Growth inhibitory properties of endothelin-1 in activated human hepatic stellate cells: a cyclic adenosine monophosphate-mediated pathway. Inhibition of both extracellular signal-regulated kinase and c-Jun kinase and upregulation of endothelin B receptors.

During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype, proliferate, and synthetize fibrosis components. We have shown that endothelin-1 (ET-1) inhibits the proliferation of activated human HSC via endothelin B (ETB) receptors. We now investigate the transduction pathway involved in the growth inhibitory effect of ET-1 in activated HSC. Endothelin-1 and the ETB receptor agonist, sarafotoxin-S6C, increased synthesis of PGI2 and PGE2, leading to elevation of cAMP. The cyclooxygenase inhibitor ibuprofen and the adenylyl cyclase inhibitor SQ22536 both blunted the growth inhibitory effect of ET-1. Analysis of early steps associated with growth inhibition indicated that: (a) similar to ET-1, forskolin decreased c-jun mRNA induction without affecting c-fos and krox 24 mRNA expression; (b) ET-1, sarafotoxin-S6C, as well as forskolin, reduced activation of both c-Jun kinase and extracellular signal-regulated kinase. Finally, forskolin, PGI2, and PGE2 raised by fivefold the number of ET binding sites after 6 h, and increased the proportion of ETB receptors from 50% in control cells to 80% in treated cells. In conclusion, ET-1 inhibits proliferation of activated HSC via ETB receptors, through a prostaglandin/cAMP pathway that leads to inhibition of both extracellular signal-regulated kinase and c-Jun kinase activities. Upregulation of ETB receptors by prostaglandin/cAMP raises the possibility of a positive feedback loop that would amplify the growth inhibitory response. These results suggest that ET-1 and agents that increase cAMP might be of interest to limit proliferation of activated HSC during chronic liver diseases.

The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release.

This study assesses the controversial role of the mitochondrial permeability transition (MPT) in apoptosis. In primary rat hepatocytes expressing an IkappaB superrepressor, tumor necrosis factor alpha (TNFalpha) induced apoptosis as shown by nuclear morphology, DNA ladder formation, and caspase 3 activation. Confocal microscopy showed that TNFalpha induced onset of the MPT and mitochondrial depolarization beginning 9 h after TNFalpha treatment. Initially, depolarization and the MPT occurred in only a subset of mitochondria; however, by 12 h after TNFalpha treatment, virtually all mitochondria were affected. Cyclosporin A (CsA), an inhibitor of the MPT, blocked TNFalpha-mediated apoptosis and cytochrome c release. Caspase 3 activation, cytochrome c release, and apoptotic nuclear morphological changes were induced after onset of the MPT and were prevented by CsA. Depolarization and onset of the MPT were blocked in hepatocytes expressing DeltaFADD, a dominant negative mutant of Fas-associated protein with death domain (FADD), or crmA, a natural serpin inhibitor of caspases. In contrast, Asp-Glu-Val-Asp-cho, an inhibitor of caspase 3, did not block depolarization or onset of the MPT induced by TNFalpha, although it inhibited cell death completely. In conclusion, the MPT is an essential component in the signaling pathway for TNFalpha-induced apoptosis in hepatocytes which is required for both cytochrome c release and cell death and functions downstream of FADD and crmA but upstream of caspase 3.

The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy.

