Oxidative stress and inflammation are fundamental for the onset of aging and appear to be causatively linked. Previously, we reported that hepatocytes from aged rats, compared with young rats, are hyperresponsive to interleukin-1b (IL-1b) stimulation and exhibit more potent c-Jun N-terminal kinase (JNK) activation and attenuated interleukin-1 receptor-associated kinase-1 (IRAK-1) degradation. An age-related increase in the activity of neutral sphingomyelinase-2 (NSMase-2), a plasma membrane enzyme, was found to be responsible for the IL-1b hyperresponsiveness. The results reported here show that increased NSMase activity during aging is caused by a 60-70% decrease in hepatocyte GSH levels. GSH, at concentrations typically found in hepatocytes from young animals, inhibits NSMase activity in a biphasic dose-dependent manner. Inhibition of GSH synthesis in young hepatocytes activates NSMase, causing increased JNK activation and IRAK-1 stabilization in response to IL-1b, mimicking the hyperresponsiveness typical for aged hepatocytes. Vice versa, increased GSH content in hepatocytes from aged animals by treatment with N-acetylcysteine inhibits NSMase activity and restores normal IL-1b response. Importantly, the GSH decline, NSMase activation, and IL-1b hyperresponsiveness are not observed in aged, calorie-restricted rats. In summary, this report demonstrates that depletion of cellular GSH during aging plays an important role in regulating the hepatic response to IL-1b by inducing NSMase-2 activity. Increased basal inflammation is a phenomenon emblematic of the aging process. It is characterized by increased concentrations of serum markers such as C-reactive protein and serum amyloid A (1) as well as by the activation of proinflammatory signaling molecules such as c-Jun N-terminal kinase (JNK) (2), nuclear factor-kB (NF-kB) (3), and CCAAT/enhancer binding proteins a and b (4). According to the oxidative stress hypothesis of aging, changes in mitochondrial functions and the deterioration of antioxidant defense mechanisms lead to an imbalance in the production and neutralization of free radicals, which in turn induces tissue damage and inflammation. Indeed, oxidative stress seems to play a fundamental role in the onset of aging-associated inflammation, because a decline in reactive oxygen species (ROS) generation brought about by calorie restriction (5) or by antioxidant supplementation decreases the expression of various inflammatory markers in aged animals. It has been proposed that the excess ROS generated during aging stimulate the secretion of proinflammatory cytokines, thus creating a proinflammatory environment (5). However, despite the fact that basal levels of some cytokines, such as tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6), seem to increase with age, these increases are modest, and it is not yet clear whether they are sufficient to evoke an inflammatory response. Moreover, the systemic concentrations of other cytokines, including interleukin-1b (IL1b), remain unchanged in healthy elder...

Aging is characterized by changes in the organism's immune functions and stress response, which in the elderly leads to increased incidence of complications and mortality following inflammatory stress. Alterations in the neuro-endocrine axes and overall decline in the immune system play an essential role in this process. Overwhelming evidence however suggests that many cellular cytokine signaling pathways are also affected, thus underscoring the idea that both, "cellular" and "systemic" changes contribute to aging. IL-1beta for example, induces more potent cellular responses in hepatocytes isolated from aged animals then in hepatocytes from young rats. This phenomenon is referred to as IL-1b hyperresponsiveness and is linked to abnormal regulation of various acute phase proteins during aging.Evidence has consistently indicated that activation of neutral sphingomyelinase and the resulting accumulation of ceramide mediate cellular responses to LPS, IL-1beta, and TNFalpha in young animals. More recent studies identified the cytokine-inducible neutral sphingomyelinase with nSMase2 (smpd3) that is localized in the plasma membrane and mediates cellular responses to IL-1beta and TNFalpha. Intriguingly, constitutive up-regulation of nSMase2 occurs in aging and it underlies the hepatic IL-1b hyperresponsiveness. The increased activity of nSMases2 in aging is caused by a substantial decline in hepatic GSH content linking thereby oxidative stress to the onset of pro-inflammatory state in liver. nSMase2 apparently follows a pattern of regulation consisting with "developmental-aging" continuum, since in animal models of delayed aging, like calorie-restricted animals, the aging-associated changes in NSMase activity and function are reversed.

