Search

Using primary fibroblasts in culture, we have investigated the signal transduction mechanisms by which phorbol esters, a class of tumor promoters, activate the 9E3 gene and its chemokine product the chicken chemotactic and angiogenic factor. This gene is highly stimulated by phorbol 12,13-dibutyrate (PDBu) via three pathways: (i) a small contribution through protein kinase C (the commonly recognized pathway for these tumor promoters), (ii) a contribution involving tyrosine kinases, and (iii) a larger contribution via pathways that can be interrupted by dexamethasone. All three of these pathways converge into the mitogen-activated protein kinases, MEK1/ERK2. Using a luciferase reporter system, we show that although both the AP-1 and PDRIIkB (a NFkappaB-like factor in chickens) response elements are capable of activation in these normal cells, regions of the 9E3 promoter containing them are unresponsive to PDBu stimulation. In contrast, we show for the first time that activation by PDBu occurs through a segment of the promoter containing Elk1 response elements; deletion and mutation of these elements abrogates 9E3/chicken chemotactic and angiogenic factor expression. Electrophoretic mobility shift assays and functional studies using PathDetect systems show that stimulation of the cells by phorbol esters leads to activation of the Elk1 transcription factor, which binds to its element in the 9E3 promoter.

Caveolae are small invaginations of the cell surface that are abundant in mature adipocytes. A recent study (Kanzaki, M., and Pessin, J. E. (2002) J. Biol. Chem. 277, 25867-25869) described novel caveolin- and actin-containing structures associated with the adipocyte cell surface that contain specific signaling proteins. We have characterized these structures, here termed "caves," using light and electron microscopy and observe that they represent surface-connected wide invaginations of the basal plasma membrane that are sometimes many micrometers in diameter. Rather than simply a caveolar domain, these structures contain all elements of the plasma membrane including clathrin-coated pits, lipid raft markers, and non-raft markers. GLUT4 is recruited to caves in response to insulin stimulation. Caves can occupy a significant proportion of the plasma membrane area and are surrounded by cortical actin. Caveolae density in caves is similar to that on the bulk plasma membrane, but because these structures protrude much deeper into the plane of focus of the light microscope molecules such as caveolin and other plasma membrane proteins appear more concentrated in caves. We conclude that the adipocyte surface membrane contains numerous wide invaginations that do not represent novel caveolar structures but rather large surface caves.

Chk1 is a serine-threonine kinase that plays an important role in the DNA damage response, including G(2)/M cell cycle control. UCN-01 (7-hydroxystaurosporine), currently in clinical trials, has recently been shown to be a potent Chk1 inhibitor that abrogates the G(2)/M checkpoint induced by DNA-damaging agents. To understand the structural basis of Chk1 inhibition by UCN-01, we determined the crystal structure of the Chk1 kinase domain in complex with UCN-01. Chk1 structures with staurosporine and its analog SB-218078 were also determined. All three compounds bind in the ATP-binding pocket of Chk1, producing only slight changes in the protein conformation. Selectivity of UCN-01 toward Chk1 over cyclin-dependent kinases can be explained by the presence of a hydroxyl group in the lactam moiety interacting with the ATP-binding pocket. Hydrophobic interactions and hydrogen-bonding interactions were observed in the structures between UCN-01 and the Chk1 kinase domain. The high structural complementarity of these interactions is consistent with the potency and selectivity of UCN-01.

Members of the tumor necrosis factor (TNF) and TNF receptor families play important roles in inducing apoptosis and mediating the inflammatory response. Activated T lymphocytes can trigger the expression of Fas-ligand on non-lymphoid tissue, such as intestinal epithelial cells (IEC), and this, in turn, can induce apoptosis in the T cells. Here, we examine the role of TNFalpha in this feedback regulation. Injection of TNFalpha into mice caused a rapid up-regulation of Fas-ligand mRNA in IEC. TNFalpha-induced activation of the Fas-ligand promoter in IEC requires NF-kappaB as this was blocked by an I-kappaBalphaM super-repressor and by mutation of an NF-kappaB site in the Fas-ligand promoter. Activation of T cells by antigen induced Fas-ligand expression in IEC in vivo in wild type, but not in TNFalpha-/- or TNFR1-/- mice. These results define a novel pathway wherein TNFalpha, produced by activated T cells in the intestine, induce Fas-ligand expression in IEC. This is the first observation that one member of the TNF superfamily mediates the regulation of another family member and represents a potential feedback mechanism controlling lymphocyte infiltration and inflammation in the small intestine.

The expression and activity of epithelial proteinases is under stringent control to prevent aberrant hydrolysis of structural proteins and disruption of tissue architecture. E-cadherin-dependent cell-cell adhesion is also important for maintenance of epithelial structural integrity, and loss of E-cadherin expression has been correlated with enhanced invasive potential in multiple tumor models. To address the hypothesis that there is a functional link between E-cadherin and proteinase expression, we have examined the role of E-cadherin in proteinase regulation. By using a calcium switch protocol to manipulate junction assembly, our data demonstrate that initiation of de novoE-cadherin-mediated adhesive contacts suppresses expression of both relative matrix metalloproteinase-9 levels and net urinary-type plasminogen activator activity. E-cadherin-mediated cell-cell adhesion increases both phosphatidylinositol 3′-kinase (PI3-kinase)-dependent AKT phosphorylation and epidermal growth factor receptor-dependent MAPK/ERK activation. Pharmacologic inhibition of the PI3-kinase pathway, but not the epidermal growth factor receptor/MAPK pathway, prevents E-cadherin-mediated suppression of proteinases and delays junction assembly. Moreover, inhibition of junction assembly with a function-blocking anti-E-cadherin antibody stimulates proteinase-dependent Matrigel invasion. As matrix metalloproteinase-9 and urinary-type plasminogen activator potentiate the invasive activity of oral squamous cell carcinoma, these data suggest E-cadherin-mediated signaling through PI3-kinase can regulate the invasive behavior of cells by modulating proteinase secretion.

Human immunodeficiency virus, type 1 (HIV-1),vprgene encodes a 14-kDa virion-associated protein, which exhibits significant effects on human cells. One important property of Vpr is its ability to induce apoptosis during infection. Apoptotic induction is likely to play a role in the pathogenesis of AIDS. However, the pathway of apoptosis is not clearly defined. In this report we investigate the mechanism of apoptosis induced by HIV-1 Vpr using a Vpr pseudotype viral infection system or adeno delivery of Vpr in primary human lymphoid cells and T-cells. With either vector, HIV-1 Vpr induced cell cycle arrest at the G2/M phase and apoptosis in lymphoid target cells. Furthermore, we observed that with both vectors, caspase 9, but not caspase 8, was activated following infection of human peripheral blood mononuclear cell with either Vpr-positive HIV virions or adeno-delivered Vpr. Activation of the caspase 9 pathway resulted in caspase 3 activation and apoptosis in human primary cells. These effects were coincident with the disruption of the mitochondrial transmembrane potential and induction of cytochrome crelease by Vpr. The Vpr-induced signaling pathway did not induce CD95 or CD95L expression. Bcl-2 overexpressing cells succumb to Vpr-induced apoptosis. These studies illustrate that Vpr induces a mitochondria-dependent apoptotic pathway that is distinct from apoptosis driven by the Fas-FasL pathway.