Using confocal microscopy, onset of the mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by the redistribution of the cytosolic fluorophore, calcein, into mitochondria. Simultaneously, mitochondria release membrane potential-indicating fluorophores like tetramethylrhodamine methylester. The MPT occurs in several forms of necrotic cell death, including oxidative stress, pH-dependent ischemia/reperfusion injury and Ca2+ ionophore toxicity. Cyclosporin A (CsA) and trifluoperazine block the MPT in these models and prevent cell killing, showing that the MPT is a causative factor in necrotic cell death. During oxidative injury induced by t-butylhydroperoxide, onset of the MPT is preceded by pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and an increase of mitochondrial free Ca2+, all changes that promote the MPT. During tissue ischemia, acidosis develops. Because of acidotic pH, anoxic cell death is substantially delayed. However, when pH is restored to normal after reperfusion (reoxygenation at pH 7.4), cell death occurs rapidly (pH paradox). This killing is caused by pH-dependent onset of the MPT, which is blocked by reperfusion at acidotic pH or with CsA. In isolated mitochondria, toxicants causing Reye's syndrome, such as salicylate and valproate, induce the MPT. Similarly, salicylate induces a CsA-sensitive MPT and killing of cultured hepatocytes. These in vitro findings suggest that the MPT is the pathophysiological mechanism underlying Reye's syndrome in vivo. Kroemer and coworkers proposed that the MPT is a critical event in the progression of apoptotic cell death. Using confocal microscopy, the MPT can be directly documented during tumor necrosis factor-alpha induced apoptosis in hepatocytes. CsA blocks this MPT and prevents apoptosis. The MPT does not occur uniformly during apoptosis. Initially, a small proportion of mitochondria undergo the MPT, which increases to nearly 100% over 1-3 h. A technique based on fluorescence resonance energy transfer can selectively reveal mitochondrial depolarization. After nutrient deprivation, a small fraction of mitochondria spontaneously depolarize and enter an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. A model is proposed in which onset of the MPT to increasing numbers of mitochondria within a cell leads progressively to autophagy, apoptosis and necrotic cell death.

Confocal microscopy of the mitochondrial permeability transition in necrotic and apoptotic cell death.

Opening of a high-conductance pore in the mitochondrial inner membrane induces onset of the mitochondrial permeability transition (mPT). Cyclosporin A and trifluoperazine inhibit this pore and block necrotic cell death in oxidative stress, Ca2+ ionophore toxicity, Reye-related drug toxicity, pH-dependent ischaemia/reperfusion injury and other models of cell injury. Confocal fluorescence microscopy directly visualizes the increased mitochondrial membrane permeability of the mPT from the movement of calcein from the cytosol into the matrix space. Pyridine nucleotide oxidation, increased mitochondrial Ca2+ and mitochondrial generation of reactive oxygen species (ROS) all contribute to the onset of the mPT in situ. Confocal microscopy also shows directly that the mPT is a critical link in apoptotic signalling by tumour necrosis factor-alpha at a point downstream of caspase 8 and upstream of caspase 3. Cyclosporin A blocks this mPT, preventing release of pro-apoptotic cytochrome c from mitochondria and subsequent apoptotic cell killing. Progression to necrosis or apoptosis after the mPT depends on the availability of ATP, which blocks necrosis but promotes the apoptotic programme. Given the pathophysiological importance of the mPT, development of agents to modulate the mPT represents an important new goal for pharmaceutical drug discovery.

Confocal microscopy of the mitochondrial permeability transition in necrotic cell killing, apoptosis and autophagy.

Onset of the cyclosporin-A-sensitive mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by laser scanning confocal microscopy. The MPT is a causative event in many types of necrotic and apoptotic cell death, including oxidative stress, ischemia/reperfusion injury, Ca2+ ionophore toxicity and tumor necrosis factor alpha (TNF alpha) induced apoptosis, and may contribute to Reye's-related drug toxicity. Pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and increased mitochondrial Ca2+ and pH can each promote onset of the MPT in situ. The MPT can also be directly visualized during TNF alpha-induced apoptosis to hepatocytes. Mitochondria spontaneously depolarize in situ after nutrient deprivation before entering an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. We propose a model in which onset of the MPT to increasing numbers of mitochondria leads progressively to autophagy, apoptosis and necrotic cell death.

Gene expression and cytokine and enzyme activation in the liver after a burn injury.

