Linear ubiquitination prevents inflammation and regulates immune signalling

Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKγ or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene (Sharpin(cpdm/cpdm)) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1β was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling.     

Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes

Following the transfer into fertilized mouse eggs of recombinant genes composed of the upstream region of the rat insulin II gene linked to sequences coding for the large-T antigen of simian virus 40, large-T antigen is detected exclusively in the beta-cells of the endocrine pancreas of transgenic mice. The alpha- and delta-cells normally found in the islets of Langerhans are rare and disordered. Well-vascularized beta-cell tumours arise in mice harbouring and inheriting these hybrid oncogenes.     

Diabetes mellitus and genetically programmed defects in beta-cell function.

The pathways that control insulin secretion and regulate pancreatic beta-cell mass are crucial in the development of diabetes mellitus. Maturity-onset diabetes of the young comprises a number of single-gene disorders affecting pancreatic beta-cell function, and the consequences of mutations in these genes are so serious that diabetes develops in childhood or adolescence. A genetic basis for the more common form of type 2 diabetes, which affects 10-20% of adults in many developed countries, is less clear cut. It is also characterized by abnormal beta-cell function, but other tissues are involved as well. However, in both forms identification of causative and susceptibility genes are providing new insight into the control of insulin action and secretion, as well as suggesting new treatments for diabetes.     

Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases

The receptor for advanced glycation end products (RAGE), a multi-ligand member of the immunoglobulin superfamily of cell surface molecules, interacts with distinct molecules implicated in homeostasis, development and inflammation, and certain diseases such as diabetes and Alzheimer's disease. Engagement of RAGE by a ligand triggers activation of key cell signalling pathways, such as p21ras, MAP kinases, NF-kappaB and cdc42/rac, thereby reprogramming cellular properties. RAGE is a central cell surface receptor for amphoterin, a polypeptide linked to outgrowth of cultured cortical neurons derived from developing brain. Indeed, the co-localization of RAGE and amphoterin at the leading edge of advancing neurites indicated their potential contribution to cellular migration, and in pathologies such as tumour invasion. Here we demonstrate that blockade of RAGE-amphoterin decreased growth and metastases of both implanted tumours and tumours developing spontaneously in susceptible mice. Inhibition of the RAGE-amphoterin interaction suppressed activation of p44/p42, p38 and SAP/JNK MAP kinases; molecular effector mechanisms importantly linked to tumour proliferation, invasion and expression of matrix metalloproteinases.     

The status of Wnt signalling regulates neural and epidermal fates in the chick embryo

The acquisition of neural fate by embryonic ectodermal cells is a fundamental step in the formation of the vertebrate nervous system. Neural induction seems to involve signalling by fibroblast growth factors (FGFs) and attenuation of the activity of bone morphogenetic protein (BMP). But FGFs, either alone or in combination with BMP antagonists, are not sufficient to induce neural fate in prospective epidermal ectoderm of amniote embryos. These findings suggest that additional signals are involved in the specification of neural fate. Here we show that the state of Wnt signalling is a critical determinant of neural and epidermal fates in the chick embryo. Continual Wnt signalling blocks the response of epiblast cells to FGF signals, permitting the expression and signalling of BMP to direct an epidermal fate. Conversely, a lack of exposure of epiblast cells to Wnt signals permits FGFs to induce a neural fate.     

S-glutathionylation uncouples eNOS and regulates its cellular and vascular function

Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O(2)(?-)), which are key mediators of cellular signalling. In the presence of Ca(2+)/calmodulin, eNOS produces NO, endothelial-derived relaxing factor, from l-arginine (l-Arg) by means of electron transfer from NADPH through a flavin containing reductase domain to oxygen bound at the haem of an oxygenase domain, which also contains binding sites for tetrahydrobiopterin (BH(4)) and l-Arg. In the absence of BH(4), NO synthesis is abrogated and instead O(2)(?-) is generated. While NOS dysfunction occurs in diseases with redox stress, BH(4) repletion only partly restores NOS activity and NOS-dependent vasodilation. This suggests that there is an as yet unidentified redox-regulated mechanism controlling NOS function. Protein thiols can undergo S-glutathionylation, a reversible protein modification involved in cellular signalling and adaptation. Under oxidative stress, S-glutathionylation occurs through thiol-disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione. Cysteine residues are critical for the maintenance of eNOS function; we therefore speculated that oxidative stress could alter eNOS activity through S-glutathionylation. Here we show that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in O(2)(?-) generation primarily from the reductase, in which two highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. We show that eNOS S-glutathionylation in endothelial cells, with loss of NO and gain of O(2)(?-) generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents, which reverse this S-glutathionylation. Thus, S-glutathionylation of eNOS is a pivotal switch providing redox regulation of cellular signalling, endothelial function and vascular tone.     

