SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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 1β (IL-1β), and we examine the factors that could be responsible for this phenomenon. IL-1β-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-1β. These age-related changes in JNK phosphorylation correlated with diminished IL-1β–induced degradation of interleukin-1 receptor–associated kinase-1 (IRAK-1). Expression levels of IL1β receptor I, total JNK, IRAK-1, and transforming growth factor-β–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-1β 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-1β 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-1β hyperresponsiveness. (HEPATOLOGY 2007.)

The process of aging is characterized by changes in the immune function and stress response,1–4 resulting in increased incidence of complications and mortality among the elderly after inflammatory stress. A decline in the adaptive component of the immune system and up-regulation of the innate immune response have been implicated in this phenomenon.5 Aging, however, also affects the cellular response to a variety of agents, including lipopolysaccharide (LPS) and tumor necrosis factor alpha, as well as the response to pharmacological inducers of oxidative stress.6–8 Peritoneal macrophages, hepatocytes, and glial cells isolated from aged animals exhibit more severe and prolonged responses to inflammatory challenges compared with cells originating from young animals.6–9 This hyperresponsiveness is manifested in altered activation patterns of cyclooxygenase-2, c-jun N-terminal kinase (JNK), nuclear factor kappa-B, CCAAT/enhancer binding protein alpha, and CCAAT/enhancer binding protein beta. Hence, changes in the cellular signaling pathways may contribute to the onset of the pro-inflammatory state in aging.

Interleukin-1-beta (IL-1β) is a prototypical inflammatory cytokine that mediates the response to infection, and its basal systemic levels are relatively constant with age.10 Various IL-1β–related functions, however, like the regulation of acute phase protein expression in liver, appear to be age dependent. Interestingly, the basal levels of acute phase reactants increase with age in a variety of species, from Drosophila melanogaster11 to mammals,1, 12 and this has been linked to aging-related disorders, such as Alzheimer's disease,13 rheumatoid arthritis,1 and cardiovascular diseases.12

Cellular responses to IL-1β are mediated through the interleukin-1 receptor type I (IL-1RI). IL-1RI and the LPS receptor are prototypical members of the toll-like receptor (TLR) family and share a conserved signaling pathway. Ligand binding to TLR results in the recruitment of several adaptor proteins including MyD88,14 followed by IL-1R–associated kinase-1 (IRAK-1) and IRAK-4 binding to the receptor complex. The subsequent IRAK-1 hyperphosphorylation facilitates binding of tumor necrosis factor–associated factor-6 and the IRAK-1/IRAK-4/ tumor necrosis factor–associated factor-6 complex then separates from the receptor and interacts with the transforming growth factor-β–activated kinase-1 (TAK-1).15 Activation of TAK-1 apparently initiates the mitogen-activated protein kinase cascade, leading to the activation of transcription factors such as activator protein-1 and nuclear factor kappa-B through JNK and inhibitory κB kinases, respectively, resulting in the induction of acute phase protein messenger ribonucleic acid (mRNA) transcription.16

IRAK-1 plays a central role in the TLR signaling cascade, and the rate of IRAK-1 degradation determines the magnitude of the response. Initially, phosphorylation of IRAK-1 is essential for its release from the receptor and the activation of downstream signaling molecules; however, IRAK-1 phosphorylation also leads to its ubiquitination and proteasome-mediated degradation, resulting in the termination of the signaling cascade.17 Suppression of IRAK-1 degradation in macrophages through the use of ubiquitin ligase inhibitors has been shown to potentiate the inflammatory response, whereas decreased stability of IRAK-1 is the hallmark of endotoxin tolerance exemplified by the muted response to secondary LPS administration.18, 19 Interferon gamma and granulocyte-macrophage colony-stimulating factor can prevent this LPS tolerance by inhibiting IRAK-1 degradation.20

Previous studies have shown that neutral sphingomyelinase-2 (NSMase-2), 1 of the 2 neutral, Mg2+-dependent sphingomyelinases in the liver, plays a role in regulating the rate of IRAK-1 degradation. Treatment of hepatocytes with IL-1β leads to increased cellular NSMase activity and accumulation of ceramide.21 Increased NSMase-2 activity attenuates IL-1β–induced IRAK-1 phosphorylation, and its subsequent ubiquitination and degradation.22 Increases in the ceramide level, however, have been shown to stimulate α-1-acid glycoprotein and C-reactive protein expression.21, 23 Ceramide generation is also involved in the activation of TAK-1, JNK, and nuclear factor kappa-B, all of which have important roles in the IL-1β cascade.24–26 Thus, the neutral sphingomyelinases and ceramide appear to be important regulators of the IL-1β signal transduction pathway.

Ceramide is an evolutionary conserved second messenger that mediates various inflammatory responses and the onset of growth arrest and cell death.27 Recent studies suggest that ceramide also plays an important role in the process of aging.28 Aging-related upregulation of various sphingomyelinases results in increased ceramide generation.29–31 In the liver, the increased ceramide production with age is paralleled by chronic and sustained increase in the activity of a plasma membrane–associated, Mg-dependent neutral sphingomyelinase.29, 31

In this study, we investigate the role that plasma membrane–associated and Mg-dependent NSMase-2 play in the aging-related changes in rat hepatic response to IL-1β.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials.

