Fumonisin-Induced Tumor Necrosis Factor-α Expression in a Porcine Kidney Cell Line Is Independent of Sphingoid Base Accumulation Induced by Ceramide Synthase Inhibition

Toxicology and Applied Pharmacology 174, 69 –77 (2001) doi:10.1006/taap.2001.9189, available online at http://www.idealibrary.com on

Fumonisin-Induced Tumor Necrosis Factor-␣ Expression in a Porcine Kidney Cell Line Is Independent of Sphingoid Base Accumulation Induced by Ceramide Synthase Inhibition Quanren He,* Ronald T. Riley,† and Raghubir P. Sharma* *Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602; and †Toxicology and Mycotoxin Research Unit, USDA–ARS, Athens, Georgia 30604 Received September 26, 2000; accepted April 10, 2001

2000; Dugyala et al., 1998; Sharma et al., 1997; Voss et al., 1998). It is carcinogenic in rats (Gelderblom et al., 1991) and mice (National Toxicology Program, 1999) and has been associated with human esophageal cancer in southern Africa (Marasas, 1996) and primary liver cancer in China (Ueno et al., 1997). Studies have demonstrated that FB 1 alters proliferation and induces apoptosis and necrosis in many types of cultured cells (Mobio et al., 2000; Ciacci-Zanella et al., 1998; Riley et al., 1999; Tolleson et al., 1996; Yoo et al., 1996a) and in liver and kidney of rodents (Sharma et al., 1997; Voss et al., 1998). Fumonisin B 1 also has been shown to stimulate proliferation in cultured Swiss 3T3 fibroblasts (Tolleson et al., 1996) and in liver and esophageal epithelial cells in vivo (Lim et al., 1996; Li et al., 2000). Fumonisin B 1 is structurally similar to sphinganine and is a potent inhibitor of ceramide synthase (sphinganine and sphingosine-N-acyl transferase), a key enzyme responsible for acylation of sphinganine in the de novo synthesis of sphingolipids and the reacylation of sphingosine (Wang et al., 1991; Merrill et al., 1993). The inhibition of ceramide synthase by FB 1 results in the accumulation of intracellular free sphinganine and sphingosine and a decrease of more complex sphingolipids (Yoo et al., 1996a). Studies in pig renal epithelial cells, colon cells, and human keratinocytes have shown that FB 1-induced apoptosis, necrosis, and inhibition of proliferation are sphinganine dependent (Riley et al., 1999; Tolleson et al., 1999; Schmelz et al., 1998; Yoo et al., 1996a). There is a close correlation between increased apoptosis in liver and kidney and the disruption of sphingolipid metabolism (Riley et al., 1994a, 2001; Tsunoda et al., 1998). We have demonstrated the role of tumor necrosis factor-␣ (TNF␣) in FB 1 toxicity in Balb/c mice using anti-TNF␣ antibodies to reduce the FB 1-induced hematological effects (Dugyala et al., 1998). Increased TNF␣ release was also measured in LPS-stimulated macrophages collected from the FB 1-treated Balb/c mice (Dugyala et al., 1998). Other studies confirm that TNF␣ plays a role in FB 1 toxicity. For example, TNF␣ mRNA expression in livers was increased in FB 1-treated mice (Sharma et al., 2000a,b) and TNF␣ induction was also observed in

