration of more nanomaterial-derived mimetic oxidase, and the extension of their applications in medical diagnosis.

Genetic determination of drought resistance in common wheat (Triticum aestivum L.)

The aim of the work is to analyze the literature data on genetic determinants and molecular mechanisms involved in the regulation of adaptation and resistance to drought in common wheat. Results. Regulation of the response to osmotic stress in common wheat is carried out through several abscisic acid-dependent or independent pathways. ABA inhibits the growth processes of aerial parts of the plant by inhibiting the action of auxins and cytokinins, increases the hydraulic conductivity of roots by modulating the activity of aquaporins - membrane water channels, changes the flow of ions in the closing cells of the stomata, which leads to their closure and a decrease in water consumption for transpiration. ABA activates a number of TFs that regulate the expression of genes, the products of which are necessary to eliminate the negative consequences of water deficit. ABA-dependent is activation of the genes of antioxidant defense enzymes - superoxide dismutase, peroxidase, catalase and enzymes of the ascorbate-glutathione cycle. Activators of their transcription are NAC, MYB, WRKY, NF-Y, ZFHD and TaERF3 TFs. Expression of LEA genes and dehydrins, which prevent protein aggregation due to dehydration, is ensured by both ABA-dependent and -independent signal transduction pathways, with the help of AREB/ABF, NAC, MYB, WRKY, AP2/EREBP and ZFHD TFs. ABA also activates the biosynthesis of proline - one of the main low-molecular osmoprotectants that accumulate in the cell and ensure the stability of its water regime. Osmolyte accumulation is regulated by MYB, WRKY, NF-Y and TaERF3 TFs. Conclusions. Thus, in the article is considered the regulatory role of ABA in the formation of drought resistance through molecular interactions involving aquaporins, dehydrins, SNRK2 protein kinases, LEA proteins and their genes, as well as genes of transcription factors NAC, MYB, WRKY, NF-Y, AP/ EREBP, ZFHD, DREB. However, due to the complexity of the wheat genome and the polygenicity of the drought resistance trait, there is currently no line of molecular genetic markers for certain alleles of drought resistance genes that would allow predicting the drought resistance of Ukrainian breeding varieties. The molecular genetic mechanisms underlying drought resistance and the identification of genes with the greatest phenotypic effect, as well as the modeling of the work of these genes at different stages of ontogenesis and the involvement of drought resistance alleles in breeding programs, currently require further research.

Neuronal Signaling Involved in Neuronal Polarization and Growth: Lipid Rafts and Phosphorylation.

Neuronal polarization and growth are developmental processes that occur during neuronal cell differentiation. The molecular signaling mechanisms involved in these events in in vivo mammalian brain remain unclear. Also, cellular events of the neuronal polarization process within a given neuron are thought to be constituted of many independent intracellular signal transduction pathways (the “tug-of-war” model). However, in vivo results suggest that such pathways should be cooperative with one another among a given group of neurons in a region of the brain. Lipid rafts, specific membrane domains with low fluidity, are candidates for the hotspots of such intracellular signaling. Among the signals reported to be involved in polarization, a number are thought to be present or translocated to the lipid rafts in response to extracellular signals. As part of our analysis, we discuss how such novel molecular mechanisms are combined for effective regulation of neuronal polarization and growth, focusing on the significance of the lipid rafts, including results based on recently introduced methods.

Control of neuronal excitability by Group I metabotropic glutamate receptors.

Metabotropic glutamate (mGlu) receptors couple through G proteins to regulate a large number of cell functions. Eight mGlu receptor isoforms have been cloned and classified into three Groups based on sequence, signal transduction mechanisms and pharmacology. This review will focus on Group I mGlu receptors, comprising the isoforms mGlu1 and mGlu5. Activation of these receptors initiates both G protein-dependent and -independent signal transduction pathways. The G-protein-dependent pathway involves mainly Gαq, which can activate PLCβ, leading initially to the formation of IP3 and diacylglycerol. IP3 can release Ca2+ from cellular stores resulting in activation of Ca2+-dependent ion channels. Intracellular Ca2+, together with diacylglycerol, activates PKC, which has many protein targets, including ion channels. Thus, activation of the G-protein-dependent pathway affects cellular excitability though several different effectors. In parallel, G protein-independent pathways lead to activation of non-selective cationic currents and metabotropic synaptic currents and potentials. Here, we provide a survey of the membrane transport proteins responsible for these electrical effects of Group I metabotropic glutamate receptors.

