Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Pulmonary inflammation is associated with production of reactive oxygen species such as superoxide (O₂ ^·), hydroxyl radicals (HO^·), and hydrogen peroxide (H₂O₂), which contribute extensively to lung injury in respiratory tract diseases. Yet the molecular mechanisms that link oxidant exposure with lung disease are poorly understood. Excessive accumulation of reactive oxidants is toxic (1), and the intracellular level of reactive oxidants is therefore tightly regulated by several antioxidants. Although antioxidant defenses are constitutively expressed in mammalian cells, additional responses are mounted when the amount of environmental oxidants exceeds a threshold level, thereby becoming a threat to overall tissue integrity. Apoptosis may be one such cellular adaptive response (2).

Apoptosis is an essential, highly conserved, and tightly regulated cellular process of cell death that is important for development, host defense, and suppression of malignant transformation and inflammatory processes (5). Keeping apoptosis in balance limits the survival of deranged cells, thus reducing inflammatory processes. However, the mechanisms by which oxidants induce apoptotic pathways remain largely unknown.

The sphingolipid ceramide is emerging as a possible regulator of apoptosis. Ceramide has been shown to potently induce apoptosis in a number of different systems (6). Tumor necrosis factor (TNF)- and other inducers of apoptosis, such as Fas ligation, serum withdrawal, some chemotherapeutic agents, and -irradiation (8, 10), have been proven to elevate cellular levels of ceramide. Moreover, the addition of cell-permeable ceramide induces apoptosis in several cell types, substantiating the possibility that ceramide generation may be the final pathway common to a variety of inducers (7, 11).

In some cases the increase in ceramide levels is due to enhanced de novo synthesis (14); in the majority, however, one or more of the cellular sphingomyelinases (SMases) are activated. Several of these sphingomyelin (SM)-specific phospholipase C activities exist in mammalian tissues (8, 12, 15). These isoenzymes differ in their catalytic properties and subcellular localization, and probably also in their modes of regulation. A lysosomal acidic SMase (aSMase) was the first SMase to be cloned and characterized on a molecular basis (19). Defects in the aSMase gene, which cause the hereditary Niemann Pick disease, result in a massive accumulation of SM in the lysosomes and death in early childhood. SM hydrolysis, triggered by the binding of extracellular ligands to cell-surface receptors or by agents that induce cellular stress, is the major source of the putative intracellular second messenger ceramide (20, 21). A Mg⁺²-dependent neutral SMase (nSMase) that resides in the plasma membrane was initially thought to provide most of the ceramide used as a second messenger (22). But very little is currently known about the regulation mechanisms of SMases.

We report herein that micromolar concentrations of H₂O₂ can induce apoptosis in normal human airway epithelial (HAE) cells. The effect is mediated by the ceramide second messenger, which is potently and rapidly generated from SM hydrolysis by nSMase. We show that exposure of the lung airway epithelium to H₂O₂ activates nSMase but not aSMase. We also demonstrate that H₂O₂ downmodulates 1,2-diacylglycerol (DAG) and protein kinase C (PKC) activity. Further, the activation of PKC by phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), minimizes the intracellular elevation of ceramide and blocks apoptosis. This suggests an antagonistic relationship between PKC and ceramide in triggering apoptosis after exposure of airway epithelial cells to H₂O₂ oxidative stress.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Culture

Airway epithelial cells were grown as described elsewhere (23). Briefly, human tracheobronchial tissues were isolated from human lung after lung resections or transplants (which were carried out at UC Davis Medical Center, Davis, CA), immersed in minimal essential medium, and treated for 24 h at 4°C with 0.1% protease. Dissociated cells were recovered by centrifugation; resuspended in growth media F12 (GIBCO) supplemented with penicillin, streptomycin, and garamycin (50 mg/ml); 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (Hepes) (15 mM, pH 7.2); transferrin (0.1 µM); insulin (10 µM); retinoic acid (0.1 µM); hydrocortisone (0.1 µM); and epidermal growth factor (EGF) (0.01 µM); and plated at a density of 1 to 5 × 10³ cells/cm². Cells were incubated at 37°C with 5% CO₂ atmosphere. The medium was changed every other day, and a final cell density of 3 to 8 × 10⁴ cells/cm² was obtained for primary cultures within 7 to 9 d of incubation. Cells were further passaged once or twice. Subcultures were performed as follows: near-confluent cultures were treated with Trypsin (0.05%)-ethylenediaminetetraacetic acid (EDTA) (1 mM) in phosphate-buffered saline (PBS), pH 7.0. After cells were detached from the plates, an equal volume of Trypsin inhibitor solution (1 mg/ml) in F12 medium was added to stop the trypsinization. Cells were recovered by centrifugation and resuspended in culture medium for plating. Cell numbers were determined using a Coulter counter (model Zf; Coulter Electronics) and verified by hemacytometer (Hausser Scientific). Cell viability was assessed by Trypan blue exclusion analysis.