Nitric oxide and S-nitrosothiols modulate a variety of important physiological activities. In vascular cells, agents that release NO and donate nitrosonium cation (NO(+)), such as S-nitrosoglutathione, are potent inducers of the antioxidant protein heme oxygenase 1 (HO-1) (Foresti, R., Clark, J. E., Green, C. J., and Motterlini, R. (1997) J. Biol. Chem. 272, 18411-18417; Motterlini, R., Foresti, R., Bassi, R., Calabrese, V., Clark, J. E., and Green, C. J. (2000) J. Biol. Chem. 275, 13613-13620). Here, we report that Angeli's salt (AS) (0.25-2 mm), a compound that releases nitroxyl anion (NO(-)) at physiological pH, induces HO-1 mRNA and protein expression in a concentration- and time-dependent manner, resulting in increased heme oxygenase activity in rat H9c2 cells. A time course analysis revealed that NO(-)-mediated HO-1 expression is transient and gradually disappears within 24 h, in accordance with the short half-life of AS at 37 degrees C (t(12) = 2.3 min). Interestingly, multiple additions of AS at lower concentrations (50 or 100 microm) over a period of time still promoted a significant increase in heme oxygenase activity. Experiments performed using a NO scavenger and the NO electrode confirmed that NO(-), not NO, is the species involved in HO-1 induction by AS; however, the effect on heme oxygenase activity can be amplified by accelerating the rate of NO(-) oxidation. N-Acetylcysteine almost completely abolished AS-mediated induction of HO-1, whereas a glutathione synthesis inhibitor (buthionine sulfoximine) significantly decreased heme oxygenase activation by AS, indicating that sulfydryl groups are crucial targets in the regulation of HO-1 expression by NO(-). We conclude that NO(-), in analogy with other reactive nitrogen species, is a potent inducer of heme oxygenase activity and HO-1 protein expression. These findings indicate that heme oxygenase can act both as a sensor to and target of redox-based mechanisms involving NO and extend our knowledge on the biological function of HO-1 in response to nitrosative stress.

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Here we report that a catalytically active derivative (termed LH(N)/A) of the type A neurotoxin from Clostridium botulinum has been coupled to a lectin obtained from Erythrina cristagalli to form a novel conjugate. This conjugate exhibits an in vitro selectivity for nociceptive afferents compared with the anatomically adjacent spinal neurons, as assessed using in vitro primary neuronal culture systems to measure inhibition of release of neurotransmitters. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LH(N)/A or recombinant LH(N)/A purified from Escherichia coli are assessed, and equivalence of the recombinant material are demonstrated. Furthermore, the dependence of inhibition of neurotransmitter release on the cleavage of SNAP-25 is demonstrated through the use of an endopeptidase-deficient LH(N)/A conjugate variant. The duration of action of inhibition of neurotransmitter released by the conjugate in vitro is assessed and is comparable with that observed with Clostridium botulinum neurotoxin. Finally, in vivo electrophysiology shows that these in vitro actions have biological relevance in that sensory transmission from nociceptive afferents through the spinal cord is significantly attenuated. These data demonstrate that the potent endopeptidase activity of clostridial neurotoxins can be selectively retargeted to cells of interest and that inhibition of release of neurotransmitters from a neuronal population of therapeutic relevance to the treatment of pain can be achieved.

Neutrophil recruitment during acute inflammation is triggered by G-protein-linked chemotactic receptors that in turn activate β2integrin (CD18), deemed a critical step in facilitating cell capture and arrest under the shear force of blood flow. A conformational switch in the I domain allosteric site (IDAS) and in CD18 regulates LFA-1 affinity for endothelial ligands including intercellular adhesion molecule 1 (ICAM-1). We examined the dynamics of CD18 activation in terms of the efficiency of neutrophil capture of ICAM-1, and we correlated this with the membrane topography of 327C, an antibody that recognizes the active conformation of CD18 I-like domain. Adhesion increased in direct proportion to chemotactic stimulus rising 7-fold over a log range of interleukin-8 (IL-8). A threshold dose of ∼75 pmIL-8, corresponding to ligation of only ∼10–100 receptors, was sufficient to activate ∼20,000 CD18 and a rapid boost in the capture efficiency on ICAM-1. This was accompanied by a rapid redistribution of active LFA-1, but not Mac-1, into membrane patches, a necessary component for optimum adhesion efficiency. Shear-resistant arrest on a monolayer of ICAM-1 was reversed within minutes of chemotactic stimulation correlating with a shift from high to low affinity CD18 and dispersal of patches of active CD18. Mobility of active CD18 into high avidity patches was dependent on phosphatidylinositol 3-kinase activity and not F-actin polymerization. The data reveal that the number of chemotactic receptors bound and the topography and lifetime of high affinity LFA-1 tightly regulate the efficiency of neutrophil capture on ICAM-1.

Purine nucleoside and nucleobase transporters are of fundamental importance for Trypanosoma brucei and related kinetoplastid parasites because these protozoa are not able to synthesize purines de novo and must salvage the compounds from their hosts. In the studies reported here, we have identified a family of six clustered genes in T. brucei that encode nucleoside/nucleobase transporters. These genes, TbNT2/927, TbNT3, TbNT4, TbNT5, TbNT6, and TbNT7, have predicted amino acid sequences that show high identity to each other and to TbNT2, a P1 type nucleoside transporter recently identified in our laboratory. Expression in Xenopus laevis oocytes revealed that TbNT2/927, TbNT5, TbNT6, and TbNT7 are high affinity adenosine/inosine transporters with K(m) values of <5 microm. In addition, TbNT5, and to a limited degree TbNT6 and TbNT7, also mediate the uptake of the nucleobase hypoxanthine. Ribonuclease protection assays showed that mRNA from all of the six members of this gene family are expressed in the bloodstream stage of the T. brucei life cycle but that TbNT2/927 and TbNT5 mRNAs are also expressed in the insect stage of the life cycle. These results demonstrate that T. brucei expresses multiple purine transporters with distinct substrate specificities and different patterns of expression during the parasite life cycle.

