Prolonged activation of ERK1,2 induces FADD-independent caspase 8 activation and cell death.

Prolonged ERK/MAPK activation has been implicated in neuronal cell death in vitro and in vivo. We found that HEK293 cells, recently reported to express neuronal markers, are exquisitely sensitive to long term ERK stimulation. Activation of an inducible form of Raf-1 (Raf-1:ER) in HEK293 cells induced massive apoptosis characterized by DNA degradation, loss of plasma membrane integrity and PARP cleavage. Cell death required MEK activity and protein synthesis and occurred via the death receptor pathway independently of the mitochondrial pathway. Accordingly, prolonged ERK stimulation activated caspase 8 and strongly potentiated Fas signaling. The death receptor adaptator FADD was found to be rapidly induced upon ERK activation. However using RNA interference and ectopic expression, we demonstrated that neither FADD nor Fas were necessary for caspase 8 activation and cell death. These findings reveal that prolonged ERK/MAPK stimulation results in caspase 8 activation and cell death.

The N-terminal 34 kDa fragment of Helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c release.

The pathogenic bacterium Helicobacter pylori produces the cytotoxin VacA, which is implicated in the genesis of gastric epithelial lesions. By transfect ing HEp-2 cells with DNAs encoding either the N-terminal (p34) or the C-terminal (p58) fragment of VacA, p34 was found localized specifically to mitochondria, whereas p58 was cytosolic. Incubated in vitro with purified mitochondria, VacA and p34 but not p58 translocated into the mitochondria. Microinjection of DNAs encoding VacA-GFP and p34-GFP, but not GFP-VacA or GFP-p34, induced cell death by apoptosis. Transient transfection of HeLa cells with p34-GFP or VacA-GFP induced the release of cytochrome c from mitochondria and activated the executioner caspase 3, as determined by the cleavage of poly(ADP-ribose) polymerase (PARP). PARP cleavage was antagonized specifically by co-transfection of DNA encoding Bcl-2, known to block mitochondria-dependent apoptotic signals. The relevance of these observations to the in vivo mechanism of VacA action was supported by the fact that purified activated VacA applied externally to cells induced cytochrome c release into the cytosol.

Independent activation of endogenous p21-activated protein kinase-3 (PAK3) and JNK by thrombin in CCL39 fibroblasts.

Thrombin, a potent mitogen for CCL39 hamster lung fibroblasts, activates the seven membrane-spanning receptor PAR1. To better understand the signaling pathways controlled by this receptor we analyzed a potential downstream effector, p21-activated protein kinase (PAK). Thrombin and PAR1 agonist peptide, as well as serum and lysophosphatidic acid, were found to stimulate HA-mPAK3 activity in CCL39 cells transfected with a plasmid encoding the epitope-tagged kinase. Similar results were obtained using antibodies developed against the endogenous kinase. PAK3 activation is sensitive to pertussis toxin, but insensitive to LY 294002, an inhibitor of phosphatidylinositol 3'-kinase. Thrombin and serum also activate c-jun amino terminal kinase (JNK). Similar to PAK3 activation, thrombin-stimulated JNK activity is inhibited by pertussis toxin, but not by LY 294002. In a CCL39-derived cell line expressing constitutively active mPAK3 in a tetracyline-dependent manner, induction of PAK activity does not lead to corresponding increases in JNK activity. Our findings indicate that PAK3 is responsive to thrombin and other G protein-coupled receptor systems. Furthermore, our data suggest that in CCL39 cells, JNK activation by thrombin occurs independently of PAK3.

Cleavage of the serum response factor during death receptor-induced apoptosis results in an inhibition of the c-FOS promoter transcriptional activity.

The c-FOS protooncogene is rapidly induced by a wide variety of extracellular stimuli including mitogenic signals. Regulation of c-FOS expression is tightly dependent on the serum response element localized within its promoter. Two transcription factors, the serum response factor (SRF) and the ternary complex factor, bind to the serum response element and play a key role in the regulation of the c-FOS promoter activity. In the present study, we show that two death effectors (CH11 and TRAIL) severely impaired the transcriptional activity of the c-FOS promoter in Jurkat T cells. This inhibition can be accounted for by the specific cleavage by caspase 3 of the SRF both in vitro and in vivo, since acetyl-DEVD-aldehyde prevented SRF cleavage and abolished the inhibitory effect of CH11 and TRAIL on the c-FOS promoter activity. Moreover, phorbol myristate acetate, a potent anti-apoptotic effector, was found to protect SRF completely from cleavage by caspase 3 and also to prevent the inhibition of the c-FOS promoter activity by death effectors. Survival factors play an essential function in the regulation of cell growth mainly by regulating the expression of immediate early gene such as c-FOS. In this line, cleavage of SRF at the onset of apoptosis could abrogate the ability of the cell to induce inappropriate survival pathways. All together, our results are consistent with a role of SRF at the interface between cell survival and death pathways.

