Alister C. Ward

Cross talk of signals between EGFR and IL-6R through JAK2/STAT3 mediate epithelial-mesenchymal transition in ovarian carcinomas.

Epidermal growth factor receptor (EGFR) is overexpressed in ovarian carcinomas, with direct or indirect activation of EGFR able to trigger tumour growth. We demonstrate significant activation of both signal transducer and activator of transcription (STAT)3 and its upstream activator Janus kinase (JAK)2, in high-grade ovarian carcinomas compared with normal ovaries and benign tumours. The association between STAT3 activation and migratory phenotype of ovarian cancer cells was investigated by EGF-induced epithelial–mesenchymal transition (EMT) in OVCA 433 and SKOV3 ovarian cancer cell lines. Ligand activation of EGFR induced a fibroblast-like morphology and migratory phenotype, consistent with the upregulation of mesenchyme-associated N-cadherin, vimentin and nuclear translocation of β-catenin. This occurred concomitantly with activation of the downstream JAK2/STAT3 pathway. Both cell lines expressed interleukin-6 receptor (IL-6R), and treatment with EGF within 1 h resulted in a several-fold enhancement of mRNA expression of IL-6. Consistent with that, EGF treatment of both OVCA 433 and SKOV3 cell lines resulted in enhanced IL-6 production in the serum-free medium. Exogenous addition of IL-6 to OVCA 433 cells stimulated STAT3 activation and enhanced migration. Blocking antibodies against IL-6R inhibited IL-6 production and EGF- and IL-6-induced migration. Specific inhibition of STAT3 activation by JAK2-specific inhibitor AG490 blocked STAT3 phosphorylation, cell motility, induction of N-cadherin and vimentin expression and IL6 production. These data suggest that the activated status of STAT3 in high-grade ovarian carcinomas may occur directly through activation of EGFR or IL-6R or indirectly through induction of IL-6R signalling. Such activation of STAT3 suggests a rationale for a combination of anti-STAT3 and EGFR/IL-6R therapy to suppress the peritoneal spread of ovarian cancer.

STAT3-mediated differentiation and survival and of myeloid cells in response to granulocyte colony-stimulating factor: role for the cyclin-dependent kinase inhibitor p27(Kip1).

The signal transducer and activator of transcription (STAT) proteins have been implicated in cytokine-regulated proliferation, differentiation and cell survival. Granulocyte colony-stimulating factor (G-CSF), a regulator of granulocytic differentiation, induces a robust and sustained activation of STAT3. Here, we show that introduction of dominant negative (DN) forms of STAT3 interferes with G-CSF-induced differentiation and survival in murine 32D cells. G-CSF induces expression of the cyclin-dependent kinase (cdk) inhibitor p27Kip1 (but not p21Cip1), which is completely blocked by DN-STAT3. The ability of tyrosine-to-phenylalanine substitution mutants of the G-CSF receptor to activate STAT3 strongly correlated with their capacity to induce p27 expression and their ability to mediate differentiation and survival, suggesting a causal relationship between STAT3 activation, p27 expression and the observed cellular responses. We identified a putative STAT binding site in the promoter region of p27 that showed both STAT3 binding in electrophoretic mobility shift assays and functional activity in luciferase reporter assays. Finally, we studied G-CSF-induced responses in primary bone marrow and spleen cells of p27-deficient mice. Compared with wild-type, myeloid progenitors from p27-deficient mice showed significantly increased proliferation and reduced differentiation in response to G-CSF. These findings indicate that STAT3 controls myeloid differentiation, at least partly, via upregulation of p27Kip1.

Multiple signals mediate proliferation, differentiation, and survival from the granulocyte colony-stimulating factor receptor in myeloid 32D cells.