Activity of a Sting and a TLR9 Agonist As an “in Situ Therapeutic Vaccination” Against Lymphomas

We have developed a therapeutic “in situ vaccination” strategy against lymphomas involving the intratumoral injection of a STING- or a TLR9 agonist. The advantage of this approach is that it does not require the isolation of tumor specific antigens since the immune system is already primed at the tumor site, but inhibited for acting.The Stimulator of Interferon Gene (STING) is part of the innate immunity involved in the intracellular pathogen recognition. More recently, it was shown to be a crucial player in the spontaneous immune response against immunogenic tumors. STING agonists are able to induce an immune response through activating dendritic cells that then produce cytokines and chemokines including type 1 IFN. Modified cyclic dinucleotides (CDNs) have enhanced ability to activate both mouse and human STING variants. When injected intratumorally, these CDNs were able to induce a tumor-specific CD8 T cell response in various tumor models. A Phase 1 clinical trial is evaluating the safety and the efficacy of intratumoral injection of CDNs in patients with advanced/metastatic solid tumors or lymphomas [NCT02675439]. Toll-like receptor 9 (TLR9) is another receptor of the innate immune system. Oligodeoxynucleotides containing unmethylated CpG motifs (CpG) are well known TLR9 agonists. Upon TLR9 stimulation, CpG can activate dendritic cells and B cells, and induce the release of multiple cytokines, such as interferons (IFNs). Intratumoral injection of CpG results in a CD8 T cell-dependent immune response against tumors. Multiple phase 1 / 2 clinical trials are evaluating the efficacy of CpG in cancer treatments. Among them, some are investigating intratumoral injection to patients with lymphomas [NCT01745354, NCT02254772, NCT00185965, and NCT00880581].STING or TLR9 activations, both result in similar immune outcomes. However, they work by independent intracellular signal transduction pathways and their expression pattern is different. TLR9 is expressed on immune cells whereas STING is expressed on a wider variety of cells among immune- and somatic cells, including endothelial cells. Therefore, we studied the properties of both, CpG and CDNs, in the context of lymphomas. We examined the direct effect of these molecules on lymphoma cells in vitro and their therapeutic effects in vivo.In vitro, both agonists were able to induce the expression of costimulatory molecules on lymphoma B cells. Interestingly, the STING agonist showed toxicity on mouse lymphoma cell lines whereas CpG showed increased proliferation on some of these same B cell lines.We studied the in vivo effects of both agonists using the A20 lymphoma injected into two different bilateral subcutaneous sites. Either the TLR9- or the STING agonist was injected in one tumor site, and both, the injected- and the uninjected tumor sites were monitored to assess the local- and the systemic effects of the treatment. As single agents, both agonists showed a similar local and systemic outcome. The treated tumor disappeared, and the growth of the distant tumor was retarded. We assumed that the failure to cure the second tumor was due to the dampening of the immune response by local immunosuppressive mechanisms. Therefore, we investigated the combination of each agonist with different checkpoint blockade strategies, with the checkpoint blocking agent being given intratumorally or systemically. Combining the TLR9- or the STING agonist with a checkpoint blocking agent induced the regression of the distant tumor. Interestingly, the preferred route of administration of the checkpoint blocking agents was different depending on TLR9 or STING stimulation. TLR9 stimulation required intratumoral checkpoint blocking, whereas STING stimulation worked better with systemic checkpoint blocking.Our results indicate that in situ stimulation of the immune system, using either STING or TLR9, combined with checkpoint blockade is a promising approach for the therapy of lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Membrane receptor cross talk in gonadotropin-, IGF-I-, and insulin-mediated steroidogenesis in fish ovary: An overview

