Authors: Kim YM, Talanian RV, Billiar TR
J. Biol. Chem. 272: 31138-31148 (1997).
Presenter: Ted Watson
Presenter's Summary
Background:
Nitric oxide was identified as the mediator formerly known as endothelial derived relaxing factor (EDRF) over ten years ago and since then the identification of its actions and sources have grown tremendously. The enzyme producing nitric oxide is called nitric oxide synthase (NOS) and three different isoforms of this enzyme have been identified; two are calcium dependent and are referred to as constitutive NOS and the third is calcium-independent and is known as the inducible NOS. One or more of these enzymes have been localized to almost all tissues of the body including both the central and peripheral nervous system, skeletal muscle, smooth muscle, liver, kidney, heart, lungs, immune cells (particularly macrophages and neutrophils), vasculature (both endothelium and smooth muscle) and are implicated in functions from host defense, long term potentiation (memory), vasodilation, and bronchodilation. Since NO is a diatomic gas with lipophilic properties it possesses the ability to cross cell membranes and does not act at cell surface receptors. The first cellular target identified for NO was guanylyl cyclase, a heme containing enzyme responsible for the intracellular production of cyclic GMP. This second messenger acts to increase the activity of cyclic GMP-dependent protein kinase, initiating a cascade of protein phosphorylation. Nitric oxide possesses the quality of being a highly reactive species which is capable of forming numerous non-enzymatic reaction products in living tissue. It is capable of reacting with reactive oxygen species (ROS) to form peroxynitrite, a molecule which has been implicated in toxic damage to cellular membranes via lipid peroxidation. Nitric oxide can also bind to sulfhydryl groups of numerous proteins forming nitrosothiols, a mechanism implicated in the transport of nitric oxide within the circulatory system. The production of nitric oxide at relatively low levels acts in many cases via the cGMP pathway to regulate vascular resistance and as a neuromodulator. Upregulation of the inducible isoform of NOS (iNOS) results in high levels of NO production, as is seen in inflammatory conditions such as asthma and rheumatoid arthritis, and this elevated NO production has been implicated in the non-specific and toxic effects of lipid peroxidation and cell necrosis. It is this dichotomous action of nitric oxide that has led to its Jekyll and Hyde reputation. High levels of NO production do provide the benefit of non-specific host defense and is strongly implicated in protection against bacterial infections. Other beneficial roles for the high levels of NO produced following stimulation have not been identified and the contribution of this increased production to cell function has not been fully elucidated.
Another area of increasing research effort is that of programmed cell death, or apoptosis. Apoptosis differs from cell necrosis in that the former involves organized, concerted destruction of the cell constituents which include membrane blebbing, specific DNA splicing and formation of apoptotic bodies. A protein known as caspase-1 was identified in 1993 and this enzyme was identified as a cysteine protease and a key mediator of the apoptosis signaling cascade. This cysteine protease family now contains at least 10 different enzymes that are involved in programmed cell death. Numerous reports have identified the caspase-3 member of this family as a mediator of apoptosis and have demonstrated that inhibition of this enzyme attenuates or eliminates the apoptotic process and it is of interest to determine whether there are endogenous regulators of this caspase-3 enzyme which contribute to the regulation of the cell.
This report examines the role of nitric oxide in regulation of this apoptosis signaling cascade and the effect that manipulation of nitric oxide production has on the activity of caspase-3.
Summary of the present work:
The authors characterized cultured hepatocyte apoptosis, identifying spontaneous apoptosis with DNA laddering after four days in culture. DNA laddering is a characteristic of apoptosis due to the fact that the DNA splicing that occurs during apoptosis is on the condensed chromatin. This condensed chromatin is wrapped around histone proteins and the only accessible cleavage sites are those that travel between adjacent histones. Since the diameter of each histone is identical the amount of DNA around a histone is the same (approximately 180 base pairs) and the DNA amounts are dependent on the number of histones between cleavage sites. Since the DNA detected with electrophoresis is conditional upon the number of histones and cleavage can only occur between histones, the resulting fragmentation appears as distinct bands in multiples of 180 base pairs, the DNA amounts surrounding a histone protein.
They confirmed induction of nitric oxide synthase by a cytokine mix, previously reported to upregulate the NOS protein and NO production, in this culture by measuring the oxygen metabolites of nitric oxide, nitrite and nitrate, and showed upregulation of both iNOS mRNA and protein in these cells. With the knowledge that the cytokine mix could increase NO production, the authors looked at the effect of this NO increase on the process of apoptosis by measuring the presence or absence of DNA laddering and found that the upregulation of NO production inhibited apoptosis completely in a L-NMMA-sensitive manner. The portion of hepatocyte survival increased four-fold compared to controls when NOS was upregulated. The addition of NO donors also inhibited the apoptosis as detected by DNA laddering. This effect of NOS upregulation on the inhibition of cell apoptosis was examined in mouse hepatocytes obtained from iNOS knockout animals. Using wild type mouse hepatocytes as controls the authors demonstrated that the NO mediated protection was in fact iNOS specific (i.e. not a result of eNOS or nNOS activity) and that this NO-dependent viability exists in multiple animal species.