The ageing systemic milieu negatively regulates neurogenesis and cognitive function

In the central nervous system, ageing results in a precipitous decline in adult neural stem/progenitor cells and neurogenesis, with concomitant impairments in cognitive functions. Interestingly, such impairments can be ameliorated through systemic perturbations such as exercise. Here, using heterochronic parabiosis we show that blood-borne factors present in the systemic milieu can inhibit or promote adult neurogenesis in an age-dependent fashion in mice. Accordingly, exposing a young mouse to an old systemic environment or to plasma from old mice decreased synaptic plasticity, and impaired contextual fear conditioning and spatial learning and memory. We identify chemokines--including CCL11 (also known as eotaxin)--the plasma levels of which correlate with reduced neurogenesis in heterochronic parabionts and aged mice, and the levels of which are increased in the plasma and cerebrospinal fluid of healthy ageing humans. Lastly, increasing peripheral CCL11 chemokine levels in vivo in young mice decreased adult neurogenesis and impaired learning and memory. Together our data indicate that the decline in neurogenesis and cognitive impairments observed during ageing can be in part attributed to changes in blood-borne factors.     

A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth

Cell growth, an increase in mass and size, is a highly regulated cellular event. The Akt/mTOR (mammalian target of rapamycin) signalling pathway has a central role in the control of protein synthesis and thus the growth of cells, tissues and organisms. A striking example of a physiological context requiring rapid cell growth is tissue repair in response to injury. Here we show that keratin 17, an intermediate filament protein rapidly induced in wounded stratified epithelia, regulates cell growth through binding to the adaptor protein 14-3-3sigma. Mouse skin keratinocytes lacking keratin 17 (ref. 4) show depressed protein translation and are of smaller size, correlating with decreased Akt/mTOR signalling activity. Other signalling kinases have normal activity, pointing to the specificity of this defect. Two amino acid residues located in the amino-terminal head domain of keratin 17 are required for the serum-dependent relocalization of 14-3-3sigma from the nucleus to the cytoplasm, and for the concomitant stimulation of mTOR activity and cell growth. These findings reveal a new and unexpected role for the intermediate filament cytoskeleton in influencing cell growth and size by regulating protein synthesis.     

Ras, PI(3)K and mTOR signalling controls tumour cell growth

All eukaryotic cells coordinate cell growth with the availability of nutrients in their environment. The mTOR protein kinase has emerged as a critical growth-control node, receiving stimulatory signals from Ras and phosphatidylinositol-3-OH kinase (PI(3)K) downstream from growth factors, as well as nutrient inputs in the form of amino-acid, glucose and oxygen availability. Notably, components of the Ras and PI(3)K signalling pathways are mutated in most human cancers. The preponderance of mutations in these interconnected pathways suggests that the loss of growth-control checkpoints and promotion of cell survival in nutrient-limited conditions may be an obligate event in tumorigenesis.     

Segregation of receptor and ligand regulates activation of epithelial growth factor receptor

Interactions between ligands and receptors are central to communication between cells and tissues. Human airway epithelia constitutively produce both a ligand, the growth factor heregulin, and its receptors--erbB2, erbB3 and erbB4 (refs 1-3). Although heregulin binding initiates cellular proliferation and differentiation, airway epithelia have a low rate of cell division. This raises the question of how ligand-receptor interactions are controlled in epithelia. Here we show that in differentiated human airway epithelia, heregulin-alpha is present exclusively in the apical membrane and the overlying airway surface liquid, physically separated from erbB2-4, which segregate to the basolateral membrane. This physical arrangement creates a ligand-receptor pair poised for activation whenever epithelial integrity is disrupted. Indeed, immediately following a mechanical injury, heregulin-alpha activates erbB2 in cells at the edge of the wound, and this process hastens restoration of epithelial integrity. Likewise, when epithelial cells are not separated into apical and basolateral membranes ('polarized'), or when tight junctions between adjacent cells are opened, heregulin-alpha activates its receptor. This mechanism of ligand-receptor segregation on either side of epithelial tight junctions may be vital for rapid restoration of integrity following injury, and hence critical for survival. This model also suggests a mechanism for abnormal receptor activation in diseases with increased epithelial permeability.     

Integrin cytoplasmic tyrosine motif is required for outside-in alphaIIbbeta3 signalling and platelet function.

Integrins not only bind adhesive ligands, they also act as signalling receptors. Both functions allow the integrin alphaIIbbeta3 to mediate platelet aggregation. Platelet agonists activate alphaIIbbeta3 (inside-out signalling) to allow the binding of soluble fibrinogen. Subsequent platelet aggregation leads to outside-in alphaIIbbeta3 signalling, which results in calcium mobilization, tyrosine phosphorylation of numerous proteins including beta3 itself, increased cytoskeletal reorganisation and further activation of alphaIIbbeta3. Thus, outside-in signals enhance aggregation, although the mechanisms and functional consequences of specific signalling events remain unclear. Here we describe a mouse that expresses an alphaIIbbeta3 in which the tyrosines in the integrin cytoplasmic tyrosine motif have been mutated to phenylalanines. These mice are selectively impaired in outside-in alphaIIbbeta3 signalling, with defective aggregation and clot-retraction responses in vitro, and an in vivo bleeding defect which is characterized by a pronounced tendency to rebleed. These data provide evidence for an important role of outside-in signalling in platelet physiology. Furthermore, they identify the integrin cytoplasmic tyrosine motif as a key mediator of beta-integrin signals and a potential target for new therapeutic agents.