Histochemical Detection of Nuclear DNA Fragmentation and Apoptotic Bodies

The terminal deoxynucleotidyl transferase end-labeling (TUNEL) technique was used in evaluation of airway epithelial cells for DNA fragmentation and the appearance of apoptotic bodies (24). Slides were stained with DNA counterstains, bis-benzimide (Hoechst 33258; Sigma), and propidium iodide. The morphologic changes in the nuclear chromatin of cells undergoing apoptosis were detected by staining with the DNA-binding fluorochrome bis-benzimide as previously described (25). In brief, 0.5 to 3.0 × 10⁶ cells were pelleted at 300 × g for 10 min and washed once with PBS. Cells were resuspended in 50 µl of 3% paraformaldehyde in PBS and incubated for 10 min at room temperature (RT). The fixative was removed and cells were washed once in PBS and resuspended in 15 µl of PBS containing 16 µg/ml bis-benzimide. After a 15-min incubation at room temperature, a 10-µl aliquot was placed on a glass slide and 500 cells per slide were scored for the incidence of apoptotic chromatin changes. The slides were viewed under a Nikon SA fluorescence microscope and view fields were captured by C-Imaging System (Compix, Cranberry Township, PA). Cells with three or more chromatin fragments were considered apoptotic.

To quantify apoptotic cells, HAE cultures were also grown in 24-well tissue culture dishes. Cells were then rinsed twice in PBS (calcium- and magnesium-free) and incubated in 70% ethanol containing 100 µg/ml Hoechst 33258 (Molecular Probes, Inc.) for 30 min at RT. This procedure served both to fix the cells remaining in the culture and to stain the DNA. After rinsing twice in PBS, the remaining liquid was aspirated and the residual fluorescence was quantified in a fluorescent plate reader.

Agarose Gel Electrophoresis for DNA Fragmentation

Oligonucleosomal fragmentation of genomic DNA was determined as previously described (26). Cells (6 to 12 × 10⁶) were lysed in 1 ml of lysis buffer (10 mM Tris [pH 7.5], 100 mM NaCl, 1 mM EDTA, 1% sodium dodecyl sulfate [SDS], and 0.5 mg/ml proteinase K). Digestion was continued for 1 to 3 h at 50°C, followed by the addition of RNase A to 0.1 mg/ml and further incubation for 1 h. Running dye (10 mM EDTA, 0.25% bromophenol blue, and 50% glycerol) was then added in a 1:6 ratio of dye:sample, and DNA preparations were electrophoresed in 1.5% agarose gels in TAE buffer (40 mM Tris-acetate and 1 mM EDTA) at 4 V/cm of gel. DNA was visualized by ethidium bromide staining.

