JAK-STAT signalling is required throughout telotrophic oogenesis and short-germ embryogenesis of the beetle Tribolium

In Drosophila, the JAK-STAT signalling pathway regulates a broad array of developmental functions including segmentation and oogenesis. Here we analysed the functions of Tribolium JAK-STAT signalling factors and of Suppressor Of Cytokine Signalling (SOCS) orthologues, which are known to function as negative regulators of JAK-STAT signalling, during telotrophic oogenesis and short-germ embryogenesis. The beetle Tribolium features telotrophic ovaries, which differ fundamentally from the polytrophic ovary of Drosophila. While we found the requirement for JAK-STAT signalling in specifying the interfollicular stalk to be principally conserved, we demonstrate that these genes also have early and presumably telotrophic specific functions. Moreover, we show that the SOCS genes crucially contribute to telotrophic Tribolium oogenesis, as their inactivation by RNAi results in compound follicles. During short-germ embryogenesis, JAK-STAT signalling is required in the maintenance of segment primordia, indicating that this signalling cascade acts in the framework of the segment-polarity network. In addition, we demonstrate that JAK-STAT signalling crucially contributes to early anterior patterning. We posit that this signalling cascade is involved in achieving accurate levels of expression of individual pair-rule and gap gene domains in early embryonic patterning.     

Tick-borne encephalitis virus NS5 associates with membrane protein scribble and impairs interferon-stimulated JAK-STAT signalling

Tick-borne encephalitis virus (TBEV) NS5 protein is a multifunctional RNA-dependent RNA polymerase that is indispensable for viral replication. TBEV is considered to be highly neurovirulent and can cause lethal encephalitis. In this study, we demonstrate a novel interaction between TBEV NS5 and the PDZ protein scribble (hScrib) affecting interferon (IFN) type I and II mediated JAK-STAT signalling. The sequence of TBEV NS5 interacting with hScrib was identified using extensive site-directed mutagenesis analysis. Two consecutive mutations in the methyltransferase (MTase) domain of NS5 were found to disrupt binding to hScrib. Colocalization studies with hScrib demonstrated that TBEV NS5 was present at the plasma membrane of mammalian cells. To address the role of viral interference with the IFN response, NS5 proteins were expressed in IFN-stimulated cells. While TBEV NS5 substantially blocked phosphorylation of STAT1, a mutated NS5 protein defective in hScrib binding failed to inhibit JAK-STAT signalling correctly. Furthermore, hScrib knock-down resulted in re-localization of NS5 to intracellular locations and abrogated the impaired STAT1 phosphorylation. These results define the TBEV NS5 protein in concert with hScrib as an antagonist of the IFN response, by demonstrating a correlation between the association and JAK-STAT interference.     

A JAK1/JAK2 chimera can sustain alpha and gamma interferon responses.

Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant gamma1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant gamma2A cells lack detectable JAK2 mRNA and protein. By using gamma2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-gamma signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-gamma. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-gammaR2 subunit requires the amino-terminal region but not the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -gamma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains.     

The Bone Marrow-Mediated Protection of Myeloproliferative Neoplastic Cells to Vorinostat and Ruxolitinib Relies on the Activation of JNK and PI3K Signalling Pathways

The classical BCR-ABL-negative Myeloproliferative Neoplasms (MPN) are a group of heterogeneous haematological diseases characterized by constitutive JAK-STAT pathway activation. Targeted therapy with Ruxolitinib, a JAK1/2-specific inhibitor, achieves symptomatic improvement but does not eliminate the neoplastic clone. Similar effects are seen with histone deacetylase inhibitors (HDACi), albeit with poorer tolerance. Here, we show that bone marrow (BM) stromal cells (HS-5) protected MPN-derived cell lines (SET-2; HEL and UKE-1) and MPN patient-derived BM cells from the cytotoxic effects of Ruxolitinib and the HDACi Vorinostat. This protective effect was mediated, at least in part, by the secretion of soluble factors from the BM stroma. In addition, it correlated with the activation of signalling pathways important for cellular homeostasis, such as JAK-STAT, PI3K, JNK, MEK-ERK and NF-κB. Importantly, the pharmacological inhibition of JNK and PI3K pathways completely abrogated the BM protective effect on MPN cell lines and MPN patient samples. Our findings shed light on mechanisms of tumour survival and may indicate novel therapeutic approaches for the treatment of MPN.     

