A high-throughput screening for mammalian cell death effectors identifies the mitochondrial phosphate carrier as a regulator of cytochrome c release.

Functional annotation of complex genomes requires the development of novel experimental platforms with increased capacity. Here, we describe a high-throughput system designed to identify cDNAs whose overexpression induces morphologically distinct cell death modalities. The methodology incorporates two robotized steps, and relies on coexpression of library clones with GFP to reveal the morphological features presented by the dying cells. By using this system we screened 135 000 cDNA clones and obtained 90 independent molecules. Interestingly, three death categories were identified, namely; apoptotic, vacuolated and autophagic. Among the pro-apoptotic clones, we found four members of the mitochondrial carrier family: the phosphate and adenine nucleotide (type 3) transporters, and the mitochondrial carrier homologs (MTCHs) 1 and 2. Expression of these molecules induced cytochrome c release and caspase-9-dependent death. One of them, the phosphate carrier, was able to interact with members of the permeability transition pore complex ANT1 and VDAC1, and its binding to ANT1 was stabilized in the presence of apoptotic activators. Depletion of this carrier by siRNA delayed cytochrome c mobilization and apoptosis. These results attribute a previously undescribed apoptotic function to the phosphate carrier and, more generally, suggest that a common property of various mitochondrial transporters was exploited during evolution to regulate apoptosis.

Lipid rafts and pseudotyping.

Specific interactions between envelope and core proteins govern the membrane assembly of most enveloped viruses. Despite this, mixed infections lead to pseudotyping, the association of the viral cores of one virus with the envelopes of another. How does this occur? We show here that the detergent-insoluble lipid rafts of the plasma membrane function as a natural meeting point for the transmembrane and core components of a phylogenetically diverse collection of enveloped viruses. As a result, viral particles preferentially incorporate both the envelope components of other viruses as well as the extra- and intracellular constituents of host cell lipid rafts, including gangliosides, glycosyl phosphatidylinositol-anchored surface proteins, and intracellular signal transduction molecules. Pharmacological disruption of lipid rafts interferes with virus production.

Regulated commitment of TNF receptor signaling: a molecular switch for death or activation.

Tumor necrosis factor receptor (TNFR) superfamily members can induce a context-dependent apoptosis or cell activation. However, the mechanisms by which these opposing programs are selected remain unclear. We show that in T cells, TNFR2 (TNFRSF1B) signaling is dramatically affected by the intracellular mediator RIP, a protein Ser/Thr kinase required for NF-kappaB activation by TNFR1 (TNFRSF1A). In the presence of RIP, TNFR2 triggers cell death, whereas in the absence of RIP, TNFR2 activates NF-kappaB. RIP is induced during IL2-driven T cell proliferation, and its inhibition reduces susceptibility to TNF-dependent apoptosis. Evidence that signaling outputs are shaped by intracellular constraints helps reconcile conflicting views of TNFR1 and TNFR2 as apoptotic mediators.

RIP mediates tumor necrosis factor receptor 1 activation of NF-kappaB but not Fas/APO-1-initiated apoptosis.

The CD95 (Fas/APO-1) and tumor necrosis factor (TNF) receptor pathways share many similarities, including a common reliance on proteins containing 'death domains' for elements of the membrane-proximal signal relay. We have created mutant cell lines that are unable to activate NF-kappaB in response to TNF. One of the mutant lines lacks RIP, a 74 kDa Ser/Thr kinase originally identified by its ability to associate with Fas/APO-1 and induce cell death. Reconstitution of the line with RIP restores responsiveness to TNF. The RIP-deficient cell line is susceptible to apoptosis initiated by anti-CD95 antibodies. An analysis of cells reconstituted with mutant forms of RIP reveals similarities between the action of RIP and FADD/MORT-1, a Fas-associated death domain protein.

Biphasic control of nuclear factor-kappa B activation by the T cell receptor complex: role of tumor necrosis factor alpha.