The p42/p44 MAP kinase pathway prevents apoptosis induced by anchorage and serum removal.

Anchorage removal like growth factor removal induces apoptosis. In the present study we have characterized signaling pathways that can prevent this cell death using a highly growth factor- and anchorage-dependent line of lung fibroblasts (CCL39). After anchorage removal from exponentially growing cells, annexin V-FITC labeling can be detected after 8 h. Apoptosis was confirmed by analysis of sub-G1 DNA content and Western blotting of the caspase substrate poly (ADP-ribose) polymerase. Growth factor withdrawal accelerates and potentiates suspension-induced cell death. Activation of Raf-1 kinase in suspension cultures of CCL39 or Madin-Darby canine kidney cells stably expressing an estrogen-inducible activated-Raf-1 construct (DeltaRaf-1:ER) suppresses apoptosis induced by growth factor and/or anchorage removal. This protective effect appears to be mediated by the Raf, mitogen- or extracellular signal-regulated kinase kinase (MEK), and mitogen-activated protein kinase module because it is sensitive to pharmacological inhibition of MEK-1 and it can be mimicked by expression of constitutively active MEK-1 in CCL39 cells. Finally, apoptosis induced by disruption of the actin cytoskeleton with the Rho-directed toxin B (Clostridium difficile) is prevented by activation of the DeltaRaf-1:ER chimeric construct. These findings highlight the ability of p42/p44 mitogen-activated protein kinase to generate survival signals that counteract cell death induced by loss of matrix contact, cytoskeletal integrity, and extracellular mitogenic factors.

p70 S6 kinase-mediated protein synthesis is a critical step for vascular endothelial cell proliferation.

In this work, we analyzed the role of the PI3K-p70 S6 kinase (S6K) signaling cascade in the stimulation of endothelial cell proliferation. We found that inhibitors of the p42/p44 MAPK pathway (PD98059) and the PI3K-p70 S6K pathway (wortmannin, Ly294002, and rapamycin) all block thymidine incorporation stimulated by fetal calf serum in the resting mouse endothelial cell line 1G11. The action of rapamycin can be generalized, since it completely inhibits the mitogenic effect of fetal calf serum in primary endothelial cell cultures (human umbilical vein endothelial cells) and another established capillary endothelial cell line (LIBE cells). The inhibitory effect of rapamycin is only observed when the inhibitor is added at the early stages of G(0)-G(1) progression, suggesting an inhibitory action early in G(1). Rapamycin completely inhibits growth factor stimulation of protein synthesis, which perfectly correlates with the inhibition of cell proliferation. In accordance with its inhibitory action on protein synthesis, activation of cyclin D1 and p21 proteins by growth factors is also blocked by preincubation with rapamycin. Expression of a p70 S6K mutant partially resistant to rapamycin reverses the inhibitory effect of the drug on DNA synthesis, indicating that rapamycin action is via p70 S6K. Thus, in vascular endothelial cells, activation of protein synthesis via p70 S6K is an essential step for cell cycle progression in response to growth factors.

The Ets2 transcription factor inhibits apoptosis induced by colony-stimulating factor 1 deprivation of macrophages through a Bcl-xL-dependent mechanism.

Bcl-xL, a member of the Bcl-2 family, inhibits apoptosis, and its expression is regulated at the transcriptional level, yet nothing is known about the transcription factors specifically activating this promoter. The bcl-x promoter contains potential Ets binding sites, and we show that the transcription factor, Ets2, first identified by its sequence identity to v-ets of the E26 retrovirus, can transactivate the bcl-x promoter. Transient expression of Ets2 results in the upregulation of Bcl-xL but not of Bcl-xS, an alternatively spliced gene product which induces apoptosis. Ets2 is ubiquitously expressed at low levels in a variety of cell types and tissues but is specifically induced to abundant levels during macrophage differentiation. Since Bcl-xL is also upregulated during macrophage differentiation, we asked whether the bcl-x could be a direct downstream target gene of Ets2 in macrophages. BAC1.2F5 macrophages, which are dependent on macrophage colony-stimulating factor 1 (CSF-1) for their growth and survival, were used in these studies. We show that CSF-1 stimulation of BAC1.2F5 macrophages results in the upregulation of expression of ets2 and bcl-xL with similar kinetics of induction. In the absence of CSF-1, these macrophages undergo cell death by apoptosis, whereas constitutive expression of Ets2 rescues these cells from cell death, and bcl-xL is upregulated. These results strongly suggest a novel role of Ets2 in affecting apoptosis through its regulation of Bcl-xL transcription.

