T3-Ti receptor triggering of T8+ suppressor T cells leads to unresponsiveness to interleukin-2

T3-associated disulphide linked heterodimers (Tin) comprised of clonally unique alpha-chains of molecular weight (MW) 49,000-54,000 and beta chains of MW 43,000 have been identified as the antigen receptors on human cytotoxic effector and inducer T-lymphocytes. Crosslinking of Ti molecules by either the appropriate nominal antigen/MHC specificity or anti-clonotypic monoclonal antibody results in clonal expansion of such cells via induction of IL-2 receptor expression, endogenous IL-2 release and IL-2-IL-2 receptor interaction. To determine whether analogous antigen receptor molecules and autocrine growth mechanisms are utilized by suppressor T-cells, we produced an anti-clonotypic monoclonal antibody against a non-cytotoxic T8+ suppressor T-cell, T8AC6, which defines a T3-associated disulphide-linked heterodimer of similar molecular weight to the above clonotypes. We find that Te-Ti triggering of suppressor clones (T8AC6, T8AC7 or T8RW) does not result in IL-2 production or T-cell proliferation and in contrast to inducer clones, also leads to a transient IL-2 unresponsive state. We suggest that such T3-Ti receptor mediated autoregulation of suppressor T-cell growth is necessary in the facilitation of initial inducer T-cell activation following antigenic perturbation.     

Human transforming growth factors induce tyrosine phosphorylation of EGF receptors

Cultured cell lines of human tumour origin as well as cells transformed by various RNA tumour viruses secrete low molecular weight polypeptide transforming growth factors (TGFs). In addition to competing with epidermal growth factor (EGF) for binding to its cellular receptor, TGFs can transform morphologically fibroblast and epithelial cells in culture. In view of accumulating evidence that tyrosine phosphorylation activity is associated with the transforming genes of various tumour viruses, we determined whether phosphotyrosine levels were elevated in these human tumour cells. We show here that TGFs produced by human tumour cells induce phosphorylation of specific tyrosine acceptor sites in the 160,000-molecular weight (160 K) EGF receptor.     

Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface.

Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional alpha-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as 'COAT-platelets', bind additional alpha-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and alpha2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an Mr of 176, and fibrinogen isolated from COAT-platelets contains covalently linked serotonin. Synthetic bovine serum albumin-(serotonin)6 binds selectively to COAT-platelets and also inhibits the retention of procoagulant proteins on COAT-platelets. These data indicate that COAT-platelets use serotonin conjugation to augment the retention of procoagulant proteins on their cell surface through an as yet unidentified serotonin receptor.     

Identity of the proliferating cell nuclear antigen and cyclin

Studies of growth regulation and cellular transformation will be assisted by the identification of proteins that are preferentially synthesized in dividing cells. The 'proliferating cell nuclear antigen' ( PCNA ), distinguished by its apparent association with cell division, is defined by reaction with an antibody found in the autoimmune disease systemic lupus erythematosus (SLE). This antibody reacts with proliferating cells including tumour cells but gives weak or undetectable immunofluorescence with resting cells of normal tissues. Peripheral blood lymphocytes are devoid of PCNA until activated by mitogen in vitro. In synchronized cultures its level and distribution fluctuate through the cell cycle, with a striking accumulation in the nucleolus late in the G1 phase and early in the S phase. Many of these properties are shared by ' cyclin '. This nuclear protein, identified by its position in a two-dimensional separation of cell proteins, is also transformation-sensitive and is preferentially synthesized in the S phase. We establish here that PCNA and cyclin are identical, and show that PCNA is an acidic nuclear protein of apparent molecular weight 35,000.     

Domains specifying thrombin-receptor interaction.

