Peter K. M. Kim

The regulatory role of nitric oxide in apoptosis

Nitric oxide (NO) is a multi-faceted molecule with dichotomous regulatory roles in many areas of biology. The complexity of its biological effects is a consequence of its numerous potential interactions with other molecules such as reactive oxygen species (ROS), metal ions, and proteins. The effects of NO are modulated by both direct and indirect interactions that can be dose-dependent and cell-type specific. For example, in some cell types NO can promote apoptosis, whereas in other cells NO inhibits apoptosis. In hepatocytes, NO can inhibit the main mediators of cell death-caspase proteases. Moreover, low physiological concentrations of NO can inhibit apoptosis, but higher concentrations of NO may be toxic. High NO concentrations lead to the formation of toxic reaction products like dinitrogen trioxide or peroxynitrite that induce cell death, if not by apoptosis, then by necrosis. Long-term exposure to nitric oxide in certain conditions like chronic inflammatory states may predispose cells to tumorigenesis through DNA damage, inhibition of DNA repair, alteration in programmed cell death, or activation of proliferative signaling pathways. Understanding the regulatory mechanisms of NO in apoptosis and carcinogenesis will provide important clues to the diagnosis and treatment of tissue damage and cancer. In this article we have reviewed recent discoveries in the regulatory role of NO in specific cell types, mechanisms of pro-apoptotic and anti-apoptotic induction by NO, and insights into the effects of NO on tumor biology.

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 α–induced hepatocyte cell death in mice, we find that activation of nuclear factor κB 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.

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 mitochon‐drial apoptosis signal via PKG/PI3K/Akt‐dependent Bad phosphorylation.—Ha, K.‐S., Kim, K. M., Kwon, Y.‐G., Bai, S.‐K., Nam, W.‐D., Yoo, Y.‐M., Kim, P. K. M., Chung, H.‐T., Billiar, T. R., Kim, Y.‐M. Nitric oxide prevents 6‐hydroxydopamine‐induced apoptosis in PC12 cells through cGMP‐dependent PI3 kinase/Akt activation. FASEB J. 17, 1036–1047 (2003)

Regulation of Caspases by Nitric Oxide

Abstract: 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‐1β 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.

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.

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.

Mechanisms of Hepatoprotection by Nitric Oxide

Abstract: 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.

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.

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

Abstract 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-κB) transcription factor is essential for SMC proliferation in vitro and is activated by vascular injury in vivo. Activation of NF-κB is controlled by several upstream regulators, including the inhibitors of kappa B (IκB). These proteins bind to and keep NF-κB inactivated. The purpose of this study was to determine whether adenoviral gene transfer of a mutated IκBα super-repressor (AdIκBα SR ) 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 AdIκBαSR or adenoviral β-galactosidase (AdLacZ). Untreated SMC or SMC infected with AdLacZ or AdIκBαSR were stimulated with 10% fetal bovine serum, interleukin-1β, or tumor necrosis factor-α. Electrophoretic mobility shift assays were used to assay for NF-κB activation. Protein levels of IκBα 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 AdIκBαSR inhibited the development of intimal hyperplasia by 49% ( P Cip1/Waf1 and p27 Kip1 protein levels. Conclusions Gene transfer of IκBα 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 IκBα may be a future therapeutic option in treatment of vascular diseases.

Inhibition of Farnesyltransferase Prevents Collagen-Induced Arthritis by Down-Regulation of Inflammatory Gene Expression through Suppression of p21ras-Dependent NF-κB Activation

Farnesylation of p21ras 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 IC50 values of 0.8 nM in vitro and 8 nM in cultured cells against p21ras farnesylation and examined the effects of this inhibitor in the settings of inflammation and arthritis. LB42708 suppressed NF-κB activation and iNOS promoter activity by suppressing the I-κB kinase activity and I-κBα degradation. The inhibitor suppressed the expression of inducible NO synthase, cyclooxygenase-2, TNF-α, and IL-1β and the production of NO and PGE2 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-α, and IL-1β 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-κB kinase activity and subsequent suppression of NF-κB-dependent inflammatory gene expression through the suppression of p21ras farnesylation. Together, these findings reveal that the inhibitory effect of LB42708 on p21ras-dependent NF-κB activation may have potential therapeutic value for arthritis and other inflammatory diseases.