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. 2013 Jan;54(1):189-201.
doi: 10.1194/jlr.M031427. Epub 2012 Nov 8.

Fenretinide inhibited de novo ceramide synthesis and proinflammatory cytokines induced by Aggregatibacter actinomycetemcomitans

Hong Yu  1 , Michael Valerio, Jacek Bielawski
Affiliations

Fenretinide inhibited de novo ceramide synthesis and proinflammatory cytokines induced by Aggregatibacter actinomycetemcomitans

Hong Yu et al. J Lipid Res. 2013 Jan.

Abstract

Ceramides play an essential role in modulating immune signaling pathways and proinflammatory cytokine production in response to infectious pathogens, stress stimuli, or chemotherapeutic drugs. In this study, we demonstrated that Aggregatibacter actinomycetemcomitans, the pathogen for aggressive periodontitis, induced de novo synthesis of ceramide in Raw 264.7 cells. In addition, we identified that fenretinide, a synthetic retinoid, suppressed the de novo synthesis of ceramide induced by A. actinomycetemcomitans. Moreover, fenretinide attenuated interleukin (IL)-1β, IL-6, and cyclooxygenase-2 mRNA expression induced by A. actinomycetemcomitans. Fenretinide also decreased IL-1β, IL-6, and prostaglandin E2 proinflammatory cytokine levels in Raw 264.7 cells induced by A. actinomycetemcomitans. However, fenretinide had no significant effects on tumor necrosis factor alpha mRNA or protein levels. Furthermore, we showed that fenretinide inhibited the janus kinase-signal transducer and activator of transcription, phosphatidylinositol 3-kinase-Akt, protein kinase C, and nuclear factor-kappaB signaling pathways, whereas fenretinide up-regulated the mitogen-activated protein kinase signaling pathways after bacterial stimulation. This study emphasizes the de novo ceramide synthesis pathway in response to bacterial stimulation and demonstrates the anti-inflammatory role of fenretinide in the bacteria-induced immune response.

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Figures

Fig. 1.
Fig. 1.
Ceramide synthesis pathways and the role of fenretinide in the de novo pathway. Ceramides can be generated by de novo, SMase, and salvage pathways. Fenretinide is an agonist for SPT, whereas it is an inhibitor for DEGS.
Fig. 2.
Fig. 2.
A.a induced de novo synthesis of ceramide in Raw 264.7 cells. Raw 264.7 cells were untreated or stimulated with A.a (100 CFUs) for various times. Purified dhSph (A), total (dhCer (B), total ceramide (C), Sph (D), S1P (E), total SM (F), dihydroC16-ceramide (dhC16-Cer) (G), dihydroC22-ceramide (dhC22-Cer) (H), dihydroC24-ceramide (dhC24-Cer) (I), dihydroC24:1-ceramide (dhC24:1-Cer) (J), C16-ceramide (K), C22-ceramide (L), C24-ceramide (M), and C24:1 ceramide (N) were analyzed and normalized by total protein. The data are representative of two separate experiments. Data are plotted as mean ± SD (n = 3; *P < 0.05, **P < 0.01).
Fig. 3.
Fig. 3.
Fenretinide induced the generation of dhSph and dhCer but inhibited the synthesis of ceramide in Raw 264.7 cells. Raw 264.7 cells were treated with fenretinide (Fen, 5 μM) or control vehicle (Veh) ethanol for 8 h. dhSph (A), total dhCer (B), total ceramide (C), Sph (D), S1P (E), total SM (F), dhCer species (G), and ceramide (H) species were analyzed and normalized by total protein. The data are representative of two separate experiments. Data are plotted as mean ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001).
Fig. 4.
Fig. 4.
A.a induced the production of dhSph, dhCer, ceramide, and Sph in vehicle-treated Raw 264.7 cells. Raw 264.7 cells were treated with vehicle (Veh) ethanol for 4 h. Then the cells were unstimulated or stimulated with A.a (100 CFUs) for 4 h. dhSph (A), total dhCer (B), total ceramide (C), Sph (D), S1P (E), total SM (F), dhCer species (G), and ceramide species (H) were analyzed and normalized by total protein. The data are representative of two separate experiments. Data are plotted as mean ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001).
Fig. 5.
Fig. 5.
Fenretinide inhibited de novo synthesis of ceramide induced by A.a in Raw 264.7 cells. Raw 264.7 cells were treated with fenretinide (Fen, 5 μM) or control vehicle (Veh) ethanol for 4 h. Then the cells were stimulated with A.a (100 CFUs) for 4 h. dhSph (A), total dhCer (B), total ceramide (C), Sph (D), S1P (E), total SM (F), dhCer species G), and ceramide (H) species were analyzed and normalized by total protein. The data are representative of two separate experiments. Data are plotted as mean ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001).
Fig. 6.
Fig. 6.
Fenretinide inhibited proinflammatory cytokine mRNA production in Raw 264.7 cells induced by A.a. Raw 264.7 cells were treated with fenretinide (2.5–5 μM) or control vehicle ethanol for 4 h. Then the cells were unstimulated or stimulated with A.a (100 CFUs) for 4 h. IL-1β mRNA (A), IL-6 mRNA (B), COX-2 mRNA (C), and TNF-α mRNA (D) levels were evaluated by quantitative real time PCR. The data are representative of three separate experiments. The mRNA levels were normalized to an endogenous control GAPDH expression and expressed as fold change compared with control groups. Data are plotted as mean ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001).
Fig. 7.
Fig. 7.
Fenretinide attenuated IL-1β, IL-6, and PGE2 cytokine expression in Raw 264.7 cells induced by A.a. Raw 264.7 cells were treated with fenretinide (2.5–5 μM) or control vehicle ethanol for 4 h. Then the cells were unstimulated or stimulated with A.a (100 CFUs) for 4 h. IL-1β (A), IL-6 (B), PGE2 (C), and TNF-α cytokines (D) were analyzed by ELISA in cell lysate. The concentration of cytokines was normalized by total protein concentration in cell lysate. The data are representative of three separate experiments. Data are plotted as mean ± SD (n = 3; **P < 0.01, ***P < 0.001).
Fig. 8.
Fig. 8.
Fenretinide attenuated p-signal transducer and activator of transcription 1 (STAT1), p-PI3K, p-Akt, p-PKC, p-IκBα, and p-NF-κBp65 in Raw 264.7 cells induced by A.a. Raw 264.7 cells were treated with fenretinide (5 μM) or control vehicle ethanol for 4 h. Then the cells were unstimulated or stimulated with A.a (100 CFUs) for various times (30–120 min). Phosphorylated (p) proteins were evaluated by Western blot assay. GAPDH served as a protein loading control. A: p-STAT1, p-PI3K, p-Akt, p-PKC, p-IκBα, p-NF-κBp65, p-JNK, p-ERK, p-p38, and GAPDH expression in Raw 264.7 cells. Protein density/mm2 of p-STAT1 (B), p-PI3K (C), p-Akt (D), p-PKC (E), p-IκBα (F), p-NF-κBp65 (G), p-JNK (H), p-ERK (I), and p-p38 (J) was normalized by GAPDH. The data are representative of three separate experiments. Data are plotted as mean ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001).
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