Distinct differences between TNF receptor 1- and TNF receptor 2-mediated activation of NFkappaB

Tumor necrosis factor (TNF) signaling is mediated via two distinct receptors, TNFR2 and TNFR1, which shows partially overlapping signaling mechanisms and biological roles. In the present study, TNFR2 and TNFR1 signal transduction mechanisms involved in activation of NFkappaB and CMV promoter-enhancer were compared with respect to their susceptibility towards inhibitors of intracellular signaling. For this, we used SW480 cells, where we have shown that TNF-signaling can occur independently through each of the two receptors. The TNFR1 response was inhibited by D609, bromophenacyl bromide (BPB), nordihydroguararetic acid (NDGA), and by sodium salicylate, while TNFR2-mediated activation of NFkappaB and CMV promoter-enhancer was resistant to these compounds. The signaling mechanisms known to be affected by these inhibitors include phospholipases as well as redox- and pH-sensitive intracellular components. Our results imply that TNFR2 signaling involved in NFkappaB activation proceeds independently of these inhibitor-sensitive signaling components, indicating distinct signaling pathways not shared with TNFR1.     

Cyclooxygenase-dependent signalling: molecular events and consequences

Non-steroidal anti-inflammatory drugs (NSAIDs) currently attract large interest. Next to pain relief, NSAIDs have important anti-thrombotic and anti-oncogenic effects. NSAIDs exert their action by inhibition of cyclooxygenase, the enzyme responsible for the production of prostanoids. Prostanoid signal transduction is still poorly understood, but it has become clear that these inflammatory lipids influence cellular physiology at three different levels: (1) activation of a 7 x transmembrane receptor coupled to heterotrimeric G proteins, (2) the inhibition of inflammation by activating corticosteroid-like receptors, (3) participation in receptor protein tyrosine kinase signal transduction. In this review prostanoid signalling at these three different levels will be reviewed and the relevance in (patho)physiological processes will be evaluated.     

Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.     

TNF signalling in tooth development

Mammalian tooth development has served as an excellent model system to investigate the intricate, interactive mechanisms of patterning, morphogenesis and cytodifferentiation during organogenesis. Teeth develop from interactions between epithelium and neural crest-derived (ecto)mesenchyme that are largely mediated by ligand-receptor signalling. It is well-established that signalling molecules of the Bmp, Fgf, Wnt and Hedgehog families, are involved at multiple stages of tooth development. Recently, however, a specific role for molecules belonging to the TNF-family of ligands in tooth morphogenesis has been identified, suggesting that this pathway, acting to activate NF-kappaB, has played an important role in the development and evolution of tooth number and shape.     

Reciprocal priming between receptor tyrosine kinases at recycling endosomes orchestrates cellular signalling outputs

Integration of signalling downstream of individual receptor tyrosine kinases (RTKs) is crucial to fine‐tune cellular homeostasis during development and in pathological conditions, including breast cancer. However, how signalling integration is regulated and whether the endocytic fate of single receptors controls such signalling integration remains poorly elucidated. Combining quantitative phosphoproteomics and targeted assays, we generated a detailed picture of recycling‐dependent fibroblast growth factor (FGF) signalling in breast cancer cells, with a focus on distinct FGF receptors (FGFRs). We discovered reciprocal priming between FGFRs and epidermal growth factor (EGF) receptor (EGFR) that is coordinated at recycling endosomes. FGFR recycling ligands induce EGFR phosphorylation on threonine 693. This phosphorylation event alters both FGFR and EGFR trafficking and primes FGFR‐mediated proliferation but not cell invasion. In turn, FGFR signalling primes EGF‐mediated outputs via EGFR threonine 693 phosphorylation. This reciprocal priming between distinct families of RTKs from recycling endosomes exemplifies a novel signalling integration hub where recycling endosomes orchestrate cellular behaviour. Therefore, targeting reciprocal priming over individual receptors may improve personalized therapies in breast and other cancers.     

Melatonin receptor pharmacology: toward subtype specificity

The recent cloning of three distinct melatonin receptor subtypes (Mel_1a, Mel_1b and Mel_1c) which are part of a new family of G-protein coupled receptors, and probably mediate the physiological actions of the hormone, has spurred interest in the design of analogues with subtype selectivity. The 5-methoxyl and N-acetyl groups of melatonin are important for binding to and activation of the receptor. The indole nucleus serves to hold these two groups at the correct distance from one another and allows them to adopt the required orientation for interaction with the receptor binding pocket. We have investigated the subtype selectivity of a number of analogues of melatonin in which the structure has systematically been modified in order to probe the similarities and differences in the interaction of ligand and receptor subtype. At all three subtypes 5-methoxyl and N-acetyl groups of melatonin are important for high affinity binding. However, replacing the 5-methoxyl group (eg with 5-H, 5-OH, 5-Me or 5-BzO) reduces affinity much less at the Mel_1b receptor subtype than at either Mel_1a or Mel_1c cloned subtypes. This suggests differences between the Mel_1b and Mel _1a/1c subtypes in the size and shape of the binding pocket or in the manner in which melatonin interacts with the receptor at this position. Further studies have revealed that analogues with longer N-acyl carbon chains behave similarly at each subtype. These observations suggest that the ‘pocket’ into which the N-acetyl group fits is very similar for each subtype. Substitutions at the 2-position on the indole ring improved affinity at each receptor subtype but did not give selective analogues. The systematic ‘mapping’ of the requirements for binding at each receptor subtype should allow the design of more selective agonists and antagonists, which will be valuable tools for the characterization and classification of functional melatonin receptors.     