Fumonisin-Induced Tumor Necrosis Factor-␣ Expression in a Porcine Kidney Cell Line Is Independent of Sphingoid Base Accumulation Induced by Ceramide Synthase Inhibition. He, Q., Riley, R. T., and Sharma, R. P. (2001). Toxicol. Appl. Pharmacol. 174, 69 –77. Previous studies have shown that fumonisin B 1 (FB 1) inhibits ceramide synthase, resulting in accumulation of free sphinganine and sphingosine. Tumor necrosis factor-␣ (TNF␣) plays an important role in FB 1 toxicity and the expression of TNF␣ mRNA in liver and kidney is increased following FB 1 exposure in mice. The objective of the current study was to investigate whether these two events (sphingoid bases accumulation and TNF␣ induction) are dependent on each other. An increase in expression of TNF␣ mRNA was detected in LLC-PK 1 cells as early as 4 h after FB 1 treatment but decreased to the control levels after 8 h. A positive linear correlation was observed between the expression of TNF␣ mRNA and FB 1 concentration. Increases of intracellular sphingoid bases were also detected after 4 h of FB 1 treatment and progressively increased until 24 h. Exposure of the cells to sphinganine or sphingosine did not significantly alter the expression of TNF␣. Inhibition of sphingoid base biosynthesis by ISP-1, a specific inhibitor of serine palmitoyltransferase, the first enzyme in de novo sphingolipid biosynthesis, efficiently blocked the accumulation of free sphingoid bases in response to FB 1, but it did not prevent the induction of TNF␣ expression. Results indicate that FB 1-induced increase in TNF␣ expression is independent of sphingoid base accumulation-induced by ceramide synthase inhibition in LLC-PK 1 cells. © 2001 Academic Press Key Words: fumonisin B 1; tumor necrosis factor-␣; sphingoid base; ISP-1; serine palmitoyltransferase inhibitor.

Fumonisins are a group of structurally related mycotoxins produced by Fusarium moniliforme (F. verticillioides), and F. proliferatum, which are commonly found on corn (Riley et al., 1993). Fumonisin B 1 (FB 1) is the most abundant fumonisin in naturally contaminated foods and feeds. It causes equine leukoencephalomalacia and porcine pulmonary edema (Marasas, 1996). Heart, liver, and kidney are the targets of FB 1 toxicity in most domestic and experimental animals (Constable et al., 69

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kidneys as early as 2 h after oral FB 1 treatment (Bhandari et al., 2000). P75 TNF␣ receptor (TNFR2) knockout mice showed a marked tolerance to FB 1 hepatotoxicity (Sharma et al., 2000a). Additionally, the inhibitor of apoptosis protein (IAP), an inhibitor of TNF␣ signaling pathway, efficiently inhibited FB 1induced apoptosis in CV-1 cells (Ciacci-Zanella and Jones, 1999). A TNF␣-like activity has also been observed in swine after FB 1 injection (Guzman et al., 1997). Taken together, these studies have demonstrated that FB 1 can induce the expression of TNF␣ and disruption of sphingolipid biosynthesis. It is not known whether these two events are independent. These observations evoked our interest to explore if free sphingoid bases accumulation is responsible for the induction of TNF␣ expression in FB 1-treated LLC-PK 1 cells. We investigated the relationship between FB 1-induced expression of TNF␣ mRNA and sphingoid bases accumulation in LLC-PK 1 cells by employing ISP-1 (myriocin), a potent specific inhibitor of serine palmitoyltransferase (SPT), the first enzyme in the sphingolipid biosynthesis pathway. Serine palmitoyltransferase inhibition prevented the accumulation of free sphingoid bases in LLC-PK 1 cells treated with FB 1 (Riley and Plattner, 1999; Riley et al., 1999). The present study shows that FB 1 increased the TNF␣ mRNA expression in LLC-PK 1 cells. Treatment of the cells with sphinganine only marginally increased the expression of TNF␣ mRNA. Inhibition of sphingoid bases accumulation by ISP-1 did not prevent FB 1-induced TNF␣ mRNA expression. These observations suggest that FB 1-induced increase in TNF␣ mRNA expression is not due to the FB 1-induced increase in free sphingoid bases in LLC-PK 1 cells. A specific inhibitor of protein kinase C (PKC), calphostin C, mimicked the FB 1-induced TNF␣ expression in LLC-PK 1 cells, suggesting that PKC may be involved in the FB 1-induced expression of TNF␣. A specific inhibitor of mitogen-activated protein kinase kinase (MAPKK), PD98059, had no significant effect on TNF␣ expression. MATERIALS AND METHODS Fumonisin and reagents. Fumonisin B 1 (purity ⬎98%), sphinganine (DLerythro-dihydrosphingosine, purity 99%), sphingosine (D-sphingosine, purity 99%), C 6-ceramide (purity 99%), and bovine serum albumin (BSA, fatty acid-free) were purchased from Sigma Chemical Co. (St. Louis, MO). C 20sphinganine standard (D-erythro-C 20-dihydro-sphingosine, purity 98%, catalog no. 1845) was purchased from Matreya Inc. (Pleasant Gap, PA). ISP-1 (myriocin) was prepared according to the method described by Riley and Plattner (1999). All other reagents were tissue culture grade or the highest purity available. Preparations of fumonisin B 1, ISP-1, and sphingolipid stock solutions. Fumonisin B 1 was dissolved in physiological-buffered saline (PBS) at 1 mM, and then diluted into growth medium and added to cultures to achieve the final concentrations. ISP-1 was dissolved first in methanol (1.097 mM) and then diluted in growth medium and added to cultures to achieve the final concentrations. Control cultures were treated with similar dilutions of the vehicle but without FB 1 or ISP-1. Free sphinganine, free sphingosine, and C 6-ceramide were first dissolved, respectively, in ethanol to a concentration of 50 mM and