Gonadal steroidogenesis is critical for survival and reproduction of all animals. The pathways that regulate gonadal steroidogenesis are therefore conserved among animals from the steroidogenic enzymes to the intracellular signaling molecules and G protein-coupled receptors (GPCRs) that mediate the activity of these enzymes. Regulation of fish ovarian steroidogenesis in vitro by gonadotropin (GtH) and GPCRs revealed interaction between adenylate cyclase and calcium/calmodulin-dependent protein kinases (CaMKs) and also MAP kinase pathway. Recent studies revealed another important pathway in GtH-induced fish ovarian steroidogenesis: cross talk between GPCRs and membrane receptor tyrosine kinases. Gonadotropin binding to Gαs-coupled membrane receptor in fish ovary leads to production of cAMP which in turn trans-activate the membrane-bound epidermal growth factor receptor (EGFR). This is followed by activation of ERK1/2 signaling that promotes steroid production. Interestingly, GtH-induced trans-activation of EGFR in the fish ovary uniquely requires matrix-metalloproteinase-mediated release of EGF. Inhibition of these proteases blocks GtH-induced steroidogenesis. Increased cAMP production in fish ovarian follicle upregulate follicular cyp19a1a mRNA expression and aromatase activity leading to increased biosynthesis of 17β-estradiol (E2). Evidence for involvement of SF-1 protein in inducing cyp19a1a mRNA and aromatase activity has also been demonstrated. In addition to GtH, insulin-like growth factor (IGF-I) and bovine insulin can alone induced steroidogenesis in fish ovary. In intact follicles and isolated theca cells, IGF-I and insulin had no effect on GtH-induced testosterone and 17a,hydroxysprogeaterone production. GtH-stimulated E2 and 17,20bdihydroxy-4-pregnane 3-one production in granulosa cells however, was significantly increased by IGF-I and insulin. Both IGF-I and insulin mediates their signaling via receptor tyrosine kinases leading to activation of PI3 kinase/Akt and MAP kinase. These kinase signals then activates steroidogenic enzymes which promotes steroid production. PI3 kinase, therefore considered to be an initial component of the signal transduction pathways which precedes MAP kinase in IGF-1 and insulininduced steroidogenesis in fish ovary. Thus, investigation on the mechanism of signal transduction regulating fish ovarian steroidogenesis have shown that multiple, apparently independent signal transduction pathways are needed to convey the message of single hormone or growth factor.

Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid

Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10−9 vs. 10−5M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10−5M, but drops to 10−9M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

Defining a novel role for the NF-κB pathway in proximal TCR signaling. (IRM10P.746)

Abstract In the accepted model of T cell activation, parallel and independent signal transduction pathways activate the transcription factors NF-κB, NFAT and AP-1 to drive clonal expansion of T cells in response to antigen. Surprisingly, we found that CD8 T cells lacking NF-κB subunits p50 and cRel fail to activate NFAT contributing to their severely impaired in vivo responsiveness. We further demonstrated that attenuated NFAT activation was due to diminished Calcium influx upon TCR engagement, and that this reduction was accomplished via sub-par phosphorylation of PLCγ. The functional relevance of impaired PLCγ activation was demonstrated by rescue of p50-/-cRel-/- T cell proliferation through pharmacological initiation of PKC and Calcium pathways. While no deficiencies in expression of TCR complex proteins were seen, we did note defects in early TCR-induced Lck and Zap70 activation, and reduced expression of IKKβ and IKKγ in p50-/-cRel-/- T cells. Beyond established roles in NF-κB activation, we show that IKKβ /γ are also crucial for TCR-induced Lck, Zap70 and PLCγ activation. These findings define a novel pathway of “retrograde signaling” in T cells whereby NF-κB subunits modulate expression of proteins which are crucial for early TCR-induced signaling. Therefore, robust T cell activation occurs in cells exhibiting baseline activation of the pro-survival NF-κB pathway either through TCR tickling or non-TCR pathways.

BMP10 as a potent inducer of trophoblast differentiation in human embryonic and induced pluripotent stem cells

Bone morphogenetic proteins (BMPs) are known to induce diverse differentiation fates in human embryonic stem cells (hESCs). In the present study, we compared the potency at which BMP5, BMP10 and BMP13, which are members of distinct BMP subgroups due to differences in sequential and structural homology, induce differentiation in hESCs and human induced pluripotent stem cells (hiPSCs). We observed, in agreement with previous BMP4 model studies, that all ligands induced differentiation to the trophoblast lineage in the absence of bFGF. However, distinct BMPs exerted differences in the kinetics of induced differentiation, with BMP10 being the most potent. hiPSCs and hESCs shared comparable expression patterns of BMP type-I and -II receptor subtypes, which might explain conserved properties with respect to ligand potency and activation of SMAD-dependent (via SMAD1/5/8) and -independent (via MAPK p38) signal transduction pathways. The tested BMPs had distinct and also conserved target genes such as CDX2, DLX3, DLX5, GATA2, GATA3, HAND1, ID2, MSX2 and TFAP2A, known to be associated with the emergence of trophoblast cells. hESCs induced expression of the BMP antagonist NOGGIN as a protection mechanism to constrict extensive BMP action. Unlike BMP4, BMP10 has been shown to be resistant to NOGGIN-induced inhibition which in part might explain its potency. BMPs, in particular BMP4, are commonly used cytokines in differentiation protocols to generate diverse mesoderm- and endoderm-derivates from human pluripotent stem cells. Our study has identified BMP10, a cardiac-specific protein, as a superior alternative to BMP4 for inducing trophoblast differentiation in human pluripotent stem cells.

