Cyclic AMP and cyclic GMP suppress TNFalpha-induced hepatocyte apoptosis by inhibiting FADD up-regulation via a protein kinase A-dependent pathway.

Cyclic AMP (cAMP) and cyclic GMP (cGMP) suppress apoptosis in many cell types, including hepatocytes. We have previously shown that membrane-permeable cAMP and cGMP analogs attenuate tumor necrosis factor alpha plus actinomycin D (TNFalpha/ActD)-induced apoptosis in hepatocytes at a step upstream of caspase activation and cytochrome c release. Recently we have also shown that FADD levels increase 10 folds in response to TNFalpha/ActD. Therefore we hypothesized that cAMP and cGMP would inhibit FADD upregulation. We show here that cyclic nucleotide analogs dibutyryl cAMP (db-cAMP) and 8-bromo-cGMP (Br-cGMP) inhibit cell death and the cleavages of multiple caspases including caspase-10, -9, -8, -3, and -2, as well as suppress FADD protein up-regulation in TNFalpha/ActD-induced apoptosis. The inhibitory effects of cAMP were seen at lower concentrations than cGMP. Both cAMP and cGMP prevented FADD overexpression and cell death in hepatocytes transfected with the FADD gene. A protein kinase A (PKA) inhibitor, KT 5720, reversed the inhibition of FADD protein levels induced by cAMP or cGMP. In conclusion, our findings indicate that cAMP and cGMP prevent TNFalpha/ActD-induced apoptosis in hepatocytes and that this occurs in association with a near complete inhibition of the upregulation of FADD via a PKA-dependent mechanism.

Inflammatory modulation of hepatocyte apoptosis by nitric oxide: in vivo, in vitro, and in silico studies.

Nitric oxide (NO*) and its reaction products are key players in the physiology and pathophysiology of inflammatory settings such as sepsis and shock. The consequences of the expression of inducible NO* synthase (iNOS, NOS-2) can be either protective or damaging to the liver. We have delineated two distinct hepatoprotective actions of NO*: the stimulation of cyclic guanosine monophosphate and the inhibition of caspases by S-nitrosation. In contrast, iNOS/NO* promotes hepatocyte death under conditions of severe redox stress, such as hemorrhagic shock or ischemia/reperfusion. Redox stress activates an unknown molecular switch that transforms NO*, which is hepatoprotective under resting conditions, into an agent that induces hepatocyte death. We hypothesize that the magnitude of the redox stress is a major determinant for the effects of NO* on cell survival by controlling the chemical fate of NO*. To address this hypothesis, we have carried out studies in relevant in vivo and in vitro settings. Moreover, we have constructed an initial mathematical model of caspase activation and coupled it to a model describing some of the reactions of NO* in hepatocytes. Our studies suggest that modulation of iron, oxygen, and superoxide may dictate whether NO* is hepatoprotective or hepatotoxic.

Nitric oxide facilitates cardiomyogenesis in mouse embryonic stem cells.

Stem cell therapy holds great promise for the replacement of damaged or dysfunctional myocardium. Nitric oxide (NO) has been shown to promote embryonic stem (ES) cell differentiation in other systems. We hypothesized that NO, through NO synthase gene transfer or exogenous NO exposure, would promote the differentiation of mouse ES cells into cardiomyocytes (CM). In our study, NO treatment increased both the number and the size of beating foci in embryoid body (EB) outgrowths. Within 2 weeks, 69% of the inducible NO synthase-transduced EB displayed spontaneously beating foci, as did 45% of the NO donor-treated EB, compared with only approximately 15% in controls. Cardiac-specific genes and protein expression were significantly increased in NO-treated ES. Electron microscopy and immunocytochemistry revealed that these NO-induced contracting cells exhibited characteristics consistent with CM. At day 7 in culture, troponin T was expressed in 45.6 +/- 20.6% of the NO-treated ES cells but in only 9.25 +/- 1.77% of control cells. Interestingly, 50.4 +/- 18.4% of NO-treated ES cells were troponin T-negative and annexin V-positive. This apoptotic phenotype was seen in <1% of the control ES cells. These data strongly support our hypothesis that mouse ES cells can be accelerated to differentiate into CM by NO treatment. NO may influence cardiac differentiation by both inducing a switch toward a cardiac phenotype and inducing apoptosis in cells not committed to cardiac differentiation.