Lipid Studies

Ceramide was quantified by the diacylglycerol kinase assay as described previously (27). In brief, after incubation with H₂O₂ cells were pelleted by centrifugation (300 × g for 10 min), washed twice with ice-cold PBS, and extracted with 1 ml of chloroform:methanol:1 N HCl (100:100:1, vol/vol/vol). Lipids in the organic-phase extract were dried under N₂ and subjected to mild alkaline hydrolysis (0.1 N methanolic KOH for 1 h at 37°C) to remove glycerophospholipids. Samples were re-extracted, and the organic phase was dried under N₂. Ceramide contained in each sample was resuspended in a 100-µl reaction mixture containing 150 µg of cardiolipin (Avanti Polar Lipids), 280 µM diethylenetriaminepentaacetic acid (Sigma), 51 mM octyl--D-glucopyranoside (Calbiochem), 50 mM NaCl, 51 mM imidazole, 1 mM EDTA, 12.5 mM MgCl₂, 2 mM dithiothreitol (DTT), 0.7% glycerol, 70 µM -mercaptoethanol, 1 mM adenosine triphosphate (ATP), 10 µCi [-³²P]ATP (3,000 Ci/mmol; Dupont New England Nuclear), and 35 µg/ml Escherichia coli diacylglycerol kinase (Calbiochem) at pH 6.5. After 30 min at RT, the reaction was stopped by extraction of lipids with 1 ml of chloroform:methanol:1 N HCl (100:100:1), 170 µl of buffered saline solution (135 mM NaCl, 1.5 mM CaCl₂, 0.5 mM MgCl₂, 5.6 mM glucose, and 10 mM Hepes [pH 7.2]), and 30 µ1 of 100 mM EDTA. The lower organic phase was dried under N₂. Ceramide 1-phosphate was resolved by thin-layer chromatography on silica gel 60 plates (Whatman) using a solvent system of chloroform:methanol:acetic acid (65:15:5) and detected by autoradiography. Incorporated ³²P was quantified by liquid scintillation counting. The level of ceramide was determined by comparison with a standard curve generated concomitantly of known amounts of ceramide (ceramide type III; Sigma). Diacylglycerol was quantified in a similar manner to ceramide, except the alkaline hydrolysis step was omitted. Changes in SM levels were measured by labeling cells to isotopic equilibrium with [³H]choline chloride (79.2 Ci/mmol; Dupont New England Nuclear) as previously described (27). Cells were incubated with [³H]choline (1.0 µCi/ml in tissue culture media) for at least three cell doublings. Incubation with H₂O₂, extraction, and alkaline hydrolysis of dried lipids were identical to those used for ceramide determinations. SM was resolved from residual phosphatidylcholine and lysophosphatidylcholine by thin-layer chromatography on silica gel 60 plates using a solvent system of chloroform:methanol:acetic acid:water (50:30:8:3), then identified by iodine vapor staining, and quantified by liquid scintillation counting. SM mass was verified by lipid phosphorus assay. In brief, SM spots were scraped and extracted three times with 500 µl of chloroform:methanol:HCl (200:100:1) and the combined extracts were dried under N₂. Samples were refluxed with 50 µl of 70% perchloric acid for 30 min at 180°C. Color reagent (1.0 ml) (0.6 M H₂SO₄, 0.25% ammonium molybdate, and 1% ascorbic acid) was added, and samples were incubated at 50°C for 1 h. Absorbance at 700 nm (A₇₀₀) was read, and phosphorous content was determined by comparison with known quantities at Na₂HPO₄.

Lipid Analogs

C2-ceramide (N-acetyl sphingosine), C6-ceramide (N-hexanoyl sphingosine), and 1,2-dioctanoyl-sn-glycerol were obtained from Matreya, and stock solutions were prepared in dimethyl sulfoxide (DMSO). C2-dihydroceramide (N-acetyl dihydrosphingosine), obtained from Matreya, and 1,2-dioctanoyl-sn-glycero-3-phosphate, obtained from Avanti Polar Lipids, were prepared as stock solutions in 100% ethanol. The final concentrations of DMSO and ethanol in the incubations were 0.2 and 0.1%, respectively, which did not induce apoptosis. All experiments involved both vehicle controls and specificity controls using biologically inactive dihydroceramide analogs or inactive L-threo stereoisomers of the active D-erythro.

SMase Assay

After stimulation with H₂O₂ for the indicated time intervals, the cells were washed once with 5 ml of PBS and harvested. The pellet was stored frozen at 70°C and resuspended in 0.5 ml of buffer (100 mM Tris-HCl [pH 7.4], 0.1% Triton X-100, 1 mM EDTA, and protease inhibitors). The cell suspension was sonicated three times (3-s bursts) using a probe sonicator and centrifuged at 500 × g at 4°C for 5 min. The supernatant was used as the enzyme source. A total of 100 µg of protein was assayed for nSMase activity in a buffer consisting of 50 mM Tris-HCl (pH 7.4), 0.1% Triton X-100, 0.1 mg bovine serum albumin (BSA), 5 mM MgCl₂, and 50 µmol of [¹⁴C]SM (12,000 disintegrations/min). The assay was incubated at 37°C for 1 h and terminated with the addition of 1 ml of 10% trichloroacetic acid. The precipitate was pelleted (1,000 × g at 4°C for 20 min) and 1 ml of the supernatant was extracted with 1 ml of anhydrous diethyl ether. A total of 0.5 ml of the aqueous phase was removed for liquid scintillation counting. aSMase activity was determined using [¹⁴C]SM resuspended in 100 mM sodium acetate, pH 5.0.