Male Fisher 344 rats were purchased from the National Institute of Aging. Animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Proteasome inhibitors Z-Leu-Leu-Nva-H aldehyde, Z-Leu-Leu-Leu-H aldehyde, and Z-Ile-Glu(OtBu)-Ala-Leu-H aldehyde were from Peptides International (Louisville, KY). Scyphostatin was provided by Sankyo (Tokyo, Japan). NSMase2 (smpd3)-specific short interfering ribonucleic acid (siRNA) (AAtgttactggctggtggacc32) and scrambled control (scr) siRNA (Cat. # 1022076) were from Qiagen (Valencia, CA). IL-1β and anti–IL-1RI were from R&D Systems (Minneapolis, MN). Anti-phospho-JNK1/2 antibody was from Cell Signaling (Beverly, MA). Anti-JNK1/2, anti-Flag peptide DYKDDDDK (anti-FLAG), anti-β-actin antibody, and secondary antibodies were from Sigma (St. Louis, MO). Anti-IRAK-1, anti-TAK-1, anti-JNK antibody (for immunoprecipitation), and protein A-Agarose beads were from Santa Cruz Biotechnology (Santa Cruz, CA). Rat recombinant GST-c-Jun (1-79) was from Biomol (Plymouth Meeting, PA). Lipids were from Avanti (Alabaster, AL).

Primary Hepatocyte Isolation, Culture, and Treatments.

Hepatocytes were isolated from ether-anesthetized young (3-4 months) and aged (20-22 months) male Fisher 344 rats by in situ collagenase perfusion and cultured in culture dishes coated with 6.3 mg/ml Matrigel in Waymouth's medium supplemented with insulin (0.15 μM) and penicillin/streptomycin (100 U/ml)21 for 5 days at 37°C in 5% CO2. Murine nSMase2 (smpd3)-specific siRNA32 was used to silence the endogenous NSMase-2. Transfections (100 pmol siRNA per dish) using Lipofectamine were performed on day 3 of cell culture. Control plates were transfected with scr control. Adenovirus expressing FLAG-tagged mouse NSMase-2 (AdNSMase-2) was used to inducibly overexpress NSMase-2.22

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Western Blotting.

The medium was aspirated from the dishes and the Matrigel was reliquefied by incubating with phosphate-buffered saline containing 5 mM ethylene diamine tetraacetic acid for 30 minutes at 4°C. The cells were pelleted by centrifugation at 500g for 4 minutes, rinsed, and lysed in 50 μl lysis buffer [1 mM ethylene diamine tetraacetic acid, 0.5% Triton X-100, 1 mM Na2VO4, 1 mM NaF, 1:200 (vol/vol) protease inhibitor cocktail, 10 mM Tris-HCl, pH 7.4] on ice for 30 minutes. Cell lysates were centrifuged at 16,000 g for 10 minutes at 4°C. Proteins (usually 80 μg/lane) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-p polyvinylidene fluoride membrane. Specified proteins were detected using the antibodies described in Materials. Protein–antibody interactions were visualized using the ECF substrate and a Storm860 fluorescent scanning instrument, and analyzed using ImageQuant5.0 software (Molecular Dynamics, Sunnyvale, CA).

NSMase Activity Assay.

Cells from each dish were harvested, resuspended in lysis buffer (see previous description) without Triton X-100, and homogenized by sonication for 5 minutes. NSMase activity was determined as described previously,22, 33 using 6-N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino-sphingomyelin (NBD-SM) as a substrate.

JNK Activity Assay.

JNK activity was measured by immune complex protein kinase assay according to Whitmarsh and Davis.34 Briefly, JNK was immunoprecipitated from cell extracts (3 × 105 cells) using 10 μl protein A–agarose beads and 2 μg anti-JNK antibody. The JNK immune complexes were washed and resuspended in 30 μl kinase assay buffer containing 2 μg glutathione S-transferase (GST)-c-Jun, 33 μM–adenosine triphosphate (ATP), and 10 μCi [γ-32P]ATP. The kinase reaction was carried out for 30 minutes at 30°C and stopped by adding SDS-PAGE loading buffer. The samples were run on a 12% SDS-PAGE minigel, and radiolabeled phospho-GST-c-Jun was detected by phosphor imager analysis.

Sphingomyelin and Ceramide Mass Measurements.

Lipids were extracted by the method of Bligh and Dyer modified as described previously35 and separated on thin-layer chromatography plates.21 The ceramide mass was quantified by high-pressure liquid chromatography35 of the long-chain bases released after an acid hydrolysis in 0.5 M HCl in methanol at 65°C for 15 hours.21 N-acetyl-C20-sphinganine was used as an internal standard. Sphingomyelin measurements were done by the method of Kahovcova and Odavic.36

Indirect Immunofluorescence.

HepG2 cells were cultured on coverslips to 50% confluence and infected with adenoviral construct overexpressing either the FLAG-tagged mouse NSMase-1 or NSMase-2. Forty-eight hours after transfection, cells were fixed with 3% paraformaldehyde and permeabilized with 0.2% Triton X-100. NSMase-1 and NSMase-2 were labeled with anti-FLAG M2 monoclonal antibody, and the immune complexes were visualized with fluorescein isothiocyanate–conjugated goat anti-mouse immunoglobulin G.

Primary hepatocytes were harvested and the Matrigel removed as described. After washing of the cells, the fixation, permeabilization, and staining with antibodies was done in suspension. The stained cells were attached to coverslips and observed using confocal microscopy.

Isolation of Microsomal and Plasma Membrane Fractions From Liver.

Plasma membrane fractions were isolated from 10% liver homogenate as previously described.37 A microsomes-enriched fraction was prepared using low-speed centrifugation to remove the nucleus followed by centrifugation at 105,000g for 60 minutes. 5′ nucleotidase and sialyl transferase were used as marker enzymes.

Statistical Analysis.

After assuming equal variance across groups, differences were assessed using Student t test. Results are presented as mean ± standard deviation (SD).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

IL-1β Induces JNK Phosphorylation More Potently in Hepatocytes From Aged Rats.