Upon establishing that increased NO production does in fact promote hepatocyte survival the details of the mechanism involved were investigated with a particular focus on caspase-3, a known mediator of programmed cell death. In isolated enzyme assays the addition of NO donors significantly and dose dependently decreased caspase-3 activity, an inhibition reversible by the addition of a compound capable of removing thiol bound nitric oxide, DTT. This DTT sensitivity suggests that NOS binds caspase-3 directly. To link this isolated enzymatic assay to biological effects caspase-3 was incubated with PARP (poly(ADP-ribose)polymerase), a known substrate of caspase-3 and a key enzyme in the DNA repair process. The co-incubation of caspase-3 with PARP produced a degradation product at 85kDa as detected by gel electrophoresis. Addition of an NO donor inhibited the degradation of PARP and this was reversed with an NO scavenger. Both thiol modifying agents and a selective inhibitor of caspase-3 also inhibited degradation of PARP by caspase-3. These same conditions were used to demonstrate the occurrence of DNA laddering in the absence of the protection afforded by NO, thiol modifying agents or the caspase-3 inhibitor. When using hepatocyte culture to assess NO inhibition of caspase-3 activity it was confirmed that NOS upregulation by cytokines inhibited caspase-3 activity by over 95%, in a L-NMMA-sensitive manner, when compared to peak levels in control cells. A key finding was that in the cell culture assay DTT, which was shown to completely reverse the NO inhibition of caspase-3 in the enzyme assay, reversed the NO dependent inhibition of caspase-3 by only 50%. In vivo experiments demonstrated similar effects of NO donors on inhibition of caspase-3 and reversibility by DTT. The partial reversibility afforded by DTT that was observed in cell culture was also present in the in vivo experiments and identified a NO dependent inhibition of caspase-3 that was not dependent on NO binding to sulfhydryl groups. The authors examined whether the known receptor for nitric oxide, guanylyl cyclase, was mediating the DTT-insensitive inhibition of caspase-3. In the hepatocyte cell culture system the addition of exogenous 8-bromo-cGMP (a non-hydrolyzable analogue of cGMP) showed significant inhibition of cell death and its associated DNA laddering. When measuring the activity of caspase-3 in the cell culture the addition of 8-bromo-cGMP inhibited its activity by approximately 60% in a DTT-insensitive manner. Using the soluble guanylyl cyclase inhibitor ODQ the caspase-3 activity was not different from the activity measured in the absence of NO donors and addition of the NO donor to the cells in the presence of ODQ reduced the caspase-3 activity by only 30%. These data suggest that stimulation of caspase-3 activity by cytokines is inhibited by both the direct action of NO on the caspase-3 enzyme and through the cGMP second messenger system stimulated by NO activation of soluble guanylyl cyclase.
The data presented in this report provide strong evidence to support the idea that high level nitric oxide production plays an important role in the inhibition of apoptosis. This is the first report describing the biological action of nitric oxide acting via two separate mechanisms to achieve the same effect. This would serve a protective role in many inflammatory conditions where the concentration of inflammatory cells and surrounding tissue would require sustained viability to complete tissue repairs. Since both macrophages and neutrophils elevate NO production when stimulated by cytokines, the presence of these cell types in inflamed tissues would serve many roles including host defense, vasodilation (to permit infiltration of additional inflammatory cells), and extend the life cycle of local cells through the elevated nitric oxide released. This multifunctional facilitation of tissue repair by NO does not occur in the CNS as it has been well characterized that high NO levels induce neurotoxicity in most cases via a NMDA-stimulated mechanism which may be unrelated to the caspase/PARP cascade.
Since high level NO production inhibits the apoptotic process in hepatocytes, is it likely that this inhibition also occurs in other tissues? In inflamed tissues there will be tissue damage and in such a case does the elevation of NO protect the tissue or organ from generalized programmed cell death in an effort to maintain the cell repair processes? There is a down side to maintaining the activity of PARP and that is the high energy demand required to repair DNA following damage. Several studies have shown that inhibition of PARP will reduce cardiac infarct size through a conservation of energy that allows general tissue repair at the expense of some unrepaired DNA damage. This does seem to present a similar Jekyll and Hyde dichotomy as seen in other conditions and does not resolve the dilemma of whether high level nitric oxide production is of a greater benefit or hindrance.
Nitric oxide (NO) has emerged as an important endogenous inhibitor of apoptosis, and here we report that NO prevents hepatocyte apoptosis initiated by the removal of growth factors or exposure to TNFalpha or anti-Fas antibody. We postulated that the mechanism of the inhibition of apoptosis by NO would include an effect on caspase-3-like protease activity. Caspase-3-like activity increased coincident with apoptosis due to all three stimuli, and treatment with the caspase-3-like protease inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde inhibited both proteolytic activity and apoptosis. Endogenous or exogenous sources of NO prevented the increase in caspase-3-like activity in hepatocytes. Exposure of purified recombinant caspase-3 to an NO or NO+ donor inhibited proteolytic activity. Dithiothreitol (DTT), but not glutathione, reversed the inhibition of recombinant caspase-3 by NO. When lysates from cells stimulated to express inducible NO synthase or cells exposed to NO donors were incubated in DTT, caspase-3-like activity increased to about 55% of cells not exposed to a source of NO. Similarly, administration of an NO donor to rats treated with TNFalpha and D-galactosamine also prevented the increase in caspase-3-like activity as measured in liver homogenates. The effect of the NO donor was reversed by about 50% if the homogenate was incubated with DTT. TNFalpha-induced apoptosis and caspase-3-like activity were also reduced in cultured hepatocytes exposed to 8-bromo-cGMP, and both effects were inhibited by the cGMP-dependent kinase inhibitor KT5823. The suppression in caspase-3-like activity in hepatocytes exposed to an NO donor was partially blocked by an inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one, (ODQ), while the incubation of these lysates in DTT almost completely restored caspase-3-like activity to the level of TNFalpha-treated controls. These data indicate that NO prevents apoptosis in hepatocytes by either directly or indirectly inhibiting caspase-3-like activation via a cGMP-dependent mechanism and by direct inhibition of caspase-3-like activity through protein S-nitrosylation.