then were prepared in 1.0 ml of 1.5 mM fatty acid-free BSA to get a concentration of 1 mM as previously described (Lambeth et al., 1988; Yoo et al., 1996a). An equal volume of absolute ethanol was added into BSA and processed as above as a BSA vehicle control. Cell culture and treatment. Pig kidney epithelial cells (LLC-PK 1, CRL 1392, passage 197) were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in 75-cm 2 culture flasks containing DMEM/Ham’s F12 (1:1) with 5% fetal bovine serum (FBS) at 37°C and 5% CO 2. For all experiments, the cells were subcultured at approximately 3000 viable cells/cm 2 in six-well plates (10 cm 2/well). Cells were allowed to attach and grow for 3 days prior to treatment with fumonisin B 1 or sphingolipids. Seeding densities were chosen according to Yoo et al. (1996a). To prevent the burst of free sphingoid bases after addition of fresh medium (Smith et al., 1997), 0.6 ml medium from each well was taken out, and 0.1 ml of FB 1 solution was added to each well containing 1.9 ml conditioned medium. In ISP-1-treated groups, 10 ␮l of ISP-1 at proper concentrations was added 1.5 h before treatment with FB 1 or sphingolipid to ensure inhibition of SPT, and thus inhibition of intracellular free sphinganine synthesis. The role of PKC was examined with 50 nM calphostin C, a specific inhibitor of PKC (Kontny et al., 2000), in the presence of light. The effect of MAP kinases was evaluated by pretreatment of cells with 50 ␮M PD98059, a selective inhibitor of MAPKK, which is upstream of MAP kinase. All experiments were repeated at least twice with similar observations. Semiquantitative analysis of TNF␣ mRNA by reverse transcriptase polymerase chain reaction (RT–PCR). Total RNA was isolated from LLC-PK 1 cells using TRI reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s protocol. Total RNA (2.5 ␮g) was transcribed to cDNA using oligo(dT) 12–18 and superscript II (Life Technologies, Grand Island, NY) at 42°C for 50 min. An aliquot of 1 ␮l (for GAPDH) or 2 ␮l (for TNF␣) was amplified by PCR using Taq DNA polymerase and 0.2 ␮M of each primer in 1⫻ PCR buffer containing 2 mM MgCl 2. PCR reactions were performed in a thermal cycler (Coy Inc., Ann Arbor, MI). The thermoamplification program consisted of an initial denaturation (5 min at 95°C), followed by 32 cycles (for TNF␣) or 34 cycles (for GAPDH) of 30 s denaturation (94°C), 30 s annealing (50°C), and 1 min elongation (72°C), with a final extension period of 1 min at 72°C. In preliminary experiments, an exponential increase in DNA was observed up to 40 cycles for both products. The sense and antisense primers were 5⬘-AAT GGC AGA GTG GGT ATG-3⬘ and 5⬘-CTT GAT GGC AGA GAG GAG-3⬘ for TNF␣, and 5⬘-TCC CTG CTT CTA CTG GTG CT-3⬘, and 5⬘-TGA GCT TGA CAA AGT GGT CG-3⬘ for GAPDH, respectively (chosen by Primer3 program; Whithead Institute, Cambridge, MA). PCR products were separated on 2% agarose gel containing 0.476 ␮M ethidium bromide and detected by a UV transilluminator (Ultra Lum Inc., Carson, CA) and photographed. The photographs were scanned and bands were quantified by using UN-SCAN-IT software (Silk Scientific Inc., Orem, UT). Density of GAPDH in the same sample was used to normalize TNF␣. In order to validate the expression of TNF␣ by RT–PCR, we attempted to compare it by measuring the TNF␣ mRNA by Northern blot. However, no message could be detected in 40 ␮g of total RNA from LLC-PK 1 cells in a Northern blot. To further determine if the TNF␣ expression measured by RT–PCR provided an accurate reflection of its increase, we compared the expression of TNF␣ message in normal and lipopolysaccharide (LPS)-treated murine macrophage (J774.A1) cells. The Northern blot (conducted as described earlier by Dugyala et al. (1998) indicated more than 700-fold increase in the TNF␣ expression by LPS treatment. On the other hand, only 200-fold increase was noted when the TNF␣ was analyzed by RT–PCR (for specific details on the method see Mathew and Sharma, 2000). These observations suggest that the RT–PCR analyses could underestimate the TNF␣ induction in LLC-PK 1 cells. Determination of intracellular free sphingoid bases. At the end of treatment, the medium was aspirated, and cells were washed once with ice-cold PBS and then were scraped into 1 ml ice-cold PBS. An aliquot (0.1 ml) of cell suspension in PBS was transferred to another tube, spun at 2000g, 4°C for 5 min. The cell pellet was solubilized using lysis buffer (1% IGEPAL CA-630;