The liver plays a critical role in the inflammatory response to injury; however, the mechanisms by which the liver is affected and how it influences the rest of the immune system are not well understood. Partial hepatectomy is a direct injury to the liver, whereas a burn is an indirect injury to liver, but both injuries appear to produce damage to the liver. In this study, we used a mouse model of 25% total body surface area and 40% total body surface area full-thickness burns to investigate the mechanism of liver damage and response to burn injury by measuring levels of c-Jun messenger (m)RNA, NFkappaB nuclear protein, interleukin-6, transaminases, and liver tissue histology over time. c-Jun and NFkappaB are 2 transcription factors that are induced by partial hepatectomy and related to hepatocyte injury and growth. In both groups of mice with burns, expression of c-Jun mRNA and NFkappaB nuclear protein was activated within 30 minutes after the burn injury, followed by increased levels of interleukin-6 and, finally, elevated enzyme levels. Liver injuries were similar in both groups despite the magnitude of the burns. We believe that these gene products are initiated in the hepatocyte injury after a burn and that they precede other inflammatory responses such as cytokine release, plasma transaminase levels, and histologic changes.

Personality traits valued by practicing nurses and measured in nursing students.

Human relationships are inherent in professional nursing practice, and are influenced by personality traits of the nurse. The purpose of this study was to develop a profile of personality traits desirable for nurses that could be used as a basis for preprofessional guidance and for teaching strategies that would promote the development of these traits. Nurses from four states indicated the level of personality traits desirable for nurses, based on Personality Research Form (PRF) definitions. The PRF was then administered to both associate degree (ADN) and baccalaureate degree (BSN) students. Although some differences were demonstrated, the level desired by the nurses was congruent with student scores, and the scores of ADN and BSN students at point of entry and on completion of their nursing education did not significantly differ for the majority of the traits measured.

PI3K inhibitors block skeletogenesis but not patterning in sea urchin embryos.

Skeletogenesis in the sea urchin embryo is a simple model of biomineralization, pattern formation, and cell-cell communication during embryonic development. The calcium carbonate skeletal spicules are secreted by primary mesenchyme cells (PMCs), but the skeletal pattern is dictated by the embryonic ectoderm. Although the process of skeletogenesis is well characterized, there is little molecular understanding of the basis of patterning within this system. In this study, we examined the contribution of phosphatidylinositide 3-kinase (PI3K)-mediated signaling to the skeletogenic process in sea urchin embryos by using the well-established PI3K inhibitors LY294002 and wortmannin. Our results show that PI3K inhibitors specifically and reversibly block skeletogenesis, and that this blockade occurs within the PMCs rather than in the ectoderm, because the inhibitors block spiculogenesis in cultured micromeres. Our results are consistent with a model in which PI3K signaling is required, not for pattern sensing or interpretation but rather for the biomineralization process itself in the sea urchin embryo.

Dominant-negative TAK1 induces c-Myc and G(0) exit in liver.

Transforming growth factor-beta (TGF-beta)-activated kinase 1 (TAK1), a serine/threonine kinase, is reported to function in the signaling pathways of TGF-beta, interleukin 1, and ceramide. However, the physiological role of TAK1 in vivo is largely unknown. To assess the function of TAK1 in vivo, dominant-negative TAK1 (dnTAK1) was expressed in the rat liver by adenoviral gene transfer. dnTAK1 expression abrogated c-Jun NH(2)-terminal kinase and c-Jun but not nuclear factor (NF)-kappaB or SMAD activation after partial hepatectomy (PH). Expression of dnTAK1 or TAM-67, a dominant-negative c-Jun, induced G(0) exit in quiescent liver and accelerated cell cycle progression after PH. Finally, dnTAK1 and TAM-67 induced c-myc expression in the liver before and after PH, suggesting that G(0) exit induced by dnTAK1 and TAM-67 is mediated by c-myc induction.

Hydrogen peroxide-induced liver cell necrosis is dependent on AP-1 activation.

To determine whether intracellular signaling events involved in apoptosis may also mediate necrosis, the role of the transcription factor AP-1 was investigated in a hepatoma cell model of cellular necrosis induced by oxidant stress. Treatment of the human hepatoma cell line HuH-7 with H2O2 caused dose-dependent necrosis as determined by light microscopy, fluorescent staining, and an absence of DNA fragmentation. H2O2 treatment led to increases in c-fos and c-jun mRNA levels, Jun nuclear kinase activity, and AP-1 DNA binding. AP-1 transcriptional activity measured with an AP-1-driven luciferase reporter gene was also increased. To determine whether this AP-1 activation contributed to H2O2-induced cell necrosis, HuH-7 cells were stably transfected with an antisense c-jun expression vector. Cells expressing antisense c-jun had decreased levels of AP-1 activation and significantly increased survival after H2O2 exposure. These data indicate that AP-1 activation occurs during oxidant-induced cell necrosis and contributes to cell death. Necrosis is therefore not always a passive process but may involve the activation of intracellular signaling pathways similar to those that mediate apoptosis.