To see whether aging affects the response to IL-1β, hepatocytes from young (3-4 months) and aged (20-22 months) rats were treated with IL-1β at different concentrations (Fig. 1A, C), or for various times (Fig. 1E, F). The activation of JNK, a major downstream target of IL-1β, was assayed by western blotting using an antibody against the phosphorylated (active) form of JNK1 and JNK2. As anticipated, treatment with IL-1β induced JNK phosphorylation in hepatocytes from both young and aged rats, but the extent of JNK phosphorylation in the latter was up to 3 times higher (Fig. 1C). Moreover, IL-1β induced substantial JNK phosphorylation in aged hepatocytes at concentrations as low as 1 ng/ml. The levels of total JNK were similar in both age groups at all IL-1β concentrations (Fig. 1A), implying that the observed differences in JNK phosphorylation were not caused by changes in its basal levels. The time course of JNK activation was not affected by aging, and JNK phosphorylation was higher in hepatocytes from aged animals at all times tested (Fig. 1E, F).

thumbnail image

Figure 1. JNK activation by IL-1β is more potent in aged hepatocytes. (A and C) Dose response of JNK phosphorylation. Hepatocytes from young (3-4 months) and aged (20-22 months) rats were treated with the indicated concentrations of IL-1β for 15 minutes. JNK phosphorylation was determined by Western blotting using an antibody specific for the dually phosphorylated forms of JNK1 and 2 (pJNK1&2). The levels of total JNK are shown as a loading control. The combined intensity of phospho JNK1 and JNK2 was quantified and normalized for the levels total JNK. Data are presented as a percentage of the phospho-JNK levels at 50 ng/ml IL-1β in young hepatocytes. Values are means ± SD, n = 3, *P < 0.01, **P < 0.001. (B, D) Dose response of JNK activation. JNK activity was assayed in vitro using [γ-32P] ATP and GST-c-jun as substrates after immunoprecipitation of total JNK. Phosphorylation of GST-c-jun was determined using phosphoimager after separation on a 12% SDS-PAGE. The data are presented as a percentage of the phosphorylated GST-c-jun in hepatocytes from young animals treated with 50 ng/ml IL-1β. Values are means ± SD, n = 3, *P < 0.01, **P < 0.001. (E, F) Time course of JNK phosphorylation. Hepatocytes were treated with IL-1β (10 ng/ml) for the indicated time, and JNK phosphorylation was determined as described for A and C. Data are presented as a percentage of the phospho-JNK level in young hepatocytes treated with IL-1β for 15 minutes.

Download figure to PowerPoint

The activity of JNK was measured in vitro by immunoprecipitation of total JNK and using [γ-32P]ATP and GST-c-jun as substrates. Preliminary studies confirmed the linearity of the assay in regards to the protein content and time (data not shown). As anticipated, the increased JNK phosphorylation observed in aged hepatocytes correlated with higher JNK activity as judged by the enhanced phosphorylation of c-jun (Fig. 1B, D).

The Effects of Aging on Expression of IL-1 Receptor, IRAK-1, and TAK-1.

To determine whether the observed age-related increases in IL-1β sensitivity are related to the expression levels of proteins that control rate-limiting steps in the IL-1β signaling cascade,38, 39 the protein levels of IL-1RI, IRAK-1, and TAK-1 were quantified (Fig. 2). No statistically significant differences were detected between young and aged hepatocytes, which suggests that the hyperresponsiveness to IL-1β is not attributable to increased expression of IL-1RI, IRAK-1, or TAK-1.

thumbnail image

Figure 2. Assessment of basal protein levels of IL-1R, IRAK-1, TAK-1, and total JNK1&2 in young and aged hepatocytes. Hepatocyte lysates from young (3-4 months) and aged (20-22 months) rats were analyzed by Western blotting using specific antibodies. The resulting bands were quantified by phosphoimager, and the signal intensity was normalized for the level of cyclophilin A. Final results are expressed as a percentage of the corresponding level for each protein in hepatocytes from young animals, and are mean ± SD, n = 4.

Download figure to PowerPoint

Attenuation of IRAK-1 Degradation Induces IL-1β Hyperresponsiveness in Hepatocytes From Young Rats.

The rate of ligand-induced proteasomal degradation of IRAK-1 plays an important role in regulation of the signaling via TLR.18, 38 Therefore, seeing whether IRAK stability also plays a role in the age-related increase in IL-1β sensitivity was of interest. IRAK-1 degradation was less pronounced in aged hepatocytes after IL-1β stimulation than in young ones (Figs. 3, 5B, and 6B). Moreover, when various proteasome inhibitors (Z-Leu-Leu-Nva-H aldehyde, Z-Leu-Leu-Leu-H aldehyde, and Z-Ile-Glu[OtBu]-Ala-Leu-H aldehyde) were used to inhibit IRAK-1 degradation in young hepatocytes, the observed stabilization of IRAK-1 (evidenced by the accumulation of both nonmodified and modified IRAK-1), was accompanied by increased phosphorylation of JNK (Fig. 3B, C, and data not shown).

thumbnail image

Figure 3. Correlation of IRAK-1 stability with aging and JNK phosphorylation. (A) Effects of aging on IRAK-1 degradation. Hepatocyte cultures from young (3-4 months) and aged (20-22 months) rats were treated with the indicated concentrations of IL-1β for 30 minutes. Levels of IRAK-1 were quantified by Western blotting and phosphoimaging. Results are expressed as a percentage of the IRAK-1 levels in untreated hepatocytes, and are means ± SD, n = 3, *P < 0.05, **P < 0.005. (B, C) Effects of IRAK-1 stabilization on JNK phosphorylation. Primary hepatocytes from young (3-4 months) rats were pretreated with proteasome inhibitor Z-Leu-Leu-Nva-H aldehyde (50 μM) for 3 hours and then incubated with IL-1β (10 ng/ml) for the indicated times. IRAK-1 and phosphoJNK were visualized by western blotting. The anti-IRAK-1 antibody recognizes both the nonmodified and modified (hyperphosphorylated/ubiquitinated) forms of IRAK-1 as indicated. Results are means ± SD, n = 4. For (A) and (C), protein levels were quantified using a phosphoimager and signal intensity was normalized for the levels of β-actin.