The process of aging has recently been shown to substantially affect the ability of cells to respond to inflammatory challenges. We demonstrate that aging leads to hepatic hyperresponsiveness to interleukin 1␤ (IL-1␤), and we examine the factors that could be responsible for this phenomenon. IL-1␤-induced phosphorylation of c-jun N-terminal kinase (JNK) in hepatocytes isolated from aged rats was 3 times more potent than that in hepatocytes from young rats. Moreover, JNK was activated by substantially lower doses of IL-1␤. These age-related changes in JNK phosphorylation correlated with diminished IL-1␤-induced degradation of interleukin-1 receptor-associated kinase-1 (IRAK-1). Expression levels of IL1␤ receptor I, total JNK, IRAK-1, and transforming growth factor-␤-activated kinase-1 (TAK-1) were not affected by aging. However, increased neutral sphingomyelinase activity was observed in hepatocytes from old animals, which we show is caused by induction of the plasma membrane localized neutral sphingomyelinase-2 (NSMase-2). We provide evidence that NSMase-2 is both required and sufficient for the onset of IL-1␤ hyperresponsiveness during aging. Overexpression of NSMase-2 in hepatocytes from young rats leads both to a reduction in IRAK-1 degradation and potentiation of JNK phosphorylation, mimicking that seen in hepatocytes from old animals. More importantly, inhibition of NSMase activity in hepatocytes from aged rats using either scyphostatin or short interfering ribonucleic acid (siRNA) leads to reversion to the "young" phenotype of IL-1␤ response. Conclusion: These results show that the process of aging causes increased basal NSMase-2 activity in hepatocytes, which in turn leads to IRAK-1 stabilization, JNK potentiation, and ultimately IL-1␤ hyperresponsiveness. (HEPATOLOGY 2007;46:1166-1176

Insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is primarily produced in the liver during inflammation and regulates biological activities of IGF-I. Here we demonstrate that interleukin-1β (IL-1β) stimulates IGFBP-1 mRNA production in a dose-dependent manner in hepatocytes from Fisher 344 rats. Employment of c-Jun N-terminal kinase (JNK) inhibitor SP600125 resulted in 3-fold reduction of IGFBP-1 mRNA and protein levels, indicating that IL-1β-induced IGFBP-1 production is mediated through JNK activation. We further show that hepatocytes from aged rats (20-22 mo), as compared to young (3-4 mo), exhibit up to 2-fold higher levels of IGFBP-1 in response to IL-1β. IL-1β-induced phosphorylation of JNK was also significantly higher in aged hepatocytes, and SP600125 treatment eliminated age-related differences in IGFBP-1 mRNA production. Moreover, glutathione depletion in hepatocytes from young rats potently activated JNK, as well as increased IL-1β-induced IGFBP-1 mRNA levels, suggesting that age-related oxidative stress underlies the upregulated JNK activation and IGFBP-1 expression.

The objective of this study is to understand the role of Neutral sphingomyelinase‐2 (NSMase‐2) in hepatic response to Interleukin 1β (IL‐1β). The IL‐1β signaling cascade is initiated by rapid phosphorylation of IL‐1β receptor activated kinase‐1 (IRAK‐1) in response to ligand binding. Subsequently, the phoshporylated IRAK‐1 is ubiquitinated and degraded and the rate of its degradation is known to affect the magnitude of IL‐1β response. We show that the activity of protein phosphatase 2A (PP2A) gradually increases in response to IL‐1β stimulation with a maximum effect seen 30 minutes after the stimulation. The PP2A catalytic subunit directly interacts with IRAK‐1, indicating that IRAK‐1 is a novel PP2A substrate. Furthermore, treatment of primary rat hepatocytes or HEK293cells overexpressing the IL‐1β receptor with PP2A inhibitor, okadaic acid, enhances the rate of IL‐1β‐induced IRAK‐1 ubiquitination and degradation.NSMase‐2 mediates the activation of PP2A by IL‐1β. Overexpression of NSMase‐2 enhances PP2A activity and delays IRAK‐1 degradation. In contrast, NSMase‐2 silencing or inhibition by pharmacological inhibitor causes faster IRAK‐1 degradation. NSMase‐2 also plays a role in IRAK‐1 intracellular translocation in response to IL‐1β. These experiment identify NSMase 2‐and PP2A as novel physiological regulators of IRAK‐1 phosphorylation.Supported by NIA RO1 AG026711.