Mechanisms of hepatic toxicity. I. TNF-induced liver injury.

Tumor necrosis factor-alpha (TNF-alpha) functions as a two-edged sword in the liver. TNF-alpha is required for normal hepatocyte proliferation during liver regeneration. It functions both as a comitogen and to induce the transcription factor nuclear factor-kappaB, which has antiapoptotic effects. On the other hand, TNF-alpha is the mediator of hepatotoxicity in many animal models, including those involving the toxins concanavalin A and lipopolysaccharide. TNF-alpha has also been implicated as an important pathogenic mediator in patients with alcoholic liver disease and viral hepatitis.

Activation of nuclear factor-kappaB during orthotopic liver transplantation in rats is protective and does not require Kupffer cells.

Reperfusion after liver transplantation results in the induction of tumor necrosis factor-alpha (TNFalpha) as well as activation of the stress-associated signaling proteins, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1), and nuclear factor-kappaB (NF-kappaB). To test the hypothesis that Kupffer cells are involved in the activation of signal transduction cascades during rat liver transplantation, Kupffer cells were depleted from donor liver using gadolinium chloride (GdCl3), and then the activation of JNK, AP-1, and NF-kappaB were assessed after transplantation. The results showed that GdCl3 treatment did not inhibit the activation of these stress signals, although transplanted livers were depleted of Kupffer cells and partially protected from reperfusion injury. Interleukin-6 (IL-6) and IL-10 messenger RNAs (mRNAs) were induced by transplantation, and the induction was suppressed by Kupffer cell depletion. The induction of TNFalpha mRNA and serum protein during liver transplantation was unaffected by GdCl3. These results show that Kupffer cells are not a major source of TNFalpha production after liver transplantation and that stress-signaling protein activation occurs independently of Kupffer cells. Transplantation strongly activates the transcription factor NF-kappaB, which blocks TNFalpha-mediated apoptosis in hepatocytes in vitro. To assess the role of NF-kappaB activation during liver transplantation, the IkappaBalpha superrepressor was expressed in donor livers using adenoviral-mediated gene transfer. Inhibition of NF-kappaB resulted in increased serum alanine aminotransferase levels after 3 hours of transplantation. In addition, the blockade of NF-kappaB resulted in increased histological tissue injury and increased hepatic terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, indicating apoptosis. These results show that NF-kappaB activation has a protective role in the transplanted liver.

TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells.

After liver injury, hepatic stellate cells (HSCs) undergo a process of activation with expression of smooth muscle alpha-actin (alpha-SMA), an increased proliferation rate, and a dramatic increase in synthesis of type I collagen. The intracellular signaling mechanisms of activation and perpetuation of the activated phenotype in HSCs are largely unknown. In this study the role of the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, were evaluated in primary cultures of rat HSCs. The effect of JNK was assessed by using an adenovirus expressing a dominant negative form of transforming growth factor beta (TGF-beta)-activated kinase 1 (TAK1) (Ad5dnTAK1) and a new selective pharmacologic inhibitor SP600125. The effect of p38 was assessed with the selective pharmacologic inhibitor SB203580. These kinases were inhibited starting either in quiescent HSCs (culture day 1) or in activated HSCs (culture day 5). Although blocking TAK1/JNK and p38 decreased the expression of alpha-SMA protein in early stages of HSC activation, no effect was observed when TAK1/JNK or p38 were inhibited in activated HSCs. JNK inhibition increased and p38 inhibition decreased collagen alpha1(I) mRNA level as measured by RNase protection assays, with maximal effects observed in early stages of HSC activation. Furthermore, TAK1/JNK inhibition decreased HSC proliferation, whereas p38 inhibition led to an increased proliferation rate of HSCs, independently of its activation status. These results show novel roles for the TAK1/JNK pathway and p38 during HSC activation in culture. Despite similar activators of TAK1/JNK and p38, their functions in HSCs are distinct and opposed.

