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RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Published on: February 13, 2013
Thrombin induces ICAM-1 expression in human lung epithelial cells via c-Src/PDGFR/PI3K/Akt-dependent NF-κB/p300
Shin-Ei Cheng1, I-Ta Lee2, Chih-Chung Lin1
1*Department of Anesthetics, Chang Gung Memorial Hospital at Lin-Kou and College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
Insights
Thrombin upregulates intercellular adhesion molecule-1 (ICAM-1) in lung epithelial cells, promoting inflammation. This study reveals a signaling pathway involving c-Src, PDGFR, PI3K, Akt, and NF-κB that mediates this effect, offering therapeutic targets.
Area of Science:
- Pulmonary Medicine
- Molecular Biology
- Cellular Signaling
Background:
- Intercellular adhesion molecule-1 (ICAM-1) upregulation is linked to lung inflammation and diseases like idiopathic pulmonary fibrosis (IPF).
- Thrombin plays a role in inflammation and lung injury by inducing adhesion molecules, but its specific mechanisms in pulmonary alveolar epithelial cells are not fully understood.
Purpose of the Study:
- To elucidate the signaling pathways responsible for thrombin-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs).
- To investigate the role of c-Src, PDGFR, PI3K/Akt, and NF-κB in mediating thrombin's effects on ICAM-1 expression and monocyte adherence.
Main Methods:
- Utilized inhibitors (PPACK, PP1, AG1296, LY294002, Bay11-7082) and siRNAs targeting key signaling molecules in HPAEpiCs.
- Assessed ICAM-1 expression, monocyte adherence, protein phosphorylation, and nuclear translocation of signaling proteins.
- Investigated NF-κB promoter activity and protein complex formation using co-immunoprecipitation and chromatin immunoprecipitation assays.
Main Results:
- Thrombin significantly increased ICAM-1 expression and monocyte adherence in HPAEpiCs.
- Inhibition or knockdown of c-Src, PDGFR, PI3K/Akt, or NF-κB components markedly reduced thrombin-induced ICAM-1 expression and monocyte adherence.
- Thrombin stimulated the phosphorylation and nuclear translocation of Akt and NF-κB, and promoted the formation of a p65-Akt-p300 complex, all of which were dependent on the identified pathway.
Conclusions:
- Thrombin induces ICAM-1 expression and monocyte adherence in HPAEpiCs through a signaling cascade involving c-Src, PDGFR, PI3K/Akt, and NF-κB.
- Understanding this pathway provides insights into lung inflammation mechanisms and suggests potential therapeutic strategies targeting ICAM-1 regulation.
Abstract:
Up-regulation of ICAM-1 (intercellular adhesion molecule-1) is frequently implicated in lung inflammation and lung diseases, such as IPF (idiopathic pulmonary fibrosis). Thrombin has been shown to play a key role in inflammation via the induction of adhesion molecules, which then causes lung injury. However, the mechanisms underlying thrombin-induced ICAM-1 expression in HPAEpiCs (human pulmonary alveolar epithelial cells) remain unclear. In the present study, we have shown that thrombin induced ICAM-1 expression in HPAEpiCs. Pre-treatment with the inhibitor of thrombin [PPACK (D-Phe-Pro-Arg-chloromethyl ketone)], c-Src (PP1), PDGFR (platelet-derived growth factor receptor) (AG1296), PI3K (phosohinositide 3-kinase) (LY294002), NF-κB (nuclear factor κB) (Bay11-7082) or p300 (GR343) and transfection with siRNAs of c-Src, PDGFR, Akt, p65 and p300 markedly reduced thrombin-induced ICAM-1 expression and monocyte adherence to HPAEpiCs challenged with thrombin. In addition, we established that thrombin stimulated the phosphorylation of c-Src, PDGFR, Akt and p65, which were inhibited by pre-treatment with their respective inhibitors PP1, AG1296, LY294002 or Bay11-7082. In addition, thrombin also enhanced Akt and NF-κB translocation from the cytosol to the nucleus, which was reduced by PP1, AG1296 or LY294002. Thrombin induced NF-κB promoter activity and the formation of the p65-Akt-p300 complex, which were inhibited by AG1296, LY294002 or PP1. Finally, we have shown that thrombin stimulated in vivo binding of p300, Akt and p65 to the ICAM-1 promoter, which was reduced by AG1296, LY294002, SH-5 or PP1. These results show that thrombin induced ICAM-1 expression and monocyte adherence via a c-Src/PDGFR/PI3K/Akt/NF-κB-dependent pathway in HPAEpiCs. Increased understanding of the signalling mechanisms underlying ICAM-1 gene regulation will create opportunities for the development of anti-inflammatory therapeutic strategies.
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