The SlyA protein from Salmonella typhimuriumis a transcription factor that contributes to virulence. It is shown that a slyAmutant is attenuated in the presence of murine macrophages compared with the parent strain. Moreover, after growth in minimal medium, survival of the slyAmutant was reduced. Altered levels of flagellin (fliC), PagC, IroN, and outer membrane proteins suggest that the slyAmutation affects the surface properties of Salmonella. The isolated SlyA protein is a cofactor-free homodimer that recognizes five sites within the promoter region of the slyAgene. One of these sites contained a near perfect inverted repeat TTAGCAAGCTAA. The other four sites contained related sequences. Occupation of the SlyA sites in the slyApromoter prevented open-complex formation, consistent with the pattern of slyA::lacZexpression parental and slyAmutant strains. By combining the footprinting data with potential SlyA binding sites recovered from a pool of random DNA sequences, a consensus was defined and used to probe the NIH Salmonellaunfinished genomes data base. These searches revealed the presence of consensus SlyA sites upstream of omp, ispA, xseB, slyA, and a gene encoding a protein with homology to a hemagglutinin. Accordingly, transcription of an omp::lacZfusion was reduced in a slyAmutant. Given the difficulties in obtaining a comprehensive picture of intracellular gene expression, the definition of the DNA sequence recognized by a transcription factor (SlyA) that is essential for survival in the macrophage environment should allow a complete regulon of genes with altered expression upon exposure to macrophages to be determined once the S. typhimuriumgenome annotation is complete.

Plakophilins are a subfamily of p120-related arm-repeat proteins that can be found in both desmosomes and the nucleus. Among the three known plakophilin members, plakophilin 1 has been linked to a genetic skin disorder and shown to play important roles in desmosome assembly and organization. However, little is known about the binding partners and functions of the most widely expressed member, plakophilin 2. To better understand the cellular functions of plakophilin 2, we have examined its protein interactions with other junctional molecules using co-immunoprecipitation and yeast two-hybrid assays. Here we show that plakophilin 2 can interact directly with several desmosomal components, including desmoplakin, plakoglobin, desmoglein 1 and 2, and desmocollin 1a and 2a. The head domain of plakophilin 2 is critical for most of these interactions and is sufficient to direct plakophilin 2 to cell borders. In addition, plakophilin 2 is less efficient than plakophilin 1 in localizing to the nucleus and enhancing the recruitment of excess desmoplakin to cell borders in transiently transfected COS cells. Furthermore, plakophilin 2 is able to associate with β-catenin through its head domain, and the expression of plakophilin 2 in SW480 cells up-regulates the endogenous β-catenin/T cell factor-signaling activity. This up-regulation by plakophilin 2 is abolished by ectopic expression of E-cadherin, suggesting that these proteins compete for the same pool of signaling active β-catenin. Our results demonstrate that plakophilin 2 interacts with a broader repertoire of desmosomal components than plakophilin 1 and provide new insight into the possible roles of plakophilin 2 in regulating the signaling activity of β-catenin.

From transcription to translation, mRNA is complexed with heterogeneous nuclear ribonucleoproteins (hnRNP proteins) that mediate mRNA processing, export from the nucleus, and delivery into the cytoplasm. Although the mechanism is unknown, export of mature mRNA from the nucleus is a critical regulatory step in gene expression. Analyses of hnRNP proteins have shown that many of these proteins are required for this essential cellular process. In this study, we characterize the Saccharomyces cerevisiaeNab2 protein, which was first identified as a poly(A) RNA-binding protein (Anderson, J. T., Wilson, S. M., Datar, K. V., and Swanson, M. S. (1993) Mol. Cell. Biol.13, 2730–2741). Our work indicates that poly(A) RNA export from the nucleus is dependent upon a functional Nab2 protein; correspondingly, export of Nab2p from the nucleus is dependent upon ongoing RNA polymerase II transcription. Furthermore, we show that Nab2p is modified within its RGG domain by the type I protein-arginine methyltransferase, Hmt1p. Our experiments demonstrate that arginine methylation is required for the export of Nab2p from the nucleus and therefore establish an in vivoeffect of this modification. Overall, these experiments provide evidence that Nab2p is an hnRNP protein that is required for poly(A) RNA export and whose export from the nucleus is regulated by Hmt1p.

Previous in vitrodata on type I collagen self-assembly into fibrils suggested that the amino acid 776–796 region of the α1(I) chain is crucial for fibril formation because it serves as the recognition site for the telopeptide of a docking collagen monomer. We used a natural collagen mutation with a deletion of amino acids 766–801 to confirm the importance of this region for collagen fibril formation. The proband has type III osteogenesis imperfecta and is heterozygous for a COL1A1 IVS 41 A+4→ C substitution. The intronic mutation causes splicing of exon 41, confirmed by sequencing of normal and shorter reverse transcriptase-PCR products. Reverse transcriptase-PCR using RNA from proband dermal fibroblasts and clonal cell lines showed the mutant cDNA was about 15% of total α1(I) cDNA. The mutant transcript is translated; structurally abnormal α chains are demonstrated in the cell layer of proband fibroblasts by SDS-urea-PAGE. The proportion of mutant chains in the secreted procollagen was determined to be 10% by resistance to digestion with MMP-1, since chains lacking exon 41 are missing the vertebral collagenase cleavage site. Secreted proband collagen was used for analysis of kinetics of binding of α1(I) C-telopeptide using an optical biosensor. Telopeptide had slower association and faster dissociation from proband than from normal collagen. Purified proband pC-collagen was used to study fibril formation. The presence of the mutant molecules decreases the rate of fibril formation. The fibrils formed in the presence of 10–15% mutant molecules have strikingly increased length compared with normal collagen, but are well organized, as demonstrated by D-periodicity. These results suggest that some collagen molecules containing the mutant chain are incorporated into fibrils and that the absence of the telopeptide binding region from even a small portion of the monomers interferes with fibril growth. Both abnormal fibrils and slower remodeling may contribute to the severe phenotype.

Galectin-7 is normally expressed in all types of stratified epithelia, but is significantly down-regulated in squamous cell carcinomas. This protein was recently found to be highly inducible by p53 in a colon carcinoma cell line, DLD-1, and designated as PIG1 (for p53-inducedgene 1). We studied transfectants of HeLa and DLD-1 cells ectopically expressing this protein and found that they were more susceptible to apoptosis than control transfectants. This was observed in apoptosis induced by mechanistically distinct stimuli, suggesting that galectin-7 acts on a common point in the apoptosis signaling pathways. Further analyses of actinomycin D-induced apoptosis demonstrated that galectin-7 expression causes enhanced caspase-3 activity and poly(ADP-ribose) polymerase cleavage, and the potentiation of apoptosis by galectin-7 was completely abrogated by a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. In addition, galectin-7 transfectants displayed accelerated mitochondrial cytochrome crelease and up-regulated JNK activity upon apoptosis induction. Several lines of evidence indicate that the effect on apoptosis is not due to the lectin functioning extracellularly through interactions with cell surface glycoconjugates. In fact, this lectin is found to localize in nuclei and cytoplasm of the transfectants and the transformed keratinocyte line HaCaT. Therefore, galectin-7 is a pro-apoptotic protein that functions intracellularly upstream of JNK activation and cytochrome crelease. DNA microarray analysis revealed genes that are differentially expressed between galectin-7 and control transfectants. Some of them are potentially contributory to this lectin's proapoptotic function and these include redox-related genes monoamine oxidase B, ryanodine receptor 2, and glutathione S-transferase Mu 3.