Carbon monoxide promotes Fas/CD95-induced apoptosis in Jurkat cells.

A properly functioning immune system is dependent on programmed cell death/apoptosis at virtually every stage of lymphocyte development and activity. Carbon monoxide (CO), an enzymatic product of heme oxyenase-1, has been shown to possess anti-apoptotic effects in a number of different model systems. The purpose of the present study was to expand on this knowledge to determine the role of CO in the well established model of Fas/CD95-induced apoptosis in Jurkat cells, and to determine the mechanism by which CO can modulate T-cell apoptosis. Exposure of Jurkat cells to CO resulted in augmentation in Fas/CD95-induced apoptosis, which correlated with CO-induced up-regulation of the pro-apoptotic protein FADD as well as activation of caspase-8, -9, and -3 while simultaneously down-regulating the anti-apoptotic protein BCL-2. These effects of CO were lost with overexpression of the small interfering RNA of FADD. CO, as demonstrated previously in endothelial cells, was also anti-apoptotic in Jurkat cells against tumor necrosis factor and etoposide. We further demonstrate that this pro-apoptotic effect of CO was independent of reactive oxygen species production and involved inhibition in Fas/CD95-induced activation of the pro-survival ERK MAPK. We conclude that in contrast to other studies showing the anti-apoptotic effects of CO, Fas/CD95-induced cell death in Jurkat cells is augmented by exposure to CO and that this occurs in part via inhibition in the activation of ERK MAPK. These data begin to elucidate specific differences with regard to the effects of CO and cell death pathways and provide important and valuable insight into potential mechanisms of action.

Inhibition of farnesyltransferase prevents collagen-induced arthritis by down-regulation of inflammatory gene expression through suppression of p21(ras)-dependent NF-kappaB activation.

Farnesylation of p21(ras) is an important step in the intracellular signaling pathway of growth factors, hormones, and immune stimulants. We synthesized a potent and selective farnesyltransferase inhibitor (LB42708) with IC(50) values of 0.8 nM in vitro and 8 nM in cultured cells against p21(ras) farnesylation and examined the effects of this inhibitor in the settings of inflammation and arthritis. LB42708 suppressed NF-kappaB activation and iNOS promoter activity by suppressing the I-kappaB kinase activity and I-kappaBalpha degradation. The inhibitor suppressed the expression of inducible NO synthase, cyclooxygenase-2, TNF-alpha, and IL-1beta and the production of NO and PGE(2) in immune-activated macrophages and osteoblasts as well as LPS-administrated mice. Furthermore, in vivo administration of LB42708 significantly decreased the incidence and severity of arthritis as well as mRNA expression of inducible NO synthase, cyclooxygenase-2, TNF-alpha, and IL-1beta in the paws of collagen-induced arthritic mice compared with controls. These observations indicate that the anti-inflammatory and antiarthritic effects of the farnesyltransferase inhibitor may be ascribed to the inhibition of I-kappaB kinase activity and subsequent suppression of NF-kappaB-dependent inflammatory gene expression through the suppression of p21(ras) farnesylation. Together, these findings reveal that the inhibitory effect of LB42708 on p21(ras)-dependent NF-kappaB activation may have potential therapeutic value for arthritis and other inflammatory diseases.

'Til cell death do us part: nitric oxide and mechanisms of hepatotoxicity.

Like many juggernauts in biology, the elusive nature of nitric oxide (NO) sprints through the fields, sometimes the savior, at other times the scimitar. In the liver, which is the metabolic center of the organism, hepatocytes and immune cells trade blows using the reactive diatomic molecule NO to induce cellular damage under toxic conditions. In response, hepatocytes can utilize several mechanisms of NO to their protective advantage by prohibiting the activation of programmed cell death, a.k.a. apoptosis. The balance of these effects in this reactive milieu set the stage for the homeostatic response to cellular injury that determines whether hepatocytes will live, die, or regenerate. Insights that we and others have gained from the liver under pathologic conditions of stress can be applied to the understanding of cellular death mechanisms in other organs and tissues.