The regulation of nuclear factor (NF)-kappa B activation by the T cell receptor (TcR)/CD3 complex in primary human T cells has been studied at various times after activation. Only p50 NF-kappa B protein bound the kappa B element of interleukin-2 receptor (IL-2R) alpha chain promoter on resting T cells. However, immediately after TcR/CD3 cross-linking (after approximately 1 h; immediate) binding of p50.p65 heterodimers was observed. p50.c-rel heterodimers were also detected bound to this sequence at early time points (7-16 h; early), and both remained active at later time points (40 h; late) after activation. This regulation takes place mainly at the level of nuclear translocation of p65 and c-rel, at immediate and early time points. Activation also induced c-rel and p105/p50 mRNA synthesis, but not p65 mRNA whose expression was constitutive. Interestingly, all those early and late events, but not the immediate ones, were inhibited by a neutralizing anti-tumor necrosis factor alpha (TNF-alpha) monoclonal antibody. Similarly, cycloheximide prevented the p65 and c-rel translocation and consequent formation of active binding heterodimers, at early and late times. Cyclosporin A impaired not only early and late, but also immediate events; however, addition of TNF-alpha prevented all inhibition. These results indicate that the regulation of NF-kappa B activation during T cell activation by TcR/CD3 signals is biphasic: TcR/CD3 triggers its immediate translocation, which is transient if no TNF-alpha is present. TNF-alpha, therefore, emerges as the main factor responsible for a second phase of NF-kappa B regulation, controlling both translocation of p65 and c-rel, and new mRNA synthesis for c-rel and p105/p50.

Regulation of interleukin-2 receptor alpha chain expression and nuclear factor.kappa B activation by protein kinase C in T lymphocytes. Autocrine role of tumor necrosis factor alpha.

The regulation of interleukin-2 receptor alpha chain (IL-2R alpha) expression and nuclear factor (NF) activation by protein kinase C (PKC) in resting T cells, has been studied. Treatment of human resting T cells with phorbol esters strongly induced the expression of IL-2R alpha and the activation of NF.kappa B. This activation was due to the translocation of p65 and c-Rel NF.kappa B proteins from cytoplasmic stores to the nucleus, where they bound the kappa B sequence of the IL-2R alpha promoter either as p50.p65 or as p50.c-Rel heterodimers. Interestingly, all of those events were largely indirect and mediated by endogenously secreted tumor necrosis factor alpha (TNF alpha), as they were strongly inhibited by a neutralizing anti-TNF alpha monoclonal antibody. Furthermore, cyclosporin A, which blocked TNF alpha production induced by PKC, strongly inhibited IL-2R alpha and NF.kappa B activation. The addition of either TNF alpha or IL-2 partially recovered cyclosporin A-induced IL-2R alpha inhibition, but only TNF alpha completely recovered NF.kappa B activation. Those results indicate that, in resting T cells, PKC activation has only a triggering role, whereas the endogenously secreted TNF alpha plays an essential role in the quantitative control of the expression of IL-2R alpha chain or NF.kappa B activation.

Control of T lymphocyte activation and IL-2 receptor expression by endogenously secreted lymphokines.