The FNR protein of Escherichia coliregulates target genes in response to anaerobiosis. Environmental oxygen is sensed by the acquisition of oxygen-labile [4Fe-4S] clusters that promote dimerization, DNA binding, and productive interactions with RNA polymerase. Three N-terminal cysteine residues (Cys20, Cys23, and Cys29) and Cys122act as ligands for the FNR iron sulfur clusters. An FNR variant, FNR-C20S, that retains only trace activity in vivocan acquire [4Fe-4S] clusters in vitrothat enhance site-specific DNA binding. Second site substitutions in activating regions AR1, AR2, and AR3 restore in vivoactivity to FNR-C20S, suggesting that the impairment in FNR-C20S activity is due to a failure to communicate with RNA polymerase effectively. Here we show that FNR-C20S can repress a simple FNR-regulated promoter in vivoand that it can form productive heterodimers with an FNR variant with altered DNA binding specificity, FNR-E209V. Transcription studies with FNR-E209V·FNR-C20S heterodimers indicate that the presence of a miscoordinated iron-sulfur cluster (FNR-C20S) in the downstream (but not the upstream) subunit of the FNR dimer impairs activation from a class II promoter and that this impairment can be overcome by amino acid substitutions known to unmask AR2 or improve AR3 in the affected subunit.

Two major signaling pathways, those triggered by estrogen (E2) and by the Wnt family, interact in the breast to cause growth and differentiation. The estrogen receptors ERαand ERβare activated by binding E2and act as ligand-dependent transcription factors. The effector for the Wnt family is the Tcf family of transcription factors. Both sets of transcription factors recognize discrete but different nucleotide sequences in the promoters of their target genes. By using transient transfections of reporter constructs for the osteopontin and thymidine kinase promoters in rat mammary cells, we show that Tcf-4 antagonizes and Tcf-1 stimulates the effects of activated ER/E2. For mutants of the former promoter, the stimulatory effects of ERα/E2can be made to be dependent on Tcf-1, and for the latter promoter the effects of the T cell factors (TCFs) are dependent on ER/E2. Direct interaction between ERs and Tcfs either at the Tcf/ERα-binding site on the DNA or in the absence of DNA is established by gel retardation assays or by coimmunoprecipitation/biosensor methods, respectively. These results show that the two sets of transcription factors can interact directly, the interaction between ERs and Tcf-4 being antagonistic and that between ERs and Tcf-1 being synergistic on the activity of the promoters employed. Since Tcf-4 is the major Tcf family member in the breast, it is suggested that the antagonistic interaction is normally dominant in vivoin this tissue.

CD47 is a unique member of the Ig superfamily with a single extracellular Ig domain followed by a multiply membrane-spanning (MMS) domain with five transmembrane segments, implicated in both integrin-dependent and -independent signaling cascades. Essentially all functions of CD47 require both the Ig and MMS domains, raising the possibility that interaction between the two domains is required for normal function. Conservation of Cys residues among CD47 homologues suggested the existence of a disulfide bond between the Ig and MMS domains that was confirmed by chemical digestion and mapped to Cys33and Cys263. Subtle changes in CD47 conformation in the absence of the disulfide were suggested by decreased binding of two anti-Ig domain monoclonal antibodies, decreased SIRPα1 binding, and reduced CD47/SIRPα1-mediated cell adhesion. Mutagenesis to prevent formation of this disulfide completely disrupted CD47 signaling independent of effects on ligand binding, as assessed by T cell interleukin-2 secretion and Ca2+responses. Loss of the disulfide did not affect membrane raft localization of CD47 or its association with αvβ3integrin. Thus, a disulfide bond between the Ig and MMS domains of CD47 is required for normal ligand binding and signal transduction.

Apoptosis via the mitochondrial pathway requires release of cytochrome cinto the cytosol to initiate formation of an oligomeric apoptotic protease-activating factor-1 (APAF-1) apoptosome. The apoptosome recruits and activates caspase-9, which in turn activates caspase-3 and -7, which then kill the cell by proteolysis. Because inactivation of this pathway may promote oncogenesis, we examined 10 ovarian cancer cell lines for resistance to cytochrome c-dependent caspase activation using a cell-free system. Strikingly, we found that cytosolic extracts from all cell lines had diminished cytochromec-dependent caspase activation compared with normal ovarian epithelium extracts. The resistant cell lines expressed APAF-1 and caspase-9, -3, and -7; however, each demonstrated diminished APAF-1 activity relative to the normal ovarian epithelium cell lines. A competitive APAF-1 inhibitor may account for the diminished APAF-1 activity because we did not detect dominant APAF-1 inhibitors, altered APAF-1 isoform expression, or APAF-1 deletion, degradation, or mutation. Lack of APAF-1 activity correlated in some but not all cell lines with resistance to apoptosis. These data suggest that regulation of APAF-1 activity may be important for apoptosis regulation in some ovarian cancers.

Tyrosine phosphorylation of junctional components has been proposed as a mechanism for modulating cell-cell adhesion. Although a correlation exists between the tyrosine phosphorylation of the adherens junction protein beta-catenin and loss of classical cadherin-mediated adhesion, the effects of tyrosine phosphorylation on the function of the adherens junction and desmosome-associated protein plakoglobin is unknown. In the present study, we investigated the effects of epidermal growth factor receptor (EGFR) tyrosine kinase activation on the subcellular distribution of plakoglobin and its association with its junctional binding partners. Long term epidermal growth factor (EGF) treatment of A431 cells revealed a modest decrease in the cytoskeleton-associated pool of plakoglobin (Pg) and a corresponding increase in the cytosolic pool of Pg. After short term EGF treatment, plakoglobin was rapidly phosphorylated, and tyrosine-phosphorylated Pg was distributed predominantly in a membrane-associated Triton X-100-soluble pool, along with a co-precipitating high molecular weight tyrosine-phosphorylated protein identified as desmoglein 2. Analysis of deletion and point mutants defined the primary EGFR-dependent targets as one or more of three C-terminal tyrosine residues. Whereas phosphorylated Pg remained associated with the desmoglein tail after both short and long term EGFR activation, no phosphorylated Pg was found associated with the N-terminal Pg-binding domain (DPNTP) of the intermediate filament-associated protein, desmoplakin. Together these results are consistent with the possibility that EGF-dependent tyrosine phosphorylation of Pg may modulate cell-cell adhesion by compromising the link between desmosomal cadherins and the intermediate filament cytoskeleton.