PKC Activity

Airway epithelial cells were harvested by scraping in cold buffer A (20 mM Tris-HCl [pH 7.5], 250 mM sucrose, 6 mM EDTA, and 0.5 mM DTT) supplemented with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, and 20 µg/ml aprotinin). The cells were sonicated for 1 min in a bath sonicator and centrifuged at 500 × g for 5 min at 4°C to remove nuclei and whole cells. The cytosolic fraction was separated from the membranes by centrifugation at 100,000 × g for 1 h. Membrane-bound PKC was solubilized by resuspending the pellet in buffer A containing 0.5% Triton X-100 for 20 min on ice, and centrifuged for 30 min at 100,000 × g to remove nonsoluble material. Both the cytoplasmic and the solubilized membrane fractions were applied to a 0.2-ml anion exchange chromatography diethylaminoethyl- cellulose (DE-52) column, washed with buffer B (20 mM Tris [pH 7.5], 2 mM EDTA, and 5 mM ethyleneglycol-bis-(-aminoethyl)- N,N,N',N'-tetraacetic acid). Bound PKC was eluted batchwise with 500 µl buffer C (buffer B containing 0.15 M NaCl). PKC activity was detected using the PKC enzyme assay kit (Amersham), according to the manufacturer's instructions, and as previously described (30). TPA was used as a positive control.

Western Blotting of PKC

Protein fractions containing PKC which were eluted from the DE-52 columns were separated on SDS 10% polyacrylamide gel electrophoresis and Western-blotted onto a nitrocellulose membrane, and membranes were blocked in 3% BSA in PBS. PKC was detected by incubating the membrane in specific antibodies directed against the various PKC isozymes (1:1,000 in PBS). Bound antibodies were detected using protein A-conjugated horseradish peroxidase. Blots were scanned with an LKB Ultrascan XL densitometer to quantify PKC immunoreactivity, as previously described (30).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

H₂O₂ Causes SM Hydrolysis to Generate Measurable Amounts of Ceramide in HAE Cells

To investigate whether H₂O₂ induces apoptosis in HAE cells through a SMase/ceramide pathway, we measured cellular SM hydrolysis and ceramide production after H₂O₂ treatments. This was compared with ceramide levels after treatment with a bacterial nSMase (Staphylococcus aureus SMase C; Sigma), a positive control for ceramide generation. Using a DAG kinase assay to measure free ceramide levels, we found that stimulation of these HAE cells with 0.2 U/ml exogenous bacterial SMase caused a large (60 to 80%) decrease in cellular SM and a corresponding increase in ceramide (Figure 1A). The changes were apparent by 5 min, maximal by 15 to 30 min, and persisted for at least 60 min. The total observed increase in cellular ceramide after treatment with exogenous SMase reached 343 ± 8% per 10⁶ HAE cells. We next used the same DAG assay to measure ceramide and SM levels in HAE cells treated with 100 µM H₂O₂ (Figure 1B). In parallel to the results with exogenous SMase treatments, stimulation of HAE cells with 100 µM H₂O₂ for 5 to 10 min caused measurable SM decrease and ceramide generation. Production of ceramide was detectable after 1 min of H₂O₂ exposure, reached a plateau at 3 min, and remained elevated for several hours. The rise in ceramide levels was 2-fold: from 127 ± 10% pmol per 10⁶ control cells to 309 ± 10% pmol of H₂O₂-treated cells. Similar quantitative results were obtained in three other studies done with H₂O₂ in the range of 50 to 100 µM. Thus, the observed increase in ceramide levels of HAE cells exposed to H₂O₂ was comparable (± 10%) to that released by the exogenous SMase treatments. These quantitative comparisons of H₂O₂ effects on SM hydrolysis with the effects of exogenous SMase suggest that this reactive oxidant activates a membrane SMase that hydrolyses the membrane pool of SM, as was also recently demonstrated in monkey airway epithelial cells (26).

View larger version (16K): [in this window] [in a new window]   Figure 1.   Changes in ceramide and SM levels of HAE cells treated with 0.2 U/ml exogenous SMase (A), or exposed to 100 µM H2O2 (B). At the indicated times, cells were extracted with chloroform:methanol:1 N HCl (100:100:1). Lipid extracts were assayed for ceramide levels by the DAG kinase reaction. For the determination of the SM levels, HAE monolayers were incubated with [3H]choline (1 µCi/ml) for three cell doublings to label cellular SM. Cells were treated with 100 µM H2O2, and at the indicated times, cells were extracted with chloroform:methanol:1 N HCl (100:100:1). Lipid extracts were subjected to mild alkaline hydrolysis, and SM was resolved by thin-layer chromatography. Baseline SM masses were determined by lipid phosphorous measurements. Values represent means ± standard error of the mean (SEM) of independent triplicate determinations from three separate studies for ceramide and four experiments for SM.