Granulocyte colony-stimulating factor (G-CSF) regulates neutrophil production through activation of its cognate receptor, the G-CSF-R. Previous studies with deletion mutants have shown that the membrane-proximal cytoplasmic domain of the receptor is sufficient for mitogenic signaling, whereas the membrane-distal domain is required for differentiation signaling. However, the function of the four cytoplasmic tyrosines of the G-CSF-R in the control of proliferation, differentiation, and survival has remained unclear. Here we investigated the role of these tyrosines by expressing a tyrosine “null” mutant and single tyrosine “add back” mutants in maturation-competent myeloid 32D cells. Clones expressing the null mutant showed only minimal proliferation and differentiation, with survival also reduced at low G-CSF concentrations. Analysis of clones expressing the add-back mutants revealed that multiple tyrosines contribute to proliferation, differentiation, and survival signals from the G-CSF-R. Analysis of signaling pathways downstream of these tyrosines suggested a positive role for STAT3 activation in both differentiation and survival signaling, whereas SHP-2, Grb2 and Shc appear important for proliferation signaling. In addition, we show that a tyrosine-independent “differentiation domain” in the membrane-distal region of the G-CSF-R appears necessary but not sufficient for mediating neutrophilic differentiation in these cells.

Regulation of granulopoiesis by transcription factors and cytokine signals.

The development of mature granulocytes from hematopoietic precursor cells is controlled by a myriad of transcription factors which regulate the expression of essential genes, including those encoding growth factors and their receptors, enzymes, adhesion molecules, and transcription factors themselves. In particular, C/EBPα, PU.1, CBF, and c-Myb have emerged as critical players during early granulopoiesis. These transcription factors interact with one another as well as other factors to regulate the expression of a variety of genes important in granulocytic lineage commitment. An important goal remains to understand in greater detail how these various factors act in concert with signals emanating from cytokine receptors to influence the various steps of maturation, from the pluripotent hematopoietic stem cell, to a committed myeloid progenitor, to myeloid precursors, and ultimately to mature granulocytes.

The Ikaros gene family: Transcriptional regulators of hematopoiesis and immunity

The Ikaros family of proteins - comprising Ikaros, Aiolos, Helios, Eos and Pegasus - are zinc finger transcription factors. These proteins participate in a complex network of interactions with gene regulatory elements, other family members and a raft of other transcriptional regulators to control gene expression including via chromatin remodelling. In this way, Ikaros family members regulate important cell-fate decisions during hematopoiesis, particularly in the development of the adaptive immune system. Mutation of several family members results in hematological malignancies,especially those of a lymphoid nature. This review describes the key roles of Ikaros proteins in development and disease, their mechanisms of action and gene targets, as well as explaining their evolutionary origins and role in the emergence of adaptive immunity.

Heterologous microarray experiments used to identify the early gene response to heat stress in a coral reef fish

Coral reef fishes are expected to experience rising sea surface temperatures due to climate change. How well tropical reef fishes will respond to these increased temperatures and which genes are important in the response to elevated temperatures is not known. Microarray technology provides a powerful tool for gene discovery studies, but the development of microarrays for individual species can be expensive and time‐consuming. In this study, we tested the suitability of a Danio rerio oligonucleotide microarray for application in a species with few genomic resources, the coral reef fish Pomacentrus moluccensis. Results from a comparative genomic hybridization experiment and direct sequence comparisons indicate that for most genes there is considerable sequence similarity between the two species, suggesting that the D. rerio array is useful for genomic studies of P. moluccensis. We employed this heterologous microarray approach to characterize the early transcriptional response to heat stress in P. moluccensis. A total of 111 gene loci, many of which are involved in protein processing, transcription, and cell growth, showed significant changes in transcript abundance following exposure to elevated temperatures. Changes in transcript abundance were validated for a selection of candidate genes using quantitative real‐time polymerase chain reaction. This study demonstrates that heterologous microarrays can be successfully employed to study species for which specific microarrays have not yet been developed, and so have the potential to greatly enhance the utility of microarray technology to the field of environmental and functional genomics.

