Volume 116, Issue 6, Pages 1330-1341 (June 1999) Helicobacter pylori–dependent ceramide production may mediate increased interleukin 8 expression in human gastric cancer cell lines  Atsushi Masamune, Tooru Shimosegawa, Osamu Masamune, Naofumi Mukaida, Masaru Koizumi, Takayoshi Toyota  Gastroenterology  Volume 116, Issue 6, Pages 1330-1341 (June 1999) DOI: 10.1016/S0016-5085(99)70497-X Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Effects of sphingolipids and PLs on IL-8 production in Kato III cells. (A) Kato III cells were treated with sphingolipids at 10 μmol/L or PLs at 200 mU/mL. IL-8 levels in conditioned media harvested after 24 hours were determined by enzyme immunoassay. (B) Kato III cells were treated with increasing concentrations of C2-ceramide in serum-free medium. After 24 hours, IL-8 levels in conditioned media were determined. (C) Kato III cells were treated with C2-ceramide (10 μmol/L) or H. pylori (1 × 108 CFU/mL). At the indicated time points, conditioned media were harvested and IL-8 levels were determined. (D) Kato III cells were pretreated with C2-ceramide (10 μmol/L) for 30 minutes, then H. pylori was added to the final concentration of 1 × 108 CFU/mL and incubation was continued. IL-8 levels in conditioned media harvested after 9 or 24 hours were determined. Data are expressed as means ± SD (n = 6 for each data point; *P < 0.05, **P < 0.01 vs. control). Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Effects of sphingolipids and PLs on IL-8 production in Kato III cells. (A) Kato III cells were treated with sphingolipids at 10 μmol/L or PLs at 200 mU/mL. IL-8 levels in conditioned media harvested after 24 hours were determined by enzyme immunoassay. (B) Kato III cells were treated with increasing concentrations of C2-ceramide in serum-free medium. After 24 hours, IL-8 levels in conditioned media were determined. (C) Kato III cells were treated with C2-ceramide (10 μmol/L) or H. pylori (1 × 108 CFU/mL). At the indicated time points, conditioned media were harvested and IL-8 levels were determined. (D) Kato III cells were pretreated with C2-ceramide (10 μmol/L) for 30 minutes, then H. pylori was added to the final concentration of 1 × 108 CFU/mL and incubation was continued. IL-8 levels in conditioned media harvested after 9 or 24 hours were determined. Data are expressed as means ± SD (n = 6 for each data point; *P < 0.05, **P < 0.01 vs. control). Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Effects of sphingolipids and PLs on IL-8 production in Kato III cells. (A) Kato III cells were treated with sphingolipids at 10 μmol/L or PLs at 200 mU/mL. IL-8 levels in conditioned media harvested after 24 hours were determined by enzyme immunoassay. (B) Kato III cells were treated with increasing concentrations of C2-ceramide in serum-free medium. After 24 hours, IL-8 levels in conditioned media were determined. (C) Kato III cells were treated with C2-ceramide (10 μmol/L) or H. pylori (1 × 108 CFU/mL). At the indicated time points, conditioned media were harvested and IL-8 levels were determined. (D) Kato III cells were pretreated with C2-ceramide (10 μmol/L) for 30 minutes, then H. pylori was added to the final concentration of 1 × 108 CFU/mL and incubation was continued. IL-8 levels in conditioned media harvested after 9 or 24 hours were determined. Data are expressed as means ± SD (n = 6 for each data point; *P < 0.05, **P < 0.01 vs. control). Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Effects of sphingolipids and PLs on IL-8 production in Kato III cells. (A) Kato III cells were treated with sphingolipids at 10 μmol/L or PLs at 200 mU/mL. IL-8 levels in conditioned media harvested after 24 hours were determined by enzyme immunoassay. (B) Kato III cells were treated with increasing concentrations of C2-ceramide in serum-free medium. After 24 hours, IL-8 levels in conditioned media were determined. (C) Kato III cells were treated with C2-ceramide (10 μmol/L) or H. pylori (1 × 108 CFU/mL). At the indicated time points, conditioned media were harvested and IL-8 levels were determined. (D) Kato III cells were pretreated with C2-ceramide (10 μmol/L) for 30 minutes, then H. pylori was added to the final concentration of 1 × 108 CFU/mL and incubation was continued. IL-8 levels in conditioned media harvested after 9 or 24 hours were determined. Data are expressed as means ± SD (n = 6 for each data point; *P < 0.05, **P < 0.01 vs. control). Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Effects of bacterial SMase on IL-8 production in Kato III cells. Kato III cells were treated with increasing concentrations of S. aureus SMase for 24 hours, and IL-8 levels in conditioned media were determined by enzyme immunoassay. Data shown are expressed as means ± SD (n = 6; *P < 0.05, **P < 0.01 vs. control). Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Effects of C2-ceramide and SMase on IL-8 production in AGS cells. AGS cell monolayers were treated with various concentrations of C2-ceramide, bacterial SMase, or H. pylori (1 × 108 CFU/mL), and IL-8 levels were determined. Data represent means ± SD (n = 6 for each data point). Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Induction of IL-8 gene transcription assessed by Northern blot analysis. (A) Kato III cells were treated with C2-ceramide at 10 μmol/L. RNAs were purified at the indicated time points, and levels of IL-8 and β-actin mRNAs were determined by Northern blot analysis. (B) Kato III cells were treated with sphingolipids at 10 μmol/L. After 4 hours of incubation, RNAs were isolated, and levels of IL-8 mRNAs were determined. (C) Kato III cells were treated with PLs at 100 mU/mL. After 4 hours of incubation, RNAs were isolated, and levels of IL-8 mRNAs were determined. Results shown are representative of 3 separate experiments. Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Induction of IL-8 gene transcription assessed by Northern blot analysis. (A) Kato III cells were treated with C2-ceramide at 10 μmol/L. RNAs were purified at the indicated time points, and levels of IL-8 and β-actin mRNAs were determined by Northern blot analysis. (B) Kato III cells were treated with sphingolipids at 10 μmol/L. After 4 hours of incubation, RNAs were isolated, and levels of IL-8 mRNAs were determined. (C) Kato III cells were treated with PLs at 100 mU/mL. After 4 hours of incubation, RNAs were isolated, and levels of IL-8 mRNAs were determined. Results shown are representative of 3 separate experiments. Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Induction of IL-8 gene transcription assessed by Northern blot analysis. (A) Kato III cells were treated with C2-ceramide at 10 μmol/L. RNAs were purified at the indicated time points, and levels of IL-8 and β-actin mRNAs were determined by Northern blot analysis. (B) Kato III cells were treated with sphingolipids at 10 μmol/L. After 4 hours of incubation, RNAs were isolated, and levels of IL-8 mRNAs were determined. (C) Kato III cells were treated with PLs at 100 mU/mL. After 4 hours of incubation, RNAs were isolated, and levels of IL-8 mRNAs were determined. Results shown are representative of 3 separate experiments. Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 C2-ceramide–induced IL-8 gene transcription through activation of NF-κB and AP-1. Kato III cells were transfected with various luciferase (luc) expression vectors and were stimulated with either C2-ceramide (■; 10 μmol/L) or H. pylori (▨; 5 × 107 CFU/mL). After 24 hours, intracellular luciferase activities were determined. Results shown are representative of 4 separate experiments. Data represent means ± SD (n = 3 for each data point). NS, not significant; mt, mutant. Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 NF-κB–binding activity was increased by C2-ceramide and SMase. (A) Electrophoretic mobility shift assay of nuclear extracts from Kato III cells treated for 1 hour with sphingolipids at 10 μmol/L or H. pylori (1 × 108 CFU/mL). Nuclear extracts were prepared from untreated cells (lane 1), C2-ceramide–treated cells (lane 2), C2-dihydroceramide–treated cells (lane 4), Sph-treated cells (lane 5), Sph-1-P–treated cells (lane 6), and H. pylori–treated cells (lane 7). Extracts were incubated with radiolabeled oligonucleotide probes for NF-κB and electrophoretically separated through 4% polyacrylamide gel. Nuclear extracts from C2-ceramide–treated cells were incubated with 100-fold excess of unlabeled oligonucleotide before the addition of radiolabeled probe in competition assay (lane 3). Arrows denote specific inducible complexes competitive with cold double-stranded NF-κB oligonucleotide. Two NF-κB–specific DNA-protein complex formations were observed with nuclear proteins extracted from Kato III cells treated with C2-ceramide or H. pylori. Sph also increased NF-κB–specific binding activity, but the activity was weaker than that induced by C2-ceramide. Neither C2-dihydroceramide nor Sph-1-P increased the binding activity. *Nonspecific bands. (B) Electrophoretic mobility shift assay of nuclear extracts from AGS cells treated for 1 hour with PLs at 200 mU/mL or H. pylori (1 × 108 CFU/mL). Nuclear extracts were prepared from untreated cells (lane 1), cells treated with secretory PLA2 from naja naja venom (lane 2), PC-PLC–treated cells (lane 3), SMase-treated cells (lane 4), and H. pylori–treated cells (lane 6). Extracts were incubated with radiolabeled oligonucleotide probes for NF-κB and