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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

Proposed model to illustrate the signaling pathways involved in ICAM-1 expression and monocyte adhesion in HPAEpiCs challenged with S1P. S1P-induced ICAM-1 expression and monocyte adhesion is mediated through Gq-/Gi-coupled receptor/PKCδ/PYK2/NADPH oxidase/ROS-dependent NF-κB activation.
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Figure 8: Proposed model to illustrate the signaling pathways involved in ICAM-1 expression and monocyte adhesion in HPAEpiCs challenged with S1P. S1P-induced ICAM-1 expression and monocyte adhesion is mediated through Gq-/Gi-coupled receptor/PKCδ/PYK2/NADPH oxidase/ROS-dependent NF-κB activation.

Mentions: Asthma, COPD, and several chronic pulmonary diseases are characterized by various extents of continuous inflammation and airway remodeling in the respiratory system. The bioactive sphingolipid metabolite, S1P, activates EDG (endothelial differentiation gene) receptors and promotes the pro-inflammatory cytokine expression to enhance allergic responses in asthma (Ammit et al., 2001). Moreover, stimulation of S1P promotes expression of cell surface adhesion molecules to accelerate lung inflammatory injury. Previous reports indicated that S1P concentration in plasma and tissue exhibits different levels according to various experimental conditions (Ammit et al., 2001; Yatomi, 2008; Price et al., 2013). The S1P concentrations have been shown to be elevated in pulmonary patients as compared with normal subjects (Ammit et al., 2001; Knapp et al., 2009, 2013). However, the molecular mechanisms of S1P induced ICAM-1 expression and infiltration of monocytes are not fully defined in HPAEpiCs. In contrast to the human, the S1P concentration is about 0.2–1 μM in the plasma of mice (Hisano et al., 2012; Kowalski et al., 2013). In this study and our previous data (Lin et al., 2015), 10 μM S1P was used to obtain the maximal responses and this induction can be manipulated with other approached methods such as the inhibitors treatment and siRNA transfection to dissect out the regulation of signal transduction pathways. The concentrations of inhibitors used were about 30-folds of their IC50 values in the literatures which allowed us to investigate the roles of signaling molecules involved in S1P-mediated responses in HPAEpiCs. The present studies demonstrated that S1P-induced ICAM-1 expression was, at least in part, mediated via Gq- and Gi/o-coupled receptors/PKCδ/PYK2/NADPH oxidase/ROS-dependent NF-κB activation (Figure 8). Genetic silencing through transfection with siRNA of PKCδ, PYK2, p47phox, or p65 and pretreatment with the inhibitor of PKCδ (Rottlerin), PYK2 (PF431396), NADPH oxidase (DPI or APO), ROS (Edaravone), or NF-κB (Bay11-7082) abrogated S1P-induced ICAM-1 expression and monocyte adhesion. Therefore, activation of S1P receptors by S1P causes inflammatory responses through ICAM-1 up-regulation.


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)

Proposed model to illustrate the signaling pathways involved in ICAM-1 expression and monocyte adhesion in HPAEpiCs challenged with S1P. S1P-induced ICAM-1 expression and monocyte adhesion is mediated through Gq-/Gi-coupled receptor/PKCδ/PYK2/NADPH oxidase/ROS-dependent NF-κB activation.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Proposed model to illustrate the signaling pathways involved in ICAM-1 expression and monocyte adhesion in HPAEpiCs challenged with S1P. S1P-induced ICAM-1 expression and monocyte adhesion is mediated through Gq-/Gi-coupled receptor/PKCδ/PYK2/NADPH oxidase/ROS-dependent NF-κB activation.
Mentions: Asthma, COPD, and several chronic pulmonary diseases are characterized by various extents of continuous inflammation and airway remodeling in the respiratory system. The bioactive sphingolipid metabolite, S1P, activates EDG (endothelial differentiation gene) receptors and promotes the pro-inflammatory cytokine expression to enhance allergic responses in asthma (Ammit et al., 2001). Moreover, stimulation of S1P promotes expression of cell surface adhesion molecules to accelerate lung inflammatory injury. Previous reports indicated that S1P concentration in plasma and tissue exhibits different levels according to various experimental conditions (Ammit et al., 2001; Yatomi, 2008; Price et al., 2013). The S1P concentrations have been shown to be elevated in pulmonary patients as compared with normal subjects (Ammit et al., 2001; Knapp et al., 2009, 2013). However, the molecular mechanisms of S1P induced ICAM-1 expression and infiltration of monocytes are not fully defined in HPAEpiCs. In contrast to the human, the S1P concentration is about 0.2–1 μM in the plasma of mice (Hisano et al., 2012; Kowalski et al., 2013). In this study and our previous data (Lin et al., 2015), 10 μM S1P was used to obtain the maximal responses and this induction can be manipulated with other approached methods such as the inhibitors treatment and siRNA transfection to dissect out the regulation of signal transduction pathways. The concentrations of inhibitors used were about 30-folds of their IC50 values in the literatures which allowed us to investigate the roles of signaling molecules involved in S1P-mediated responses in HPAEpiCs. The present studies demonstrated that S1P-induced ICAM-1 expression was, at least in part, mediated via Gq- and Gi/o-coupled receptors/PKCδ/PYK2/NADPH oxidase/ROS-dependent NF-κB activation (Figure 8). Genetic silencing through transfection with siRNA of PKCδ, PYK2, p47phox, or p65 and pretreatment with the inhibitor of PKCδ (Rottlerin), PYK2 (PF431396), NADPH oxidase (DPI or APO), ROS (Edaravone), or NF-κB (Bay11-7082) abrogated S1P-induced ICAM-1 expression and monocyte adhesion. Therefore, activation of S1P receptors by S1P causes inflammatory responses through ICAM-1 up-regulation.

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