Download figure to PowerPoint

thumbnail image

Figure 5. NSMase-2 is sufficient to induce the aging phenotype. (A) Activation of NSMase-2 by IL-1β. Primary hepatocytes from young (3-4 months) rats were infected with AdNSMase-2, and expression of NSMase-2 was induced with doxycycline (black circles, left-hand scale). The controls were infected, non-induced hepatocytes (white circles, right-hand scale). All cells were treated with 25 ng/ml IL-1β for the indicated times and NSMase activity was assayed as described. Values are means ± SD, n = 3, *P < 0.05. (B) Effects of NSMase-2 overexpression in young hepatocytes on IRAK stability and JNK phosphorylation. Hepatocytes from young rats were infected with AdNSMase-2, and the expression of NSMase-2 was induced for 72 hours. The cells were then treated with the indicated concentrations of IL-1β for 30 minutes. IRAK-1 and phospho-JNK levels were determined by western blotting using specific antibodies. Anti-FLAG M2 monoclonal antibody was used to monitor for expression of the FLAG-tagged NSMase-2. β-Actin levels were used to control for uniform loading. Data are representative of 5 independent hepatocyte preparations.

Download figure to PowerPoint

thumbnail image

Figure 6. Inhibition of NSMase activity in aged hepatocytes diminishes IL-1β hyperresponsiveness. (A). Scyphostatin inhibition of endogenous and overexpressed NSMase-2 in hepatocytes from aged (20-22 months) rats. NSMase-2 overexpression was achieved by infecting the cells with AdNSMase-2 (moi 2-5) and inducing expression for 48 hours. NSMase activity was measured in vitro, in the presence of the indicated concentrations of scyphostatin. Results are expressed as a percentage of the activity in samples without scyphostatin and are means ± SD, n = 6. (B, C) IRAK-1 degradation and JNK phosphorylation in aged hepatocytes after scyphostatin treatment. Hepatocytes from aged rats were pre-incubated with scyphostatin or with vehicle control (ethanol, 0.5% final concentration) for 1 hour, and then treated with the indicated concentrations of IL-1β for 30 minutes. IRAK-1 and phospho-JNK were visualized by Western blotting. (D) NSMase-2 silencing by siRNA. Hepatocytes from young (3-4 months) animals overexpressing FLAG-tagged NSMase-2 were transfected with either NSMase-2 siRNA or scrRNA for 48 hours. The efficiency of silencing was checked by western blotting using anti-FLAG antibody. (E-H). Effects of NSMase-2 silencing on IRAK-1 degradation (E, G) and JNK phosphorylation (F, H). Hepatocytes from aged rats were transfected with either NSMase-2 siRNA scrRNA for 48 hours and then treated with the indicated concentrations of IL-1β for 30 minutes. The levels of IRAK and Phospho-JNK were determined by western blotting and phosphoimaging. Results are presented as a percentage of the IRAK-1 levels in hepatocytes not treated with IL-1β (G) and as a percentage of the JNK phosphorylation measured in hepatocytes transfected with scrRNA and treated with 10 ng/ml IL-1β (H). Results are representative of 2 independent hepatocyte preparations, where each treatment was performed in duplicates. Values are means ± SD, n = 4. For all western blots, β-actin levels were used to control for uniform loading.

Download figure to PowerPoint

NSMase-2 Is Responsible for the Aging-Related Increases in Liver NSMase Activity.

We have previously shown that increased NSMase-2 activity inhibits IL-1β–induced IRAK-1 phosphorylation, leading to IRAK-1 stabilization and enhanced JNK activation.22 Because NSMase activity in liver increases with aging,29, 31 seeing which of the 2 known neutral sphingomyelinases, NSMase-1 or NSMase-2, is responsible for this phenomenon was of interest.

Total cellular NSMase activity in hepatocytes from aged rats is 2 times higher than that in hepatocytes from young animals (Fig. 4A). Accordingly, the cellular content of ceramide is approximately 30% higher in aged hepatocytes (Fig. 4B). Because previous studies have suggest that the NSMase activity that is upregulated during aging is associated with the plasma membrane,29, 31 we measured the mass of sphingomyelin in plasma membranes and in microsomal preparations from livers of young and aged rats. Sphingomyelin is the substrate that NSMases hydrolyze to ceramide. In the aged rats, a decrease in sphingomyelin was found in the plasma membrane fraction but not in the microsomal one (Fig. 4C). This confirms that the aging-induced increases in NSMase activity are limited to the plasma membrane. To identify which of the 2 NSMases is localized at the plasma membrane, we performed indirect immunofluorescence studies with HepG2 cells overexpressing FLAG-tagged mouse NSMase-1 or NSMase-2. Western blot analyses and in vitro NSMase activity assays were used to monitor for the inducible expression of functionally active proteins.22 NSMase-1 exhibits diffuse cytosolic localization (Fig. 4D), whereas NSMase-2 was localized exclusively at the plasma membrane. Neither aging nor IL-1β affected this localization of NSMase-2 (Fig. 4F, G). To show even more conclusively that NSMase-2 is the enzyme affected by aging, hepatocytes from aged rats were transfected either with siRNA against NSMase-2 or with control scrambled siRNA (scrRNA). As shown in Fig. 4E, the NSMase-2 siRNA inhibited most of the aging-associated increase in NSMase activity, whereas the scrRNA had no effect. Ceramide mass decreased accordingly by approximately 45%. Taken together, these data show that the observed increases in basal liver NSMase activity during aging are likely attributable to upregulation of NSMase-2.