Treatment of HAE cells with the membrane-permeant C6-ceramide also resulted in rapid intracellular accumulation of ceramide (not shown), consistent with results in other cells (31), and suggesting that both membrane-permeant ceramide and H₂O₂ activate a membrane nSMase. Both the effects of the synthetic ceramide analogs and those of H₂O₂ on the increase in cellular ceramide levels were specific, in that none of them induced an increase in the level of the lipid second messenger DAG. In fact, a decrease in endogenous DAG levels has recently been shown with H₂O₂ treatment of tracheobronchial epithelial cells from nonhuman primates (26).

We then directly measured the effects of H₂O₂ on SMase activity. Treatment of HAE cells with 75 µM H₂O₂ induced a greater than 2.5-fold activation of nSMase activity within 5 to 10 min, but not of aSMase activity (Figure 2). Similar results were obtained with 50 µM C6-ceramide, but not with 50 µM of the immediate precursor of ceramide, dihydroceramide, which lacks the trans double bond C4-C5 of the sphingoid base backbone. At the same time, aSMase activity remained unchanged throughout all experiments, suggesting that in airway epithelial cells only nSMase and not aSMase is activated by H₂O₂ and by membrane-permeant ceramides.

View larger version (24K): [in this window] [in a new window]   Figure 2.   H2O2 activates nSMase (open symbols) but not aSMase ( filled symbols) in HAE cells. (A) Time response for treatments with 75 µM H2O2 (squares), 50 µM C6-ceramide (circles), and 50 µM dihydroceramide (triangles). (B) Dose response for H2O2 exposures of 20 min. At the indicated times, cells were harvested in PBS, pelleted, and frozen. Cells were subsequently lysed as described in MATERIALS AND METHODS. nSMase (darker bars) and aSMase assays (lighter bars) were performed in triplicate. Values represent means ± SEM of independent triplicate determinations from three separate studies.

Ceramide Mimics H₂O₂ in Induction of Apoptotic DNA Degradation in HAE Cells

Figure 3 shows that treatment of HAE cells with 100 µM H₂O₂ for 12 h produced typical cell morphology changes of apoptotic nuclear condensation and segmentation. Similar changes were observed when the cells were treated with 50 µM C2-ceramide but not with dihydroceramide, the immediate precursor of ceramide. In addition to the typical morphologic changes, H₂O₂ and ceramide-induced apoptosis was demonstrated by agarose gel electrophoresis of DNA extracted from treated HAE cells (Figure 4). As shown, 12-h treatments with increasing doses of C6- ceramide (25 to 75 µM) produced a typical ladder pattern of oligonucleosomal fragments, similar to that triggered by exposure to increasing concentrations of H₂O₂ (50 to 200 µM). In contrast, treatment with 50 µM of dihydroceramide resulted in no apoptotic response (Figure 4, lane 5). Further, other cell-permeable analogs of lipid second messengers, including 1,2-dioctanoyl-sn-glycerol (the analog of DAG), and 1,2-dioctanoyl-sn-glycero-3-phosphate (the analog of phosphatidic acid) did not induce apoptosis (Figure 5). Importantly, ceramide-induced cell death is stereospecific because only the D-erythro isomer but not the inactive L-threo isomer of C6-ceramide induced epithelial cell apoptosis in a dose-dependent manner (Figure 5).

View larger version (196K): [in this window] [in a new window]   Figure 3.   Effects of H2O2, C2-ceramide, and C2-dihydroceramide on cell morphology in HAE cells. HAE monolayers were (A) untreated; (B) treated with 50 µM C2-dihydroceramide; (C) treated with 100 µM H2O2; and (D) treated with 50 µM C2-ceramide. Treatments were terminated after 12 h by fixing the cells in 2% glutaraldehyde. Shown: (A and B) Control-treated and C2-dihydroceramide-treated cells; (C) nucleus heavily fragmented, cytoplasmic vacuoles, membrane blebbing; (D) nucleus fragmented, extensive membrane blebbing. This experiment represents one of three similar studies.

View larger version (111K): [in this window] [in a new window]   Figure 4.   Agarose gel electrophoresis of DNA ladders induced by H2O2 and by a synthetic ceramide analog (C6-ceramide) in HAE cells. HAE monolayers were treated for 12 h with various doses of H2O2 or ceramides. DNA was extracted and subjected to agarose gel electrophoresis and analyzed by Southern blot hybridization with total DNA. Lane 1, control; lane 2, 50 µM H2O2; lane 3, 100 µM H2O2; lane 4, 200 µM H2O2; lane 5, 50 µM C6- dihydroceramide; lane 6, 25 µM C6-ceramide; lane 7, 50 µM C6-ceramide; and lane 8, 75 µM C6-ceramide. Arrowheads mark 500- and 1000-base pair locations based on ethidium bromide staining of DNA size markers run on the original gel. This experiment represents one of three similar studies.