An anchorage-dependent signal distinct from p42/44 MAP kinase activation is required for cell cycle progression.

Most normal cells require both mitogens and integrin-mediated attachment for growth. It is generally accepted that the p42/p44 MAP kinase module, which can be activated by both growth factors and adhesion, plays a critical role in G0 to S phase progression of quiescent cells. Studies on various cultured fibroblasts have shown that removal of anchorage leads to cell cycle arrest in G1 and it has been proposed that adhesion-dependent G1 progression requires the joint regulation of p42/p44 MAP kinase by integrins and growth factors. In quiescent CCL39 lung fibroblasts, MAP kinase activation in response to serum becomes compromised when cells are placed in suspension. Under these conditions, serum-stimulated cells arrest their growth in mid-G1 with reduced cyclin D1 expression and increased p21Cip/Waf1 expression, as compared to their attached counterparts. To determine whether a casual link exists between suboptimal activation of MAP kinase in non-adherent cells and the observed G1 block, we used a variant of CCL39 stably expressing an estrogen-inducible activated-Raf-1 construct (deltaRaf-1:ER). We found that even strong and sustained activation of MAP kinase with estradiol, in addition to serum, is not able to boost cyclin D1 expression levels or stimulate hyperphosphorylation of pRb in suspended CCL39-deltaRaf-1:ER cells. These results indicate that p42/p44 MAP kinase activation is not a limiting factor for G1 to S phase transit in absence of anchorage. Thus, at least one adhesion-mediated signalling event, distinct from MAP kinase activation is required for maximal cyclin D1 induction and hyperphosphorylation of pRb.

A reliable way of obtaining stable inducible clones.

Inducible gene expression systems provide a powerful tool for the analysis of gene product functions. The 'Tetracycline (Tc) expression system' has been widely and successfully used in many instances. However, this system remains somewhat tedious to use due to: (i) the establishment of a primary cell line constitutively and stably expressing the Tc-regulated transactivator and (ii) the obtention of a secondary line expressing the gene of interest in a Tc-dependent manner. In order to facilitate these two critical steps, we devised an efficient and molecular biology-free strategy allowing the successful selection of clones expressing any cDNA under tight regulation.

Post-translational and activation-dependent modifications of the G protein-coupled thrombin receptor.

The purpose of the present study was to analyze the post-translational and activation-dependent modifications of the G protein-coupled thrombin receptor. A human receptor cDNA was engineered to encode an epitope tag derived from the vesicular stomatitis virus glycoprotein at the COOH terminus of the receptor and expressed in human embryonic kidney 293 cells. We show here that the mature receptor is a glycosylated protein with an apparent molecular mass ranging from 68 to 80 kDa by SDS-polyacrylamide gel electrophoresis. Removal of asparagine-linked oligosaccharides with N-glycosidase F leads to the appearance of a 36-40-kDa receptor species. The current model for receptor activation by thrombin involves specific hydrolysis of the arginine-41/serine-42 (Arg-41/Ser-42) peptide bond. Cleavage of the receptor by thrombin was demonstrated directly by Western analyses performed on membranes and glycoprotein-enriched lysates from transfected cells. Whereas thrombin treatment of cells results in increased mobility of the receptor in SDS-polyacrylamide gel electrophoresis, we found that their treatment with the thrombin receptor agonist peptide leads to a decrease in thrombin receptor mobility due, in part, to phosphorylation. The serine proteases trypsin and plasmin also cleave and activate the receptor similar to thrombin, whereas chymotrypsin cleaves the receptor at a site distal to Arg-41, thus rendering it unresponsive to thrombin while still responsive to thrombin receptor agonist peptide.

Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation.