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20 mM Tris–HCl, pH 8.0; 150 mM NaCl; 1.5 ␮g/ml aprotinin; 1 ␮g/ml leupeptin; 0.5 mM phenlmethylsulfonyl fluoride; and 0.5 mM dithiothreitol) and stored at ⫺85°C until analysis of total protein by Bradford reagent (Bio-Rad Laboratories, Hercules, CA) using a 96-well plate according to the manufacturer’s protocol. Free sphingoid bases were extracted from the remainder of cells by using the modified method of Yoo et al. (1996b). The relative amounts of free sphinganine and sphingosine in base-treated cell extracts were determined by high-performance liquid chromatography (HPLC) utilizing a modification (Yoo et al., 1996b) of the method originally described by Merrill et al. (1988). Sphingoid bases were quantitated based on the recovery of a C 20 sphinganine internal standard. A complete description of the HPLC apparatus and derivatization procedures has been provided in Riley et al. (1994b); except the fluorescence detector used in this study was Luminescence Spectrometer LS30 (Perkin–Elmer Inc., Norwalk, CT). The instrument limit of detection for C 20 was 26.8 fmol/assay (equivalent to 1 fmol/␮g protein). Cytotoxicity assay for ISP-1. The cytotoxicity of ISP-1 in LLC-PK 1 cells was determined by 3-(4,5-dimethyl-thiazol-yl-2)-2,5-diphenyl tetrazolium bromide assay (Dugyala et al., 1998) and lactate dehydrogenase release (Yoo et al., 1996a). No evidence of increased cytotoxicity was detected in LLC-PK 1 cells by up to 1000 nM of ISP-1 treatment for 5.5 h (data not presented). Statistical analysis of data. The results were expressed as means ⫾ SE. Differences among treatments were analyzed statistically by one-way analysis of variance followed by Fisher’s protected least significance difference test for post hoc multiple comparison unless stated otherwise. Probabilities ⱕ0.05 were considered to be statistically significant.

