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

Chih-Chung Lin,Chuen-Mao Yang,Rou-Ling Cho,Chien-Chung Yang,Chen-Yu Wang,Li-Der Hsiao

doi:10.3389/fphar.2016.00080

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, p47phox, 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.

Highlights

Sustained stimulation and inflammation in respiratory system are pivotal events in pathogenic progressing of asthma or other chronic obstructive pulmonary diseases
We suggested that PKCδ and PYK2 may be required for the S1P-induced VCAM-1 expression in HPAEpiCs
Experiments were undertaken to investigate the effects of S1P on expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte adhesion on HPAEpiCs. These findings suggest that the increased expression of ICAM-1 and monocyte adhesion on S1P-challenged HPAEpiCs are mediated through Gq- and Gi/ocoupled receptor/PKCδ/PYK2/NADPH oxidase/ROS-dependent NF-κB activation

Summary

Introduction

Sustained stimulation and inflammation in respiratory system are pivotal events in pathogenic progressing of asthma or other chronic obstructive pulmonary diseases. Initiated complicated interaction of the intracellular molecules and recruited serious intercellular interactions such as lung resident cells, vascular endothelium and circulating polymorphonuclear cells (PMNs) (Lin et al, 2015). Previous studies indicate that pro-inflammatory cytokine-induced expression of adhesion molecules on the endothelial cells surface is implicated in the inflammatory responses and facilitate (or guidance) PMNs infiltration to the inflammatory area. ICAM-1 and VCAM-1 are belong to the Ig superfamily (Lee and Yang, 2013). In the regulation of transendothelial migration, ICAM-1 expression can mediate the tight adhesiveness of PMNs. PMNs were arrested at non-junctional locations to penetrate the vascular endothelium barrier and attached on the resident cells (Yang et al, 2005; Lee et al, 2013b)

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