Platelet activation by the coagulation protease thrombin is central to arterial thrombosis, a major cause of morbidity and mortality. We recently isolated a complementary DNA encoding the platelet thrombin receptor. The extracellular amino-terminal extension of this seven transmembrane domain receptor contains the putative thrombin cleavage site LDPR/S which is critical for receptor activation. By replacing this cleavage site with the cleavage site for enterokinase, we have created a functional enterokinase receptor. This result demonstrates that all information necessary for receptor activation is provided by receptor proteolysis. Nanomolar enterokinase concentrations are required to activate this new receptor, in contrast to the picomolar thrombin concentrations that activate wild-type thrombin receptor. We identified a receptor domain critical for thrombin's remarkable potency at its receptor. This domain resembles the carboxyl tail of the leech anticoagulant hirudin and functions by binding to thrombin's anion-binding exosite. Our studies thus define a model for thrombin-receptor interaction. The utility of this model was demonstrated by the design of novel thrombin inhibitors based on receptor peptides.     

PPARγ激动剂诱导HT-29凋亡及周期阻滞的作用

PPAR属于核受体超家族,与特异配体结合后调控一些基因的表达,这些受调控的基因涉及脂质的代谢,糖尿病以及肿瘤等多个方面.目的是研究PPAR γ激动剂罗格列酮诱导结肠癌细胞HT-29凋亡及细胞周期阻滞的作用,并对其机制做相应的探讨.试验结果显示,罗格列酮可诱导HT-29细胞发生凋亡,并阻滞细胞于G1期,此效果伴随着Bcl-2的表达降低,p21的表达升高.罗格列酮在升高PPAR γ表达的同时,也激活了细胞内ERK的传导通路.因此,罗格列酮是通过诱导结肠癌细胞凋亡及周期阻滞而发挥其抗肿瘤作用,此作用为PPAR γ依赖的,并且与激活ERK通路有关.这些研究结果提示PPAR γ有望成为结肠癌治疗的分子靶点.     

Dendritic cell PAR1-S1P3 signalling couples coagulation and inflammation

Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.     

An artefact explains the apparent association of the transferrin receptor with a ras gene product

There is substantial evidence implicating ras genes in a number of human neoplasms. The ras genes of several human tumours display mutational changes which are likely to be responsible for their transforming activity. Normal cells also express ras genes, over-expression of which can induce cellular transformation. ras genes encode proteins of approximately 21,000 molecular weight (MW) (p21) that are localized to the inner surface of the plasma membrane. Much effort is being focused on the elucidation of the physiological function of ras-encoded proteins in normal and transformed cells, concentrating on interactions between p21 and other cellular elements. Recently, Finkel and Cooper reported that p21 in extracts of human bladder carcinoma cells is involved in a molecular complex with the transferrin receptor of these cells. This report aroused considerable interest, particularly as expression of the transferrin receptor has been linked to cell proliferation. I present here evidence that the apparent association of p21 and the transferrin receptor is an artefact of the immunoprecipitation technique.     

Effect of thrombin on the apoptosis of hippocampal neurons in vitro

Hippocampal neurons were treated by thrombin and thrombin receptor activating peptides (TRAP). Cell survival rate was decreased in a dose-dependent manner by MTT assay. The numbers of apoptotic cell and apoptotic rate of hippocampal neurons treated by different concentrations of thrombin were increased in a dose-dependent manner by terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end-labeling (TUNEL) method and Flow Cytometry. When the concentration of thrombin is 40 U/mL, TUNEL positive cells and apoptotic rate of hippocampal neurons reached peak value, were 27.3±4.0 and (29.333±4.633)%, respectively. Immunocytochemistry assay show that Bcl-2 protein expression was down-regulated and Bax protein expression was up-regulated with the concentration of thrombin increased. TRAP can mimic the effect of thrombin to induce apoptosis on hippocampal neurons. These data demonstrated that thrombin induced hippocampal neuron apoptosis in a dose-dependent manner through activating protease-activated protein-1 (PAR-1). The change in expression of Bcl-2 and Bax was related with the effect of high concentration thrombin induced apoptosis on hippocampal neurons. Foundation item: Supported by the Natural Science Foundation of Hainan Province (N30215) Biography: YANG Wen-qiong (1968-), female, Ph.D. candidate, research direction: cerebrovascular disease.     

