NAMPT-dependent NAD+ salvage is crucial for the decision between apoptotic and necrotic cell death under oxidative stress.

Oxidative stress is a state in which the accumulation of reactive oxygen species exceeds the capacity of cellular antioxidant systems. Both apoptosis and necrosis are observed under oxidative stress, and we have reported that these two forms of cell death are induced in H2O2-stimulated HeLa cells depending on the concentration of H2O2. Weak H2O2 stimulation induces apoptosis, while strong H2O2 stimulation induces necrosis. However, the detailed mechanisms controlling the switching between these forms of cell death depending on the level of oxidative stress remain elusive. Here, we found that NAD+ metabolism is a key factor in determining the form of cell death in H2O2-stimulated HeLa cells. Under both weak and strong H2O2 stimulation, intracellular nicotinamide adenine dinucleotide (NAD+) was depleted to a similar extent by poly (ADP-ribose) (PAR) polymerase 1 (PARP1)-dependent consumption. However, the intracellular NAD+ concentration recovered under weak H2O2 stimulation but not under strong H2O2 stimulation. NAD+ recovery was mediated by nicotinamide (NAM) phosphoribosyltransferase (NAMPT)-dependent synthesis via the NAD+ salvage pathway, which was suggested to be impaired only under strong H2O2 stimulation. Furthermore, downstream of NAD+, the dynamics of the intracellular ATP concentration paralleled those of NAD+, and ATP-dependent caspase-9 activation via apoptosome formation was thus impaired under strong H2O2 stimulation. Collectively, these findings suggest that NAD+ dynamics balanced by PARP1-dependent consumption and NAMPT-dependent production are important to determine the form of cell death activated under oxidative stress.

Expression and Localization of Paneth Cells and Their α-Defensins in the Small Intestine of Adult Mouse.

Paneth cells contribute to intestinal innate immunity by sensing bacteria and secreting α-defensin. In Institute of Cancer Research (ICR) mice, α-defensin termed cryptdin (Crp) in Paneth cells consists of six major isoforms, Crp1 to 6. Despite accumulating evidences that α-defensin functions in controlling the intestinal microbiota, topographical localization of Paneth cells in the small intestine in relation to functions of α-defensin remains to be determined. In this study, we examined the expression level of messenger RNA (mRNA) encoding six Crp-isoforms and Crp immunoreactivities using singly isolated crypts together with bactericidal activities of Paneth cell secretions from isolated crypts of duodenum, jejunum, and ileum. Here we showed that levels of Crp mRNAs in the single crypt ranged from 5 x 103 to 1 x 106 copies per 5 ng RNA. For each Crp isoform, the expression level in ileum was 4 to 50 times higher than that in duodenum and jejunum. Furthermore, immunohistochemical analysis of isolated crypts revealed that the average number of Paneth cell per crypt in the small intestine increased from proximal to distal, three to seven-fold, respectively. Both Crp1 and 4 expressed greater in ileal Paneth cells than those in duodenum or jejunum. Bactericidal activities in secretions of ileal Paneth cell exposed to bacteria were significantly higher than those of duodenum or jejunum. In germ-free mice, Crp expression in each site of the small intestine was attenuated and bactericidal activities released by ileal Paneth cells were decreased compared to those in conventional mice. Taken together, Paneth cells and their α-defensin in adult mouse appeared to be regulated topographically in innate immunity to control intestinal integrity.

The regulatory and signaling mechanisms of the ASK family.

Apoptosis signal-regulating kinase 1 (ASK1) was identified as a MAP3K that activates the JNK and p38 pathways, and subsequent studies have reported ASK2 and ASK3 as members of the ASK family. The ASK family is activated by various intrinsic and extrinsic stresses, including oxidative stress, ER stress and osmotic stress. Numerous lines of evidence have revealed that members of the ASK family are critical for signal transduction systems to control a wide range of stress responses such as cell death, differentiation and cytokine induction. In this review, we focus on the precise signaling mechanisms of the ASK family in response to diverse stressors.