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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

Gq-coupled receptor and Gi/o-coupled receptor play key roles in S1P-induced ICAM-1 expression. (A) Cells were pretreated with GPA2 or GPA2A 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 GPA2 (1 μM) or GPA2A (1 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 GPA2 (1 μM) or GPA2A (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 < 0.01, as compared with the cells exposed to S1P alone.
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Figure 2: Gq-coupled receptor and Gi/o-coupled receptor play key roles in S1P-induced ICAM-1 expression. (A) Cells were pretreated with GPA2 or GPA2A 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 GPA2 (1 μM) or GPA2A (1 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 GPA2 (1 μM) or GPA2A (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 < 0.01, as compared with the cells exposed to S1P alone.

Mentions: Sphingosine-1-phosphate regulates numerous cellular responses, including formation of adherent junctions, proliferation, migration, and survival (Ryan and Spiegel, 2008; Takashima et al., 2008; Li et al., 2009). These myriad effects are partly elicited by binding of S1P to a family of five G protein–coupled receptors, termed S1PR1–5. S1PR1, S1PR2, and S1PR3 are ubiquitously expressed, whereas the levels of S1PR4 and S1PR5 expression are predominantly existed in immune cells, CNS, and some organs. Indeed, S1PR1, 2 and 3 are expressed on HPAEpiCs (Lin et al., 2015). Thus, we further investigated whether Gq- or Gi/o-coupled receptor was involved in S1P-induced ICAM-1 expression. As shown in Figures 2A,B, our data indicated that pretreatments of GPA2A (Gq-coupled receptor antagonist) and GPA2 (Gi/o-coupled receptor antagonist) concentration-dependently block the S1P-induced ICAM-1 expression in protein (∼63%) and mRNA (∼68%), respectively. The analysis of promoter activity (∼62%) also exhibit consistent results. Finally, we observed that S1P-enhanced monocyte adhesion was also reduced to approximate 70% of control by these two inhibitors (Figure 2C). Taken together, we suggested that Gq- and Gi/o-coupled receptors play key roles in S1P-induced 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)

Gq-coupled receptor and Gi/o-coupled receptor play key roles in S1P-induced ICAM-1 expression. (A) Cells were pretreated with GPA2 or GPA2A 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 GPA2 (1 μM) or GPA2A (1 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 GPA2 (1 μM) or GPA2A (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 < 0.01, as compared with the cells exposed to S1P alone.
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Related In: Results  -  Collection

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

Figure 2: Gq-coupled receptor and Gi/o-coupled receptor play key roles in S1P-induced ICAM-1 expression. (A) Cells were pretreated with GPA2 or GPA2A 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 GPA2 (1 μM) or GPA2A (1 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 GPA2 (1 μM) or GPA2A (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 < 0.01, as compared with the cells exposed to S1P alone.
Mentions: Sphingosine-1-phosphate regulates numerous cellular responses, including formation of adherent junctions, proliferation, migration, and survival (Ryan and Spiegel, 2008; Takashima et al., 2008; Li et al., 2009). These myriad effects are partly elicited by binding of S1P to a family of five G protein–coupled receptors, termed S1PR1–5. S1PR1, S1PR2, and S1PR3 are ubiquitously expressed, whereas the levels of S1PR4 and S1PR5 expression are predominantly existed in immune cells, CNS, and some organs. Indeed, S1PR1, 2 and 3 are expressed on HPAEpiCs (Lin et al., 2015). Thus, we further investigated whether Gq- or Gi/o-coupled receptor was involved in S1P-induced ICAM-1 expression. As shown in Figures 2A,B, our data indicated that pretreatments of GPA2A (Gq-coupled receptor antagonist) and GPA2 (Gi/o-coupled receptor antagonist) concentration-dependently block the S1P-induced ICAM-1 expression in protein (∼63%) and mRNA (∼68%), respectively. The analysis of promoter activity (∼62%) also exhibit consistent results. Finally, we observed that S1P-enhanced monocyte adhesion was also reduced to approximate 70% of control by these two inhibitors (Figure 2C). Taken together, we suggested that Gq- and Gi/o-coupled receptors play key roles in S1P-induced 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