PAR3 is a cofactor for PAR4 activation by thrombin

Identification of the mechanisms by which the coagulation protease thrombin activates platelets is critical for understanding haemostasis and thrombosis. Thrombin activates cells at least in part by cleaving protease-activated G-protein-coupled receptors (PARs). PAR3 and PAR4 are thrombin receptors expressed in mouse platelets. Inhibition of thrombin binding to mPAR3 (ref. 4) and knockout of the mPAR3 gene inhibited mouse platelet activation at low but not high concentrations of thrombin. Thus PAR3 is important for thrombin signalling in mouse platelets. Expression of human PAR3 in heterologous expression systems reliably resulted in responsiveness to thrombin. Curiously, despite its importance for the activation of mouse platelets by thrombin, mouse PAR3 (mPAR3) did not lead to thrombin signalling even when overexpressed. We now report that mPAR3 and mPAR4 interact in a novel way: mPAR3 does not itself mediate transmembrane signalling but instead functions as a cofactor for the cleavage and activation of mPAR4 by thrombin. This establishes a paradigm for cofactor-assisted PAR activation and for a G-protein-coupled receptor's acting as an accessory molecule to present ligand to another receptor.     

The molecular mechanism of different sensitivity of breast cancer cell lines to TRAIL

Although Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of various cancer cells, some caner cell lines are resistant to TRAIL-induced cell death. To investigate the molecular mechanisms underlying TRAIL-resistance, two human breast cancer cell lines, MCF-7 (resistant to TRAIL) and MDA-MB-231 (sensitive to TRAIL), were used as a model system to analyze the different sensitivities to TRAIL cytotoxicity. PKCδ inhibitor rottlerin, but not MEK and ERK1/2 inhibitor U0126 nor PI3K inhibitor LY294002, was shown to enhance TRAIL-induced apoptosis in MCF-7 cells significantly, suggesting that PKCδ might play an important role in the resistance of MCF-7 cells to TRAIL. In contrast, rottlerin, U0126, and Ly294002 had no effect on MDA-MB-231 apoptosis induced by TRAIL under the same conditions. Further experiment showed that the combination of rottlerin and TRAIL cleaved PARP in the MCF-7 cells synergistically, but not in the MDA-MB-231 cells. The role of PKCδ in TRAIL-resistant MCF-7 cells was confirmed by knocking down the endogenous PKCδ expression using RNAi technology. Furthermore, caspase-3 reconstitution in MCF-7 cells was unable to alter PKCδ expression, suggesting that innate caspase-3 deficient in the cells does not cause PKCδ high expression. These data provide evidence for the first time that PKCδ plays a critical role in breast cancer cell lines to TRAIL cytotoxicity.     

The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3

CD2 (known also as T11 (ref. 1), LFA-2 (ref. 2) and the erythrocyte rosette receptor (ref. 3] is a functionally important T lymphocyte surface glycoprotein of relative molecular mass 50,000 to 58,000 (Mr 50-58 K) which appears early in thymocyte ontogeny and is present on all mature T cells. Monoclonal antibodies to CD2 inhibit cytotoxic T-lymphocyte (CTL)-mediated killing by binding to the T lymphocyte and blocking adhesion to the target cell. Such antibodies also inhibit T helper cell responses including antigen-stimulated proliferation, interleukin-2 (IL-2) secretion, and IL-2 receptor expression. Certain combinations of monoclonal antibodies to CD2 epitopes trigger proliferation of peripheral blood T lymphocytes, cytotoxic effector function and expression of IL-2 receptors by thymocytes, resulting in thymocyte proliferation in the presence of exogenous IL-2 (ref. 11). These findings suggest that CD2 can function in signalling as well as being an adhesion molecule. To understand the role of CD2 in T-cell adhesion and activation, it is essential to define its natural ligand. Our previous observation that purified CD2 inhibits rosetting of T lymphocytes with sheep erythrocytes and can be absorbed by sheep erythrocytes suggested it also might bind with detectable affinity to human cells. We now report that CD2 binds to a cell-surface antigen known as lymphocyte function-associated antigen-3 (LFA-3) with high affinity, and can mediate adhesion of lymphoid cells via interaction with LFA-3.     