RESULTS

Time course of change in TNF␣ mRNA expression. The TNF␣ mRNA expression was significantly increased at 4 h after 10 ␮M FB 1 treatment (Fig. 1). No change was noticed at other time points up to 24 h. The change in control groups over time was not significant. TNF␣ mRNA expression in relation to FB 1 concentration. The effect of FB 1 on the expression of TNF␣ mRNA was concentration dependent (Fig. 2). A positive linear correlation between the expression of TNF␣ mRNA in LLC-PK 1 cells and the concentrations of FB 1 exposure was observed (r 2 ⫽ 0.975, df ⫽ 3, p ⬍ 0.01). Fumonisin B 1-induced accumulation of intracellular free sphingoid bases. To correlate the effects of FB 1 on TNF␣ expression and sphingolipid metabolism, we investigated the accumulation of intracellular free sphingoid bases over a short period of FB 1 treatment. Figure 3 shows the accumulation of free sphinganine and sphingosine in FB 1 (10 ␮M)-treated LLC-PK 1 cells as a function of time. The results indicated that free sphingosine and sphinganine significantly increased at 2 and 4 h, respectively; the increases were time dependent until 24 h of FB 1 treatment. Intracellular sphingoid bases also showed a concentration-dependent increase in response to FB 1 at 4 h (Fig. 4). ISP-1 inhibited de novo biosynthesis of free sphinganine. ISP-1 is a potent and selective inhibitor of SPT. We used this inhibitor to block the biosynthesis of free sphinganine (Fig. 5) and investigated if the induction of TNF␣ expression in FB 1exposed cells could be prevented. The levels of free sphinganine and sphingosine in cells treated with only FB 1 (10 ␮M)

FIG. 1. Time course for the expression of TNF␣ mRNA in LLC-PK 1 cells after FB 1 exposure. The cells were incubated with or without 10 ␮M FB 1 for up to 24 h. The total RNA from the cells was transcribed into cDNA and analyzed by RT–PCR as described under the Materials and Methods. *Significantly different ( p ⬍ 0.05) from the concurrent control (student’s t test). Data are the mean ⫾ SE (n ⫽ 3) from a representative experiment.

for 4 h were 12–26 and 30 – 40 fmol/␮g protein, respectively. After pretreatment with 150 nM ISP-1, these values were reduced 10- and 2-fold, respectively, compared to FB 1-treated cultures (Fig. 5). Free sphinganine was not detected in cultures treated with greater than 300 nM ISP-1, and sphingosine was undetectable in 1000 nM ISP-1-pretreated cells. These results showed that ISP-1 efficiently inhibited FB 1-induced accumulation of intracellular free sphingoid bases. ISP-1 could not prevent FB 1-induced TNF␣ mRNA expression. It has been shown that elevated sphingoid bases are responsible for some or all of the increased apoptosis and altered proliferation induced by FB 1 (Riley et al., 1999; Tolleson et al., 1999; Schmelz et al., 1998; Yoo et al., 1996a). To determine whether the increases of intracellular free sphingoid bases account for TNF␣ induction, LLC-PK 1 cells were pretreated with ISP-1 for 1.5 h to inhibit the accumulation of free sphingoid bases before being treated with FB 1. Treatment of the cells with 10 ␮M FB 1 for 4 h increased TNF␣ gene expression significantly (Fig. 6A). No change was observed in 150 nM ISP-1-treated cells for 5.5 h compared to the control. Cotreatment with 150 to 1000 nM ISP-1 plus FB 1 did not reduce the expression of TNF␣ mRNA compared to the treatment with FB 1 only (Fig. 6). Effects of sphinganine, sphingosine, and C 6-ceramide on TNF␣ mRNA expression in cells. To further determine if sphinganine or sphingosine is responsible for the induction of

