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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

NF-κB plays a key role in mediating S1P-induced ICAM-1 expression. (A) Cells were pretreated with Bay11-7082 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 Bay11-7082 (100 nM) 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 Bay11-7082 (100 nM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled or p65, and then incubated with 10 μM S1P for 16 h. The levels of p65 and ICAM-1 proteins were determined by Western blot. (E) Cells were pretreated with GPA2A, Rottlerin, PF431396, DPI, APO, Edaravone, Bay11-7082 for 1 h or the vehicle control (Basal), and then incubated with 10 μM S1P for 30 min. Cells were fixed and then labeled with an anti-p65 antibody and then FITC-conjugated secondary antibody. Individual cells were enlarged with 1000x and imaged. The nuclear regions are indicated with solid arrows and the cytoplasmic regions are indicated with dashed arrows. (F) Cells were treated with 10 μM S1P for the indicated times or (G) pretreated with Bay11-7082, Rottlerin, PF431396, Edaravone, or DPI for 1 h, and then incubated with 10 μM S1P for the indicated times. The levels of phospho-p65 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 6: NF-κB plays a key role in mediating S1P-induced ICAM-1 expression. (A) Cells were pretreated with Bay11-7082 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 Bay11-7082 (100 nM) 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 Bay11-7082 (100 nM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled or p65, and then incubated with 10 μM S1P for 16 h. The levels of p65 and ICAM-1 proteins were determined by Western blot. (E) Cells were pretreated with GPA2A, Rottlerin, PF431396, DPI, APO, Edaravone, Bay11-7082 for 1 h or the vehicle control (Basal), and then incubated with 10 μM S1P for 30 min. Cells were fixed and then labeled with an anti-p65 antibody and then FITC-conjugated secondary antibody. Individual cells were enlarged with 1000x and imaged. The nuclear regions are indicated with solid arrows and the cytoplasmic regions are indicated with dashed arrows. (F) Cells were treated with 10 μM S1P for the indicated times or (G) pretreated with Bay11-7082, Rottlerin, PF431396, Edaravone, or DPI for 1 h, and then incubated with 10 μM S1P for the indicated times. The levels of phospho-p65 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: Activation of the NF-κB is dependent on the phosphorylation and degradation of IκB (Baeuerle and Baltimore, 1996; Shih et al., 2015). Upon degradation of IκB, the NF-κB complex translocates into the nucleus to turn on the transcription of specific genes that have the κB sites in their promoter regions. To examine whether NF-κB participates in S1P-imediated ICAM-1 expression, Bay11-7082 (an inhibitor of NF-κB) was used to study VCAM-1 expression in HPAEpiCs. We found that pretreatment with Bay11-7082 significantly reduced S1P-induced ICAM-1 protein in a dose-dependent manner, by approximate 71% (Figure 6A). We also obtained the consistent results at transcriptional analysis that pretreatment of Bay11-7082 attenuates both of the S1P-induced VCAM-1 mRNA expression approximate 71% and promoter activity about 77%, respectively (Figure 6B). Importantly, Bay11-7082 also attenuated monocyte adhesion to S1P-challenged HPAEpiCs (Figure 6C). To further confirm that NF-κB is a targeted transcription factor in S1P-induced ICAM-1 expression in HPAEpiCs, transfection of p65 siRNA was used to clarify the role of NF-κB in the S1P-mediated response. The results of Western blotting indicated that transfection with p65 siRNA significantly reduced p65 protein expression by approximate 51%, and then blocked S1P-induced ICAM-1 expression from 3.0-fold to 1.6-fold in HPAEpiCs (Figure 6D). Moreover, in our study, we showed that S1P markedly induced NF-κB 65 nuclear translocation, which was inhibited by GPA2A, Rottlerin, PF431396, DPI, APO, Edaravone, or Bay11-7082 (Figure 6E). On the other hand, we also found that 10 μM S1P stimulated NF-κB p65 phosphorylation in a time-dependent manner (Figure 6F, within 30 min). This induction was also reduced by pretreatment with Bay11-7082, Rottlerin, PF431396, Edaravone, and DPI (Figure 6G). Based on these results, we demonstrated that S1P initiates ROS-dependent NF-κB pathway to induce ICAM-1 expression on HPAEpiCs and resulted in the monocyte attachment.