The mitogen-activated protein kinases (MAP kinases) p42mapk and p44mapk are serine/threonine kinases rapidly activated in cells stimulated with various extracellular signals by dual phosphorylation of tyrosine and threonine residues. They are thought to play a pivotal role in integrating and transmitting transmembrane signals required for growth and differentiation. Here we demonstrate that activation of these ubiquitously expressed MAP kinases is essential for growth. To specifically suppress MAP kinase activation in fibroblasts, we transiently expressed either the entire p44mapk antisense RNA or p44mapk kinase-deficient mutants (T192A or Y194F). As expected, and through independent mechanisms, both approaches strongly inhibited MAP kinase activation. The antisense reduced the expression of endogenous p42mapk and p44mapk by 90%, whereas overexpression of the T192A mutant inhibited growth factor activation of both endogenous MAP kinases by up to 70%. As a consequence, we found that the antisense as well as the T192A mutant of p44mapk inhibited growth factor-stimulated gene transcription (collagenase promoter assay with chloramphenicol acetyltransferase reporter) and cell growth. These effects were proportional to the extent of MAP kinase inhibition and reversed by coexpression of the wild-type p44mapk. Therefore we conclude that growth factor activation of p42mapk and p44mapk is an absolute requirement for triggering the proliferative response.

Various modes of gene regulation by nuclear receptors for steroid and thyroid hormones.

AP-1 is a transcriptional activator composed of homo- and heterodimers of Jun and Fos proteins. It is involved in activation of genes, such as collagenase, stromelysin, IL-2 and TGF beta 1, by tumour promoters, growth factors and cytokines. AP-1 activity is also elevated in response to transforming oncogenes and is required for cell proliferation. AP-1 activity is subject to complex regulation both transcriptionally and post-transcriptionally. Transcriptional control of jun and fos gene expression determines the amount and composition of the AP-1 complex. The jun and fos genes are regulated both positively and negatively and are highly inducible in response to extracellular stimuli. Post translational control is also important. Both cJun and cFos are subject to regulated phosphorylation. In the case of cJun, phosphorylation of sites near the DNA-binding domain inhibits DNA-binding, while dephosphorylation reverses this inhibition. Phosphorylation of cJun on sites within the N-terminal activation domain increases its ability to activate transcription. The protein kinase phosphorylating these sites is stimulated by cytokines and growth factors. Another mechanism modulating AP-1 activity is transcriptional interference by members of the nuclear receptor family and is relevant for the pathophysiology of rheumatoid arthritis (RA). In RA, chronic inflammation leads to increased AP-1 activity in T cells,macrophages and synoviocytes as a response to secretion of cytokines such as IL-1 and TNF alpha. While the IL-2 gene plays a major role in T cell activation, another AP-1 target gene encodes an enzyme, collagenase, responsible for destruction of bone and tendon.(ABSTRACT TRUNCATED AT 250 WORDS)

Biphasic increase in c-jun mRNA is required for induction of AP-1-mediated gene transcription: differential effects of muscarinic and thrombin receptor activation.

Activation of either muscarinic cholinergic or thrombin receptors increases phosphoinositide turnover, Ca2+ mobilization, and redistribution of protein kinase C and induces rapid transient increases in c-fos mRNA and c-jun mRNA in 1321N1 cells. To determine whether the increases in c-fos and c-jun mRNA induced by carbachol and thrombin are sufficient to stimulate AP-1-mediated transactivation, 1321N1 cells were transfected with a reporter carrying two copies of the tetradecanoyl phorbol acetate response element and the firefly luciferase gene. Thrombin was significantly more effective than carbachol at stimulating AP-1-mediated transactivation. To identify the factors underlying the difference in AP-1 activity induced by carbachol and thrombin, members of the fos and jun families which encode components of AP-1 were examined. Carbachol and thrombin have similar effects on expression of c-fos, fosB, fra-2, junB, and junD, both acutely and over a 24-h time course. However, whereas carbachol leads only to transient induction of c-jun (maximal at 0.5 h), thrombin induces a biphasic increase in c-jun mRNA--an initial peak at 0.5 h and a second, more-prolonged increase at 12 h. Thrombin but not carbachol also induces a late increase in fra-1 mRNA, which peaks at 12 h. The secondary increase in c-jun mRNA is associated with marked increases in c-Jun protein levels and AP-1 DNA-binding activity. The late induction of c-jun and fra-1 mRNA can be prevented by adding the antagonist hirudin 30 min after thrombin, which results in loss of thrombin-stimulated increases in c-Jun protein, AP-1 DNA-binding activity, and AP-1-mediated transactivation. These findings suggest that rapid and transient conduction of c-fos and c-jun mRNA is insufficient to induce prominent changes in gene transcription, while the sustained increase in c-jun mRNA and perhaps the late induction of fra-1 mRNA are required for generation of AP-1 DNA-binding activity and transactivation through AP-1.

Fos and Jun repress transcriptional activation by myogenin and MyoD: the amino terminus of Jun can mediate repression.