Evolution of Class I cytokine receptors

BackgroundThe Class I cytokine receptors have a wide range of actions, including a major role in the development and function of immune and blood cells. However, the evolution of the genes encoding them remains poorly understood. To address this we have used bioinformatics to analyze the Class I receptor repertoire in sea squirt (Ciona intestinalis) and zebrafish (Danio rerio).ResultsOnly two Class I receptors were identified in sea squirt, one with homology to the archetypal GP130 receptor, and the other with high conservation with the divergent orphan receptor CLF-3. In contrast, 36 Class I cytokine receptors were present in zebrafish, including representative members for each of the five structural groups found in mammals. This allowed the identification of 27 core receptors belonging to the last common ancestor of teleosts and mammals.ConclusionThis study suggests that the majority of diversification of this receptor family occurred after the divergence of urochordates and vertebrates approximately 794 million years ago (MYA), but before the divergence of ray-finned from lobe-finned fishes around 476 MYA. Since then, only relatively limited lineage-specific diversification within the different Class I receptor structural groups has occurred.

Zebrafish granulocyte colony-stimulating factor receptor signaling promotes myelopoiesis and myeloid cell migration

Granulocyte colony-stimulating factor receptor (GCSFR) signaling participates in the production of neutrophilic granulocytes during normal hematopoietic development, with a particularly important role during emergency hematopoiesis. This study describes the characterization of the zebrafish gcsf and gcsfr genes, which showed broad conservation and similar regulation to their mammalian counterparts. Morpholino-mediated knockdown of gcsfr and overexpression of gcsf revealed the presence of an anterior population of myeloid cells during primitive hematopoiesis that was dependent on GCSF/GCSFR for development and migration. This contrasted with a posterior domain that was largely independent of this pathway. Definitive myelopoiesis was also partially dependent on a functional GCSF/GCSFR pathway. Injection of bacterial lipopolysaccharide elicited significant induction of gcsf expression and emergency production of myeloid cells, which was abrogated by gcsfr knockdown. Collectively, these data demonstrate GCSF/GCSFR to be a conserved signaling system for facilitating the production of multiple myeloid cell lineages in both homeostatic and emergency conditions, as well as for early myeloid cell migration, establishing a useful experimental platform for further dissection of this pathway.

Virulence of influenza A virus for mouse lung.

The experimental infection of mouse lung with influenza A virus has proven to be an invaluable model for studying the mechanisms of viral adaptation and virulence. These investigations have identified critical roles for the haemagglutinin (HA) and matrix (M) genes of the virus in determining virulence for mouse lung. For the HA gene, the loss of glycosylation sites from the encoded polypeptide or changes which may affect the pH of HA-mediated endosome fusion have been observed following adaptation. These alterations also have the potential to impact on receptor specificity, β inhibitor sensitivity and activation cleavage which may act in concert to account for the increased virulence of adapted strains. For the M gene, two specific changes in the M1 protein have been identified in strains adapted to, or virulent for, mouse lung. These changes are likely to affect pH-dependent association/dissociation of M1 with the viral ribonucleoprotein, and control virulence as well as growth. The role of other genes in mouse lung virulence remains unknown.

Signaling mechanisms of cytokine receptors and their perturbances in disease

Cytokines regulate the proliferation and differentiation of cells through their interaction with specific receptors on the surface of target cells which are coupled to intracellular signal transduction pathways. The cytokine receptor class I superfamily, characterized by structural homology in the extracellular domain, includes receptors for many interleukins and hematopoietic growth factors, but also those of growth hormone, leptin, ciliary neurotrophic factor (CNTF), oncostatin M (OSM), leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1). The receptors for interferons are structurally distinct and have therefore been categorized separately (class II cytokine receptors). The discovery of the JAK/STAT pathway in the early 1990s has been an important step forward in deciphering cytokine mediated signaling. This pathway connects activation of the receptor complexes directly to transcription of genes. Studies of humans and mice, deficient for one of the JAKs or STATs, have revealed crucial roles of these molecules in embryonic development, blood cell formation and immune responses. In addition, recent studies have revealed some of the mechanisms that control the activation of the JAKs and STATs, which contribute to signal intensity and specificity. In this review we will summarize these recent insights and discuss their implications for a variety of pathological conditions.