The mitochondrial permeability transition augments Fas-induced apoptosis in mouse hepatocytes.

Tumor necrosis factor-alpha receptor 1 and Fas recruit overlapping signaling pathways. To clarify the differences between tumor necrosis factor alpha (TNFalpha) and Fas pathways in hepatocyte apoptosis, primary mouse hepatocytes were treated with TNFalpha or an agonist anti-Fas antibody after infection with an adenovirus expressing an IkappaB superrepressor (Ad5IkappaB). Treatment with TNFalpha induced apoptosis in Ad5IkappaB-infected mouse hepatocytes, as we previously reported for rat hepatocytes. Ad5IkappaB plus anti-Fas antibody or actinomycin D plus anti-Fas antibody rapidly induced apoptosis, whereas anti-Fas antibody alone produced little cytotoxicity. The proteasome inhibitor (MG-132) and a dominant-negative mutant of nuclear factor-kappaB-inducing kinase also promoted TNFalpha- and Fas-mediated apoptosis. Expression of either crmA or a dominant-negative mutant of the Fas-associated death domain protein prevented TNFalpha- and Fas-mediated apoptosis. In addition, the caspase inhibitors, DEVD-cho and IETD-fmk, inhibited TNFalpha- and Fas-mediated apoptosis. In Ad5IkappaB-infected hepatocytes, caspases-3 and -8 were activated within 2 h after treatment with anti-Fas antibody or within 6 h after TNFalpha treatment. Confocal microscopy demonstrated onset of the mitochondrial permeability transition (MPT) and mitochondrial depolarization by 2-3 h after anti-Fas antibody treatment and 8-10 h after TNFalpha treatment, followed by cytochrome c release. The combination of the MPT inhibitors, cyclosporin A, and trifluoperazine, protected Ad5IkappaB-infected hepatocytes from TNFalpha-mediated apoptosis. After anti-Fas antibody, cyclosporin A and trifluoperazine decreased cytochrome c release but did not prevent caspase-3 activation and cell-death. In conclusion, nuclear factor-kappaB activation protects mouse hepatocytes against both TNFalpha- and Fas-mediated apoptosis. TNFalpha and Fas recruit similar but nonidentical, pathways signaling apoptosis. The MPT is obligatory for TNFalpha-induced apoptosis. In Fas-mediated apoptosis, the MPT accelerates the apoptogenic events but is not obligatory for them.

Corn oil rapidly activates nuclear factor-kappaB in hepatic Kupffer cells by oxidant-dependent mechanisms.

N-6 polyunsaturated fatty acids (N-6 PUFAs), major constituents of corn oil and natural ligands for peroxisome proliferator-activated receptors, increase the rate of growth of established tumors. It has been proposed that chemical peroxisome proliferators increase hepatocyte proliferation by mechanisms involving activation of nuclear factor-kappaB (NF-kappaB) and production of low levels of tumor necrosis factor alpha (TNFalpha) by Kupffer cells; however, how N-6 PUFAs are involved in increased cell proliferation in liver is not well understood. Here, the hypothesis that N-6 PUFAs increase production of mitogens by activation of Kupffer cell NF-kappaB was tested. A single dose of corn oil (2 ml/kg, i.g.), but not olive oil or medium-chain triglycerides (saturated fat), caused an approximately 3-fold increase in hepatocyte proliferation. Similarly, when activity of NF-kappaB in whole rat liver or isolated hepatocytes and Kupffer cells was measured at various time intervals for up to 36 h, only corn oil activated NF-kappaB. Corn oil increased NF-kappaB activity approximately 3-fold 1-2 h after treatment exclusively in the Kupffer cell fraction. In contrast, increases were small and only occurred after approximately 8 h in hepatocytes. The activation of NF-kappaB at 2 h and increases in cell proliferation at 24 h due to corn oil were prevented almost completely when rats were pretreated for 4 days with either dietary glycine (5% w/w), an agent that inactivates Kupffer cells, or the NADPH oxidase inhibitor, diphenyleneiodonium (s.c., 1 mg/kg/day). Furthermore, arachidonic acid (100 microM) activated superoxide production approximately 4-fold when added to isolated Kupffer cells in vitro. This phenomenon was not observed with oleic or linoleic acids. Interestingly, a single dose of corn oil increased TNFalpha mRNA nearly 2-fold 8 h after treatment. It is concluded that corn oil rapidly activates NF-kappaB in Kupffer cells via oxidant-dependent mechanisms. This triggers production of low levels of TNFalpha which is mitogenic in liver and promotes growth of hepatocytes.