Members of the transforming growth factor-β (TGF-β) superfamily signal through unique cell membrane receptor serine-threonine kinases to activate downstream targets. TRAP1 is a previously described 96-kDa cytoplasmic protein shown to bind to TGF-β receptors and suggested to play a role in TGF-β signaling. We now fully characterize the binding properties of TRAP1, and show that it associates strongly with inactive heteromeric TGF-β and activin receptor complexes and is released upon activation of signaling. Moreover, we demonstrate that TRAP1 plays a role in the Smad-mediated signal transduction pathway, interacting with the common mediator, Smad4, in a ligand-dependent fashion. While TRAP1 has only a small stimulatory effect on TGF-β signaling in functional assays, deletion constructs of TRAP1 inhibit TGF-β signaling and diminish the interaction of Smad4 with Smad2. These are the first data to identify a specific molecular chaperone for Smad4, suggesting a model in which TRAP1 brings Smad4 into the vicinity of the receptor complex and facilitates its transfer to the receptor-activated Smad proteins.

Tissue factor pathway inhibitor (TFPI) contains three Kunitz-type proteinase inhibitor domains and is a potent inhibitor of tissue factor-mediated coagulation. Here, we report that TFPI inhibits the proliferation of basic fibroblast growth factor-stimulated endothelial cells. A truncated form of TFPI, containing only the first two Kunitz-type proteinase inhibitor domains, has very little antiproliferative activity, suggesting that the carboxyl-terminal region of TFPI is responsible for this activity. Binding studies revealed that full-length TFPI, but not the truncated TFPI molecule, is recognized by the very low density lipoprotein receptor (VLDL receptor) indicating that this receptor is a novel high affinity endothelial cell receptor for TFPI. The antiproliferative activity of TFPI on endothelial cells is inhibited by the receptor-associated protein, a known antagonist of ligand binding by the VLDL receptor, and by anti-VLDL receptor antibodies. These results confirm that the antiproliferative activity of TFPI is mediated by the VLDL receptor and suggest that this receptor-ligand system may be a useful target for the development of new anti-angiogenic and antitumor agents.

Cytotoxic T lymphocytes kill virus-infected and tumor cell targets through the concerted action of proteins contained in cytolytic granules, primarily granzyme B and perforin. Granzyme B, a serine proteinase with substrate specificity similar to the caspase family of apoptotic cysteine proteinases, is capable of cleaving and activating a number of death proteins in target cells. Despite the ability to engage the death pathway at multiple entry points, the preferred mechanism for rapid induction of apoptosis by granzyme B has yet to be clearly established. Here we use time lapse confocal microscopy to demonstrate that mitochondrial cytochromecrelease is the primary mode of granzyme B-induced apoptosis and that Bcl-2 is a potent inhibitor of this pivotal event. Caspase activation is not required for cytochrome crelease, an activity that correlates with cleavage and activation of Bid, which we have found to be cleaved more readily by granzyme B than either caspase-3 or caspase-8. Bcl-2 blocks the rapid destruction of targets by granzyme B by blocking mitochondrial involvement in the process.

CD47 is a ubiquitously expressed membrane protein with an extracellular Ig domain and a multiple membrane-spanning domain that can synergize with antigen to induce interleukin (IL)-2 secretion by T lymphocytes. Ligation of CD47 induced actin polymerization and increased protein kinase Cθ (PKCθ) association with the cytoskeleton independent of antigen receptor ligation, but ligation of mutant forms of the molecule missing either the Ig domain or the multiple membrane-spanning domain did not. Simultaneous ligation of CD47 and CD3 led to additive effects on F-actin and synergistic effects on PKCθ cytoskeletal association. Disruption of membrane rafts by removal of cholesterol with cyclodextrin blocked CD47-induced actin polymerization, and mutant forms of CD47 that localized poorly to rafts failed to effect cytoskeletal rearrangement. However, raft association alone was not sufficient, because a raft-localized CD47 Ig domain bound to the membrane by a glycan phosphoinositol anchor was unable to induce actin polymerization. A mutant form of CD47 without its Ig domain that did not induce actin polymerization or localize to rafts still enhanced T cell receptor (TCR)-dependent tyrosine phosphorylation of PLCγ and associated Ca2+signaling but did not augment IL-2 secretion. Thus, CD47 synergy with TCR to increase [Ca2+]iis independent of actin and rafts but is insufficient to explain CD47 cooperation with TCR in IL-2 synthesis. Full synergy with TCR requires CD47 localization to membrane rafts where ligation leads to TCR-independent signals causing actin polymerization and PKCθ translocation.

The protein specified by the Saccharomyces cerevisiaenuclear gene PET111specifically activates translation of the mitochondrially coded mRNA for cytochromecoxidase subunit II (Cox2p). We found Pet111p specifically in mitochondria of both wild-type cells and cells expressing a chromosomal gene for a functional epitope-tagged form of Pet111p. Pet111p was associated with mitochondrial membranes and was highly resistant to extraction with alkaline carbonate. Pet111p was protected from proteolytic digestion by the mitochondrial inner membrane. Thus, it is exposed only on the matrix side, where it could participate directly in organellar translation and localize Cox2p synthesis by virtue of its functional interaction with the COX2mRNA 5′-untranslated leader. We also found that Pet111p is present at levels limiting the synthesis of Cox2p by examining the effect of alteredPET111gene dosage in the nucleus on expression of a reporter gene, cox2::ARG8m, that was inserted into mitochondrial DNA. The level of the reporter protein, Arg8p, was one-half that of wild type in a diploid strain heterozygous for a pet111deletion mutation, whereas it was increased 2.8-fold in a strain bearing extra copies of PET111on a high-copy plasmid. Thus, Pet111p could play dual roles in both membrane localization and regulation of Cox2p synthesis within mitochondria.