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centration–response study showed that sphinganine did not significantly increase the expression of TNF␣ in LLC-PK 1 cells, although 1 ␮M sphinganine marginally increased TNF␣ mRNA (0.05 ⬍ p ⬍ 0.1; Fig. 8) and the level of TNF␣ expression at 1 ␮M sphinganine was not significantly different from the FB 1 only group. Effect of PKC or MAP kinase inhibition on FB 1-induced TNF␣ expression. Addition of the PKC inhibitor calphostin C to LLC-PK 1 cells induced the expression of TNF␣ to an extent similar to that increased by FB 1 (Fig. 9). No further increase was observed when the cells were treated both with FB 1 and calphostin C. The treatment of LLC-PK 1 cells with an inhibitor of MAPK kinase had no significant effect on TNF␣ expression, either alone or in combination with FB 1 (data not shown). DISCUSSION FIG. 2. Concentration-dependent effects of FB 1 on the expression of TNF␣ mRNA in LLC-PK 1 cells. The cells were treated with FB 1 at different concentrations for 4 h. The cellular total RNA was analyzed by RT–PCR. Data are the mean ⫾ SE (n ⫽ 4). *Significantly different ( p ⬍ 0.05) from the concurrent control.

TNF␣ expression, we treated the cells with sphinganine or sphingosine, respectively, and the TNF␣ mRNA was analyzed. The results showed that neither treatment could increase TNF␣ expression (Fig. 7). Sphinganine and sphingosine can be metabolized to ceramide, so we also determined the effect of C 6-ceramide on TNF␣ expression. The result showed that C 6-ceramide did not alter the expression of TNF␣ mRNA. Cotreatment of the cells with FB 1 plus sphinganine (1 ␮M) or FB 1 plus sphingosine (1 ␮M), or FB 1 plus ISP-1 and sphinganine, or sphingosine did not reduce the expression of TNF␣ compared to cultures treated with FB 1 only (Fig. 7). A con-

This study demonstrated that FB 1 induced TNF␣ mRNA expression in LLC-PK 1 cells. Inhibition of de novo sphinganine biosynthesis by ISP-1 did not prevent the induction of TNF␣ mRNA in response to FB 1 exposure. It appears that FB 1-induced expression of TNF␣ mRNA in LLC-PK 1 cells is independent of the FB 1-induced intracellular free sphingoid bases accumulation or SPT inhibition. The induced expression of TNF␣ mRNA was observed at 4 h in response to FB 1 treatment, but, thereafter, it reduced to the control levels. In our preliminary study, we found that FB 1 (10 ␮M) increased TNF␣ mRNA between 4 and 6 h (data not shown). These observations indicate that the expression of TNF␣ mRNA in response to FB 1 in LLC-PK 1 cells is an early and transient event, whereas the FB 1 inhibition of ceramide synthase is persistent for up to 72 h after removal of FB 1 (Riley et al., 1998). The increases of both TNF␣ mRNA expression and free sphingoid bases in FB 1-treated LLC-PK 1 cells began almost at

FIG. 3. Time course of accumulation of free sphingosine (A) and sphinganine (B) in FB 1-treated LLC-PK 1 cells. Data are the mean ⫾ SE (n ⫽ 3). *Significantly different ( p ⬍ 0.05) from the concurrent control.

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FIG. 4. Concentrations of free sphingosine (A) and sphinganine (B) after exposure of LLC-PK 1 cells to different FB 1 concentrations for 4 h. Data are the mean ⫾ SE (n ⫽ 3). *Significantly different ( p ⬍ 0.05) from the concurrent control.

the same time, and both were concentration dependent. Exogenously added sphinganine produced a nonsignificant increase in TNF␣ mRNA expression at 1 ␮M ( p ⬎ 0.05). However, inhibition of free sphingoid bases accumulation by ISP-1 did not block the induction of TNF␣ mRNA by FB 1, which indicated that sphinganine generated de novo was not the cause of the increased TNF␣ expression. Sphingosine had no effect on the basal as well as the FB 1-induced expression of TNF␣ in