Teneurin C-terminal associated peptides (TCAP): modulators of corticotropin-releasing factor (CRF) physiology and behavior

The existence of the teneurin C-terminal associated peptides (TCAP) was reported in 2004 after screening a rainbow trout hypothalamic cDNA for corticotropin-releasing factor (CRF)-related homologs. In vertebrates, there are four TCAP paralogs, where each peptide is associated with a teneurin transmembrane protein. The TCAPs are 40 or 41 amino acids in length and possess less than 20% residue identity with the CRF family of paralogs. Orthologs of TCAP are found in all metazoans with the possible exception of poriferans and cnidarians. Recent evidence indicates that TCAP and the teneurins may have been introduced into the Metazoa via horizontal gene transfer from prokaryotes into a basal protistan. Thus, the origin of the TCAPs likely predated that of the CRF family. In the mammalian brain, TCAP-1 is transcribed independently from teneurin-1. Moreover, TCAP-1 acts on neurons by a CRF-receptor independent signal transduction pathway to regulate cellular cytoskeletal function to stimulate cell activity. Administration of synthetic TCAP-1 to rodents inhibits a number of CRF- and stress-associated behaviors via a hypothalamic–pituitary–adrenal (HPA) axis-independent mechanism.

The effect of synthetic inducers of systemic resistance in inhibiting grey mold development on geranium and poinsettia

Several chemical activators of resistance were tested to evaluate their efficiency in restricting development caused by <i>Botryotinia fuckeliana (Botrytis cinerea)</i> on geranium and poinsettia leaves. The used signal pathway inducers of salicylic acid, such as DL-â-aminobutyric acid (BABA), g-aminobutyric acid (GABA) and benzothiadiazole (BTH), at the applied rates markedly inhibited the development of grey mould in the both plant species. The mentioned inducers showed no toxic or restricting effect on the fungus growth and development under <i>in vitro</i> conditions. Only methyl jasmonate (Me-JA), a potential activator of independent signal transduction pathway, at the applied quantities (O,1-1,0 mg·ml<sup>-1</sup>), noticeably enhanced the disease spot development probably through stimulation of ethylene synthesis in plant. Me-JA added to medium with <i>B.cinerea</i> showed a strong antifungal action, whereas ethylene significantly stimulated mycelium growth in the <i>in vitro</i> cultures.

The Anti-Inflammatory Fungal Compound (S)-Curvularin Reduces Proinflammatory Gene Expression in an In Vivo Model of Rheumatoid Arthritis

In previous studies, we identified the fungal macrocyclic lactone (S)-curvularin (SC) as an anti-inflammatory agent using a screening system detecting inhibitors of the Janus kinase/signal transducer and activator of transcription pathway. The objective of the present study was to investigate whether SC is able to decrease proinflammatory gene expression in an in vivo model of a chronic inflammatory disease. Therefore, the effects of SC and dexamethasone were compared in the model of collagen-induced arthritis (CIA) in mice. Total genomic microarray analyses were performed to identify SC target genes. In addition, in human C28/I2 chondrocytes and MonoMac6 monocytes, the effect of SC on proinflammatory gene expression was tested at the mRNA and protein level. In the CIA model, SC markedly reduced the expression of a number of proinflammatory cytokines and chemokines involved in the pathogenesis of CIA as well as human rheumatoid arthritis (RA). In almost all cases, the effects of SC were comparable with those of dexamethasone. In microarray analyses, we identified additional new therapeutic targets of SC. Some of them, such as S100A8, myeloperoxidase, or cathelicidin, an antimicrobial peptide, are known to be implicated in pathophysiological processes in RA. Similar anti-inflammatory effects of SC were also observed in human C28/I2 chondrocyte cells, which are resistant to glucocorticoid treatment. These data indicate that SC and glucocorticoid effects are mediated via independent signal transduction pathways. In summary, we demonstrate that SC is a new effective anti-inflammatory compound that may serve as a lead compound for the development of new drugs for the therapy of chronic inflammatory diseases.