The IL-1 receptor-associated kinase (IRAK/mPLK) is linked to the regulation of nuclear factor-κB (NF-κB)-dependent gene expression. Here we describe a novel binding partner of IRAK/mPLK that we term SIMPL (signaling molecule that associates with the mouse pelle-like kinase). Overexpression of SIMPL leads to the activation of NF-κB-dependent promoters, and inactivation of SIMPL inhibits IRAK/mPLK as well as tumor necrosis factor receptor type I-induced NF-κB activity. Dominant inhibitory alleles of IκB kinase (IKKα or IKKβ) block the activation of NF-κB by IRAK/mPLK and SIMPL. Furthermore, SIMPL binds IRAK/mPLK and the IKKs in vitroand in vivo. In the presence of antisense mRNA to SIMPL, the physical association between IRAK/mPLK and IKKβ but not IRAK/mPLK and IKKα is greatly diminished. Moreover, dominant-negative SIMPL blocks IKKα- or IKKβ-induced NF-κB activity. These results lead us to propose a model in which SIMPL functions to regulate NF-κB activity by linking IRAK/mPLK to IKKβ/α-containing complexes.AF093135

Protein kinase CK2 (CK2) has long been implicated in the regulation of cell growth and proliferation. Its activity is generally elevated in rapidly proliferating tissues, and nuclear matrix (NM) is an important subnuclear locale of its functional signaling. In the prostate, nuclear CK2 is rapidly lost commensurate with induction of receptor-mediated apoptosis after growth stimulus withdrawal. By contrast, chemical-induced apoptosis in prostate cancer and other cells (by etoposide and diethylstilbestrol) evokes an enhancement in CK2 associated with the NM that appears to be because of translocation of CK2 from the cytoplasmic to the nuclear compartment. This shuttling of CK2 to the NM may reflect a protective response to chemical-mediated apoptosis. Supporting evidence for this was obtained by employing cells that were transiently transfected with various expression plasmids of CK2 (thereby expressing additional CK2) prior to treatment with etoposide or diethylstilbestrol. Cells transfected with the CK2α or CK2αβ showed significant resistance to chemical-mediated apoptosis commensurate with the corresponding elevation in CK2 in the NM. Transfection with CK2β did not demonstrate this effect. These results suggest, for the first time, that besides the commonly appreciated function of CK2 in cell growth, it may also have a role in protecting cells against apoptosis.

We demonstrate that the signal peptides of prepro-α-factor and preinvertase must be cleaved before Asn-X-Ser/Thr acceptor tripeptides located near the signal peptides of these precursors can be efficiently glycosylated within the endoplasmic reticulum of the yeast Saccharomyces cerevisiae. The data support a model whereby the interaction of a signal peptide with the membrane prevents an acceptor tripeptide juxtaposed to the signal peptide from accessing the oligosaccharyltransferase active site until the signal peptide is cleaved.

Nocturnin is a vertebrate circadian clock-regulated gene, and in Xenopus laevisits mRNA is specifically expressed in retinal photoreceptor cells. We have investigated the transcriptional regulatory mechanism that drives this precise spatial expression pattern of the nocturnin gene. A deletion series of the nocturnin 5′-flanking sequence driving the green fluorescence protein (GFP) reporter was used to generate transgenicXenopustadpoles. We found that a construct containing 2.6 kilobase pairs of 5′-flanking sequence targeted high level GFP reporter expression specifically to photoreceptor cells, in a pattern identical to endogenous nocturnin. This photoreceptor-specific expression pattern was maintained with several further deletions of 5′-upstream sequence, including a short 59-base pair fragment. Within this region of 59 base pairs, three perfect repeats of a novel protein binding site were identified by electrophoretic mobility shift assay. Competitions using varying oligonucleotide sequences demonstrated that the sequence required for protein binding is CAGACAGGCTATA, designated photoreceptor-conserved element II (PCE II). The protein complex that binds to this element is enriched in retinal extracts, and mutations of PCE II which fail to bind the protein complex also fail to direct GFP reporter expression to photoreceptors. These results indicate that the PCE II in the proximal promoter of the nocturnin gene is sufficient for driving the photoreceptor-specific expression of nocturnin.

The novel pore-forming toxin hemolysin E (HlyE, ClyA, or SheA) consists of a long four-helix bundle with a subdomain (β tongue) that interacts with target membranes at one pole and an additional helix (αG) that, with the four long helices, forms a five-helix bundle (tail domain) at the other pole. Random amino acid substitutions that impair hemolytic activity were clustered mostly, but not exclusively, within the tail domain, specifically amino acids within, adjacent to, or interacting with αG. Deletion of amino acids downstream of αGdid not affect activity, but deletions encompassing αGyielded insoluble and inactive proteins. In the periplasm Cys-285 (αG) is linked to Cys-87 (αB) of the four-helix bundle via an intramolecular disulfide. Oxidized HlyE did not form spontaneously in vitrobut could be generated by addition of Cu(II) or mimicked by treatment with Hg(II) salts to yield inactive proteins. Such treatments did not affect binding to target membranes nor assembly into non-covalently linked octameric complexes once associated with a membrane. However, gel filtration analyses suggested that immobilizing αGinhibits oligomerization in solution. Thus once associated with a membrane, immobilizing αGinhibits HlyE activity at a late stage of pore formation, whereas in solution it prevents aggregation and consequent inactivation.

Application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to PC12 cells under resting conditions evoked quantal catecholamine secretion, as detected amperometrically. This effect was not mimicked by 4alpha-phorbol-12,13-didecanoate, another phorbol ester, which is inactive with respect to protein kinase C activation, and was prevented by the protein kinase C inhibitor bisindolylmaleimide. TPA also caused a rise of [Ca(2+)](i) in Fura-2-loaded PC12 cells, and again this was not mimicked by 4alpha-phorbol-12,13-didecanoate and could be blocked by bisindolylmaleimide. TPA-evoked secretion was entirely dependent on extracellular Ca(2+) and was fully abolished by nifedipine, as were TPA-induced rises of [Ca(2+)](i). Resting membrane potential, monitored using perforated patch recordings, was unaffected by TPA. However, a small (6-8 mV) hyperpolarizing shift in the voltage dependence of Ca(2+) channel currents (determined using whole-cell patch clamp recordings) was induced by TPA, and this could be fully prevented by nifedipine. In contrast to results with depolarizing stimuli, which evoke exocytosis because of Ca(2+) influx through N-type channels in these cells, the present results indicate that protein kinase C activation leads directly to quantal catecholamine secretion in the absence of depolarizing stimuli via a selective shift in the activation of L-type Ca(2+) channels.

FNR is an Escherichia coli transcription factor that activates gene expression in response to anaerobiosis at a large number of promoters by making direct contacts with RNA polymerase. At class II FNR-dependent promoters, where the DNA site for FNR overlaps the -35 element, activating region 1 of FNR is proposed to interact with the C-terminal domain of the RNA polymerase alpha-subunit. Using a model class II FNR-dependent promoter, FF(-41.5), we have performed in vivo and in vitro experiments to investigate the role of this interaction. Our results show that FNR, carrying substitutions in activating region 1, is compromised in its ability to promote open complex formation and thus to activate transcription. Abortive initiation assays were used to assess the contribution of activating region 1 of FNR to open complex formation. A new method for the purification of the FNR protein is also described.