TNF receptor-associated factor-2 is involved in both IL-1 beta and TNF-alpha signaling cascades leading to NF-kappa B activation and IL-8 expression in human intestinal epithelial cells.

Cytokine signaling involves the participation of many adaptor proteins, including the docking protein TNF receptor-associated factor-2 (TRAF-2), which is believed to transmit the TNF-alpha signal through both the I kappa B/NF-kappa B and c-Jun N-terminal kinase (JNK)/stress-related protein kinase (SAPK) pathways. The physiological role of TRAF proteins in cytokine signaling in intestinal epithelial cells (IEC) is unknown. We characterized the effect of a dominant-negative TRAF-2 delivered by an adenoviral vector (Ad5dnTRAF-2) on the cytokine signaling cascade in several IEC and also investigated whether inhibiting the TRAF-2-transmitting signal blocked TNF-alpha-induced NF-kappa B and IL-8 gene expression. A high efficacy and level of Ad5dnTRAF-2 gene transfer were obtained in IEC using a multiplicity of infection of 50. Ad5dnTRAF-2 expression prevented TNF-alpha-induced, but not IL-1 beta-induced, I kappa B alpha degradation and NF-kappa B activation in NIH-3T3 and IEC-6 cells. TNF-alpha-induced JNK activation was also inhibited in Ad5dnTRAF-2-infected HT-29 cells. Induction of IL-8 gene expression by TNF-alpha was partially inhibited in Ad5dnTRAF-2-transfected HT-29, but not in control Ad5LacZ-infected, cells. Surprisingly, IL-1 beta-mediated IL-8 gene expression was also inhibited in HT-29 cells as measured by Northern blot and ELISA. We concluded that TRAF-2 is partially involved in TNF-alpha-mediated signaling through I kappa B/NF-kappa B in IEC. In addition, our data suggest that TRAF-2 is involved in IL-1 beta signaling in HT-29 cells. Manipulation of cytokine signaling pathways represents a new approach for inhibiting proinflammatory gene expression in IEC.

Glycine and uridine prevent D-galactosamine hepatotoxicity in the rat: role of Kupffer cells.