Similarities and differences of innate immune responses elicited by smooth and rough LPS

The lipopolysaccharide is the major component of Gram-negative bacteria outer membrane. LPS comprises three covalently linked regions: the lipid A, the rough core oligosaccharide, and the O-antigenic side chain determining serotype specificity. Wild-type LPS (sLPS) contains the O-antigenic side chain and is referred to as smooth. Rough LPS (rLPS) does not contain the O-side chain. Most wt bacteria and especially wt Enterobacteriaceae express prevalently the sLPS form although some truncated rLPS molecules always reach the external membrane. The two sLPS and rLPS forms are used almost indistinctly to study the effects on innate immune cells. Nevertheless, there is evidence that their mechanism of action may be different. For instance, while sLPS requires CD14 for the initiation of both MyD88-dependent and independent signal transduction pathways at least at low doses, rLPS leads to MyD88-dependent responses in the absence of CD14 even at low doses. Here we have identified additional differences in the signaling capacity of the two LPS species in the mouse. We have found that rLPS, diversely from sLPS, is capable of activating in dendritic cells (DCs) the Ca2+/calcineurin and NFAT pathway in a CD14-independent manner, moreover it is also capable per se of activating the inflammasome and eliciting IL-1β secretion independent of the presence of additional stimuli required instead for sLPS. The ability of rLPS of activating the inflammasome in vitro has as a direct consequence a higher efficiency of rLPS-exposed DCs in activating natural killer (NK) cells compared to sLPS-exposed DCs. However, diversely from possible predictions, we found that the different efficiencies of the two LPS species in eliciting innate responses are almost nullified in vivo. Therefore, sLPS and rLPS induce nearly similar in vivo innate responses but with different mechanisms of signaling.

Effects of NVP-BSK805, a Novel JAK2 Inhibitor, on BCR-ABL and JAK2V617F Positive Cell Lines

Abstract 5002 Background:Janus kinases are critical components of cytokine signaling pathways that regulate hematopoiesis, growth, immunity, inflammation, and development. Oncogenic mutations of the non-receptor tyrosine kinase JAK2 are found in many Philadelphia chromosome negative myeloproliferative neoplasms. The V617F mutation in JAK2 occurs in 95% of patients with polycythemia vera, 50% of those with essential thrombocythemia and 50% of primary myelofibrosis patients. Preclinical results strongly support that JAK2 inhibitors could be effectively used in these three indications. Replacement of valine 617 with phenylalanine upregulates the tyrosine kinase activity of JAK2, causing constitutive activation of the JAK-STAT pathway and growth factor-independent cell proliferation. JAK2 has also been postulated to play an important role in BCR-ABL signal transduction. Therefore, inhibitors of the tyrosine kinase activity of JAK2 are under investigation as new therapy strategies for CMPNs. In this study the role of the novel JAK2 inhibitor, NVP-BSK805 (Novartis Pharmaceuticals), has been investigated in cells expressing either BCR-ABL or mutant JAK2. Possible synergistic effects between NVP-BSK805 and the already established tyrosine kinase inhibitors imatinib and nilotinib were assessed. Methods:The in vitro activity of NVP-BSK805 was analyzed in 12 hematopoietic cell lines, including 7 BCR-ABL positive (K562, KCL22, KU812, Lama87, BV173, EM3, SUP-B15), 4 JAK2 mutated (CHRF288, SET2, UKE1, HEL), the T-cell leukemia cell line Jurkat, and the neuroendocrine colonic tumour line LCC-18. Concentration kinetics from 0 up to 25 μM were established using XTT proliferation assays and flow cytometry for measuring apoptosis. Protein levels of JAK2, phospho-JAK2, STAT5, phospho-STAT5 and BCR-ABL were analyzed using Western blotting. NVP-BSK805 was also tested in combination with imatinib and nilotinib. JAK2 was sequenced in all cell lines in order to detect possible mutations in the gene. Results:Of the JAK2 mutated cell lines tested, 3 of 4 (CHRF288, SET2, UKE1) showed a significant reduction of proliferation, as well as viability, compared to the other cell lines. CHRF288 responded best to NVP-BSK805 with an IC50 value of 0.22 ± 0.04 μM. UKE1 and SET2 had similar values of 0.35 ± 0.03 μM and 0.37 ± 0.05 μM. Interestingly, HEL (V617F positive) cells showed only an IC50 value (1.8 ± 0.17 μM) for NVP-BSK805, comparable with that of the non-mutated BCR-ABL positive cell lines (1.5 to 2.7 μM). LCC-18 showed the weakest response of all cell lines tested, with an IC50 value of 9.93 ± 0.202 μM. Each cell line responded to concentrations higher than 5 μM with a strong reduction of proliferation due to inhibition of various kinases. Combination of the JAK2 inhibitor with imatinib and nilotinib showed no significant additive or synergistic effects, although all BCR-ABL positive cell lines responded well to both CML therapeutic agents. Western blotting of proteins of the JAK-STAT pathway confirmed the results of the proliferation and apoptosis tests showing a strong reduction of phoshorylated STAT5 in CHRF288 cells after a 30 min incubation even with NVP-BSK805 concentrations as low as 0.01 μM. UKE-1 and SET-2 showed reduction of pSTAT5 from 0.1 μM. Levels of total STAT5 were not affected. In all the other cell lines no changes were detected in any of the proteins tested. Conclusions:Here, we tested a novel JAK2 inhibitor in cells carrying the V617F mutation. Interestingly, not every cell line with the JAK2 V617F mutation showed a good response upon JAK2 inhibition, indicating that there are additional factors determining response. On the other hand, clinical trials with JAK inhibitors in myelofibrosis have shown responses in V617F-mutated and non-mutated patients, warranting further research to identify predictors of response. In BCR-ABL mutant cells not harbouring JAK2 mutations no significant inhibition of proliferation or apoptosis was detected following JAK2 inhibition, indicating that there are JAK2 independent signal transduction pathways of BCR-ABL to avoid apoptosis. Disclosures:le Coutre:Novartis Pharmaceuticals: Honoraria, Research Funding.

