Expression of neutral sphingomyelinase‐2 (NSMase‐2) in primary rat hepatocytes modulates IL‐β‐induced JNK activation

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Abstract
SPECIFIC AIMS Neutral plasma membrane sphingomyelinase (NSMase) has been suggested to participate in numerous cellular responses, including inflammation, cell growth arrest, and apoptosis. Until recently, however, the proteins responsible for this activity were not purified or sequenced. Based on homology searches with bacterial SMase, a novel putative NSMase (NSMase-2) was identified and characterized. The goal of this study was to test whether NSMase-2 plays a role in signal transduction, particularly in IL-1β signaling. PRINCIPAL FINDINGS 1. Molecular cloning and functional characterization of NSMase-2 The I.M.A.G.E consortium clone 2076661, which is identical to the published mouse NSMase-2 cDNA (AJ250461), was used. NSMase-2 was PCR amplified, sequenced, FLAG-tagged at the carboxyl terminus, and cloned into the shuttle vector pAd.trac.teton under doxycycline-inducible promoter. The resulting construct, pAd.trac.teton-NSMase-2, was used for the initial characterization of NSMase-2 in HEK 293 cells. NSMase-2 appeared on a Western blot as a single band of apparent molecular mass of 73 kDa. The induction of expression resulted in a significant increase in the initial velocity of NSMase activity, from 1 pmol/min in the noninduced cells to 30 pmol/min in induced cells. HPLC analyses confirmed that only one product, NBD-ceramide, was generated at the expected molar ratio of 1:1. Addition of 0.5% Triton X-100 or 50 mM EDTA to the assay mixture suppressed the inducible NSMase activity. Recombinant adenovirus expressing NSMase-2 (AdNSMase-2) was generated and used to infect primary hepatocytes. As in HEK cells, induction of NSMase-2 expression led to the production of a 73 kDa protein with significant NSMase activity. More important, overexpression of NSMase-2 resulted in increases in cellular ceramide levels by 30%, 62%, and 73% at 16, 48, and 72 h. This corresponds to an increase by 0.5, 1.25, and 1.3 nmol/mg protein, respectively, and indicates that the expressed NSMase-2 hydrolyses endogenous substrate and generates ceramide in the cells. No statistically significant differences were found in the viability of hepatocytes among the groups. 2. Subcellular localization of NSMase-2 Neutral sphingomyelinase activity is associated mainly with the plasma membrane, and the predicted amino acid sequence for NSMase-2 reveals two putative transmembrane domains at the amino terminus. Subcellular localization of the FLAG-tagged protein was determined by confocal microscopy using anti-FLAG antibody in Hep G2 cells, showing that NSMase-2 was localized mainly at the plasma membrane (Fig. 1 ⤻ ). Figure 1. Subcellular localization of NSMase-2 determined by indirect immunofluorescence and confocal microscopy. Download figure Download PowerPoint Taken together, these results indicate that the cloned NSMase-2 encodes a functionally active Mg2+-dependent and plasma membrane-bound NSMase. 3. Effects of NSMase-2 expression on IL-1β-induced JNK activation Our previous studies showed that IL-1β stimulates NSMase activity in primary hepatocytes, so it was of interest to see whether NSMase-2 has a role in IL-1β signaling. One of the most important targets of IL-1β is JNK, a member of the MAP kinase family. As anticipated, treatment with IL-1β increased transiently the level of the active, phosphorylated form of JNK. (Fig. 2 ⤻ A, B). NSMase-2 overexpression enhanced up to threefold the potential of IL-1β to induce JNK phosphorylation whereas in the absence of IL-1β, NSMase-2 overexpression had only a small effect on JNK. A dose response study (Fig. 2C, D⤻ ) confirmed that the magnitude of IL-1β-induced JNK phosphorylation was substantially increased in the presence of NSMase-2 over a broad range of IL-1β concentrations. Figure 2. Overexpression of NSMase-2 augments IL-1β-induced phosphorylation of JNK in primary hepatocytes. A, C) AdNSMase-2 infected primary rat hepatocytes were cultured in the presence or absence of doxycycline (2 μg/mL) for 72 h, then treated with 25 ng/mL of IL-1β (A) or the indicated doses for 15 min (C). Cells were harvested, lysed, and phosphorylation of JNK was determined by Western blot using an antibody specific for the dually phosphorylated active form of JNK. B, D) Quantification of the intensity of the pJNK bands from panels A and C was done using Storm™860 scanning instrument. Values represent combined intensity of JNK1 and JNK 2. β-Actin was used as a control for uniform loading. Download figure Download PowerPoint 4. The potentiation effects of NSMase-2 on JNK phosphorylation are not affected by the tyrosine kinase inhibitor AG126 The IL-1β receptor-associated kinase IRAK has a central role in the IL-1β signaling cascade. Upon receptor activation, it is hyperphosphorylated by itself and by other kinase(s), such as IRAK-4, which triggers the release of a multiprotein signaling complex and activation of the MAP kinase cascade (Fig. 3 ⤻ ). To test whether NSMase-2 overexpression affects IL-1β signaling cascade up- or downstream of IRAK, we used AG126. This putative tyrosine phosphorylation inhibitor has been found to prevent lipopolysaccharide-induced IRAK activation. Control experiments showed that AG126 inhibited IL-1β-induced phosphorylation of IRAK by 50% at most, probably because AG126, a tyrosine kinase inhibitor, cannot block IRAK autophosphorylation that occurs at serine or threonine residues. The addition of AG126 also prevented IL-1β-induced JNK phosphorylation, but only when NSMase-2 expression was not induced. In contrast, AG 126 had only a partial effect in doxycycline-induced cells, not being able to inhibit the “potentiation” effect of NSMase-2. Figure 3....