LLC-PK 1 cells. It has been reported that sphingosine inhibited the expression of TNF␣ mRNA and TNF␣ release in rabbit alveolar macrophages in response to LPS (Lo et al., 1999). Thus, it is unlikely that accumulation of sphingosine can induce the expression of TNF␣ mRNA in FB 1-treated cells. ISP-1 is a potent specific inhibitor of SPT, which is a key enzyme responsible for the de novo biosynthesis of sphinganine (Riley and Plattner, 1999). In our current study, ISP-1

FIG. 5. ISP-1 inhibition of FB 1-induced sphingoid bases accumulation in LLC-PK 1 cells. The cells were pretreated with the indicated concentration of ISP-1 or 2 ␮l methanol (the solvent for ISP-1 stock) for 1.5 h and then were exposed to FB 1 for 4 h in the presence of ISP-1. Each bar represents the mean ⫾ SE (n ⫽ 3) for sphingosine (A and C) and sphinganine (B and D). ND, the sphingoid bases in these samples were not detectable. *Significantly different ( p ⬍ 0.05) from the concurrent control. ¶Significantly different ( p ⬍ 0.01) from the FB 1-treated group.

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FIG. 6. Effects of ISP-1 on FB 1-induced overexpression of TNF␣ mRNA in LLC-PK 1 cells. The cells were pretreated with the indicated concentration of ISP-1 or 2 ␮l methanol (the dissolvent for ISP-1 stock) for 1.5 h and then were exposed to FB 1 for 4 h in the presence of ISP-1. Each bar represents the mean ⫾ SE (n ⫽ 3). *Significantly different ( p ⬍ 0.05) from the concurrent control.

treatment resulted in decreases of sphingoid bases to levels as low as 2- to 10 times less than control cultures. It also efficiently prevented the accumulation of free sphingoid bases as a result of FB 1 treatment. The decrease in sphinganine was not due to its cytotoxic effect in LLC-PK 1 cells because neither the cell viability nor LDH release was altered by the addition of up to 1 ␮M ISP-1 for 5.5 h. ISP-1 did not change the basal

expression of TNF␣ nor did it decrease the induced expression of TNF␣ following FB 1 exposure in the cells. Even when the accumulation of free sphingoid bases was completely inhibited by ISP-1, the expression of TNF␣ was still induced following FB 1 treatment. Irrespective of sphinganine’s effects on TNF␣ mRNA expression in LLC-PK 1 cells, the fact that inhibition of free sphingoid bases biosynthesis does not prevent the induc-

FIG. 7. Effects of sphinganine (Sa), sphingosine (So), and C 6-ceramide (Cer) on TNF␣ mRNA expression in LLC-PK 1 cells. Cells were treated with medium as control (C), bovine serum albumin (BSA), FB 1 (10 ␮M), Sa (1 ␮M), Sa (2 ␮M), Sa (1 ␮M) ⫹ FB 1 (10 ␮M), Sa (1 ␮M) ⫹ FB 1 (10 ␮M) ⫹ 150 nM ISP-1, So (1 ␮M), So (1 ␮M) ⫹ FB 1 (10 ␮M), So (1 ␮M) ⫹ FB 1 (10 ␮M) ⫹ 150 nM ISP-1, or C 6-ceramide (1 ␮M) for 4 h. ISP-1 was added 1.5 h before FB 1 plus Sa or So treatment. Data are the mean ⫾ SE (n ⫽ 4). *Significantly different ( p ⬍ 0.05) from the control.