Regulation of Cdc45 in the cell cycle and after DNA damage

The Cdc (cell division cycle) 45 protein has a central role in the regulation of the initiation and elongation stages of eukaryotic chromosomal DNA replication. In addition, it is the main target for a Chk1 (checkpoint kinase 1)-dependent Cdc25/CDK2 (cyclin-dependent kinase 2)-independent DNA damage checkpoint signal transduction pathway following low doses of BPDE (benzo[a]pyrene dihydrodiol epoxide) treatment, which causes DNA damage similar to UV-induced adducts. Cdc45 interacts physically and functionally with the putative eukaryotic replicative DNA helicase, the MCM (mini-chromosome maintenance) complex, and forms a helicase active 'supercomplex', the CMG [Cdc45-MCM2-7-GINS (go-ichi-ni-san)] complex. These known protein-protein interactions, as well as unknown interactions and post-translational modifications, may be important for the regulation of Cdc45 and the initiation of DNA replication following DNA damage. Future studies will help to elucidate the molecular basis of this newly identified S-phase checkpoint pathway which has Cdc45 as a target.

Mechanisms of chemokine and antigen-dependent T-lymphocyte navigation

T-lymphocyte trafficking is targeted to specific organs by selective molecular interactions depending on their differentiation and functional properties. Specific chemokine receptors have been associated with organ-specific trafficking of memory and effector T-cells, as well as the recirculation of naïve T-cells to secondary lymphoid organs. In addition to the acquisition of tissue-selective integrins and chemokine receptors, an additional level of specificity for T-cell trafficking into the tissue is provided by specific recognition of antigen displayed by the endothelium involving the TCRs (T-cell antigen receptors) and co-stimulatory receptors. Activation of PI3K (phosphoinositide 3-kinase) is a robust signalling event shared by most chemokine receptors as well as the TCR and co-stimulatory receptors, contributing to several aspects of T-lymphocyte homing as well as actin reorganization and other components of the general migratory machinery. Accordingly, inhibition of PI3K has been considered seriously as a potential therapeutic strategy by which to combat various T-lymphocyte-dependent pathologies, including autoimmune and inflammatory diseases, as well as to prevent transplant rejection. However, there is substantial evidence for PI3K-independent mechanisms that facilitate T-lymphocyte migration. In this regard, several other signalling-pathway components, including small GTPases, PLC (phospholipase C) and PKC (protein kinase C) isoforms, have also been implicated in T-lymphocyte migration in response to chemokine stimulation. The present review will therefore examine the PI3K-dependent and -independent signal-transduction pathways involved in T-cell migration during distinct modes of T-cell trafficking in response to either chemokines or the TCR and co-stimulatory molecules.