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)

NF-κB plays a key role in mediating S1P-induced ICAM-1 expression. (A) Cells were pretreated with Bay11-7082 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 Bay11-7082 (100 nM) 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 Bay11-7082 (100 nM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled or p65, and then incubated with 10 μM S1P for 16 h. The levels of p65 and ICAM-1 proteins were determined by Western blot. (E) Cells were pretreated with GPA2A, Rottlerin, PF431396, DPI, APO, Edaravone, Bay11-7082 for 1 h or the vehicle control (Basal), and then incubated with 10 μM S1P for 30 min. Cells were fixed and then labeled with an anti-p65 antibody and then FITC-conjugated secondary antibody. Individual cells were enlarged with 1000x and imaged. The nuclear regions are indicated with solid arrows and the cytoplasmic regions are indicated with dashed arrows. (F) Cells were treated with 10 μM S1P for the indicated times or (G) pretreated with Bay11-7082, Rottlerin, PF431396, Edaravone, or DPI for 1 h, and then incubated with 10 μM S1P for the indicated times. The levels of phospho-p65 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 6: NF-κB plays a key role in mediating S1P-induced ICAM-1 expression. (A) Cells were pretreated with Bay11-7082 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 Bay11-7082 (100 nM) 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 Bay11-7082 (100 nM) for 1 h, and then incubated with 10 μM S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled or p65, and then incubated with 10 μM S1P for 16 h. The levels of p65 and ICAM-1 proteins were determined by Western blot. (E) Cells were pretreated with GPA2A, Rottlerin, PF431396, DPI, APO, Edaravone, Bay11-7082 for 1 h or the vehicle control (Basal), and then incubated with 10 μM S1P for 30 min. Cells were fixed and then labeled with an anti-p65 antibody and then FITC-conjugated secondary antibody. Individual cells were enlarged with 1000x and imaged. The nuclear regions are indicated with solid arrows and the cytoplasmic regions are indicated with dashed arrows. (F) Cells were treated with 10 μM S1P for the indicated times or (G) pretreated with Bay11-7082, Rottlerin, PF431396, Edaravone, or DPI for 1 h, and then incubated with 10 μM S1P for the indicated times. The levels of phospho-p65 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: Activation of the NF-κB is dependent on the phosphorylation and degradation of IκB (Baeuerle and Baltimore, 1996; Shih et al., 2015). Upon degradation of IκB, the NF-κB complex translocates into the nucleus to turn on the transcription of specific genes that have the κB sites in their promoter regions. To examine whether NF-κB participates in S1P-imediated ICAM-1 expression, Bay11-7082 (an inhibitor of NF-κB) was used to study VCAM-1 expression in HPAEpiCs. We found that pretreatment with Bay11-7082 significantly reduced S1P-induced ICAM-1 protein in a dose-dependent manner, by approximate 71% (Figure 6A). We also obtained the consistent results at transcriptional analysis that pretreatment of Bay11-7082 attenuates both of the S1P-induced VCAM-1 mRNA expression approximate 71% and promoter activity about 77%, respectively (Figure 6B). Importantly, Bay11-7082 also attenuated monocyte adhesion to S1P-challenged HPAEpiCs (Figure 6C). To further confirm that NF-κB is a targeted transcription factor in S1P-induced ICAM-1 expression in HPAEpiCs, transfection of p65 siRNA was used to clarify the role of NF-κB in the S1P-mediated response. The results of Western blotting indicated that transfection with p65 siRNA significantly reduced p65 protein expression by approximate 51%, and then blocked S1P-induced ICAM-1 expression from 3.0-fold to 1.6-fold in HPAEpiCs (Figure 6D). Moreover, in our study, we showed that S1P markedly induced NF-κB 65 nuclear translocation, which was inhibited by GPA2A, Rottlerin, PF431396, DPI, APO, Edaravone, or Bay11-7082 (Figure 6E). On the other hand, we also found that 10 μM S1P stimulated NF-κB p65 phosphorylation in a time-dependent manner (Figure 6F, within 30 min). This induction was also reduced by pretreatment with Bay11-7082, Rottlerin, PF431396, Edaravone, and DPI (Figure 6G). Based on these results, we demonstrated that S1P initiates ROS-dependent NF-κB pathway to induce ICAM-1 expression on HPAEpiCs and resulted in the monocyte attachment.

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