The relative contribution of endogenously secreted lymphokines, TNF-alpha and IL-2, to T cell activation has been studied. Interestingly, a neutralizing anti-TNF-alpha mAb strongly inhibited the proliferation, IL-2R alpha expression, and activation of the nuclear factor (NF)-kappa B induced on human purified T lymphocytes by an immobilized anti-CD3 mAb (imm.anti-CD3). Furthermore, the addition of exogenous TNF-alpha to T cells activated by imm.anti-CD3 or phorbol esters strongly stimulated all those activities. Similarly, neutralizing anti-IL-2R alpha Abs inhibited the cell proliferation and the IL-2R alpha expression induced by imm.anti-CD3, whereas exogenous addition of IL-2 enhanced both activities. However, exogenous addition of IL-2 did not affect NF-kappa B activation. Cyclosporin A, which prevented lymphokine mRNA transcription, inhibited more than 90% of the IL-2R alpha and NF-kappa B levels induced by TCR/CD3-mediated activation. Exogenous TNF-alpha and IL-2 were equally important to the partial recovery of this IL-2R alpha inhibition, and high doses of both lymphokines together completely abolished the inhibitory effect of cyclosporin A. However, only TNF-alpha (but not IL-2) efficiently recovered the nuclear NF-kappa B levels. Those results indicate that the autocrine secretion of TNF-alpha and IL-2, and not the primary stimulus itself, contribute mostly to the regulation of IL-2R; thereby playing a very important role in quantitative control of T cell proliferation and leaving to the specifically TCR/CD3-derived signals the triggering role in T cell activation.

CD4 gene transcription is transiently repressed during differentiation of myeloid cells to macrophage-like cells.

The CD4 glycoprotein, which serves as receptor for human immunodeficiency virus (HIV), is expressed in several types of cells of hematopoietic origin, including T lymphocytes and monocytes. Triggering differentiation of peripheral blood monocytes, monocytic U-937 or promyelocytic HL-60 precursor cells to macrophage-like cells by phorbol ester treatment transiently induced both a rapid reduction in surface CD4, demonstrated by flow-cytometry analysis, and a gradual loss of CD4 mRNA, revealed by Northern-blot analysis. Experiments in HL-60 cells to determine the cause of the observed decay in CD4 mRNA levels suggested that the half-life of CD4 transcripts did not diminish but increased after phorbol ester stimulation. Direct measurement of CD4 gene transcription by run-on analysis indicated that the rate of synthesis of new CD4 mRNA molecules was reduced approximately 10-fold after phorbol ester stimulation, whereas the rate of synthesis of c-fos mRNA resulted in a 2.5-fold increase. These data suggest that phorbol ester treatment specifically reduces CD4 mRNA levels by repressing CD4 gene transcription. These findings may be relevant to understand the regulation of CD4 gene expression during differentiation.

Differential effect of tumour necrosis factor on human thymocyte subpopulations.

We have studied the effect of tumour necrosis factor (TNF) on purified human thymocyte subpopulations. For this purpose human thymocytes were purified by negative selection with three rounds of several antibodies plus complement. TNF was able to co-stimulate in a dose-response manner the proliferation of single positive (SP) CD3+ CD4+ or CD3+ CD8+ thymocytes in the presence of optimal doses of interleukin-2 (IL-2), phytohaemagglutinin (PHA), anti-CD3 antibodies or phorbol esters. However, CD1+ CD3low CD4+ CD8+ cortical thymocytes did not proliferate significantly in response to any stimulus alone or in combination. The TNF proliferative effect on SP thymocytes was blocked by an anti-IL-2R alpha antibody. In addition, TNF enhanced the expression of the IL-2R alpha but not IL-2R beta on the cell surface of CD1- CD3+ SP thymocytes over the levels induced by the other primary stimuli, inducing as a consequence, an increase in the number of high affinity IL-2R. Furthermore, TNF was able to increase IL-2R alpha mRNA levels on SP thymocytes. On the other hand, TNF was mitogenic in the absence of any other stimulus for CD1- CD3- CD4- CD8- prethymocytes, as was IL-2, and this proliferation was not blocked by anti-IL-2R alpha antibodies. Furthermore, the proliferation of this subset in response to IL-2 and TNF was additive. TNF was able to increase directly the cell surface expression of both chains, IL-2R beta and IL-2R alpha, and the IL-2R alpha messenger RNA (mRNA) levels of CD1- CD3- CD4- CD8- prethymocytes. In summary, our results suggest that TNF may have an important role as a co-stimulatory signal in some human thymocyte subpopulations by inducing the expression of IL-2R.