Fidelity in planar cell polarity signalling

The polarity of Drosophila wing hairs displays remarkable fidelity. Each of the approximately 30,000 wing epithelial cells constructs an actin-rich prehair that protrudes from its distal vertex and points distally. The distal location and orientation of the hairs is virtually error free, thus forming a nearly perfect parallel array. This process is controlled by the planar cell polarity signalling pathway. Here we show that interaction between two tiers of the planar cell polarity signalling mechanism results in the observed high fidelity. The first tier, mediated by the cadherin Fat, dictates global orientation by transducing a directional signal to individual cells. The second tier, orchestrated by the 7-pass transmembrane receptor Frizzled, aligns each cell's polarity with that of its neighbours through the action of an intercellular feedback loop, enabling polarity to propagate from cell to cell. We show that all cells need not respond correctly to the presumably subtle signal transmitted by Fat. Subsequent action of the Frizzled feedback loop is sufficient to align all the cells cooperatively. This economical system is therefore highly robust, and produces virtually error-free arrays.     

Sortilin is essential for proNGF-induced neuronal cell death

Sortilin (approximately 95 kDa) is a member of the recently discovered family of Vps10p-domain receptors, and is expressed in a variety of tissues, notably brain, spinal cord and muscle. It acts as a receptor for neurotensin, but predominates in regions of the nervous system that neither synthesize nor respond to this neuropeptide, suggesting that sortilin has additional roles. Sortilin is expressed during embryogenesis in areas where nerve growth factor (NGF) and its precursor, proNGF, have well-characterized effects. These neurotrophins can be released by neuronal tissues, and they regulate neuronal development through cell survival and cell death signalling. NGF regulates cell survival and cell death via binding to two different receptors, TrkA and p75NTR (ref. 10). In contrast, proNGF selectively induces apoptosis through p75NTR but not TrkA. However, not all p75NTR-expressing cells respond to proNGF, suggesting that additional membrane proteins are required for the induction of cell death. Here we report that proNGF creates a signalling complex by simultaneously binding to p75NTR and sortilin. Thus sortilin acts as a co-receptor and molecular switch governing the p75NTR-mediated pro-apoptotic signal induced by proNGF.     

A C. elegans Ror receptor tyrosine kinase regulates cell motility and asymmetric cell division.

Ror kinases are a family of orphan receptors with tyrosine kinase activity that are related to muscle specific kinase (MuSK), a receptor tyrosine kinase that assembles acetylcholine receptors at the neuromuscular junction. Although the functions of Ror kinases are unknown, similarities between Ror and MuSK kinases have led to speculation that Ror kinases regulate synaptic development. Here we show that the Caenorhabditis elegans gene cam-1 encodes a member of the Ror kinase family that guides migrating cells and orients the polarity of asymmetric cell divisions and axon outgrowth. We find that tyrosine kinase activity is required for some of the functions of CAM-1, but not for its role in cell migration. CAM-1 is expressed in cells that require its function, and acts cell autonomously in migrating neurons. Overexpression and loss of cam-1 function result in reciprocal cell-migration phenotypes, indicating that levels of CAM-1 influence the final positions of migrating cells. Our results raise the possibility that Ror kinases regulate cell motility and asymmetric cell division in organisms as diverse as nematodes and mammals.     

The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.     