Extrahepatic factors, such as increased gut permeability and bacteria from the gut, have been shown to play a role in D-galactosamine toxicity in rats. Because bacterial endotoxin activates Kupffer cells, the purpose of this study was to clarify the role of Kupffer cells in the mechanism of D-galactosamine hepatotoxicity in rats and determine whether uridine, a compound that rescues animals from D-galactosamine toxicity, affects Kupffer cells. Rats were fed control or glycine (5%) containing diets to prevent Kupffer cell activation or treated with gadolinium chloride (GdCl3, 20 mg/kg) to destroy Kupffer cells selectively before injection of D-galactosamine (500 mg/kg, intraperitoneally). D-galactosamine caused panlobular focal hepatocellular necrosis, polymorphonuclear cell infiltration, and increased serum transaminases significantly at 24 hours. Dietary glycine or pretreatment with GdCl3 prevented these effects. D-galactosamine caused a transient increase in circulating endotoxin that was maximal at 1 hour and was blunted significantly by dietary glycine. Additionally, antisera to tumor necrosis factor-alpha (TNF-alpha) prevented hepatotoxicity caused by D-galactosamine. Moreover, apoptosis in hepatocytes caused by D-galactosamine occurred before necrosis (6 hours) and was prevented by glycine, GdCl3, TNF-alpha antiserum, and uridine. Thus, it was hypothesized that TNF-alpha from Kupffer cells causes apoptosis after D-galactosamine administration in the rat. Indeed, increases in TNF-alpha messenger RNA (mRNA) were detected as early as 2.5 hours after D-galactosamine treatment. Previous work proposed that uridine blocks D-galactosamine toxicity by preventing inhibition of mRNA synthesis. In view of these results, the possibility that uridine might affect Kupffer cells was investigated. Uridine significantly blunted the increase in [Ca2+]i and release of TNF-alpha caused by endotoxin in isolated Kupffer cells and prevented apoptosis caused by D-galactosamine treatment in vivo. These data support the hypothesis that uridine prevents D-galactosamine hepatotoxicity not only by rescuing the hepatocyte in the late phases of the injury but also preventing TNF-alpha release from Kupffer cells thereby blocking apoptosis that occurs early after D-galactosamine treatment. Taken together, these data strongly support the role of Kupffer cell activation by endotoxin early after D-galactosamine treatment as an important event in the mechanism of hepatotoxicity in the rat.

The focal adhesion kinase suppresses transformation-associated, anchorage-independent apoptosis in human breast cancer cells. Involvement of death receptor-related signaling pathways.

The focal adhesion kinase (FAK) is a mediator of cell-extracellular matrix signaling events and is overexpressed in tumor cells. In order to rapidly down-regulate FAK function in normal and transformed mammary cells, we have used adenoviral gene transduction of the carboxyl-terminal domain of FAK (FAK-CD). Transduction of adenovirus containing FAK-CD in breast cancer cells caused loss of adhesion, degradation of p125(FAK), and induced apoptosis. Furthermore, breast tumor cells that were viable without matrix attachment also underwent apoptosis upon interruption of FAK function, demonstrating that FAK is a survival signal in breast tumor cells even in the absence of matrix signaling. In addition, both anchorage-dependent and anchorage-independent apoptotic signaling required Fas-associated death domain and caspase-8, suggesting that a death receptor-mediated apoptotic pathway is involved. Finally, FAK-CD had no effect on adhesion or viability in normal mammary cells, despite the loss of tyrosine phosphorylation of p125(FAK). These results indicate that FAK-mediated signaling is required for both cell adhesion and anchorage-independent survival and the disruption of FAK function involves the Fas-associated death domain and caspase-8 apoptotic pathway.

Reperfusion after liver transplantation in rats differentially activates the mitogen-activated protein kinases.

The injury resulting from cold ischemia and warm reperfusion during liver transplantation is a major clinical problem that limits graft success. Kupffer cell activation plays a pivotal role in reperfusion injury, and Kupffer cell products, including free radicals and tumor necrosis factor alpha (TNF-alpha), are implicated as damaging agents. However, the second messengers and signaling pathways that are activated by the stress of hepatic ischemia/reperfusion remain unknown. The purpose of this study is to assess the activation of the three known vertebrate mitogen activated protein kinase (MAPKs) and the activating protein 1 (AP-1) transcription factor in response to ischemia and reperfusion in the transplanted rat liver. There was a potent, sustained induction of c-jun N-terminal kinase (JNK), but not of the related MAPKs extracellular signal-regulated kinases (ERK) or p38, upon reperfusion after transplantation. TNF-alpha messenger RNA (mRNA) levels and transcription factors AP-1 and nuclear factor-kappaB (NF-kappaB) were induced in the liver after 60 minutes of reperfusion. Finally, there was an elevation of ceramide, but not diacylglycerol or sphingosine, in the transplanted liver. Ceramide is a second messenger generated by TNF-alpha treatment and is an activator of JNK. Because JNK activation preceded the elevations in ceramide and TNF-alpha mRNA, these results suggest that increased hepatic TNF-alpha and ceramide may perpetuate JNK induction, but that they are not the initiating signals of JNK activation during reperfusion injury in the transplanted liver.