Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na(+)- or H(+)-linked myo-inositol transporters. While Na(+)-coupled myo-inositol transporters are found exclusively in the plasma membrane, H(+)-linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H(+)-linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol.

Tyrosine kinase inhibitor-induced CD70 expression mediates drug resistance in leukemia stem cells by activating Wnt signaling.

In chronic myelogenous leukemia (CML), oncogenic BCR-ABL1 activates the Wnt pathway, which is fundamental for leukemia stem cell (LSC) maintenance. Tyrosine kinase inhibitor (TKI) treatment reduces Wnt signaling in LSCs and often results in molecular remission of CML; however, LSCs persist long term despite BCR-ABL1 inhibition, ultimately causing disease relapse. We demonstrate that TKIs induce the expression of the tumor necrosis factor (TNF) family ligand CD70 in LSCs by down-regulating microRNA-29, resulting in reduced CD70 promoter DNA methylation and up-regulation of the transcription factor specificity protein 1. The resulting increase in CD70 triggered CD27 signaling and compensatory Wnt pathway activation. Combining TKIs with CD70 blockade effectively eliminated human CD34(+) CML stem/progenitor cells in xenografts and LSCs in a murine CML model. Therefore, targeting TKI-induced expression of CD70 and compensatory Wnt signaling resulting from the CD70/CD27 interaction is a promising approach to overcoming treatment resistance in CML LSCs.

In vivo TCR Signaling in CD4(+) T Cells Imprints a Cell-Intrinsic, Transient Low-Motility Pattern Independent of Chemokine Receptor Expression Levels, or Microtubular Network, Integrin, and Protein Kinase C Activity

Intravital imaging has revealed that T cells change their migratory behavior during physiological activation inside lymphoid tissue. Yet, it remains less well investigated how the intrinsic migratory capacity of activated T cells is regulated by chemokine receptor levels or other regulatory elements. Here, we used an adjuvant-driven inflammation model to examine how motility patterns corresponded with CCR7, CXCR4, and CXCR5 expression levels on ovalbumin-specific DO11.10 CD4(+) T cells in draining lymph nodes. We found that while CCR7 and CXCR4 surface levels remained essentially unaltered during the first 48-72 h after activation of CD4(+) T cells, their in vitro chemokinetic and directed migratory capacity to the respective ligands, CCL19, CCL21, and CXCL12, was substantially reduced during this time window. Activated T cells recovered from this temporary decrease in motility on day 6 post immunization, coinciding with increased migration to the CXCR5 ligand CXCL13. The transiently impaired CD4(+) T cell motility pattern correlated with increased LFA-1 expression and augmented phosphorylation of the microtubule regulator Stathmin on day 3 post immunization, yet neither microtubule destabilization nor integrin blocking could reverse TCR-imprinted unresponsiveness. Furthermore, protein kinase C (PKC) inhibition did not restore chemotactic activity, ruling out PKC-mediated receptor desensitization as mechanism for reduced migration in activated T cells. Thus, we identify a cell-intrinsic, chemokine receptor level-uncoupled decrease in motility in CD4(+) T cells shortly after activation, coinciding with clonal expansion. The transiently reduced ability to react to chemokinetic and chemotactic stimuli may contribute to the sequestering of activated CD4(+) T cells in reactive peripheral lymph nodes, allowing for integration of costimulatory signals required for full activation.

Efficient T-cell priming and activation requires signaling through prostaglandin E2 (EP) receptors

Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings.

H19 lncRNA alters stromal cell growth via IGF signaling in the endometrium of women with endometriosis

Endometriosis affects approximately 15% of reproductive aged women and is associated with chronic pelvic pain and infertility. However, the molecular mechanisms by which endometriosis impacts fertility are poorly understood. The developmentally regulated, imprinted H19 long noncoding RNA (lncRNA) functions to reduce the bioavailability of microRNA let-7 by acting as a molecular sponge. Here we report that H19 expression is significantly decreased in the eutopic endometrium of women with endometriosis as compared to normal controls. We show that decreased H19 increases let-7 activity, which in turn inhibits Igf1r expression at the post-transcriptional level, thereby contributing to reduced proliferation of endometrial stromal cells. We propose that perturbation of this newly identified H19/Let-7/IGF1R regulatory pathway may contribute to impaired endometrial preparation and receptivity for pregnancy in women with endometriosis. Our finding represents the first example of a lncRNA-based mechanism in endometriosis and its associated infertility, thus holding potential in the development of novel therapeutics for women with endometriosis and infertility.