T cells can distinguish between allogeneic major histocompatibility complex products on different cell types

In the response of T cells to foreign antigens, the ligand for the T cell alpha/beta receptor is presented on a cell surface as a fragment of antigen complexed to one of the membrane molecules encoded in the major histocompatibility complex (MHC). The receptor apparently interacts via its variable elements (V beta, D beta, J beta, V alpha and J alpha) with residues within both the antigen and MHC portion of the ligand. The frequency of T cells responding to a conventional antigen plus self MHC is usually quite low, presumably reflecting the relative rarity of receptors with the particular combination of variable elements to match the antigen/MHC ligand. T cells also respond to allogeneic forms of MHC molecules in the absence of added antigen. In this case the frequency of responding T cells is very high. One hypothesis to explain this observation is that, in the absence of foreign antigen, MHC molecules are complexed to a large array of peptides derived from self-proteins. In this case the combination of the polymorphic MHC amino acid residues and many different self peptides presents so many possible ligands that the likelihood of recognition by a given T cell receptor is quite high. The recent crystallography experiments which revealed a dramatic binding cleft on the face of a human MHC molecule have given impetus to this view, but as yet there is no direct supporting evidence. We have recently described a close association between murine T cell receptors utilizing the V beta 17a element and reactivity to various allogeneic forms of the murine MHC molecule, I-E (ref. 8). In this paper, we show that this I-E ligand is detected on B cells, but not on I-E+ macrophages or fibroblasts expressing a transfected I-E gene. These results strongly suggest a B cell specific product combines with I-E to form the allogeneic ligand for V beta 17a+ receptors and thus support the concept of alloreactivity described above.     

Growth inhibition by protein kinase C late in mitogenesis

The importance of alpha-thrombin in the clotting cascade is well-known, but it is also a potent mitogen. Like many other mitogens, thrombin causes receptor-mediated activation of a phosphatidylinositol-specific phospholipase C (PLC), leading to the release of diacylglycerol and the subsequent activation of protein kinase C (refs 3-6). Protein kinase C is probably important in cell proliferation, as activation of this enzyme by phorbol esters promotes growth in many systems. Some growth factors have tyrosine kinase activity and function without activation of PLC or protein kinase C. In this report we show that alpha-thrombin retains its mitogenicity in vascular smooth muscle cells depleted of protein kinase C. Phorbol-12-myristate-13-acetate (PMA) is found to be a potent growth inhibitor when added to vascular smooth muscle cells with alpha-thrombin. Moreover, growth inhibition is maximal when protein kinase C is activated 4 hours after exposure to thrombin, long after the completion of 'early events' induced by thrombin. Thus, PMA probes an event late in the G1 phase of the cell cycle or at the G1-S transition.     

Social controls on cell survival and cell death.

Programmed cell death occurs in most animal tissues at some stage of their development, but the molecular mechanism by which it is executed is unknown. For some mammalian cells, programmed death seems to occur by default unless suppressed by signals from other cells. Such dependence on specific survival signals provides a simple way to eliminate misplaced cells, for regulating cell numbers and, perhaps, for selecting the fittest cells. But how general is this dependence on survival signals?     

Insulin rapidly stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact cells

Phosphotyrosine-containing proteins are minor components of normal cells which appear to be associated primarily with the regulation of cellular metabolism and growth. The insulin receptor is a tyrosine-specific protein kinase, and one of the earliest detectable responses to insulin binding is activation of this kinase and autophosphorylation of its beta-subunit. Tyrosine autophosphorylation activates the phosphotransferase in the beta-subunit and increases its reactivity toward tyrosine phosphorylation of other substrates. When incubated in vitro with [gamma-32P]ATP and insulin, the purified insulin receptor phosphorylates various proteins on their tyrosine residues. However, so far no proteins other than the insulin receptor have been identified as undergoing tyrosine phosphorylation in response to insulin in an intact cell. Here, using anti-phosphotyrosine antibodies, we have identified a novel phosphotyrosine-containing protein of relative molecular mass (Mr) 185,000 (pp185) which appears during the initial response of hepatoma cells to insulin binding. In contrast to the insulin receptor, pp185 does not adhere to wheat-germ agglutininagarose or bind to anti-insulin receptor antibodies. Phosphorylation of pp185 is maximal within seconds after exposure of the cells to insulin and exhibits a dose-response curve similar to that of receptor autophosphorylation, suggesting that this protein represents the endogenous substrate for the insulin receptor kinase.