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Sphingosine 1-Phosphate-Induced ICAM-1 Expression via NADPH Oxidase/ROS-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells.

Lin CC, Yang CC, Cho RL, Wang CY, Hsiao LD, Yang CM - Front Pharmacol (2016)

Bottom Line: In addition, S1P induced NADPH oxidase activation and intracellular ROS generation, which were also reduced by Rottlerin or PF431396.In the S1P-treated mice, we found that the levels of ICAM-1 protein and mRNA in the lung fractions, the pulmonary hematoma and leukocyte count in bronchoalveolar lavage fluid were enhanced through a PKCδ/PYK2/NADPH oxidase/ROS/NF-κB signaling pathway.We concluded that S1P-accelerated lung damage is due to the ICAM-1 induction associated with leukocyte recruitment.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthetics, Chang Gung Memorial Hospital at LinkouTaoyuan, Taiwan; College of Medicine, Chang Gung UniversityTaoyuan, Taiwan.

ABSTRACT
The intercellular adhesion molecule-1 (ICAM-1) expression is frequently correlated with the lung inflammation. In lung injury, sphingosine-1-phosphate (S1P, bioactive sphingolipid metabolite), participate gene regulation of adhesion molecule in inflammation progression and aggravate tissue damage. To investigate the transduction mechanisms of the S1P in pulmonary epithelium, we demonstrated that exposure of HPAEpiCs (human pulmonary alveolar epithelial cells) to S1P significantly induces ICAM-1 expression leading to increase monocyte adhesion on the surface of HPAEpiCs. These phenomena were effectively attenuated by pretreatments with series of inhibitors such as Rottlerin (PKCδ), PF431396 (PYK2), diphenyleneiodonium chloride (DPI), apocynin (NADPH oxidase), Edaravone (ROS), and Bay11-7082 (NF-κB). Consistently, knockdown with siRNA transfection of PKCδ, PYK2, p47 (phox) , and p65 exhibited the same results. Pretreatment with both Gq-coupled receptor antagonist (GPA2A) and Gi/o-coupled receptor antagonist (GPA2) also blocked the upregulation of ICAM-1 protein and mRNA induced by S1P. We observed that S1P induced PYK2 activation via a Gq-coupled receptor/PKCδ-dependent pathway. In addition, S1P induced NADPH oxidase activation and intracellular ROS generation, which were also reduced by Rottlerin or PF431396. We demonstrated that S1P induced NF-κB p65 phosphorylation and nuclear translocation in HPAEpiCs. Activated NF-κB was blocked by Rottlerin, PF431396, APO, DPI, or Edaravone. Besides, the results of monocyte adhesion assay indicated that S1P-induced ICAM-1 expression on HPAEpiCs can enhance the monocyte attachments. In the S1P-treated mice, we found that the levels of ICAM-1 protein and mRNA in the lung fractions, the pulmonary hematoma and leukocyte count in bronchoalveolar lavage fluid were enhanced through a PKCδ/PYK2/NADPH oxidase/ROS/NF-κB signaling pathway. We concluded that S1P-accelerated lung damage is due to the ICAM-1 induction associated with leukocyte recruitment.

No MeSH data available.


Related in: MedlinePlus

Sphingosine-1-phosphate-induced ICAM-1 expression requires ongoing transcription and translation. (A) Cells were pretreated with Actinomycin D (Act. D) or CHI for 1 h, and then incubated with 10 μM S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with Act. D (10 nM) or CHI (1 μM) for 1 h, and then incubated with 10 μM S1P for 4 h. The ICAM-1 mRNA expression was determined by real-time PCR. (C) Cells were pretreated with Act. D (10 nM) or CHI (1 μM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. Data are expressed as mean ± SEM of three independent experiments. ∗P < 0.05; #P < 0.01, as compared with the cells exposed to S1P alone.
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Figure 1: Sphingosine-1-phosphate-induced ICAM-1 expression requires ongoing transcription and translation. (A) Cells were pretreated with Actinomycin D (Act. D) or CHI for 1 h, and then incubated with 10 μM S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with Act. D (10 nM) or CHI (1 μM) for 1 h, and then incubated with 10 μM S1P for 4 h. The ICAM-1 mRNA expression was determined by real-time PCR. (C) Cells were pretreated with Act. D (10 nM) or CHI (1 μM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. Data are expressed as mean ± SEM of three independent experiments. ∗P < 0.05; #P < 0.01, as compared with the cells exposed to S1P alone.

Mentions: Our previous report indicated that the concentration of S1P (10 μM) used to obtain a maximal ICAM-1 expression which was applied in this study (Lin et al., 2015). To examine the mechanisms of S1P-induced ICAM-1 expression, HPAEpiCs were stimulated with S1P (10 μM) in the pretreatment with (or without) (Act. D, transcriptional inhibitor) or cycloheximide (CHI, translational inhibitor) and then Western blotting was performed to analyze ICAM-1 protein expression. Our data indicated that both of Act. D and CHI treatments can attenuate the S1P-mediated ICAM-1 induction in a dose-dependent manner (Figure 1A). Besides, Act. D, but not CHI, significantly reduced S1P-induced ICAM-1 mRNA expression in HPAEpiCs (Figure 1B). Next, we found that adhesion of THP-1 to HPAEpiCs was enhanced by S1P challenge about fivefold, which was also obviously inhibited by pretreatment with Act. D or CHI (Figure 1C). Taken together, we suggested that S1P-induced ICAM-1 expression depends on de novo protein synthesis in HPAEpiCs.