IRF-1 expression induces apoptosis and inhibits tumor growth in mouse mammary cancer cells in vitro and in vivo.

Interferon regulatory factor-1 (IRF-1) is a nuclear transcription factor that mediates interferon and other cytokine effects and appears to have antitumor activity in vitro and in vivo in cancer cells. We have constructed a recombinant adenoviral vector (Ad-IRF-1) that infects mammary cells with high efficiency and results in high levels of functional IRF-1 protein in transfected cells. Overexpression of IRF-1 in two mouse breast cancer cell lines, C3-L5 and TS/A, resulted in apoptosis in these cell lines as assessed by Annexin V staining. The involvement of caspases was confirmed by significant inhibition of apoptosis by a caspase inhibitor, and by demonstration of caspase-3 activity, cleavage of caspase-3, and PARP cleavage. Interestingly, the growth of nonmalignant breast cell lines C127I and NMuMG did not appear to be inhibited by IRF-1 overexpression. Suppression of growth for breast cancer cell lines in vivo was demonstrated by both preinfection of breast cancer cells ex vivo and by intratumoral injection of Ad-IRF-1 into established tumors in their natural hosts. The mechanism of apoptosis may involve the transcriptional upregulation of bak, caspase-8, and caspase-7 expression. These data support the antitumor potential of IRF-1 and the use of agents that increase IRF-1 in breast cancer.

Carbon monoxide protects against liver failure through nitric oxide-induced heme oxygenase 1.

Carbon monoxide (CO) and nitric oxide (NO) each have mechanistically unique roles in various inflammatory disorders. Although it is known that CO can induce production of NO and that NO can induce expression of the cytoprotective enzyme heme oxygenase 1 (HO-1), there is no information whether the protective effect of CO ever requires NO production or whether either gas must induce expression of HO-1 to exert its functional effects. Using in vitro and in vivo models of tumor necrosis factor alpha-induced hepatocyte cell death in mice, we find that activation of nuclear factor kappaB and increased expression of inducible NO are required for the protective effects of CO, whereas the protective effects of NO require up-regulation of HO-1 expression. When protection from cell death is initiated by CO, NO production and HO-1 activity are each required for the protective effect showing for the first time an essential synergy between these two molecules in tandem providing potent cytoprotection.

Overexpression of mutated IkappaBalpha inhibits vascular smooth muscle cell proliferation and intimal hyperplasia formation.

PURPOSE: Vascular injury and inflammation are associated with elaboration of a number of cytokines that signal through multiple pathways to act as smooth muscle cell (SMC) mitogens. Activation of the nuclear factor-kappa B (NF-kappaB) transcription factor is essential for SMC proliferation in vitro and is activated by vascular injury in vivo. Activation of NF-kappaB is controlled by several upstream regulators, including the inhibitors of kappa B (IkappaB). These proteins bind to and keep NF-kappaB inactivated. The purpose of this study was to determine whether adenoviral gene transfer of a mutated IkappaBalpha super-repressor (AdIkappaBalphaSR) could inhibit development of intimal hyperplasia in vivo and to investigate how over-expression of this construct influences in vitro SMC proliferation and cell cycle regulatory proteins. METHODS: A rat carotid injury model was used to study prevention of intimal hyperplasia. Arteries were assayed 14 days after injury and infection with AdIkappaBalphaSR or adenoviral beta-galactosidase (AdLacZ). Untreated SMC or SMC infected with AdLacZ or AdIkappaBalphaSR were stimulated with 10% fetal bovine serum, interleukin-1beta, or tumor necrosis factor-alpha. Electrophoretic mobility shift assays were used to assay for NF-kappaB activation. Protein levels of IkappaBalpha and cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27(Kip1) were determined with Western blot analysis. Proliferation was measured with (3)H-thymidine incorporation assays. RESULTS: AdIkappaBalphaSR inhibited the development of intimal hyperplasia by 49% (P <.05). Infection with AdIkappaBalphaSR significantly suppressed in vitro SMC proliferation when stimulated with serum, interleukin 1, or tumor necrosis factor alpha, and did not result in cell death. Inhibition of proliferation was associated with increased p21(Cip1/Waf1) and p27(Kip1) protein levels. CONCLUSIONS: Gene transfer of IkappaBalpha super-repressor inhibited development of intimal hyperplasia in vivo and SMC proliferation in vitro. The antiproliferative activity may be related to cell cycle arrest through upregulation of the cyclin-dependent kinase inhibitors p21 and p27. Overexpression of IkappaBalpha may be a future therapeutic option in treatment of vascular diseases.