Hes binding to STAT3 mediates crosstalk between Notch and JAK-STAT signalling

Although the Notch and JAK-STAT signalling pathways fulfill overlapping roles in growth and differentiation regulation, no coordination mechanism has been proposed to explain their relationship. Here we show that STAT3 is activated in the presence of active Notch, as well as the Notch effectors Hes1 and Hes5. Hes proteins associate with JAK2 and STAT3, and facilitate complex formation between JAK2 and STAT3, thus promoting STAT3 phosphorylation and activation. Furthermore, suppression of endogenous Hes1 expression reduces growth factor induction of STAT3 phosphorylation. STAT3 seems to be essential for maintenance of radial glial cells and differentiation of astrocytes by Notch in the developing central nervous system. These results suggest that direct protein-protein interactions coordinate cross-talk between the Notch-Hes and JAK-STAT pathways.     

An ES-like pluripotent state in FGF-dependent murine iPS cells

Recent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a naïve LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGF-dependent primed pluripotent state represented by murine and rat epiblast stem cells (EpiSCs). We find that derivation of induced pluripotent stem cells (iPSCs) under EpiSC culture conditions yields FGF-dependent iPSCs from hereon called FGF-iPSCs) which, unexpectedly, display naïve ES-like/ICM properties. FGF-iPSCs display X-chromosome activation, multi-lineage differentiation, teratoma competence and chimera contribution in vivo. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions.Characterization of the key molecular signalling pathways revealed FGF-iPSCs to depend on the Activin/Nodal and FGF pathways, while signalling through the JAK-STAT pathway is not required for FGF-iPS cell maintenance. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions.     

Combined pharmacophore,virtual screening and molecular dynamics studies to identify Bruton’s tyrosine kinase inhibitors

Bruton’s tyrosine Kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase expressed in hematopoietic cells. BTK plays a critical role in many cellular signalling pathways making it a potential target to treat autoimmune diseases and cancer. BTK signalling is important for the production of arthritis-associated antibodies, and inhibiting BTK will help the system to block the production of disease-associated antibodies. In this study, we have implemented ligand-based pharmacophore modelling and virtual screening against natural compounds followed by molecular docking, density functional theory and molecular dynamics studies for 50 ns. Four compounds with high affinity towards BTK were identified, and it could be used as a potent lead molecule for designing BTK inhibitor.     

The role of cytoplasmic nanospaces in smooth muscle cell Ca2+ signalling

We address the importance of cytoplasmic nanospaces in Ca(2+) transport and signalling in smooth muscle cells and how quantitative modelling can shed significant light on the understanding of signalling mechanisms. Increasingly more convincing evidence supports the view that these nanospaces--nanometre-scale spaces between organellar membranes, hosting cell signalling machinery--are key to Ca(2+) signalling as much as Ca(2+) transporters and Ca(2+) storing organelles. Our research suggests that the origin of certain diseases is to be sought in the disruption of the proper functioning of cytoplasmic nanospaces. We begin with a historical perspective on the study of smooth muscle cell plasma membrane-sarcoplasmic reticulum nanospaces, including experimental evidence of their role in the generation of asynchronous Ca(2+) waves. We then summarize how stochastic modelling approaches have aided and guided our understanding of two basic functional steps leading to healthy smooth muscle cell contraction. We furthermore outline how more sophisticated and realistic quantitative stochastic modelling is now being employed not only to deepen our understanding but also to aid in the hypothesis generation for further experimental investigation.     

Post-lactational mammary gland regression: molecular basis and implications for breast cancer

During pregnancy, there is a massive increase in the number of luminal epithelial cells in the breast, which are destined to become the milk factories after birth. These cells are no longer required when the young are weaned, and are removed in a carefully orchestrated event called involution. In this process, the secretory epithelial cells die and are replaced by adipocytes, which redifferentiate as the epithelium is removed. It is essential that the gland is properly remodelled to a pre-pregnant state so that successful lactation can occur following a subsequent pregnancy. Furthermore, failure to remove unnecessary lactational alveoli during weaning could result in inflammation and tissue damage. Recently, it has been shown that components in the fatty stroma in involuting breast can promote metastasis. Thus, it is important to understand the molecular mechanisms that regulate involution, how these can fail, the consequences of the remodelling process, and how this knowledge can inform us about breast cancer. In this review, I discuss the roles of the JAK-STAT, NF-kappaB and other signalling pathways in the regulation of apoptosis and tissue remodelling during involution.     

A novel type II membrane receptor up-regulated by IFN-alpha in fibroblasts functions in cell proliferation through the JAK-STAT signalling pathway

A type II membrane protein similar to CD69 (TIIMPSC) has been isolated in human embryo fibroblasts treated with IFN-alpha. Structural analysis and immunofluorescence detection has suggested that this protein is located on the surface of fibroblasts, generally considered, a receptor. Cell proliferation assay has revealed that activation of TIIMPSC elevates the level of fibroblast proliferation. Further, examination of signal transduction has indicated that expression of this protein is up-regulated by IFN-alpha stimulation, and that it is involved in the regulation of fibroblast growth through the JAK-STAT signalling pathway.     

