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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

NADPH oxidase/ROS play key roles in mediating S1P-induced ICAM-1 expression. (A) Cells were pretreated with APO, DPI, or Edaravone 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 APO (10 μM), DPI (1 μM), or Edaravone (10 μM) for 1 h, and then incubated with 10 μM S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with APO (10 μM), DPI (1 μM), or Edaravone (10 μM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled or p47phox, and then incubated with 10 μM S1P for 16 h. The levels of p47phox and ICAM-1 proteins were determined by Western blot. (E) Cells were treated with 10 μM S1P for the indicated times. The ROS production and NADPH oxidase activity were measured. (F) Cells were pretreated with DPI, APO, Rottlerin, or PF431396 for 1 h, and then incubated with 10 μM S1P for 60 min or 10 min. The ROS production and NADPH oxidase activity were measured. Data are expressed as mean ± SEM of three independent experiments. #P < 0.01, as compared with the cells exposed to S1P alone (A–C,F) or vehicle alone (E).
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Figure 5: NADPH oxidase/ROS play key roles in mediating S1P-induced ICAM-1 expression. (A) Cells were pretreated with APO, DPI, or Edaravone 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 APO (10 μM), DPI (1 μM), or Edaravone (10 μM) for 1 h, and then incubated with 10 μM S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with APO (10 μM), DPI (1 μM), or Edaravone (10 μM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled or p47phox, and then incubated with 10 μM S1P for 16 h. The levels of p47phox and ICAM-1 proteins were determined by Western blot. (E) Cells were treated with 10 μM S1P for the indicated times. The ROS production and NADPH oxidase activity were measured. (F) Cells were pretreated with DPI, APO, Rottlerin, or PF431396 for 1 h, and then incubated with 10 μM S1P for 60 min or 10 min. The ROS production and NADPH oxidase activity were measured. Data are expressed as mean ± SEM of three independent experiments. #P < 0.01, as compared with the cells exposed to S1P alone (A–C,F) or vehicle alone (E).

Mentions: Excessive production of ROS by NADPH oxidase from tissue injury is associated with a range of respiratory inflammatory diseases (Lee and Yang, 2012). Our previous studies also indicated that ICAM-1 induction by TNF-α-treatment is mediated by the NADPH oxidase activation and ROS overproduction (Lee and Yang, 2012). Moreover, our data indicated that all of the inhibitors of NADPH oxidase (DPI and APO) and ROS (Edaravone) markedly reduced S1P-induced ICAM-1 protein (∼70, 57, and 60%) and mRNA levels (∼74, 67, and 66%), as well as promoter activity (∼55, 50, and 41%) (Figures 5A,B). In addition, these three inhibitors also reduced monocyte adhesion to HPAEpiCs challenged with 10 μM S1P (Figure 5C). Activated NADPH oxidase is dependent on a multimeric protein complex formation, in which consists with at least three cytosolic subunits (p47phox, p67phox, and p40phox) interaction. Because conformational change of phosphorylated p47phox associated with p22phox, p47phox plays a role as an “organizer subunit” to organize other cytosolic factor translocations (Lee and Yang, 2012). We further examined the role of p47phox in S1P-induced ICAM-1 expression. As shown in Figure 5D, p47phox siRNA transfection knocked down the total p47phox protein by approximate 60%, and then inhibited S1P-enhanced ICAM-1 expression by approximate 41%. Moreover, S1P time-dependently induced both of the NADPH oxidase activity and intracellular ROS generation (Figure 5E). Consistently, activation of NADPH oxidase activity and ROS generation were inhibited by DPI, APO, Rottlerin, or PF431396 (Figure 5F). Taken together, we suggested that S1P induces NADPH oxidase/ROS-dependent ICAM-1 expression 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)

NADPH oxidase/ROS play key roles in mediating S1P-induced ICAM-1 expression. (A) Cells were pretreated with APO, DPI, or Edaravone 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 APO (10 μM), DPI (1 μM), or Edaravone (10 μM) for 1 h, and then incubated with 10 μM S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with APO (10 μM), DPI (1 μM), or Edaravone (10 μM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled or p47phox, and then incubated with 10 μM S1P for 16 h. The levels of p47phox and ICAM-1 proteins were determined by Western blot. (E) Cells were treated with 10 μM S1P for the indicated times. The ROS production and NADPH oxidase activity were measured. (F) Cells were pretreated with DPI, APO, Rottlerin, or PF431396 for 1 h, and then incubated with 10 μM S1P for 60 min or 10 min. The ROS production and NADPH oxidase activity were measured. Data are expressed as mean ± SEM of three independent experiments. #P < 0.01, as compared with the cells exposed to S1P alone (A–C,F) or vehicle alone (E).
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Figure 5: NADPH oxidase/ROS play key roles in mediating S1P-induced ICAM-1 expression. (A) Cells were pretreated with APO, DPI, or Edaravone 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 APO (10 μM), DPI (1 μM), or Edaravone (10 μM) for 1 h, and then incubated with 10 μM S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with APO (10 μM), DPI (1 μM), or Edaravone (10 μM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled or p47phox, and then incubated with 10 μM S1P for 16 h. The levels of p47phox and ICAM-1 proteins were determined by Western blot. (E) Cells were treated with 10 μM S1P for the indicated times. The ROS production and NADPH oxidase activity were measured. (F) Cells were pretreated with DPI, APO, Rottlerin, or PF431396 for 1 h, and then incubated with 10 μM S1P for 60 min or 10 min. The ROS production and NADPH oxidase activity were measured. Data are expressed as mean ± SEM of three independent experiments. #P < 0.01, as compared with the cells exposed to S1P alone (A–C,F) or vehicle alone (E).
Mentions: Excessive production of ROS by NADPH oxidase from tissue injury is associated with a range of respiratory inflammatory diseases (Lee and Yang, 2012). Our previous studies also indicated that ICAM-1 induction by TNF-α-treatment is mediated by the NADPH oxidase activation and ROS overproduction (Lee and Yang, 2012). Moreover, our data indicated that all of the inhibitors of NADPH oxidase (DPI and APO) and ROS (Edaravone) markedly reduced S1P-induced ICAM-1 protein (∼70, 57, and 60%) and mRNA levels (∼74, 67, and 66%), as well as promoter activity (∼55, 50, and 41%) (Figures 5A,B). In addition, these three inhibitors also reduced monocyte adhesion to HPAEpiCs challenged with 10 μM S1P (Figure 5C). Activated NADPH oxidase is dependent on a multimeric protein complex formation, in which consists with at least three cytosolic subunits (p47phox, p67phox, and p40phox) interaction. Because conformational change of phosphorylated p47phox associated with p22phox, p47phox plays a role as an “organizer subunit” to organize other cytosolic factor translocations (Lee and Yang, 2012). We further examined the role of p47phox in S1P-induced ICAM-1 expression. As shown in Figure 5D, p47phox siRNA transfection knocked down the total p47phox protein by approximate 60%, and then inhibited S1P-enhanced ICAM-1 expression by approximate 41%. Moreover, S1P time-dependently induced both of the NADPH oxidase activity and intracellular ROS generation (Figure 5E). Consistently, activation of NADPH oxidase activity and ROS generation were inhibited by DPI, APO, Rottlerin, or PF431396 (Figure 5F). Taken together, we suggested that S1P induces NADPH oxidase/ROS-dependent ICAM-1 expression 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