Fas-associating death domain protein overexpression induces apoptosis in lung cancer cells.

OBJECTIVES: Non-small cell lung cancers commonly develop resistance to radiation and chemotherapy, and they often present at stages beyond surgical resectability. Because current treatment modalities are inadequate, novel therapies are necessary to reduce the effects of the increasing incidence in pulmonary neoplasms. Fas-associating death domain protein is a central mediator of death receptor-initiated apoptosis that directly activates the caspase-8 protease. We hypothesized that overexpression of Fas-associating death domain protein would effectively eradicate lung cancer cells by induction of apoptosis. METHODS: Cultured A549 alveolar carcinoma and NCI-H226 squamous carcinoma cells were exposed to increasing multiplicities of infection of a replication-deficient, adenoviral vector that expresses the wild-type murine Fas-associating death domain protein gene or control virus for 4 hours. Twenty-four hours later, cells were assessed for viability by crystal violet staining and caspase activation by microscopic analysis. Protein lysates were examined by Western blotting for expression of Fas-associating death domain protein and activated caspase-8. RESULTS: Adenoviral infection with the wild-type murine Fas-associating death domain protein gene in A549 cells resulted in a dose-dependent expression of Fas-associating death domain protein and the appearance of cleaved, activated caspase-8. Increasing multiplicities of infection of the wild-type murine Fas-associating death domain protein gene, but not control adenovirus, was associated with increased cell death in A549 and NCI-H226 cells. The wild-type murine Fas-associating death domain protein gene infection of A549 cells at multiplicities of infection of 50 induced at least 10-fold increase in Fas-associating death domain protein levels and decreased viability by > 50% (n = 3; P <.001). CONCLUSION: Overexpression of Fas-associating death domain protein induced dose-dependent cell death in A549 and NCI-H226 lung epithelial cancer cells. Expression of Fas-associating death domain protein results in activation of caspases, a hallmark of apoptosis. Delivery of the wild-type murine Fas-associating death domain protein gene to drug- and radiation-resistant lung cancer may be a novel method for therapy of non-small cell lung cancer.

Nitric oxide prevents 6-hydroxydopamine-induced apoptosis in PC12 cells through cGMP-dependent PI3 kinase/Akt activation.

Nitric oxide (NO) functions not only as an important signaling molecule in the brain by producing cGMP, but also regulates neuronal cell apoptosis. The mechanism by which NO regulates apoptosis is unclear. In this study, we demonstrated that NO, produced either from the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) or by transfection of neuronal NO synthase, suppressed 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells by inhibiting mitochondrial cytochrome c release, caspase-3 and -9 activation, and DNA fragmentation. This protection was significantly reversed by the soluble guanylyl cyclase inhibitor 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalon-1-one, indicating that cGMP is a key mediator in NO-mediated anti-apoptosis. Moreover, the membrane-permeable cGMP analog 8-Br-cGMP inhibited 6-OHDA-induced apoptosis. These anti-apoptotic effects of SNAP and 8-Br-cGMP were suppressed by cGMP-dependent protein kinase G (PKG) inhibitor KT5823, indicating that PKG is a downstream signal mediator in the suppression of apoptosis by NO and cGMP. Both SNAP and 8-Br-cGMP induced endogenous Akt activation and Bad phosphorylation, resulting in the inhibition of Bad translocation to mitochondria; these effects were inhibited by KT5823 and the phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and Wortmannin. Our data suggest that the NO/cGMP pathway suppresses 6-OHDA-induced PC12 cell apoptosis by suppressing the mitochondrial apoptosis signal via PKG/PI3K/Akt-dependent Bad phosphorylation.