The lactose permease is an integral membrane protein that cotransports H+and lactose into the bacterial cytoplasm. Previous work has shown that bulky substitutions at glycine 64, which is found on the cytoplasmic edge of transmembrane segment 2 (TMS-2), cause a substantial decrease in the maximal velocity of lactose uptake without significantly affecting the Kmvalues (Jessen-Marshall, A. E., Parker, N. J., and Brooker, R. J. (1997) J. Bacteriol.179, 2616–2622). In the current study, mutagenesis was conducted along the face of TMS-2 that contains glycine-64. Single amino acid substitutions that substantially changed side-chain volume at codons 52, 57, 59, 63, and 66 had little or no effect on transport activity, whereas substitutions at codons 49, 53, 56, and 60 were markedly defective and/or had lower levels of expression. According to helical wheel plots, Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64 form a continuous stripe along one face of TMS-2. Several of the TMS-2 mutants (S56Y, S56L, S56Q, Q60A, and Q60V) were used as parental strains to isolate mutants that restore transport activity. These mutations were either first-site mutations or second-site suppressors in TMS-1, TMS-2, TMS-7 or TMS-11. A kinetic analysis showed that the suppressors had a higher rate of lactose transport compared with the corresponding parental strains. Overall, the results of this study are consistent with the notion that a face on TMS-2, containing Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64, plays a critical role in conformational changes associated with lactose transport. We hypothesize that TMS-2 slides across TMS-7 and TMS-11 when the lactose permease interconverts between the C1 and C2 conformations. This idea is discussed within the context of a revised model for the structure of the lactose permease.

Interleukin 6 (IL6) plays key roles in hematopoiesis, immune, and acute phase responses. Dysregulated IL6 expression is implicated in diseases such as atherosclerosis and arthritis. We have examined the functional effect of four polymorphisms in the IL6 promoter (−597G→A, −572G→C, −373AnTn, −174G→C) by identifying the naturally occurring haplotypes and comparing their effects on reporter gene expression. The results indicate different transcriptional regulation in the ECV304 cell line compared with the HeLa cell line, suggesting cell type-specific regulation of IL6 expression. The haplotypes showed functional differences in the ECV304 cell line; transcription was higher from the GG9/11G haplotype and lower from the AG8/12G allele. The differences suggest that more than one of the polymorphic sites is functional; the base differences at distinct polymorphic sites do not act independently of one another, and one polymorphism influences the functional effect of variation at other polymorphic sites. These results show that genetic polymorphisms in the promoter influence IL6 transcription not by a simple additive mechanism but rather through complex interactions determined by the haplotype.

Nuclear mutants of Saccharomyces cerevisiae assigned to complementation group G34 are respiratory-deficient and lack cytochrome oxidase activity and the characteristic spectral peaks of cytochromes a and a(3). The corresponding gene was cloned by complementation, sequenced, and identified as reading frame YGR062C on chromosome VII. This gene was named COX18. The COX18 gene product is a polypeptide of 316 amino acids with a putative amino-terminal mitochondrial targeting sequence and predicted transmembrane domains. Respiratory chain carriers other than cytochromes a and a(3) and the ATPase complex are present at near wild-type levels in cox18 mutants, indicating that the mutations specifically affect cytochrome oxidase. The synthesis of Cox1p and Cox3p in mutant mitochondria is normal whereas Cox2p is barely detected among labeled mitochondrial polypeptides. Transcription of COX2 does not require COX18 function, and a chimeric COX3-COX2 mRNA did not suppress the respiratory defect in the null mutant, indicating that the mutation does not impair transcription or translation of the mRNA. Western analysis of cytochrome oxidase subunits shows that inactivation of the COX18 gene greatly reduces the steady state amounts of subunit 2 and results in variable decreases in other subunits of cytochrome oxidase. A gene fusion expressing a biotinylated form of Cox18p complements cox18 mutants. Biotinylated Cox18p is a mitochondrial integral membrane protein. These results indicate Cox18p to be a new member of a group of mitochondrial proteins that function at a late stage of the cytochrome oxidase assembly pathway.

Islet amyloid polypeptide (IAPP) and insulin are expressed in the β-cells of the islets of Langerhans. They are co-secreted in response to changes in glucose concentration, and their mRNA levels are also regulated by glucose. The promoters of both genes share similar cis-acting sequence elements, and both bind the homeodomain transcription factor PDX1, which plays an important role in the regulation of the insulin promoter and insulin mRNA levels by glucose. Here we examine the role of PDX1 in the regulation of the human IAPP promoter by glucose. The experiments were facilitated by the availability of a human β-cell line (NES2Y) that lacks PDX1. NES2Y cells also lack operational KATPchannels, resulting in a loss of control of calcium signaling. We have previously used these cells to show that glucose regulation of the insulin gene is dependent on PDX1, but not calcium. In the mouse β-cell line Min6, glucose (16 mm) stimulated a 3.5–4-fold increase in the activity of a −222 to +450 IAPP promoter construct compared with values observed in 0.5 mmglucose. In NES2Y cells, glucose failed to stimulate transcriptional activation of the IAPP promoter. Overexpression of PDX1 in NES2Y cells failed to reinstate glucose-responsive control of the IAPP promoter. Glucose effects on the IAPP promoter were observed only in the presence of PDX1 when normal calcium signaling was restored by overexpression of the two KATPchannel subunits SUR1 and Kir6.2. The importance of calcium was further emphasized by an experiment in which glucose-stimulated IAPP promoter activity was inhibited by the calcium channel blocker verapamil (50 μm). Verapamil was further shown to inhibit the stimulatory effect of glucose on IAPP mRNA levels. These results demonstrate that like the insulin promoter, glucose regulation of the IAPP promoter is dependent on the activity of PDX1, but unlike the insulin promoter, it additionally requires the activity of another, as yet uncharacterized factor(s), the activity of which is calcium-dependent.

Nuclear mutants of Saccharomyces cerevisiaeassigned to complementation group G34 are respiratory-deficient and lack cytochrome oxidase activity and the characteristic spectral peaks of cytochromes aanda3. The corresponding gene was cloned by complementation, sequenced, and identified as reading frame YGR062C on chromosome VII. This gene was named COX18. TheCOX18gene product is a polypeptide of 316 amino acids with a putative amino-terminal mitochondrial targeting sequence and predicted transmembrane domains. Respiratory chain carriers other than cytochromes aand a3and the ATPase complex are present at near wild-type levels in cox18mutants, indicating that the mutations specifically affect cytochrome oxidase. The synthesis of Cox1p and Cox3p in mutant mitochondria is normal whereas Cox2p is barely detected among labeled mitochondrial polypeptides. Transcription of COX2does not requireCOX18function, and a chimericCOX3-COX2mRNA did not suppress the respiratory defect in the null mutant, indicating that the mutation does not impair transcription or translation of the mRNA. Western analysis of cytochrome oxidase subunits shows that inactivation of theCOX18gene greatly reduces the steady state amounts of subunit 2 and results in variable decreases in other subunits of cytochrome oxidase. A gene fusion expressing a biotinylated form of Cox18p complements cox18mutants. Biotinylated Cox18p is a mitochondrial integral membrane protein. These results indicate Cox18p to be a new member of a group of mitochondrial proteins that function at a late stage of the cytochrome oxidase assembly pathway.

