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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

PKCδ is involved in S1P-induced ICAM-1 expression. (A) Cells were pretreated with Rottlerin 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 Rottlerin (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 transfected with siRNA of scrambled or PKCδ, and then incubated with 10 μM S1P for 16 h. The levels of PKCδ and ICAM-1 proteins were determined by Western blot. (D) Cells were pretreated with Rottlerin (10 μM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. (E) Cells were pretreated without or with Rottlerin, GPA2A, or GPA2 for 1 h, and then incubated with 10 μM S1P for the indicated time intervals. The levels of phospho-PKCδ were determined by Western blot. 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 3: PKCδ is involved in S1P-induced ICAM-1 expression. (A) Cells were pretreated with Rottlerin 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 Rottlerin (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 transfected with siRNA of scrambled or PKCδ, and then incubated with 10 μM S1P for 16 h. The levels of PKCδ and ICAM-1 proteins were determined by Western blot. (D) Cells were pretreated with Rottlerin (10 μM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. (E) Cells were pretreated without or with Rottlerin, GPA2A, or GPA2 for 1 h, and then incubated with 10 μM S1P for the indicated time intervals. The levels of phospho-PKCδ were determined by Western blot. Data are expressed as mean ± SEM of three independent experiments. #P < 0.01, as compared with the cells exposed to S1P alone.

Mentions: Several lines of evidence indicate that PKCδ activity plays critical roles to regulate cellular physiological functions (Kikkawa et al., 2002). We also obtained the similar results that pretreatment of Rottlerin (selective PKCδ inhibitor) significantly attenuate S1P-induced ICAM-1 protein expression by approximate 85% (Figure 3A). This inhibitory phenomenon was also exhibited in mRNA by approximate 95% (Figure 3B) and promoter activity by approximate 55% (Figure 3B). To confirm that PKCδ is involved in S1P-induced ICAM-1 expression, siRNA transfection was used to knockdown PKCδ expression. The results of Western blotting indicated that PKCδ siRNA transfection reduces PKCδ protein by approximate 55%, and then inhibits the S1P-induced ICAM-1 expression by approximate 40% (Figure 3C). Consistently, pretreatment with Rottlerin also decreased monocyte adhesion to the cell surface of HPAEpiCs challenged with S1P by approximate 65% (Figure 3D). Especially, we found that S1P can trigger PKCδ phosphorylation with a time-dependent manner, which was reduced by pretreatment of Rottlerin and GPA2A, but not GPA2 (Figure 3E). Taken together, we demonstrated that the Gq-coupled receptor/PKCδ activation mediate S1P stimulated ICAM-1 expression in these cells.


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)

PKCδ is involved in S1P-induced ICAM-1 expression. (A) Cells were pretreated with Rottlerin 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 Rottlerin (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 transfected with siRNA of scrambled or PKCδ, and then incubated with 10 μM S1P for 16 h. The levels of PKCδ and ICAM-1 proteins were determined by Western blot. (D) Cells were pretreated with Rottlerin (10 μM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. (E) Cells were pretreated without or with Rottlerin, GPA2A, or GPA2 for 1 h, and then incubated with 10 μM S1P for the indicated time intervals. The levels of phospho-PKCδ were determined by Western blot. 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 3: PKCδ is involved in S1P-induced ICAM-1 expression. (A) Cells were pretreated with Rottlerin 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 Rottlerin (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 transfected with siRNA of scrambled or PKCδ, and then incubated with 10 μM S1P for 16 h. The levels of PKCδ and ICAM-1 proteins were determined by Western blot. (D) Cells were pretreated with Rottlerin (10 μM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. (E) Cells were pretreated without or with Rottlerin, GPA2A, or GPA2 for 1 h, and then incubated with 10 μM S1P for the indicated time intervals. The levels of phospho-PKCδ were determined by Western blot. Data are expressed as mean ± SEM of three independent experiments. #P < 0.01, as compared with the cells exposed to S1P alone.
Mentions: Several lines of evidence indicate that PKCδ activity plays critical roles to regulate cellular physiological functions (Kikkawa et al., 2002). We also obtained the similar results that pretreatment of Rottlerin (selective PKCδ inhibitor) significantly attenuate S1P-induced ICAM-1 protein expression by approximate 85% (Figure 3A). This inhibitory phenomenon was also exhibited in mRNA by approximate 95% (Figure 3B) and promoter activity by approximate 55% (Figure 3B). To confirm that PKCδ is involved in S1P-induced ICAM-1 expression, siRNA transfection was used to knockdown PKCδ expression. The results of Western blotting indicated that PKCδ siRNA transfection reduces PKCδ protein by approximate 55%, and then inhibits the S1P-induced ICAM-1 expression by approximate 40% (Figure 3C). Consistently, pretreatment with Rottlerin also decreased monocyte adhesion to the cell surface of HPAEpiCs challenged with S1P by approximate 65% (Figure 3D). Especially, we found that S1P can trigger PKCδ phosphorylation with a time-dependent manner, which was reduced by pretreatment of Rottlerin and GPA2A, but not GPA2 (Figure 3E). Taken together, we demonstrated that the Gq-coupled receptor/PKCδ activation mediate S1P stimulated ICAM-1 expression in these cells.

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