Down-regulation by tumor necrosis factor-alpha of neutrophil cell surface expression of the sialophorin CD43 and the hyaluronate receptor CD44 through a proteolytic mechanism.

Adhesion of human neutrophils to endothelial cells is a crucial step during migration to the extravascular sites of inflammation. A large number of molecules, including the CD44 and LAM-1 antigens, have been described to participate in this process. We have investigated the regulation by human recombinant tumor necrosis factor-alpha (TNF-alpha) of human neutrophil plasma membrane expression of both CD44 and LAM-1 adhesion molecules, as well as that of CD43 sialophorin, which has been involved in adhesion and activation of leukocytes. The expression of these three antigens was down-regulated in neutrophils upon TNF-alpha treatment, as determined by immunofluorescence and immunoprecipitation experiments. However, the expression of other cell surface molecules, such as CD45 or CD11b, was up-regulated. Similar regulatory effects were also observed upon neutrophil treatment with other activating agents such as the chemoattractant peptide formyl-Met-Leu-Phe, the calcium ionophore A23187, or the phorbol ester phorbol 12-myristate 13-acetate. Protease inhibitors virtually abrogated the TNF-alpha-induced down-regulation of CD43 and CD44 expression, but not that of LAM-1, suggesting the involvement of a protease activity in this process. These results underline the role of TNF-alpha on the differential regulation of cell surface expression of neutrophil adhesion molecules, thus implying modifications in the neutrophil adhesive properties.

Role of soluble cytokines on the restricted replication of poliovirus in the monocytic U937 cell line.

Poliovirus infection of monocytic U937 cells is characterized by an incomplete shut-off of host protein synthesis occurring at late times postinfection and by a slow accumulation of viral mRNA which is not efficiently translated. By using exogenous cytokines, neutralizing antibodies to different cytokines and specific cDNA probes, we have studied the involvement of soluble factors on the restricted replication of poliovirus in U937 cells. Our results show that both IFN-gamma and TNF-alpha mRNA levels increased in U937 cells upon poliovirus infection. Moreover, treatment of infected U937 cells with antibodies to IFN-gamma induced a remarkably stronger shut-off of host protein synthesis than that found in control cultures. A role of IFN-gamma in protecting the infected cells from an extensive blockade of host mRNA translation is proposed.

Synergy of tumor necrosis factor with protein kinase C activators on T cell activation.

The ability of tumor necrosis factor (TNF)-alpha to activate T lymphocytes in combination with other stimuli has been studied. TNF was strongly co-mitogenic with low doses of anti-CD3 antibodies or phorbol esters (those which are strong activators of protein kinase C, PKC) but poorly with phytohemagglutinin or concanavalin A. No synergism was seen with the calcium ionophore A23187. TNF was co-mitogenic with several phorbol esters known to activate PKC but was uneffective with inactive phorbol esters such as methyl-phorbol 12-myristate 13-acetate. Furthermore, H-7 a known inhibitor of PKC, inhibited the proliferative response of T cells induced by esters plus TNF. This effect took place at low doses of TNF and was also observed with purified T lymphocytes indicating that the effect of TNF was not dependent on accessory cells. This proliferative effect of TNF was inhibited by an anti-interleukin 2 receptor (IL2R) antibody, MAR 108, which blocks IL2 binding to its receptor. Although PKC activation induced CD25 (IL2R) expression but very little IL2 synthesis, TNF did not synergize by augmenting the synthesis of this lymphokine in peripheral blood lymphocytes stimulated with phorbol esters. By contrast, TNF strongly increased the membrane level of CD25 and to a lesser extent that of the activation antigen, 4F2, over the levels already induced by phorbol esters on T cells. More interestingly, TNF significantly increased the number of high-affinity IL2R on purified T cells in the presence of phorbol 12,13-dibutyrate. Our results indicate that TNF is co-mitogenic with those stimuli which strongly activate PKC and suggest that TNF may play a role on T cell activation increasing the number of effective IL2/IL2R interactions when these are limiting.