The stress protein heme oxygenase-1 (HO-1) is induced in endothelial cells exposed to nitric oxide (NO)-releasing agents, and this process is finely modulated by thiols (Foresti, R., Clark, J. E., Green, C. J., and Motterlini R. (1997) J. Biol. Chem. 272, 18411-18417). Here, we report that up-regulation of HO-1 in aortic endothelial cells by severe hypoxic conditions (pO(2)

Eosinophils, the major immune effector cells contributing to allergic inflammation and asthma, are profoundly affected by interleukin (IL) 5 with respect to their differentiation, viability, recruitment, and cytotoxic effector functions. IL-5 enhances eosinophil responsiveness to a variety of chemotactic factors via a process called priming, although the molecular mechanism is unknown. In this study, we report that, following IL-5 priming of eosinophils, chemotactic agents including fMet-Leu-Phe, IL-8, and RANTES, promote vigorous transient activation of ERK1 and ERK2. In contrast, these chemotactic factors stimulate weak or indiscernible ERK activation in unprimed eosinophils. Furthermore, this intracellular marker of priming is selective for IL-5-related cytokines, in that it is observed following exposure to IL-5 and granulocyte macrophage-colony stimulating factor but not to interferon-γ, stem cell factor, tumor necrosis factor α, or IL-4. Interestingly, priming of chemoattractant-induced ERK activation is accompanied by an increase in association of tyrosine-phosphorylated proteins with the adapter protein Grb2. The biological relevance of ERK activation to IL-5 priming is supported by the observation that inhibition of ERK activity by treatment with the MEK inhibitors PD98059 or U0126 inhibited the release of leukotriene C4stimulated by fMet-Leu-Phe in IL-5-primed eosinophils. These data provide evidence for a previously undescribed fundamental mechanism by which stimulation of IL-5 family receptors induces a rapid phenotypic alteration in the signal transduction pathways of chemotactic receptors, enabling their activation of the ERK1 and ERK2 pathway and contributing to the capacity of these cells to synthesize LTC4.

Cell death due to thymine (dThd) deficiency, associated with the cytotoxic action of 5-fluorouracil in colon cancer, is regulated in thymidylate synthase-deficient (TS(-)) human colon carcinoma cells via the Fas (CD95, APO-1) death receptor. This was demonstrated by inhibiting the loss in clonogenicity of TS(-) cells by anti-FasL and in enhanced survival of TS(-) clones selected for resistance to Fas-mediated apoptosis, following dThd deprivation. During thymineless stress in TS(-) cells, Fas ligand (FasL) is expressed, and its promoter (hFasLPr) is activated. Transactivation of hFasLPr, dependent upon dThd deficiency, was inhibited following mutation of the binding sites for NF-kappaB or AP-1 and by preventing NF-kappaB or AP-1 activation, which inhibited expression of FasL and enhanced clonogenic survival in stable transformants expressing IkappaBalphaM or DN-MEKK, respectively. These results demonstrate the crucial roles for NF-kappaB and AP-1 in the regulation of FasL in Fas-mediated thymineless death of colon carcinoma cells.

Cell death due to thymine (dThd) deficiency, associated with the cytotoxic action of 5-fluorouracil in colon cancer, is regulated in thymidylate synthase-deficient (TS−) human colon carcinoma cells via the Fas (CD95, APO-1) death receptor. This was demonstrated by inhibiting the loss in clonogenicity of TS−cells by anti-FasL and in enhanced survival of TS−clones selected for resistance to Fas-mediated apoptosis, following dThd deprivation. During thymineless stress in TS−cells, Fas ligand (FasL) is expressed, and its promoter (hFasLPr) is activated. Transactivation of hFasLPr, dependent upon dThd deficiency, was inhibited following mutation of the binding sites for NF-κB or AP-1 and by preventing NF-κB or AP-1 activation, which inhibited expression of FasL and enhanced clonogenic survival in stable transformants expressing IκBαM or DN-MEKK, respectively. These results demonstrate the crucial roles for NF-κB and AP-1 in the regulation of FasL in Fas-mediated thymineless death of colon carcinoma cells.

There is growing evidence that CD36 has an important physiological function in the uptake of oxidized low density lipoprotein (OxLDL) by macrophages. However, the ligand specificity and the nature of the ligands on OxLDL that mediate the binding to CD36 remain ill defined. Results from recent studies suggested that some of the macrophage scavenger receptors involved in the uptake of OxLDL recognized boththe lipid and the protein moieties of OxLDL, but there was no conclusive direct evidence for this. The present studies were undertaken to test whether a single, well characterized OxLDL receptor, CD36, could bind both the lipid and protein moieties of OxLDL. COS-7 cells transiently transfected with mouse CD36 cDNA bound intact OxLDL with high affinity. This binding was very effectively inhibited (∼50%) both by the reconstituted apoB from OxLDL and by microemulsions prepared from OxLDL lipids. The specific binding of both moieties to CD36 was further confirmed by direct ligand binding analysis and by demonstrating reciprocal inhibition, i.e.apoB from OxLDL inhibited the binding of the OxLDL lipids and vice versa. Furthermore, a monoclonal mouse antibody that recognizes oxidation-specific epitopes in OxLDL inhibited the binding of intact OxLDL and also that of its purified protein and lipid moieties to CD36. This antibody recognizes the phospholipid 1-palmitoyl 2-(5′-oxovaleroyl) phosphatidylcholine. This model of an oxidized phospholipid was also an effective competitor for the CD36 binding of both the resolubilized apoB and the lipid microemulsions from OxLDL. Our results demonstrate that oxidized phospholipids in the lipid phase or covalently attached to apoB serve as ligands for recognition by CD36 and, at least in part, mediate the high affinity binding of OxLDL to macrophages.

The transcription factor c-Myc is important for the control of cell cycle progression, neoplasia, and apoptotic cell death. c-Myc dimerizes with its partner Max to form an active transcription factor complex. Little is known, however, about the transcriptional targets of c-Myc and their roles in c-Myc-induced cell death. Here we demonstrate that T cell activation-induced expression of Fas ligand (FasL, CD95-L, APO-1-L), which can induce apoptotic cell death in many different cell types, is regulated by c-Myc. Down-modulation of c-Myc protein via antisense oligonucleotides blocked activation-induced FasL mRNA and protein expression and functional FasL expression in activated T cells and T cell lines. Further, FasL promoter activity in T cells is driven by overexpression of c-Myc and inhibited by expression of dominant-negative mutants of c-Myc and Max. Our findings indicate that c-Myc controls apoptotic cell death in T cells through regulation of FasL expression.