Modelling dynamic plant cells

A major challenge in plant biology is to understand how functions in plant cells emerge from interactions between molecular components. Computational and mathematical modelling can encapsulate the relationships between molecular components and reveal how biological functions emerge. We review recent progress in modelling in metabolism, growth, signalling and circadian rhythms in plant cells. We discuss challenges and opportunities for future directions.     

Does efficiency sensing unify diffusion and quorum sensing?

Quorum sensing faces evolutionary problems from non-producing or over-producing cheaters. Such problems are circumvented in diffusion sensing, an alternative explanation for quorum sensing. However, both explanations face the problems of signalling in complex environments such as the rhizosphere where, for example, the spatial distribution of cells can be more important for sensing than cell density, which we show by mathematical modelling. We argue that these conflicting concepts can be unified by a new hypothesis, efficiency sensing, and that some of the problems associated with signalling in complex environments, as well as the problem of maintaining honesty in signalling, can be avoided when the signalling cells grow in microcolonies.     

Tumour-secreted miR-9 promotes endothelial cell migration and angiogenesis by activating the JAK-STAT pathway

Angiogenesis plays a crucial role during tumorigenesis and much progress has been recently made in elucidating the role of VEGF and other growth factors in the regulation of angiogenesis. Recently, microRNAs (miRNAs) have been shown to modulate a variety of physiogical and pathological processes. We identified a set of differentially expressed miRNAs in microvascular endothelial cells co-cultured with tumour cells. Unexpectedly, most miRNAs were derived from tumour cells, packaged into microvesicles (MVs), and then directly delivered to endothelial cells. Among these miRNAs, we focused on miR-9 due to the strong morphological changes induced in cultured endothelial cells. We found that exogenous miR-9 effectively reduced SOCS5 levels, leading to activated JAK-STAT pathway. This signalling cascade promoted endothelial cell migration and tumour angiogenesis. Remarkably, administration of anti-miR-9 or JAK inhibitors suppressed MV-induced cell migration in vitro and decreased tumour burden in vivo. Collectively, these observations suggest that tumour-secreted miRNAs participate in intercellular communication and function as a novel pro-angiogenic mechanism.     

The role of cytoplasmic nanospaces in smooth muscle cell Ca<Superscript>2+</Superscript> signalling

We address the importance of cytoplasmic nanospaces in Ca^2?+? transport and signalling in smooth muscle cells and how quantitative modelling can shed significant light on the understanding of signalling mechanisms. Increasingly more convincing evidence supports the view that these nanospaces—nanometre-scale spaces between organellar membranes, hosting cell signalling machinery—are key to Ca^2?+? signalling as much as Ca^2?+? transporters and Ca^2?+? storing organelles. Our research suggests that the origin of certain diseases is to be sought in the disruption of the proper functioning of cytoplasmic nanospaces. We begin with a historical perspective on the study of smooth muscle cell plasma membrane–sarcoplasmic reticulum nanospaces, including experimental evidence of their role in the generation of asynchronous Ca^2?+? waves. We then summarize how stochastic modelling approaches have aided and guided our understanding of two basic functional steps leading to healthy smooth muscle cell contraction. We furthermore outline how more sophisticated and realistic quantitative stochastic modelling is now being employed not only to deepen our understanding but also to aid in the hypothesis generation for further experimental investigation.     

Data-driven modelling of receptor tyrosine kinase signalling networks quantifies receptor-specific potencies of PI3K- and Ras-dependent ERK activation

Signal transduction networks in mammalian cells, comprising a limited set of interacting biochemical pathways, are accessed by various growth factor and cytokine receptors to elicit distinct cell responses. This raises the question as to how specificity of the stimulus-response relationship is encoded at the molecular level. It has been proposed that specificity arises not only from the activation of unique signalling pathways, but also from quantitative differences in the activation and regulation of shared receptor-proximal signalling proteins. To address such hypotheses, data sets with greater precision and coverage of experimental conditions will need to be acquired, and rigorous frameworks that codify and parameterize the inherently non-linear relationships among signalling activities will need to be developed. In the present study we apply a systematic approach combining quantitative measurements and mathematical modelling to compare the signalling networks accessed by FGF (fibroblast growth factor) and PDGF (platelet-derived growth factor) receptors in mouse fibroblasts, in which the ERK (extracellular-signal-regulated kinase) cascade is activated by Ras- and PI3K (phosphoinositide 3-kinase)-dependent pathways. We show that, whereas the FGF stimulation of PI3K signalling is relatively weak, this deficiency is compensated for by a more potent Ras-dependent activation of ERK. Thus, as the modelling would predict, the ERK pathway is activated to a greater extent in cells co-stimulated with FGF and PDGF, relative to the saturated levels achieved with either ligand alone. It is envisaged that similar approaches will prove valuable in the elucidation of quantitative differences among other closely related receptor signalling networks.