Stress-induced sphingolipid signaling: role of type-2 neutral sphingomyelinase in murine cell apoptosis and proliferation

Abstract

Background: Sphingomyelin hydrolysis in response to stress-inducing agents, and subsequent ceramide generation, are implicated in various cellular responses, including apoptosis, inflammation and proliferation, depending on the nature of the different acidic or neutral sphingomyelinases. This study was carried out to investigate whether the neutral Mg(2+)-dependent neutral sphingomyelinase-2 (nSMase2) plays a role in the cellular signaling evoked by TNFalpha and oxidized LDLs, two stress-inducing agents, which are mitogenic at low concentrations and proapoptotic at higher concentrations.

Methodology and principal findings: For this purpose, we used nSMase2-deficient cells from homozygous fro/fro (fragilitas ossium) mice and nSMase2-deficient cells reconstituted with a V5-tagged nSMase2. We report that the genetic defect of nSMase2 (in fibroblasts from fro/fro mice) does not alter the TNFalpha and oxidized LDLs-mediated apoptotic response. Likewise, the hepatic toxicity of TNFalpha is similar in wild type and fro mice, thus is independent of nSMase2 activation. In contrast, the mitogenic response elicited by low concentrations of TNFalpha and oxidized LDLs (but not fetal calf serum) requires nSMase2 activation.

Conclusion and significance: nSMase2 activation is not involved in apoptosis mediated by TNFalpha and oxidized LDLs in murine fibroblasts, and in the hepatotoxicity of TNFalpha in mice, but is required for the mitogenic response to stress-inducing agents.

Figure 1. TNFα and oxLDLs induce dose-dependent apoptotic or mitogenic effects in wt murine fibroblasts.

Wild-type (wt) murine fibroblasts were starved in serum-free medium for 24 h, then were treated for 24 h with the indicated concentration of TNFα (0–50 ng/ml) or of LDL non oxidized (LDLs) and oxidized (oxLDLs) (0–200 µg/ml). DNA synthesis was quantified by [³H]thymidine incorporation and was expressed as % of untreated control (A,D). Apoptosis and necrosis were visualized by fluorescence microscopy of cells stained by Syto13/PI (B,C,E). Primary apoptosis was characterized by condensed picnotic or fragmented nuclear stained green/yellow by Syto13, whereas post-apoptotic necrosis exhibited the same nuclear morphology but was stained red by PI (permeabilization of the plasma membrane in a late step of apoptosis). Only few cells exhibited the feature of primary necrosis, i.e. loose chromatin stained red. In, A,B,D,E, Mean ± SEM of 4 experiments. *: p<0.05.

Figure 2. TNFα and oxLDLs induce nSMase activation in wt but not in fro/fro fibroblasts.

A,B - Sphingomyelin hydrolysis was monitored after metabolic labeling using [³H]choline chloride (0.5 mCi/ml) of cells, as described in the Method section. Then, cells were stimulated with oxLDLs (200 µg/ml) (A) or TNFα (50 ng/ml) (B), and the level of cellular sphingomyelin was determined at the indicated time. Results are expressed as % of control (time 0 h of stimulation). C,D -Basal nSMase activity in untreated cells (C) and in cells treated with TNFα (50 ng/ml) or oxLDLs (200 µg/ml) for the indicated time (D). Mean ± SEM of 3 to 5 separated experiments. * p<0.05.

Figure 3. TNFα, oxLDLs and staurosporine induce cell apoptosis in wt and fro/fro fibroblasts.

Fibroblasts were incubated with TNFα (50 ng/ml, 48 h), oxLDLs (200 µg/ml, 24 h) or staurosporine (100 nM, 6 h). Cell viability was evaluated by the MTT assay (A–C) and by counting apoptotic cells after syto13/PI labeling (D,E), as in Fig. 1. Caspase 3 activation was determined by western blot showing pro-caspase (32 kDa) and cleaved active caspase (17 kDa) (F). Time-course of DEVDase activity were measured using the fluorogenic substrate Ac-DEVD-AMC in cells treated by oxLDLs, TNFα and staurosporin, respectively (G–I). The results are mean ± SEM of 3 to 5 separated experiments. * p<0.05 for apoptotic cell counting and DEVDase activity measurement (comparison between cells treated with or without agonist).

Figure 4. Effects of TNFα and oxLDLs on fro/fro fibroblasts transfected with active V5-nSMase2 vector.

The fro/fro fibroblasts were stably transfected using pEF-6 plasmid containing V5-nSMase2 cDNA. After clone selection, the expression of V5-nSMase2 was evaluated by western blot using an anti-V5 antibody (A), immunocytochemistry (B) and by enzymatic determination of nSMase activity, under basal conditions (C) and after activation by TNFα (50 ng/ml) or oxLDLs (200 µg/ml) (D). Time course of sphingomyelin hydrolysis induced by TNFα (50 ng/ml) or oxLDLs (200 µg/ml) were determined in V5-transfected cells under the conditions indicated in the legend to Figure 2 (E). Caspase activity was evaluated by western blot of caspase-3 (32 KDa) (F), and by fluorometric determination of DEVDase activity (G), under the conditions of Fig. 3. Apoptosis triggered by TNFα (50 ng/ml, 48 h incubation) or oxLDLs (200 µg/ml, 24 h incubation) was evaluated by fluorescence microscopy counting of cells labeled by syto13/PI, under conditions of Fig. 1 and 3 (H). In Fig. 4C–E, and G,H, the data are expressed as mean ± SEM of 3 to 5 separated experiments * p<0.05; ns, not significant.

Figure 5. Time course of TNFα-induced toxicity in wt and fro/fro mice.

Mice (10 wt or 10 fro/fro, 5 females and 5 males) were intraperitoneally injected with D-galactosamine (20 mg) and then injected intravenously with PBS or TNFα (40 µg/kg of body weight). A -Time course of survival of wt (red symbols) and fro/fro (green) mice treated or not by D-galactosamine and TNFα. B - Histological analysis of hematoxylin-eosin stained liver sections from wt or fro/fro mice injected with D-galactosamine/PBS (sacrificed 48 hours after the injection) or D-galactosamine/TNFα (immediately taken off after mice death). Representative microscopy pictures of liver sections from wt and fro/fro mice (magnification, ×400).

Figure 6. Proliferation mediated by TNFα or oxLDLs requires the activation of the nSMase2.

A,B - TNFα (5 ng/ml) and oxLDLs (50 µg/ml) induced nSMase activation (A) and ERK1/2 phosphorylation (B) in wt and in V5-fro) but not in fro/fro fibroblasts. It may be noted that FCS (10%) induce ERK1/2 phosphorylation in the 3 cell types, independently of nSMase2 deficiency (B). C,D - Proliferation was evaluated by DNA synthesis ([³H]thymidine incorporation after 24 h incubation with the agonists) and cell count (performed after 48 h incubation with the agonists), in wt, fro/fro and V5-fro fibroblasts, after treatment by TNFα, oxLDLs and FCS, as in Fig. 6A,B. In A,C,D, mean ± SEM of 3 to 5 separated experiments. * p<0.05 (comparison between cells treated with or without TNFα, as indicated).