View larger version (20K): [in this window] [in a new window]   Figure 5.   Ceramide-induced apoptosis in airway epithelial cells is stereospecific. HAE cell monolayers were treated as indicated with active 20 µM C2-D-erythro-ceramide (open squares), 20 µM C6-L-threo-ceramide (open diamonds), 50 µM C6-dihydroceramide (open circles), 20 µM 1, 2-dioctanoyl-sn-glycerol (open triangles), or 20 µM 1,2 dioctanoyl-sn-glycero-3-phosphate (sectioned squares) for 24 h. The cells were then fixed and stained with Hoechst 33258, and residual fluorescence was quantified in a fluorescent plate reader. The data are presented as percent of control, which represents the residual fluorescence (arbitrary units) in treated wells/residual fluorescence in control wells × 100. Values represent means ± SEM of independent triplicate determinations from three separate studies.

A time course of nuclear fragmentation demonstrated an increase in the number of apoptotic cells, which became apparent 6 to 12 h after the addition of 100 µM H₂O₂ to the culture medium. At 12 h, nearly 60% of the cells demonstrated apoptotic changes by TUNEL (Figure 6), and at 24 h, nearly 80% of the cells demonstrated apoptotic changes. The morphologic changes after treatment with ceramide analogs developed more rapidly than those induced by H₂O₂. At 6 h of treatment with C6-ceramide, about 60% of the airway epithelial cells were TUNEL-positive, and at 12 h, nearly 80% of the cells demonstrated apoptotic changes by TUNEL. However, the effects at 18 to 24 h were quantitatively comparable both for ceramide analogs and for H₂O₂.

View larger version (70K): [in this window] [in a new window]   Figure 6.   TUNEL assays of HAE cells treated with H2O2 and with a synthetic ceramide analog (C6-Ceramide). Upper panel: HAE monolayers were untreated (Control) or treated with 100 µM H2O2 or with 20 µM synthetic ceramide analog (C6-ceramide). Treatments were terminated after 12 h by fixing the cells on slides for the TUNEL assay as described in MATERIALS AND METHODS. Lower panel: To quantify TUNEL-positive HAE cells, 500 cells per slide were scored for the incidence of apoptotic chromatin changes. The slides were viewed under a Nikon SA fluorescence microscope and view fields were captured by C-Imaging System (Compix). Cells with three or more chromatin fragments were considered apoptotic. Values reflect the means ± SEM of quadruplicate determinations.

Countereffects of PKC and Ceramide in H₂O₂-Induced Apoptosis of HAE Cells

PKC activation is often antiapoptotic, although in a few cell types PKC initiates apoptosis by an unknown mechanism (32). Exposure of HAE cells to 100 µM H₂O₂ inhibited PKC activity after its translocation from the membrane to the cytosol (Figure 7A). Similarly, treatments with 15 µM C6-ceramide also triggered PKC translocation from the membrane to the cytosol (Figure 7B). On the other hand, exposure to TPA or to a synthetic DAG analog (not shown) caused activation of PKC after its translocation to the membrane (Figure 7B). The mechanism of PKC inhibition by ceramide is not yet known, but H₂O₂ may inhibit PKC in airway epithelial cells both by reducing DAG levels (26) and by activating the nSMase, thereby increasing ceramide levels.

View larger version (16K): [in this window] [in a new window]   Figure 7.   PKC inhibition by H2O2 and C6-ceramide, and activation by phorbol ester (TPA). Airway epithelial cells were treated with (A) 100 µM H2O2 or with (B) 15 µM C6-ceramide or 50 ng/ ml TPA. At the indicated times, cells were collected, cytoplasmic and membranal PKC was purified, and PKC activity was measured. Values represent means ± SEM of independent triplicate determinations from three separate studies.