Myogenin and MyoD belong to a family of muscle-specific helix-loop-helix (HLH) proteins that have the potential to activate muscle-specific genes in nonmyogenic cells. Peptide growth factors can block the ability of myogenin and MyoD to activate their target genes. Here, we show that the growth factor-inducible proto-oncogenes c-fos, c-jun, and junB mimic the effects of exogenous growth factors and suppress trans-activation of the muscle creatine kinase (MCK) enhancer by myogenin and MyoD. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, is an inefficient inhibitor of the trans-activating capacity of myogenin and MyoD. Transcriptional repression by Fos and Jun is specific to myogenic HLH proteins and is not observed with the widely expressed HLH protein E47, which recognizes the same DNA sequence. Repression of the MCK enhancer by Fos and Jun is targeted at the myogenin and MyoD DNA recognition sequence and can be mediated by the amino terminus of c-Jun. Comparison of several myogenin mutants for their responsiveness to Fos and Jun shows that repression is directed at the basic-HLH region. These results indicate that members of the Jun family can be distinguished on the basis of their effects on muscle-specific transcription and suggest there is cross talk between transcription factors that control myogenesis and those involved in cell proliferation.

Cloning, functional expression and role in cell growth regulation of a hamster 5-HT2 receptor subtype.

We have isolated a hamster fibroblast cDNA clone that encodes a serotoninergic receptor whose deduced amino acid sequence displays 94% identity with the rat brain serotonin (5-HT) type 2 receptor. When expressed in Xenopus oocytes, the hamster receptor efficiently couples to the phosphoinositide second messenger system and leads to intracellular Ca2+ mobilization in response to 5-HT. To determine the pharmacological properties of this receptor, and to evaluate the role of phospholipase C (PLC) activation in growth modulation by 5-HT, we have expressed it in hamster fibroblasts. Transfected cells that express 5-HT receptors were selected using a novel method based on coexpression of the Na+/H+ antiporter gene as a selectable marker. After co-transfection of the 5-HT receptor and Na+/H+ antiporter cDNAs in fibroblasts lacking antiporter activity (variants of the CCL39 line), 50% of the clones resistant to an acute acid load express functional receptors. The pharmacological profile of the transfected receptor is consistent with it being of the 5-HT2 subtype, and the extent of 5-HT-stimulated PLC activation in independent clones correlates with their relative level of cRNA expression. In cells in where addition of 5-HT leads to strong activation of PLC, and inhibition of adenylate cyclase via endogenous 5-HT1b receptors, 5-HT alone has little effect on DNA synthesis stimulation. Thus we conclude that activation of the PLC signalling pathway in these cells is not sufficient to trigger G0/G1 to S phase transition. Strong activation of PLC via 5-HT2 receptors does however contribute to the synergy observed between 5-HT (Gi-coupled pathway) and fibroblast growth factor (tyrosine kinase-activated pathway) on DNA synthesis reinitiation in transfected cells.

Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction.

Glucocorticoids are potent inhibitors of collagenase induction by phorbol esters and inflammatory mediators. The target for this negative effect is the AP-1 site within the collagenase promoter, which also mediates its induction. Negative regulation is due to repression of AP-1 activity by the glucocorticoid receptor (GCR). While the GCR is a potent inhibitor of AP-1 activity (Jun/Fos), both c-Jun and c-Fos are potent repressors of GCR activity. In vitro experiments using purified GCR and c-Jun proteins suggest that mutual repression is due to direct interaction between the two. Direct interaction between GCR and either c-Jun or c-Fos is demonstrated by cross-linking and coimmunoprecipitation. These findings reveal a cross talk between two major signal transduction systems used to control gene transcription in response to extracellular stimuli, and a novel mechanism for transcriptional repression.

Functional expression of Ca2(+)-mobilizing alpha-thrombin receptors in mRNA-injected Xenopus oocytes.

alpha-Thrombin (TH) initiates a program of intracellular events that lead to DNA replication in quiescent CCL39 Chinese hamster lung fibroblasts via membrane receptors that have yet to be characterized at a molecular level. Functional TH receptors were expressed in Xenopus laevis oocytes following injection of poly(A)+ RNA from TH-responsive CCL39 cells; their presence was demonstrated by TH-stimulated 45Ca2+ efflux or Ca2(+)-dependent Cl- channel activation. In voltage clamp experiments on microinjected oocytes a Ca2(+)-activated Cl- current was detected in response to TH (0.2-10 U/ml). The TH response was blocked by a specific TH inhibitor, and potentiated by addition of FGF or intracellular injection of GTP-gamma-S.