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tive effect of FB 1 on TNF␣ mRNA strongly supports the conclusion that the accumulation of free sphingoid bases is not required for the induction of TNF␣ in FB 1-exposed cells. Previous studies have shown that accumulation of intracellular free sphingoid bases is responsible for the cytotoxic effects of FB 1 (Riley et al., 1999; Tolleson et al., 1999; Yoo et al., 1996a; Schmelz et al., 1998; Tsunoda et al., 1998). Sphingoid bases can induce cellular apoptosis and regulate cell growth and differentiation (Merrill et al., 1997). Elevated endogenous sphinganine has been shown to affect the activity of PKC (Smith et al., 1997). Thus, disruption of sphingolipid metabolism exposure to FB 1 can account for some of its toxic effects. However, it has been suggested that FB 1-induced activation of mitogen-activated protein kinase (MAP kinase) (Wattenberg et al., 1996; Pinelli et al., 1999) and translocation of PKC from cytosol to membrane, which in turn catalytically activates PKC (Yeung et al., 1996) following FB 1 exposure, was not dependent on the accumulation of intracellular sphingoid bases. The mechanism for the overexpression of TNF␣ mRNA By FB 1 is unknown. However, it may be related to the disruption of MAP kinase and/or PKC activities in response to FB 1. Previous work has shown that FB 1 induces PKC translocation from cytosol to membrane in rat cerebrocortical slices via direct interaction with diacylglycerol binding site (Yeung et al., 1996). This translocation can induce catalytic activation of PKC, which is required for the production of TNF␣ in response to LPS or phorbol ester treatment in human peripheral monocytes (Kontny et al., 2000). However, it has been reported that FB 1 can directly inhibit PKC (Huang et al., 1995) and the PKC inhibition is mediated via accumulation of intracellular free sphingoid bases (Merrill et al., 1997). However, inhibition of PKC by calphostin C also increased the TNF␣ expression in the present study, suggesting that PKC inhibition may be the

FIG. 8. Concentration effects of sphinganine on the expression of TNF␣ mRNA in LLC-PK 1 cells. Cells were exposed to 10 ␮M FB 1 or 0.5 ␮M, 1 ␮M, or 2 ␮M sphinganine for 4 h. Each bar represents the mean ⫾ SE (n ⫽ 3). *Significantly different ( p ⬍ 0.05) from the control.

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FIG. 9. Effects of calphostin C on FB 1-induced TNF␣ expression in LLC-PK 1 cells. The cells were pretreated with 50 nM calphostin C (Cal) for 2 h, and the incubation was continued with or without FB 1 (10 ␮M) for 4 h in the presence of the inhibitor. Data are the mean ⫾ SE (n ⫽ 3). *Significantly different ( p ⬍ 0.05) from control.

cause of TNF␣ induction following FB 1 treatment. It is noteworthy that TNF␣ itself also inhibits PKC via de novo ceramide production in L929 cells; the effect was counteracted by the addition of FB 1 (Lee et al., 2000). Other important pathways, which involve the regulation of TNF␣ production, are the MAP kinase pathways. Previous studies have shown that LPS-induced TNF␣ mRNA as well as TNF␣ production are dependent on activation of the MAP kinase pathways (Means et al., 2000). Mitogen-activated protein kinase activity is stimulated by FB 1 in Swiss 3T3 cells and human bronchial epithelial cells as early as several minutes following FB 1 exposure (Wattenberg et al., 1996; Pinelli et al., 1999) and decreased after 4 h of treatment (Pinelli et al., 1999). Pinelli et al. (1999) further demonstrated that the stimulation of MAP kinase occurred before the accumulation of free sphingoid base was detected. However, in LLC-PK 1 cells, a role for MAP kinase involvement in FB 1-induced TNF␣ expression was not apparent. In conclusion, FB 1 can induce TNF␣ expression in LLCPK 1 cells, and this increase is sphingoid base independent. The expression of TNF␣ is complex and involves a series of regulatory sequences, such as binding of NF-␬B to a TNF␣ promoter. To fully understand the molecular bases for the overexpression of TNF␣ by FB 1, the roles of PKC and MAP kinase activation following FB 1 exposure in TNF␣ expression remain to be investigated. Studies on the regulatory elements function of TNF␣ expression following FB 1 exposure may help to further elucidate FB 1 toxic mechanisms. The significance of TNF␣ production in response to FB 1 is the subject of ongoing studies in our laboratory. ACKNOWLEDGMENTS The authors gratefully acknowledge the analytical help of Ms. Jency Showker at the Toxicology and Mycotoxin Research Unit, USDA, Athens,

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GA. This work was supported by Grant ES09403 from the National Institute of Environmental Health Sciences, NIH.

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