To address the role of PKC in the regulation of ceramide production and apoptosis, we evaluated the impact of the PKC activator TPA on the H₂O₂-induced ceramide production (Figure 8) and on the apoptotic signaling pathway triggered by H₂O₂ and ceramide (Figure 9). As shown, pretreatment of HAE cells with TPA (50 ng/ml) for 30 min abolished 75 to 100 µM H₂O₂-induced nSMase activation and SM hydrolysis to ceramide. Similar results were obtained with a 15-min or 1-h pretreatment with TPA. Figure 9 shows that pretreatment of HAE cells with TPA also eliminated the ability of 100 µM H₂O₂ to induce apoptosis in these cells. Although H₂O₂ enhanced apoptotic DNA laddering (Figure 9B, lane 2), preincubation with TPA eliminated it (Figure 9B, lane 3). Hence, activation of PKC by TPA appears to block both the generation of ceramide (Figure 9A) and the induction of apoptosis caused by H₂O₂ exposure (Figure 9B). Additional experiments were performed to examine whether selective restoration of ceramide would overcome this inhibition. HAE cells were first treated for 30 min with 50 ng/ml TPA, then exposed to 100 µM H₂O₂ and subsequently incubated with increasing concentrations of C2-ceramide (25 to 75 µM) (Figure 9, lanes 4 - 6). The later step restored the apoptotic response, as demonstrated by the increase in the DNA laddering, suggesting that apoptotic signaling can be produced via ceramide generation by H₂O₂ exposure and that ceramide-mediated apoptosis may be subjected to a transmodulatory control via PKC (35). Therefore, we conclude that H₂O₂-induced generation of ceramide is a critical and obligatory event in the H₂O₂ induction of the apoptotic cascade in HAE cells.

View larger version (20K): [in this window] [in a new window]   Figure 8.   TPA inhibits H2O2-induced nSMase activation. HAE cells were treated with 75 µM H2O2 as described in the caption to Figure 2 with or without 30 min pretreatment with 50 ng/ml TPA. At the indicated times, cells were harvested in PBS, pelleted, and frozen. Cells were subsequently lysed as described in MATERIALS AND METHODS. nSMase assays were performed in triplicate as described. Values represent means ± SEM of independent triplicate determinations from three separate studies.

View larger version (30K): [in this window] [in a new window]   Figure 9.   Phorbol esters inhibit H2O2-induced ceramide production and apoptosis (DNA laddering) whereas ceramide analogs restore apoptosis. Cells were cultured as described in the caption to Figure 1, except that 50 ng/ml TPA or the diluent DMSO was added for 30 min before the cells were exposed to 100 µM H2O2. SM and ceramide levels were quantified as described in Figure 1. (A) Ceramide levels in H2O2-treated and TPA-pretreated cells. Values are derived from triplicate determinations from two experiments. The mean ranges of values for SM and ceramide were 2 and 7%, respectively. Data are presented only for ceramide levels. (B) Agarose analyses were run as described in the caption to Figure 4 after restoration of apoptosis by ceramide treatment of airway epithelial cells that were pretreated with H2O2 and TPA. The treatments were as follows: lane 1, control; lane 2, 100 µM H2O2; lane 3, 100 µM H2O2 + 50 ng/ml TPA; lane 4, 100 µM H2O2 + 50 ng/ml TPA + 25 µM C2-ceramide; lane 5, 100 µM H2O2 + 50 ng/ml TPA + 50 µM C2-ceramide; lane 6, 100 µM H2O2 + 50 ng/ ml TPA + 75 µM C2-ceramide. This experiment represents one of three similar studies.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Inflammatory diseases of the respiratory tract such as asthma, bronchiectasis, and adult respiratory distress syndrome (ARDS) are characterized by a large increase in inflammatory oxidants such as H₂O₂, which largely contribute to lung injury typified by increased epithelial cell permeability and decreased lung function (36). Reactive oxygen species have been implicated as mediators of lung injury in both ARDS (alveolar damage) and asthma (airway epithelial damage). Indeed, elevated levels of H₂O₂ were found in expired breath condensate of patients with asthma, and an oxidant-antioxidant imbalance may also play a role in the damage found in the bronchial epithelium in the airways of patients with cystic fibrosis (39, 40).

We postulated that H₂O₂, the agent commonly produced during lung inflammatory processes, induces epithelial apoptosis, which is mediated by the ceramide signal transduction pathway.

Signaling pathways involved in apoptosis induction remain largely unknown. The SM pathway, initiated by hydrolysis of SM in the cell membrane to generate the second messenger ceramide (6, 27), is thought to mediate apoptosis in response to TNF- (7, 41), to Fas ligand (13), and to X-rays (11). It is not known whether ceramide plays a role in the stimulation of other forms of stress-induced apoptosis. Our recent studies have suggested that reactive oxygen intermediates may be involved in cellular signaling pathways via plasma membrane-anchored receptors and enzymes. We have shown that H₂O₂ activates phosphorylation of EGF receptor tyrosine but not threonine (42), whereas peroxynitrite (ONOO) affects EGF receptor dimerization (43). Because lung airway epithelial cells are extensively exposed to reactive oxidants, we initiated studies to address whether these cells are capable of entering apoptosis when exposed to physiologic micromolar concentrations of H₂O₂ and whether the process is mediated by ceramide as a second messenger.