electrophoretically separated through 4% polyacrylamide gel. Nuclear extracts from SMase-treated cells were incubated with 100-fold excess of unlabeled oligonucleotide before the addition of radiolabeled probe in competition assay (lane 5). Arrows denote specific inducible complexes competitive with cold double-stranded NF-κB oligonucleotide. SMase, but neither PLA2 nor PLC, increased NF-κB–specific binding activity. *Nonspecific bands. (C) For super shift assays, nuclear extracts from C2-ceramide– or H. pylori–treated Kato III cells were incubated for 1 hour at 4°C with rabbit polyclonal antibodies against p50, p65, or c-Rel subunits of NF-κB complexes or without antibody (lane “no Ab”) before incubation with the radiolabeled probe. Arrows denote further gel retardations (super shifts). Specific antibodies to p50 and p65 retarded both of the NF-κB complexes and the upper one, respectively. In contrast, anti-c-Rel antibody failed to affect complex formation. Results shown are representative of 3 separate experiments. Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 NF-κB–binding activity was increased by C2-ceramide and SMase. (A) Electrophoretic mobility shift assay of nuclear extracts from Kato III cells treated for 1 hour with sphingolipids at 10 μmol/L or H. pylori (1 × 108 CFU/mL). Nuclear extracts were prepared from untreated cells (lane 1), C2-ceramide–treated cells (lane 2), C2-dihydroceramide–treated cells (lane 4), Sph-treated cells (lane 5), Sph-1-P–treated cells (lane 6), and H. pylori–treated cells (lane 7). Extracts were incubated with radiolabeled oligonucleotide probes for NF-κB and electrophoretically separated through 4% polyacrylamide gel. Nuclear extracts from C2-ceramide–treated cells were incubated with 100-fold excess of unlabeled oligonucleotide before the addition of radiolabeled probe in competition assay (lane 3). Arrows denote specific inducible complexes competitive with cold double-stranded NF-κB oligonucleotide. Two NF-κB–specific DNA-protein complex formations were observed with nuclear proteins extracted from Kato III cells treated with C2-ceramide or H. pylori. Sph also increased NF-κB–specific binding activity, but the activity was weaker than that induced by C2-ceramide. Neither C2-dihydroceramide nor Sph-1-P increased the binding activity. *Nonspecific bands. (B) Electrophoretic mobility shift assay of nuclear extracts from AGS cells treated for 1 hour with PLs at 200 mU/mL or H. pylori (1 × 108 CFU/mL). Nuclear extracts were prepared from untreated cells (lane 1), cells treated with secretory PLA2 from naja naja venom (lane 2), PC-PLC–treated cells (lane 3), SMase-treated cells (lane 4), and H. pylori–treated cells (lane 6). Extracts were incubated with radiolabeled oligonucleotide probes for NF-κB and electrophoretically separated through 4% polyacrylamide gel. Nuclear extracts from SMase-treated cells were incubated with 100-fold excess of unlabeled oligonucleotide before the addition of radiolabeled probe in competition assay (lane 5). Arrows denote specific inducible complexes competitive with cold double-stranded NF-κB oligonucleotide. SMase, but neither PLA2 nor PLC, increased NF-κB–specific binding activity. *Nonspecific bands. (C) For super shift assays, nuclear extracts from C2-ceramide– or H. pylori–treated Kato III cells were incubated for 1 hour at 4°C with rabbit polyclonal antibodies against p50, p65, or c-Rel subunits of NF-κB complexes or without antibody (lane “no Ab”) before incubation with the radiolabeled probe. Arrows denote further gel retardations (super shifts). Specific antibodies to p50 and p65 retarded both of the NF-κB complexes and the upper one, respectively. In contrast, anti-c-Rel antibody failed to affect complex formation. Results shown are representative of 3 separate experiments. Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 NF-κB–binding activity was increased by C2-ceramide and SMase. (A) Electrophoretic mobility shift assay of nuclear extracts from Kato III cells treated for 1 hour with sphingolipids at 10 μmol/L or H. pylori (1 × 108 CFU/mL). Nuclear extracts were prepared from untreated cells (lane 1), C2-ceramide–treated cells (lane 2), C2-dihydroceramide–treated cells (lane 4), Sph-treated cells (lane 5), Sph-1-P–treated cells (lane 6), and H. pylori–treated cells (lane 7). Extracts were incubated with radiolabeled oligonucleotide probes for NF-κB and electrophoretically separated through 4% polyacrylamide gel. Nuclear extracts from C2-ceramide–treated cells were incubated with 100-fold excess of unlabeled oligonucleotide before the addition of radiolabeled probe in competition assay (lane 3). Arrows denote specific inducible complexes competitive with cold double-stranded