Sphingosine 1-Phosphate-Induced ICAM-1 Expression via NADPH Oxidase/ROS-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells.

Lin CC, Yang CC, Cho RL, Wang CY, Hsiao LD, Yang CM - Front Pharmacol (2016)

Sphingosine-1-phosphate-induced ICAM-1 expression requires ongoing transcription and translation. (A) Cells were pretreated with Actinomycin D (Act. D) or CHI for 1 h, and then incubated with 10 μM S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with Act. D (10 nM) or CHI (1 μM) for 1 h, and then incubated with 10 μM S1P for 4 h. The ICAM-1 mRNA expression was determined by real-time PCR. (C) Cells were pretreated with Act. D (10 nM) or CHI (1 μM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. Data are expressed as mean ± SEM of three independent experiments. ∗P < 0.05; #P < 0.01, as compared with the cells exposed to S1P alone.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4815023&req=5

Figure 1: Sphingosine-1-phosphate-induced ICAM-1 expression requires ongoing transcription and translation. (A) Cells were pretreated with Actinomycin D (Act. D) or CHI for 1 h, and then incubated with 10 μM S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with Act. D (10 nM) or CHI (1 μM) for 1 h, and then incubated with 10 μM S1P for 4 h. The ICAM-1 mRNA expression was determined by real-time PCR. (C) Cells were pretreated with Act. D (10 nM) or CHI (1 μM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. Data are expressed as mean ± SEM of three independent experiments. ∗P < 0.05; #P < 0.01, as compared with the cells exposed to S1P alone.
Mentions: Our previous report indicated that the concentration of S1P (10 μM) used to obtain a maximal ICAM-1 expression which was applied in this study (Lin et al., 2015). To examine the mechanisms of S1P-induced ICAM-1 expression, HPAEpiCs were stimulated with S1P (10 μM) in the pretreatment with (or without) (Act. D, transcriptional inhibitor) or cycloheximide (CHI, translational inhibitor) and then Western blotting was performed to analyze ICAM-1 protein expression. Our data indicated that both of Act. D and CHI treatments can attenuate the S1P-mediated ICAM-1 induction in a dose-dependent manner (Figure 1A). Besides, Act. D, but not CHI, significantly reduced S1P-induced ICAM-1 mRNA expression in HPAEpiCs (Figure 1B). Next, we found that adhesion of THP-1 to HPAEpiCs was enhanced by S1P challenge about fivefold, which was also obviously inhibited by pretreatment with Act. D or CHI (Figure 1C). Taken together, we suggested that S1P-induced ICAM-1 expression depends on de novo protein synthesis in HPAEpiCs.

Bottom Line: In addition, S1P induced NADPH oxidase activation and intracellular ROS generation, which were also reduced by Rottlerin or PF431396.In the S1P-treated mice, we found that the levels of ICAM-1 protein and mRNA in the lung fractions, the pulmonary hematoma and leukocyte count in bronchoalveolar lavage fluid were enhanced through a PKCδ/PYK2/NADPH oxidase/ROS/NF-κB signaling pathway.We concluded that S1P-accelerated lung damage is due to the ICAM-1 induction associated with leukocyte recruitment.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthetics, Chang Gung Memorial Hospital at LinkouTaoyuan, Taiwan; College of Medicine, Chang Gung UniversityTaoyuan, Taiwan.

ABSTRACT
The intercellular adhesion molecule-1 (ICAM-1) expression is frequently correlated with the lung inflammation. In lung injury, sphingosine-1-phosphate (S1P, bioactive sphingolipid metabolite), participate gene regulation of adhesion molecule in inflammation progression and aggravate tissue damage. To investigate the transduction mechanisms of the S1P in pulmonary epithelium, we demonstrated that exposure of HPAEpiCs (human pulmonary alveolar epithelial cells) to S1P significantly induces ICAM-1 expression leading to increase monocyte adhesion on the surface of HPAEpiCs. These phenomena were effectively attenuated by pretreatments with series of inhibitors such as Rottlerin (PKCδ), PF431396 (PYK2), diphenyleneiodonium chloride (DPI), apocynin (NADPH oxidase), Edaravone (ROS), and Bay11-7082 (NF-κB). Consistently, knockdown with siRNA transfection of PKCδ, PYK2, p47 (phox) , and p65 exhibited the same results. Pretreatment with both Gq-coupled receptor antagonist (GPA2A) and Gi/o-coupled receptor antagonist (GPA2) also blocked the upregulation of ICAM-1 protein and mRNA induced by S1P. We observed that S1P induced PYK2 activation via a Gq-coupled receptor/PKCδ-dependent pathway. In addition, S1P induced NADPH oxidase activation and intracellular ROS generation, which were also reduced by Rottlerin or PF431396. We demonstrated that S1P induced NF-κB p65 phosphorylation and nuclear translocation in HPAEpiCs. Activated NF-κB was blocked by Rottlerin, PF431396, APO, DPI, or Edaravone. Besides, the results of monocyte adhesion assay indicated that S1P-induced ICAM-1 expression on HPAEpiCs can enhance the monocyte attachments. In the S1P-treated mice, we found that the levels of ICAM-1 protein and mRNA in the lung fractions, the pulmonary hematoma and leukocyte count in bronchoalveolar lavage fluid were enhanced through a PKCδ/PYK2/NADPH oxidase/ROS/NF-κB signaling pathway. We concluded that S1P-accelerated lung damage is due to the ICAM-1 induction associated with leukocyte recruitment.

No MeSH data available.


Related in: MedlinePlus