thumbnail image

Figure 4. NSMase-2 is responsible for the age-related increase in hepatic NSMase activity. (A) Basal NSMase activity in primary hepatocytes from young (3-4 months) and aged (20-22 months) rats. NSMase activity in cell homogenates was measured in vitro using NBD-sphingomyelin as exogenous substrate. Results are presented as specific activity (pmol/mg protein/minute), and are mean ± SD, n = 3, *P < 0.03. (B) Ceramide mass in hepatocytes from young and aged rats measured by thin-layer chromatography/high-pressure liquid chromatography. Data are presented as nanomole of ceramide per milligram of cellular protein (mean ± SD, *P < 0.05). (C) Sphingomyelin levels in plasma membranes and in microsomes. Microsomal and plasma membrane fractions were isolated from the pooled livers of 5 young and 5 aged rats, and sphingomyelin mass was measured. Data are presented as a percentage of the total phospholipid mass (mean ± SD, n = 3; *P < 0.05). (D) Subcellular localization of NSMase-1 and NSMase-2. Hep G2 cells were infected with either AdNSMase-1 or AdNSMase-2 (moi 2-5), and expression was induced for 48 hours. The NSMases were visualized using antibodies against the FLAG tag and confocal microscopy. (E) Inhibition of aging-related increase in NSMase activity using siRNA against NSMase-2. Hepatocytes were isolated from young and aged rats, and the latter were transfected with either NSMase-2-specific siRNA or scrRNA. NSMase activity was measured in cell homogenates after 48 hours using NBD-SM as a substrate. Values are mean ±SD, n = 3, *P < 0.03. (F, G) Subcellular localization of NSMase-2 in primary hepatocytes from young (3-4 months) and aged (20-22 months) rats. The cells were infected with AdNSMase-2 for 48 hours. Hepatocytes were incubated with IL-1β (10 ng/ml) or vehicle for 30 minutes. The NSMase-2 was visualized using antibodies against the FLAG tag and confocal microscopy.

Download figure to PowerPoint

NSMase-2 Is Activated by IL-1β, and Increases in Its Basal Levels Are Sufficient to Induce the Aging Phenotype.

We next tested whether induction of NSMase-2 activity in hepatocytes from young rats would be sufficient to induce the hyperresponsive phenotype. For this purpose, young hepatocytes were infected with AdNSMase-2.22 No adenovirus-associated toxicity was observed for up to 72 hours after infection (data not shown). Overexpression of NSMase-2 resulted in an increase in the level of endogenous ceramide from 1.6 ± 0.2 to 2.7 ± 0.2 nmol/mg protein. To ascertain that the overexpressed enzyme is functionally similar to the endogenous one, its inducibility by IL-1β was assessed. As anticipated, IL-1β treatment stimulated both, the endogenous NSMase activity and the activity attributable to the overexpressed enzyme (Fig. 5A). Young hepatocytes overexpressing NSMase-2 on IL-1β stimulation showed decreased IRAK-1 degradation and increased JNK phosphorylation (Fig. 5B), similar to the response observed in hepatocytes from aged animals.

Downregulation of NSMase Activity in Aged Hepatocytes Restores Normal Response to IL-1β.

To further confirm these observations, we used scyphostatin and siRNA to suppress NSMase-2 in aged hepatocytes. Scyphostatin is an NSMase inhibitor40, 41 that has been shown to efficiently inhibit the plasma membrane-localized, Mg2+-dependent NSMase.42 The addition of scyphostatin to hepatocyte homogenates from aged rats led to in vitro inhibition of NSMase activity in a concentration-dependent manner (Fig. 6A). Inhibition was observed not only for the endogenous NSMase activity, but also, more importantly, more than 75% of the overexpressed NSMase-2 activity was suppressed. Hence, scyphostatin is a very potent inhibitor of NSMase-2. Addition of scyphostatin to the cell cultures did not result in any significant toxicity for up to 48 hours (data not shown).

The addition of scyphostatin to cultured hepatocytes from aged animals resulted in accelerated IRAK-1 degradation after IL-1β treatment (Fig. 6B). Scyphostatin also reduced the potency of IL-1β to induce JNK phosphorylation (Fig. 6C).

NSMase-2–specific siRNA was employed as an alternative approach to downregulate NSMase-2 activity in hepatocytes from aged rats. In cells overexpressing the FLAG-tagged NSMase-2, over 75% expression inhibition was achieved with the siRNA (Fig 6D). In noninfected hepatocytes, the siRNA inhibited 20%-35% of the endogenous NSMase activity (data not shown, and Fig. 4E). This would imply 40%-80% inhibition of endogenous NSMase-2 activity based on the fact that according to recent studies43 NSMase-2 accounts for approximately 25%-50% of the total Mg2+-dependent NSMase activity in liver, with the rest being attributed to NSMase-1, an enzyme that has no known role in cell signaling. Most significantly, NSMase-2 inhibition with siRNA enhanced IRAK-1 degradation on IL-1β treatment (Fig. 6E, G) and also suppressed the IL-1β–induced JNK phosphorylation (Fig. 6F, H). This is similar to the results obtained with scyphostatin.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

These studies provide an insight into the aging-associated changes in the hepatic IL-1β response and could further our understanding of the mechanisms behind the increased inflammatory response observed in the elderly. The increased response with age to various inducers of stress and inflammation usually has been attributed to abnormal regulation of the levels of systemic mediators. Our findings, however, along with those of other studies,6–8 implicate the cell signaling machinery in the phenomenon. We show that IL-1β evokes a more potent JNK response in hepatocytes isolated from aged rats than in hepatocytes isolated from young rats. Importantly, concentrations of IL-1β that are too low to appreciably affect JNK activation in young animals are capable of inducing significant JNK phosphorylation in aged animals. If this holds true for humans as well, then plasma concentrations of IL-1β (and possibly other cytokines) that are considered subclinical in young individuals might prove to be clinically important in the elderly.