Results from the present study provide evidence that ceramide contributes to H₂O₂-induced cell death. Ceramide production preceded the onset of DNA fragmentation and cell death. The time courses of ceramide generation and HAE cell death in response to H₂O₂ were consistent with the contention that ceramide is an early messenger for this process. The mechanism of ceramide accumulation after H₂O₂ treatment appears to be secondary to the activation of SMase pathway(s). These same pathways have been shown to respond to TNF-, Fas, and -irradiation- initiated apoptosis (11, 13, 41), wherein ceramide accumulation is concurrent with SM hydrolysis by activated SMases. However, whereas in the TNF- cascade both the membrane-associated neutral and the acidic forms of SMase are activated by TNF- receptors through different domains, our present studies demonstrated that only nSMase and not aSMase is affected by H₂O₂.

The mechanism by which H₂O₂ stimulates SM hydrolysis to ceramide is unknown, and very little is known about the regulatory mechanisms of SMases. We have recently found that depletion of glutathione (GSH) from airway epithelial cells by DL-buthionine-[S,R]-sulfoximine caused a dose- and time-dependent increase in ceramide and apoptosis (44). It has also been shown by others (45) that partially purified magnesium-dependent, neutral pH-optimum, and membrane-associated nSMase is inhibited in vitro by GSH. Inasmuch as GSH depletion is observed in a variety of cells in the process of cellular apoptosis, it is possible that depletion of GSH may be an important mechanism in the activation of nSMase. Therefore, it is conceivable that H₂O₂ activates nSMase by releasing it from GSH inhibition (44), thereby coupling oxidative stress and signaling via products of SM hydrolysis to induce apoptosis.

The specificity of various lipids in inducing apoptosis in lung epithelial cells was determined by treatments with various permeable ceramide synthetic analogs. Isomers, such as dihydro C6-ceramide (which lacks the 4, 5 double bond), did not elicit apoptosis. Moreover, the phospholipid DAG (physiologic activator of PKC) counteracted ceramide-induced apoptosis, indicating that the context of the ceramide signal determines the ultimate biologic response, and that ceramide-mediated apoptosis may be subject to transmodulatory control through DAG/PKC. Therefore, PKC activation may provide an antiapoptotic mechanism in lung epithelial cells.

Previous studies have yielded conflicting observations regarding the involvement of PKC in apoptosis, pointing to a great functional variability depending on cell type, agent or condition causing apoptosis, and intracellular signaling pathways. PKC activation is often antiapoptotic: it has been shown in the current studies in HAE cells, and also in other cells (46), that activation of PKC by DAG or phorbol esters induces its translocation from the cytosol to the membrane (49) and inhibits ceramide-induced apoptosis (7, 8, 35). However, the mechanisms by which PKC activators inhibit ceramide-induced apoptosis are still unknown. On the other hand, in a few cell types PKC actually initiates apoptosis by an unknown mechanism. Recent investigations showed activation of PKC- by TPA-induced apoptosis in LNCaP prostate cancer cells (32).

It has been reported that ceramide has no effect on PKC activity in vitro (46), but it remained unclear whether ceramide has any effect on PKC in vivo. Indeed, recent in vivo studies reported that both C2- and C6-ceramide inhibited PKC- activity, whereas C2- and C6-dihydroceramides did not (47). In addition, SMase treatment of mouse epidermal or human skin fibroblast cells, or incubation of these cells with C2-ceramide, blocked PKC-'s translocation to the membrane and thus inhibited its activity. Similarly, our present studies also demonstrated that H₂O₂ in HAE cells induced ceramide generation and blocked PKC translocation to the membrane, whereas the exposure of HAE cells to the membrane-permeant ceramide analog C6-ceramide blocked membrane PKC translocation. Together, these observations support a model of a balance between upregulation of apoptosis via H₂O₂-induction of the SM/ceramide pathway, and downregulation of apoptosis by natural suppresser mechanisms through PKC.

In summary, the ceramide generated by H₂O₂ activation of SMase has been shown to serve as a second messenger in initiating an apoptotic response in HAE cells. PKC activation represents an upstream antiapoptotic checkpoint at the SMase level as well as a checkpoint downstream of ceramide generation. The balance between these pro- and antiapoptotic systems may determine the magnitude of the observed apoptotic response.

Further identification of the molecular mechanisms leading to ceramide generation and an improved understanding of the molecular basis of apoptosis may have important implications for pharmacologic intervention in the signaling processes that regulate oxidant-mediated inflammatory diseases in the lung.