NF-κB oligonucleotide. Two NF-κB–specific DNA-protein complex formations were observed with nuclear proteins extracted from Kato III cells treated with C2-ceramide or H. pylori. Sph also increased NF-κB–specific binding activity, but the activity was weaker than that induced by C2-ceramide. Neither C2-dihydroceramide nor Sph-1-P increased the binding activity. *Nonspecific bands. (B) Electrophoretic mobility shift assay of nuclear extracts from AGS cells treated for 1 hour with PLs at 200 mU/mL or H. pylori (1 × 108 CFU/mL). Nuclear extracts were prepared from untreated cells (lane 1), cells treated with secretory PLA2 from naja naja venom (lane 2), PC-PLC–treated cells (lane 3), SMase-treated cells (lane 4), and H. pylori–treated cells (lane 6). Extracts were incubated with radiolabeled oligonucleotide probes for NF-κB and electrophoretically separated through 4% polyacrylamide gel. Nuclear extracts from SMase-treated cells were incubated with 100-fold excess of unlabeled oligonucleotide before the addition of radiolabeled probe in competition assay (lane 5). Arrows denote specific inducible complexes competitive with cold double-stranded NF-κB oligonucleotide. SMase, but neither PLA2 nor PLC, increased NF-κB–specific binding activity. *Nonspecific bands. (C) For super shift assays, nuclear extracts from C2-ceramide– or H. pylori–treated Kato III cells were incubated for 1 hour at 4°C with rabbit polyclonal antibodies against p50, p65, or c-Rel subunits of NF-κB complexes or without antibody (lane “no Ab”) before incubation with the radiolabeled probe. Arrows denote further gel retardations (super shifts). Specific antibodies to p50 and p65 retarded both of the NF-κB complexes and the upper one, respectively. In contrast, anti-c-Rel antibody failed to affect complex formation. Results shown are representative of 3 separate experiments. Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 AP-1–binding activity was increased by C2-ceramide and H. pylori. Electrophoretic mobility shift assay of nuclear extracts from Kato III cells treated for 1 hour with C2-ceramide (10 μmol/L) or H. pylori (1 × 108 CFU/mL). Nuclear extracts were prepared from untreated cells (lane 1), C2-ceramide–treated cells (lane 2), C2-dihydroceramide–treated cells (lane 3), and H. pylori–treated cells (lane 4). Extracts were incubated with radiolabeled oligonucleotide probes for (A) AP-1 and (B) NF-IL6 and electrophoretically separated. Arrows denote specific complexes competitive with cold double-stranded oligonucleotide. C2-ceramide and H. pylori increased AP-1–specific binding activity by 2.1- and 2.3-fold, respectively. In contrast, NF-IL6–binding activity was not altered by the treatment. Results shown are representative of 3 separate experiments. Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 8 H. pylori infection increased intracellular ceramide levels. (A) Kato III and (B) AGS cells were treated with H. pylori (1 × 108 CFU/mL), and intracellular ceramide levels at indicated time points were determined by diacylglycerol kinase assay. Results represent means ± SD from 3 separate experiments. Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 8 H. pylori infection increased intracellular ceramide levels. (A) Kato III and (B) AGS cells were treated with H. pylori (1 × 108 CFU/mL), and intracellular ceramide levels at indicated time points were determined by diacylglycerol kinase assay. Results represent means ± SD from 3 separate experiments. Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 9 Direct contact with live H. pylori was required for ceramide generation. Kato III cells were incubated for 1 hour with live H. pylori (1 × 108 CFU/mL), H. pylori culture supernatants, heat-killed H. pylori, water-soluble extracts of H. pylori, or lipopolysaccharides (LPS) from Escherichia coli or were cocultured with H. pylori but separated by a 0.1-μm filter that prevented direct contact between the bacteria and the epithelial cells. Intracellular ceramide levels were determined by diacylglycerol kinase assay. Results represent means ± SD from 3 separate experiments. Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 10 Transient exposure to C2-ceramide resulted in significant production of IL-8. Kato III cells were incubated with C2-ceramide (at 10 μmol/L) for the time indicated, then culture supernatants were replaced with fresh medium (without C2-ceramide). After 24 hours of incubation, conditioned media were harvested, and IL-8 levels were determined. Data shown are expressed as means ± SD (n = 6 for each data point). Gastroenterology 1999 116, 1330-1341DOI: (10.1016/S0016-5085(99)70497-X) Copyright © 1999 American Gastroenterological Association Terms and Conditions