JNK belongs to the mitogen-activated protein kinase family and is activated by dual phosphorylation on Thr183 and Tyr185 in response to cellular stress caused by osmotic shock or by agents such as ultraviolet radiation, LPS, and certain cytokines such as IL-1β and tumor necrosis factor alpha.44 Often, however, JNK activation is regulated in a synergistic manner and possibly integrates the input of distinct signal transduction pathways activated during stress. The potentiation of JNK phosphorylation during aging may result from such integration of signaling involving the major cellular stress response pathway of ceramide and the IL-1β signaling pathway. Moreover, our results indicate that the aging-associated increases in the basal activity of NSMase-2, which leads to ceramide generation, underlie the observed IL-1β hyperresponsiveness.

The stabilization of IRAK-1 seemingly also plays an important role in IL-1β hyperresponsiveness. A similar role for IRAK-1 has been shown in the signaling pathways of other ligands of the TLR family.18, 20 Apparently, by exhibiting higher basal NSMase activity (and higher ceramide content at the plasma membrane), hepatocytes from aged animals slow down IRAK-1 degradation, keeping IRAK-1 available for recruitment to various signaling complexes, which in turn could result in potentiation of the downstream cascades leading to JNK phosphorylation. This stabilization of IRAK-1 may involve a phosphatase, such as the ceramide-activated protein phosphatase, protein phosphatase 2A. Indications to that extent come from our previous studies,22 in which increased IRAK-1 stability in IL-1β–stimulated cells overexpressing NSMase-2 was protein phosphatase 2A dependent and correlated with the inhibition of IRAK-1 phosphorylation. We are currently trying to determine whether IRAK-1 is a direct substrate of protein phosphatase 2A.

If NSMase-2 is, indeed, as the studies presented here seem to indicate, an upstream modulator of IRAK-1 stability and JNK phosphorylation, then the age-related increase in NSMase activity observed in liver might play a crucial role in the onset of the proinflammatory state that epitomizes the process of aging. Indeed, we show that overexpression of NSMase-2 in hepatocytes from young rats causes reduced IRAK-1 degradation and increased JNK phosphorylation, mimicking the “old hepatocyte” phenotype. Conversely, inhibition of NSMase activity in hepatocytes from aged rats restores the “young” phenotype of IL-1β response. However, neither aging nor elevation in NSMase-2 activity in young hepatocytes induces JNK phosphorylation in the absence of IL-1β, suggesting that although NSMase-2 and aging modulate the amplitude of IL-1β signal, they are not sufficient to mimic its effects on JNK. At this time, we are trying to establish the factors behind the observed increases in the basal NSMase activity during aging. No differences were found between the NSMase-2 mRNA expression levels in the livers of young and aged animals (data not shown). Another possibility stems from a report showing that reduced glutathione is a potent inhibitor of NSMase.45 This observation could be of relevance because a substantial decrease in the levels of reduced glutathione has been found in livers of aged animals.46 Indeed, in studies just recently completed we provide evidence that the decline in rat liver glutathione content during aging underlies the increase in NSMase activity and the onset of IL-1β hyperresponsiveness (Rutkute et al., in preparation).2

Recent studies have delineated a much broader role for the ceramide-metabolizing enzymes in the biology of aging. For example, although thorough longevity studies have yet to be performed, NSMase-2 knockout mice exhibit dwarfism and delayed puberty, which are indicators of delayed aging.43 In D. melanogaster, mutation in the neutral ceramidase gene leads to increased cellular ceramide content and a rapid onset of aging-associated photoreceptor degeneration.47 And lastly, but most importantly, convincing evidence for ceramide's involvement in the process of aging comes from studies in yeast, where the longevity assurance genes, LAG1 and LAC1, in yeast that participate in de novo ceramide biosynthesis, have been shown to determine yeast lifespan.48

In summary, we present evidence that NSMase-2 plays a crucial role in the regulation of hepatic functions during aging and mediates aging-associated hyperresponsiveness to IL-1β (Fig. 7). Pharmacological inhibitor of NSMase-2 was able to restore the normal pattern of IL-1β signaling, and this makes NSMase-2 a potential target for aging-specific anti-inflammatory treatments. Most importantly, our data, together with other published reports, suggests that onset of the chronic inflammatory state in aged organisms may result from the combined effects of increased cellular responsiveness to stress and subclinical increases in the levels of systemic or local mediators of inflammation.

thumbnail image

Figure 7. Proposed mechanism for the age-related hyperresponsiveness to IL-1β. Binding of IL-1β to the IL-1RI induces the formation of a signaling complex containing IRAK-1, IRAK-4, MyD88, and other adapter molecules. IRAK-4 and other, unidentified kinases phosphorylate IRAK-1 at multiple residues, thus activating its own kinase activity, leading to further autophosphorylation. The hyperphosphorylation of IRAK-1 leads to disassociation of the complex from the IL-1β receptor and facilitates its interaction with TAK-1, which is upstream of JNK. Phosphorylation of JNK ultimately leads to activation of AP-1 transcription factor that regulates acute phase protein transcription through the acute phase response element (APRE) in the promoters. Phosphorylation of IRAK-1 also serves as a signal for its ubiquitination and rapid degradation in proteasomes, which effectively terminates the signaling cascade. IL-1β binding to its receptor, however, also transiently activates the plasma membrane-localized NSMase-2, which regulates the rate of IRAK degradation. During aging, the constitutive activity of NSMase-2 is increased, leading to attenuation of IRAK-1 degradation and increased phosphorylation of JNK (indicated with “+” and “−” signs). NSMase-2 may exert its stabilizing effect on IRAK-1 by maintaining IRAK-1 in a less phosphorylated state,22 possibly through the activation of a phosphatase.