Nonendothelial Mesenchymal Cell–Derived MCP-1 Is Required for FGF-2–Mediated Therapeutic Neovascularization

Monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine that is known as an inflammatory/arteriogenic factor. Angiogenesis contributes to the inflammatory process; however, the molecular and cellular mechanisms of the links among the inflammatory pathway, arteriogenesis, and angiogenesis have not been well elucidated. Using murine models of fibroblast growth factor-2 (FGF-2)-mediated therapeutic neovascularization, we here show that FGF-2 targets nonendothelial mesenchymal cells (NEMCs) enhancing both angiogenic (vascular endothelial growth factor [VEGF]) and arteriogenic (MCP-1) signals via independent signal transduction pathways. Severe hindlimb ischemia stimulated MCP-1 expression that was strongly enhanced by FGF-2 gene transfer, and a blockade of MCP-1 activity via a dominant negative mutant as well as a deficiency of its functional receptor CCR2 resulted in the diminished recovery of blood flow attributable to adaptive and therapeutic neovascularization. Tumor necrosis factor (TNF)-alpha stimulated MCP-1 expression in all cell types tested, whereas FGF-2-mediated upregulation of MCP-1 was found only in NEMCs but not in others, a finding that was not affected by VEGF in vitro and in vivo. These results indicate that FGF-2 targets NEMCs independently, enhancing both angiogenic (VEGF) as well as inflammatory/arteriogenic (MCP-1) pathways. Therefore, MCP-1/CCR2 plays a critical role in adaptive and FGF-2-mediated therapeutic neovascularization.

Pseudomonas aeruginosa autoinducer modulates host cell responses through calcium signalling

The opportunistic pathogen Pseudomonas aeruginosa utilizes a cell density-dependent signalling phenomenon known as quorum sensing (QS) to regulate several virulence factors needed for infection. Acylated homoserine lactones, or autoinducers, are the primary signal molecules that mediate QS in P. aeruginosa. The autoinducer N-3O-dodecanoyl-homoserine lactone (3O-C12) exerts effects on mammalian cells, including upregulation of pro-inflammatory mediators and induction of apoptosis. However, the mechanism(s) by which 3O-C12 affects mammalian cell responses is unknown. Here we report that 3O-C12 induces apoptosis and modulates the expression of immune mediators in murine fibroblasts and human vascular endothelial cells (HUVEC). The effects of 3O-C12 were accompanied by increases in cytosolic calcium levels that were mobilized from intracellular stores in the endoplasmic reticulum (ER). Calcium release was blocked by an inhibitor of phospholipase C, suggesting that release occurred through inositol triphosphate (IP3) receptors in the ER. Apoptosis, but not immunodulatory gene activation, was blocked when 3O-C12-exposed cells were co-incubated with inhibitors of calcium signalling. This study indicates that 3O-C12 can activate at least two independent signal transduction pathways in mammalian cells, one that involves increases in intracellular calcium levels and leads to apoptosis, and a second pathway that results in modulation of the inflammatory response.

Expression profiling of host pathogen interactions: how Mycobacterium tuberculosis and the macrophage adapt to one another

It has recently become feasible to quantify all mRNAs encoded by the genomes of bacterial pathogens and their eukaryotic host cells and to apply this approach to study the interaction of Mycobacterium tuberculosis with its primary host cell, the macrophage. These studies helped to identify regulatory circuits which mediate adaptation of the M. tuberculosis transcriptome to intraphagosomal environments and stimulated hypotheses for the function of these circuits in human tuberculosis. The macrophage transcriptome reacts to infections with the induction of a pathogen-unspecific expression program as well as the induction of pathogen-specific expression signatures, both of which contribute to the immunologic activation of the infected cell. M. tuberculosis induced changes in the macrophage transcriptome are mediated by Toll-like receptor dependent and Toll-like receptor independent signal transduction pathways. This response is shaped by macrophage produced reactive nitrogen and oxygen molecules and affected by viability and virulence of the pathogen.