Ultraviolet irradiation increases FADD protein in apoptotic human keratinocytes.

Ultraviolet irradiation (UV) can induce keratinocyte apoptosis by activating death receptors that recruit the intracellular adaptor molecule FADD/MORT1 (Fas-associating death domain protein/mediator of receptor-induced toxicity). We hypothesized that UV could alter FADD expression levels to augment UV-induced keratinocyte apoptosis. In a dose-dependent manner UV B irradiation increased the expression of FADD protein in a human keratinocyte cell line (CCD-1106) with a corresponding increase in caspase-8 cleavage and cellular apoptosis. FADD overexpression induced cell death in 80% of cells compared with 10% spontaneous cell death in controls. Inhibition of FADD protein by adenoviral expression of anti-sense FADD reduced keratinocyte apoptosis. Regulation of FADD expression by UV may serve to enhance death receptor-mediated keratinocyte death.

A DNA microarray study of nitric oxide-induced genes in mouse hepatocytes: implications for hepatic heme oxygenase-1 expression in ischemia/reperfusion.

Nitric oxide (NO) can modulate numerous genes directly; however, some genes may be modulated only in the presence of the inflammatory stimuli that increase the expression of the inducible nitric oxide synthase (iNOS). One method by which to examine changes in NO-mediated gene expression is to carry out a gene array analysis on NO-nai;ve cells. Herein, we report a gene array analysis on mRNA from iNOS-null (iNOS(-/-)) mouse hepatocytes harvested from mice exposed to NO by infection with an adenovirus expressing human iNOS (Ad-iNOS). Of the 6500 genes on this array, only approximately 200 were modulated either up or down by the increased iNOS activity according to our criteria for significance. Several clearly defined families of genes were modulated, including genes coding for proinflammatory transcription factors, cytokines, cytokine receptors, proteins associated with cell proliferation and cellular energetics, as well as proteins involved in apoptosis. Our results suggest that iNOS has a generally anti-inflammatory and anti-apoptotic role in hepatocytes but also acts to suppress proliferation and protein synthesis. The expression of iNOS results in increased expression of stress-related proteins, including heme oxygenase-1 (HO-1). We used HO-1 to confirm that a significant change identified by an analysis could be demonstrated as significant in cells and tissues. The elevation of HO-1 was confirmed at the protein level in hepatocytes in vitro. Furthermore, iNOS(-/-) mice experienced greatly increased liver injury subsequent to intestinal ischemia/reperfusion injury, associated with an inability to upregulate HO-1. This is the first study to address the global gene changes induced by iNOS in any cell type, and the findings presented herein may have clinical relevance for conditions such as septic or hemorrhagic shock in which hepatocytes, NO, and HO-1 play a crucial role.

Nitric oxide (NO) pretreatment increases cytokine-induced NO production in cultured rat hepatocytes by suppressing GTP cyclohydrolase I feedback inhibitory protein level and promoting inducible NO synthase dimerization.