Download figure to PowerPoint

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Dr. Toshio Takatsu (Sankyo, Japan) for the gift of scyphostatin, and the Cardiovascular Research Group (University of Kentucky) for valuable comments.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Rosenthal CJ, Franklin EC. Variation with age and disease of an amyloid A protein-related serum component. J Clin Invest 1975; 55: 746753.
  • 2
    Krabbe KS, Bruunsgaard H, Hansen CM, Moller K, Fonsmark L, Qvist J, et al. Ageing is associated with a prolonged fever response in human endotoxemia. Clin Diagn Lab Immunol 2001; 8: 333338.
  • 3
    Ballou SP, Lozanski FB, Hodder S, Rzewnicki DL, Mion LC, Sipe JD, et al. Quantitative and qualitative alterations of acute-phase proteins in healthy elderly persons. Age Ageing 1996; 25: 224230.
  • 4
    Yamamoto K, Shimokawa T, Yi H, Isobe K, Kojima T, Loskutoff DJ, et al. Aging accelerates endotoxin-induced thrombosis: increased responses of plasminogen activator inhibitor-1 and lipopolysaccharide signaling with aging. Am J Pathol 2002; 161: 18051814.
  • 5
    Han D, Hosokawa T, Aoike A, Kawai K. Age-related enhancement of tumor necrosis factor (TNF) production in mice. Mech Ageing Dev 1995; 84: 3954.
  • 6
    Wu D, Marko M, Claycombe K, Paulson KE, Meydani SN. Ceramide-induced and age-associated increase in macrophage COX-2 expression is mediated through up-regulation of NF-kappa B activity. J Biol Chem 2003; 278: 1098310992.
  • 7
    Hsieh CC, Xiong W, Xie Q, Rabek JP, Scott SG, An MR, et al. Effects of age on the posttranscriptional regulation of CCAAT/enhancer binding protein alpha and CCAAT/enhancer binding protein beta isoform synthesis in control and LPS-treated livers. Mol Biol Cell 1998; 9: 14791494.
  • 8
    Hsieh CC, Rosenblatt JI, Papaconstantinou J. Age-associated changes in SAPK/JNK and p38 MAPK signaling in response to the generation of ROS by 3-nitropropionic acid. Mech Ageing Dev 2003; 124: 733746.
  • 9
    Claycombe KJ, Wu D, Nikolova-Karakashian M, Palmer H, Beharka A, Paulson KE, et al. Ceramide mediates age-associated increase in macrophage cyclooxygenase-2 expression. J Biol Chem 2002; 277: 3078430791.
  • 10
    Di Iorio A, Ferrucci L, Sparvieri E, Cherubini A, Volpato S, Corsi A, et al. Serum IL-1beta levels in health and disease: a population-based study. ‘The InCHIANTI study’. Cytokine 2003; 22: 198205.
  • 11
    Zerofsky M, Harel E, Silverman N, Tatar M. Aging of the innate immune response in Drosophila melanogaster. Aging Cell 2005; 4: 103108.
  • 12
    Berk BC, Weintraub WS, Alexander RW. Elevation of C-reactive protein in “active” coronary artery disease. Am J Cardiol 1990; 65: 168172.
  • 13
    Abraham CR, Selkoe DJ, Potter H. Immunochemical identification of the serine protease inhibitor alpha 1-antichymotrypsin in the brain amyloid deposits of Alzheimer's disease. Cell 1988; 52: 487501.
  • 14
    Wesche H, Henzel WJ, Shillinglaw W, Li S, Cao Z. MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 1997; 7: 837847.
  • 15
    Cao Z, Xiong J, Takeuchi M, Kurama T, Goeddel DV. TRAF6 is a signal transducer for interleukin-1. Nature 1996; 383: 443446.
  • 16
    Ninomiya-Tsuji J, Kishimoto K, Hiyama A, Inoue J, Cao Z, Matsumoto K. The kinase TAK1 can activate the NIK-I kappaB as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature 1999; 398: 252256.
  • 17
    Yamin TT, Miller DK. The interleukin-1 receptor-associated kinase is degraded by proteasomes following its phosphorylation. J Biol Chem 1997; 272: 2154021547.
  • 18
    Cuschieri J, Gourlay D, Garcia I, Jelacic S, Maier RV. Implications of proteasome inhibition: an enhanced macrophage phenotype. Cell Immunol 2004; 227: 140147.
  • 19
    Li L, Cousart S, Hu J, McCall CE. Characterization of interleukin-1 receptor-associated kinase in normal and endotoxin-tolerant cells. J Biol Chem 2000; 275: 2334023345.
  • 20
    Adib-Conquy M, Cavaillon JM. Gamma interferon and granulocyte/monocyte colony-stimulating factor prevent endotoxin tolerance in human monocytes by promoting interleukin-1 receptor-associated kinase expression and its association to MyD88 and not by modulating TLR4 expression. J Biol Chem 2002; 277: 2792727934.
  • 21
    Chen J, Nikolova-Karakashian M, Merrill AH Jr, Morgan ET. Regulation of cytochrome P450 2C11 (CYP2C11) gene expression by interleukin-1, sphingomyelin hydrolysis, and ceramides in rat hepatocytes. J Biol Chem 1995; 270: 2523325238.
  • 22
    Karakashian AA, Giltiay NV, Smith GM, Nikolova-Karakashian MN. Expression of neutral sphingomyelinase-2 (NSMase-2) in primary rat hepatocytes modulates IL-beta-induced JNK activation. FASEB J 2004; 18: 968970.
  • 23