Targeting the PI3K/AKT/mTOR signaling pathway in medulloblastoma

Medulloblastoma is the most common malignant childhood brain tumor and is associated with a poor outcome. There is an urgent need to develop novel targeted therapeutic approaches for medulloblastoma, which will arise from an enhanced understanding of the disease at the molecular level. Medulloblastoma has been recognized to be a heterogeneous disease, and no recurrent cancer gene mutations have been found, although many of the mutations described so far affect key intracellular signaling pathways, such as sonic hedgehog (SHH) and Wnt/β-catenin. The PI3K/AKT/mTOR (PAM) signaling pathway controls key cellular responses, such as cell growth and proliferation, survival, migration and metabolism. Over the last decades, it has been recognized that this intracellular signaling pathway is frequently activated by genetic and epigenetic alterations in malignant brain tumors, including medulloblastoma. Clinical trials have started to evaluate the safety and efficacy of agents targeting this pathway in malignant brain tumors. Due to the complexity of the PAM signaling pathway, there remain significant difficulties in the development of novel therapeutic approaches. The future challenges in developing effective treatments for cancer patients include the development of predictive biomarkers and combinatorial approaches to effectively target multiple signal transduction pathways. In this review article, we will summarize the current knowledge about the role of PAM signaling in medulloblastoma and discuss the strategies that are currently being evaluated with targeted agents against this pathway.

The Osr1 and Osr2 genes act in the pronephric anlage downstream of retinoic acid signaling and upstream of Wnt2b to maintain pectoral fin development.

Vertebrate odd-skipped related genes (Osr) have an essential function during the formation of the intermediate mesoderm (IM) and the kidney structures derived from it. Here, we show that these genes are also crucial for limb bud formation in the adjacent lateral plate mesoderm (LPM). Reduction of zebrafish Osr function impairs fin development by the failure of tbx5a maintenance in the developing pectoral fin bud. Osr morphant embryos show reduced wnt2b expression, and increasing Wnt signaling in Osr morphant embryos partially rescues tbx5a expression. Thus, Osr genes control limb bud development in a non-cell-autonomous manner, probably through the activation of Wnt2b. Finally, we demonstrate that Osr genes are downstream targets of retinoic acid (RA) signaling. Therefore, Osr genes act as a relay within the genetic cascade of fin bud formation: by controlling the expression of the signaling molecule Wnt2ba in the IM they play an essential function transmitting the RA signaling originated in the somites to the LPM.

Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-βs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-β is essential for the formation of cartilage-like tissue and that alterations within the TGF-β signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-β1 was required to induce the formation of cartilage-like tissue. When TGF-β signaling was blocked by inhibiting the TGF-β receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-β receptors 1 and 2 and TGF-β2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-β. We propose that a decrease in the expression of the chondrogenic growth factor TGF-β2 and of the TGF-β receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-β signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation.

Redox modulation of STIM-ORAI signaling.

STIM1 and ORAI1 constitute the core machinery of the ubiquitous store-operated calcium entry pathway and loss of function in these proteins is associated with severe immune and muscular disorders. Other isoforms-STIM1L, STIM2, ORAI2 and ORAI3 exhibit varied expression levels in different cell types along with several other interaction partners and thereby play different roles to facilitate, regulate and fine-tune the calcium entry. STIM proteins convey the Ca(2+) store-depletion message to the PM and thereby participate in refilling of the ER by physically interacting with the Ca(2+)-selective ORAI channels at the PM. STIM and ORAI are exposed to oxidative modifications in the ER, the cytosol, and at the cell surface, and redox-mediated alterations in STIM/ORAI coupling might contribute to autoimmune disorders and cancer progression. This review discusses the redox reactivity of cysteine residues in STIM and ORAI isoforms, focusing on the oxidative modifications of STIM and ORAI proteins by which STIM-ORAI signaling can be modulated.

A fungal endophyte helps plants to tolerate root herbivory through changes in gibberellin and jasmonate signaling

Plant–microbe mutualisms can improve plant defense, but the impact of root endophytes on below-ground herbivore interactions remains unknown. We investigated the effects of the root endophyte Piriformospora indica on interactions between rice (Oryza sativa) plants and its root herbivore rice water weevil (RWW; Lissorhoptrus oryzophilus), and how plant jasmonic acid (JA) and GA regulate this tripartite interaction. Glasshouse experiments with wild-type rice and coi1-18 and Eui1-OX mutants combined with nutrient, jasmonate and gene expression analyses were used to test: whether RWW adult herbivory above ground influences subsequent damage caused by larval herbivory below ground; whether P. indica protects plants against RWW; and whether GA and JA signaling mediate these interactions. The endophyte induced plant tolerance to root herbivory. RWW adults and larvae acted synergistically via JA signaling to reduce root growth, while endophyte-elicited GA biosynthesis suppressed the herbivore-induced JA in roots and recovered plant growth. Our study shows for the first time the impact of a root endophyte on plant defense against below-ground herbivores, adds to growing evidence that induced tolerance may be an important root defense, and implicates GA as a signal component of inducible plant tolerance against biotic stress.

Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling

Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neurite initiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present NeuriteTracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth.

A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space

Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.

Spatio-temporal Rho GTPase signaling - where are we now?