Nitric oxide (NO) regulates the biological activity of many enzymes and other functional proteins as well as gene expression. In this study, we tested whether pretreatment with NO regulates NO production in response to cytokines in cultured rat hepatocytes. Hepatocytes were recovered in fresh medium for 24 h following pretreatment with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and stimulated to express the inducible NO synthase (iNOS) with interleukin-1beta and interferon-gamma or transfected with the human iNOS gene. NO pretreatment resulted in a significant increase in NO production without changing iNOS expression for both conditions. This effect, which did not occur in macrophages and smooth muscle cells, was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO(2)(-), or NO(3)(-) did not increase the cytokine-induced NO production. SNAP pretreatment increased cytosolic iNOS activity measured only in the absence of exogenous tetrahydrobiopterin (BH(4)). SNAP pretreatment suppressed the level of GTP cyclohydrolase I (GTPCHI) feedback regulatory protein (GFRP) and increased GTPCHI activity without changing GTPCHI protein level. SNAP pretreatment also increased total cellular levels of biopterin and active iNOS dimer. These results suggest that SNAP pretreatment increased NO production from iNOS by elevating cellular BH(4) levels and promoting iNOS subunit dimerization through the suppression of GFRP levels and subsequent activation of GTPCHI.

Hepatocyte Fas-associating death domain protein/mediator of receptor-induced toxicity (FADD/MORT1) levels increase in response to pro-apoptotic stimuli.

We examined the regulation of Fas-associating death domain (FADD) protein as an important adaptor molecule in apoptosis signaling and hypothesized that the regulation of FADD could contribute to hepatocyte death. FADD/mediator of receptor-induced toxicity (MORT1) is required for activation of several signaling pathways of cell death. In this study we report the interesting and unexpected result that actinomycin D increased the expression of FADD protein, and we demonstrate that other cellular stresses like ultraviolet irradiation or heat shock could also increase FADD levels in hepatocytes. In cells treated with actinomycin D, FADD levels were elevated homogeneously in the cytoplasm. The increase in cytoplasmic FADD protein by actinomycin D or FADD overexpression alone both correlated with cell death, and specific antisense inhibition of FADD expression consistently diminished approximately 30% of the cell death induced by actinomycin D. These data indicate that FADD protein expression can increase rapidly in hepatocytes exposed to broadly cytotoxic agents.

Regulation of caspases by nitric oxide.

Nitric oxide can prevent or induce apoptosis depending on its concentration, cell type, and the oxidative milieu. Nitric oxide inhibits apoptosis and inflammation by S-nitrosylation of the active site cysteine of caspases, the central effector molecules of cell death as well as maturation of IL-1beta and IL-18. The ability of nitric oxide to S-nitrosylate caspases depends on multiple factors including the presence of free iron and intracellular redox potential. There are no known direct effects of nitric oxide on promoting caspase activation or activity. However, nitric oxide has been shown to promote apoptotic pathways in numerous cell types through the indirect activation of caspases. In this article we review the relationship of nitric oxide and caspase activity, modulation of this effect by iron, and clinical implications for the use of nitric oxide in regulating inflammation and apoptosis.

Mechanisms of hepatoprotection by nitric oxide.

Nitric oxide (NO) exerts numerous antiapoptotic effects on hepatocytes in settings of inflammation and tissue damage. These actions of NO are modulated by a variety of mechanisms under both physiologic and pathologic conditions. Nitric oxide inhibits cell death or apoptosis by modulation of heat shock proteins, S-nitrosylation of caspases at their catalytic site cysteine residue, triggering of the cGMP pathway, and prevention of mitochondrial dysfunction. Our preliminary studies also suggest that NO can modulate apoptosis-related genes in a manner consistent with an antiapoptotic effect. This review focuses on these molecular mechanisms of cytoprotection by NO.

Regulation of apoptosis by nitrosative stress.

Nitrosative stress can prevent or induce apoptosis. It occurs via S-nitrosylation by the interaction of nitric oxide (NO) with the biological thiols of proteins. Cellular redox potential and non-heme iron content determine S-nitrosylation. Apoptotic cell death is inhibited by S-nitrosylation of the redox-sensitive thiol in the catalytic site of caspase family proteases, which play an essential role in the apoptotic signal cascade. Nitrosative stress can also promote apoptosis by the activation of mitochondrial apoptotic pathways, such as the release of cytochrome c, an apoptosis-inducing factor, and endonuclease G from mitochondria, as well as the suppression of NF-kB activity. In this article we reviewed the mechanisms whereby S-nitrosylation and nitrosative stress regulate the apoptotic signal cascade.