    Lozanski G, Berthier F, Kushner I. The sphingomyelin-ceramide pathway participates in cytokine regulation of C-reactive protein and serum amyloid A, but not alpha-fibrinogen. Biochem J 1997; 328(Pt 1): 271275.
  • 24
    Shirakabe K, Yamaguchi K, Shibuya H, Irie K, Matsuda S, Moriguchi T, et al. TAK1 mediates the ceramide signaling to stress-activated protein kinase/c-Jun N-terminal kinase. J Biol Chem 1997; 272: 81418144.
  • 25
    Westwick JK, Bielawska AE, Dbaibo G, Hannun YA, Brenner DA. Ceramide activates the stress-activated protein kinases. J Biol Chem 1995; 270: 2268922692.
  • 26
    Verheij M, Bose R, Lin XH, Yao B, Jarvis WD, Grant S, et al. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 1996; 380: 7579.
  • 27
    Hannun YA, Obeid LM. The ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind. J Biol Chem 2002; 277: 2584725850.
  • 28
    Obeid LM, Hannun YA. Ceramide, stress, and a “LAG” in aging. Sci Aging Knowledge Environ 2003; 2003: PE27.
  • 29
    Lightle SA, Oakley JI, Nikolova-Karakashian MN. Activation of sphingolipid turnover and chronic generation of ceramide and sphingosine in liver during aging. Mech Ageing Dev 2000; 120: 111125.
  • 30
    Cutler RG, Kelly J, Storie K, Pedersen WA, Tammara A, Hatanpaa K, et al. Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease. Proc Natl Acad Sci U S A 2004; 101: 20702075.
  • 31
    Petkova DH, Momchilova-Pankova AB, Markovska TT, Koumanov KS. Age-related changes in rat liver plasma membrane sphingomyelinase activity. Exp Gerontol 1988; 23: 1924.
  • 32
    Marchesini N, Osta W, Bielawski J, Luberto C, Obeid LM, Hannun YA. Role for mammalian neutral sphingomyelinase 2 in confluence-induced growth arrest of MCF7 cells. J Biol Chem 2004; 279: 2510125111.
  • 33
    Nikolova-Karakashian M, Morgan ET, Alexander C, Liotta DC, Merrill AH, Jr. Bimodal regulation of ceramidase by interleukin-1beta. Implications for the regulation of cytochrome p450 2C11. J Biol Chem 1997; 272: 1871818724.
  • 34
    Whitmarsh AJ, Davis RJ. Analyzing JNK and p38 mitogen-activated protein kinase activity. Methods Enzymol 2001; 332: 319336.
  • 35
    Merrill AH JrWE, Mullins RE, Jamison WC, Nimkar S, Liotta DC. Quantitation of free sphingosine in liver by high-performance liquid chromatography. Anal Biochem 1988; 171: 373381.
  • 36
    Kahovcova J, Odavic R. A simple method for the quantitative analysis of phospholipids separated by thin layer chromatography. J Chromatogr 1969; 40: 9096.
  • 37
    Hubbard AL, Wall DA, Ma A. Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains. J Cell Biol 1983; 96: 217229.
  • 38
    Janssens S, Beyaert R. Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members. Mol Cell 2003; 11: 293302.
  • 39
    Sato S, Sanjo H, Takeda K, Ninomiya-Tsuji J, Yamamoto M, Kawai T, et al. Essential function for the kinase TAK1 in innate and adaptive immune responses. Nat Immunol 2005; 6: 10871095.
  • 40
    Nara F, Tanaka M, Hosoya T, Suzuki-Konagai K, Ogita T. Scyphostatin, a neutral sphingomyelinase inhibitor from a discomycete, Trichopeziza mollissima: taxonomy of the producing organism, fermentation, isolation, and physico-chemical properties. J Antibiot (Tokyo) 1999; 52: 525530.
  • 41
    Nara F, Tanaka M, Masuda-Inoue S, Yamasato Y, Doi-Yoshioka H, Suzuki-Konagai K, et al. Biological activities of scyphostatin, a neutral sphingomyelinase inhibitor from a discomycete, Trichopeziza mollissima. J Antibiot (Tokyo) 1999; 52: 531535.
  • 42
    Bernardo K, Krut O, Wiegmann K, Kreder D, Micheli M, Schafer R, Sickman A, et al. Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain. J Biol Chem 2000; 275: 76417647.
  • 43
    Stoffel W, Jenke B, Block B, Zumbansen M, Koebke J. Neutral sphingomyelinase 2 (smpd3) in the control of postnatal growth and development. Proc Natl Acad Sci U S A 2005; 102: 45544559.
  • 44
    Davis RJ. Signal transduction by the c-Jun N-terminal kinase. Biochem Soc Symp 1999; 64: 112.
  • 45
    Liu B, Hannun YA. Inhibition of the neutral magnesium-dependent sphingomyelinase by glutathione. J Biol Chem 1997; 272: 1628116287.
  • 46
    Hagen TM, Yowe DL, Bartholomew JC, Wehr CM, Do KL, Park JY, et al. Mitochondrial decay in hepatocytes from old rats: membrane potential declines, heterogeneity and oxidants increase. Proc Natl Acad Sci U S A 1997; 94: 30643069.
  • 47
    Acharya U, Patel S, Koundakjian E, Nagashima K, Han X, Acharya JK. Modulating sphingolipid biosynthetic pathway rescues photoreceptor degeneration. Science 2003; 299: 17401743.
  • 48
    Schorling S, Vallee B, Barz WP, Riezman H, Oesterhelt D. Lag1p and Lac1p are essential for the Acyl-CoA-dependent ceramide synthase reaction in Saccharomyces cerevisae. Mol Biol Cell 2001; 12: 34173427.