Negative receptor signalling

Binding of external factors to cell membrane receptors triggers intracellular signalling pathways that ultimately determine if the cell proliferates, differentiates or undergoes apoptosis. Activated receptors also initiate a cascade of events, called negative receptor signalling, that decreases the amplitude of positive signals and modulates the level of cell stimulation. Recent studies have revealed that negative signalling by receptor tyrosine kinases involves coordinated action of ubiquitin ligases (i.e. Cbl), adaptor proteins (i.e. Grb2 and CIN85), inhibitory molecules (i.e. Sprouty), cytoplasmic kinases (i.e. activated Cdc42-associated kinase) and phosphoinositol metabolites. These inhibitory signals are essential for normal cell functioning, and their deregulation often results in human diseases.     

毒蕈碱乙酰胆碱受体新成员:孤儿受体m5

m5受体是M受体家族一个新成员,与其它亚型相比,由于缺乏高选择性配体和标记条件以及富含m5受体的组织来源,目前对其分子结构、结合特性、组织分布、生理功能等知之甚少。随着免疫沉淀、原位杂交和药理学标记方法的发展,现已证实m5受体主要分布于大脑皮质最外层,海马齿状回、CAl、CA2区,嗅结节,横核以及外周血淋巴细胞、单核细胞、睫状肌等部位,提示m5受体在调节大脑和外周组织胆碱能神经活动方面具有独特而重要的作用,有可能成为治疗帕金森病、阿尔采末病、局灶性脑缺血等疾病新的药物靶点。     

Characterisation of monoclonal antibodies to the TNF and TNF receptor families

Tumour necrosis factor (TNF) family ligands and their corresponding receptors play important roles in the immune system and are involved in immune regulation such as lymphoid development, cell proliferation, differentiation, activation and death. Antibodies against these ligands and receptors together with Fc-fusion proteins, have been particularly useful as immunological tools in addressing the underlying involvement of these proteins in these contexts and furthermore, have given us hope in using them as potential therapeutic agents. Over last few years, there have been many additions to these ever-growing TNF family ligands and their receptors. Here, we have generated and characterised a set of monoclonal antibodies, together with mAbs from the HLDA workshop, against DcR1, DcR2, DR4, DR5, TRAIL, APRIL, BAFF, BAFF-R, BCMA, and TACI, which may be useful in phenotypic and functional studies of the role of TNF and TNF receptor family in immune function and regulation in relation to health and disease.     

Differential role of TNF receptors in cellular trafficking of intact TNF.

BACKGROUND/AIMS: Although ligand signaling and degradation within the cell have received much attention, few studies have quantified the role of receptors on the transcytosis of ligand into and out of the cell in intact form. Accordingly, we determined the differential role of the two receptors for tumor necrosis factor alpha (TNFR1, TNFR2) on cellular transcytosis. METHODS: TNFR1 and TNFR2 were overexpressed in HEK293 cells by transient transfection. Cell surface binding, endocytosis, and exocytosis of (125)I-TNF were quantified. Degradation was determined by acid precipitation and size-exclusion chromatography. RESULTS: TNFR1- mediated uptake of TNF was faster than TNFR2-mediated uptake of TNF. TNFR2, however, exhibited greater capacity, leading to a higher percentage release of TNF into the exocytosis medium. Rather than being degraded, most of the TNF inside the cell remained intact for 1 h. Both receptors exerted protective roles against degradation, but there was no cooperativity between them. CONCLUSION: The effects of TNFR1 and TNFR2 in shepherding TNF across the cell illustrate the differential roles of receptors on the cellular trafficking of the ligand in intact form so as to facilitate its biological effects.     

Interplay between mitochondria and cellular calcium signalling

Mitochondria are increasingly ascribed central roles in vital cell signalling cascades. These organelles are now recognised as initiators and transducers of a range of cell signals, including those central to activation and amplification of apoptotic cell death. Moreover, as the main source of cellular ATP, mitochondria must be responsive to fluctuating energy demands of the cell. As local and global fluctuations in calcium concentration are ubiquitous in eukaryotic cells and are the common factor in a dizzying array of intra- and inter-cellular signalling cascades, the relationships between mitochondrial function and calcium transients is currently a subject of intense scrutiny. It is clear that mitochondria not only act as local calcium buffers, thus shaping spatiotemporal aspects of cytosolic calcium signals, but that they also respond to calcium uptake by upregulating the tricarboxylic acid cycle, thus reacting metabolically to local signalling. In this chapter we review current knowledge of mechanisms of mitochondrial calcium uptake and release and discuss the consequences of mitochondrial calcium handling for cell function, particularly in conjunction with mitochondrial oxidative stress.