Rho-family GTPases are molecular switches that transmit extracellular cues to intracellular signaling pathways. Their regulation is likely to be highly regulated in space and in time, but most of what is known about Rho-family GTPase signaling has been derived from techniques that do not resolve these dimensions. New imaging technologies now allow the visualization of Rho GTPase signaling with high spatio-temporal resolution. This has led to insights that significantly extend classic models and call for a novel conceptual framework. These approaches clearly show three things. First, Rho GTPase signaling dynamics occur on micrometer length scales and subminute timescales. Second, multiple subcellular pools of one given Rho GTPase can operate simultaneously in time and space to regulate a wide variety of morphogenetic events (e.g. leading-edge membrane protrusion, tail retraction, membrane ruffling). These different Rho GTPase subcellular pools might be described as 'spatio-temporal signaling modules' and might involve the specific interaction of one GTPase with different guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and effectors. Third, complex spatio-temporal signaling programs that involve precise crosstalk between multiple Rho GTPase signaling modules regulate specific morphogenetic events. The next challenge is to decipher the molecular circuitry underlying this complex spatio-temporal modularity to produce integrated models of Rho GTPase signaling.

Hypersensitivity of an Arabidopsis Sugar Signaling Mutant toward Exogenous Proline Application

In transgenic Arabidopsis a patatin class I promoter from potato is regulated by sugars and proline (Pro), thus integrating signals derived from carbon and nitrogen metabolism. In both cases a signaling cascade involving protein phosphatases is involved in induction. Other endogenous genes are also regulated by both Pro and carbohydrates. Chalcone synthase (CHS) gene expression is induced by both, whereas the Pro biosynthetic Δ1-pyrroline-5-carboxylate synthetase (P5CS) is induced by high Suc concentrations but repressed by Pro, and Pro dehydrogenase (ProDH) is inversely regulated. The mutantrsr1-1, impaired in sugar dependent induction of the patatin promoter, is hypersensitive to low levels of external Pro and develops autofluorescence and necroses. Toxicity of Pro can be ameliorated by salt stress and exogenously supplied metabolizable carbohydrates. The rsr1-1 mutant shows a reduced response regarding sugar induction of CHS andP5CS expression. ProDH expression is de-repressed in the mutant but still down-regulated by sugar. Pro toxicity seems to be mediated by the degradation intermediate Δ1-pyrroline-5-carboxylate. Induction of the patatin promoter by carbohydrates and Pro, together with the Pro hypersensitivity of the mutant rsr1-1, demonstrate a new link between carbon/nitrogen and stress responses.

Novel mechanisms for PKC family enzymes to regulate PI3K signaling pathway

Phosphatidylinositol 3-kinase (PI3K) generates membrane phospholipids that serve as second messengers to recruit signaling proteins to plasma membrane consequently regulating cell growth and survival. PI3K is a heterodimer consisting of a catalytic p110 subunit and a regulatory p85 subunit. Association of the p85 with other signal proteins is critical for induced PI3K activation. Activated PI3K, in turn, leads to signal flows through a variety of PI3K effectors including PDK1, AKT, GSK3, BAD, p70 S6K and NFκB. The PI3K pathway is under regulation by multiple signal proteins representing cross-talk between different signaling cascades. In this study, we have evaluated the role of protein kinase C family kinases on signaling through PI3K at multiple levels. Firstly, we observed that the action of PKC specific inhibitors like Ro-31-8220 and GF109203X was associated with an increased AKT phosphorylation and activity, suggesting that PKC kinases might play a negative role in the regulation of PI3K pathway. Then, we demonstrated the stimulation of AKT by PKC inhibition was dependent on functional PI3K enzyme and able to be transmitted to the AKT effector p70 S6K. Furthermore, we showed an inducible physical association between the PKCζ isotype and AKT, which was accompanied by an attenuated AKT activity. However, a kinase-dead form of PKC failed to affect AKT. In the second part of our research we revealed the ability of a different PKC family member, PKCδ to bind to the p85 subunit of PI3K in response to oxidative stress, a process requiring the activity of src tyrosine kinases. The interaction was demonstrated to be a direct and specific contact between the carboxyl terminal SH2 domain of p85 and tyrosine phosphorylated PKCδ. Several different types of agonists were capable to induce this association including tyrosine kinases and phorbol esters with PKCδ tyrosine phosphorylation being integral components. Finally, the PKCδ-PI3K complex was related to a reduction in the AKT phosphorylation induced by src. A kinase-deficient mutant of PKCδ was equally able to inhibit AKT signal as the wild type, indicative of a process independent of PKCδ catalytic activity. Altogether, our data illustrate different PKC isoforms regulating PI3K pathway at multiple levels, suggesting a mechanism to control signal flows through PI3K for normal cell activities. Although further investigation is required for full understanding of the regulatory mechanism, we propose that complex formation of signal proteins in PI3K pathway and specific PKC isoforms plays important role in their functional linkage. ^