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HO-1 induction by CO-RM2 attenuates TNF-α-induced cytosolic phospholipase A2 expression via inhibition of PKCα-dependent NADPH oxidase/ROS and NF-κB.

Chi PL, Liu CJ, Lee IT, Chen YW, Hsiao LD, Yang CM - Mediators Inflamm. (2014)

Bottom Line: Here, we reported that TNF-α-induced cPLA2 expression was mediated through TNFR1/PKCα-dependent signaling pathways, including NADPH oxidase (NOX) activation/ROS production and NF-κB activation.CO-RM2 significantly suppressed TNF-α-induced cPLA2 expression by inhibiting the ROS generation and the phosphorylation of NF-κB p65 and IKK α/β, but not the phosphorylation of p38 MAPK and JNK1/2.These results were further confirmed by a ChIP assay to detect the NF-κB DNA-binding activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 333, Taiwan.

ABSTRACT
Rheumatoid arthritis (RA) is characterized by chronic inflammatory infiltration of the synovium and elevation of proinflammatory cytokines. Cytosolic phospholipase A2 (cPLA2) is involved in the development of inflammatory diseases. Heme oxygenase-1 (HO-1) has been shown to possess anti-inflammatory properties. The objective of the study was to investigate the detailed mechanisms of TNF-α-induced cPLA2 expression and to determine whether carbon monoxide releasing molecule-2 (CO-RM2) suppresses TNF-α-induced expression of NF-κB-related proinflammatory genes, including cPLA2, via HO-1 induction in RA synovial fibroblasts (RASFs). Here, we reported that TNF-α-induced cPLA2 expression was mediated through TNFR1/PKCα-dependent signaling pathways, including NADPH oxidase (NOX) activation/ROS production and NF-κB activation. CO-RM2 significantly suppressed TNF-α-induced cPLA2 expression by inhibiting the ROS generation and the phosphorylation of NF-κB p65 and IKK α/β, but not the phosphorylation of p38 MAPK and JNK1/2. These results were further confirmed by a ChIP assay to detect the NF-κB DNA-binding activity. Our results demonstrated that induction of HO-1 by CO-RM2 exerted anti-inflammatory and antioxidant effects which were required in concert to prevent the activation of NF-κB leading to induction of various inflammatory genes implicated in the pathogenesis of RA.

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Effect of CORM-2 on TNF-α-induced NF-κB activation and cPLA2 expression. (a) Cells were pretreated with CORM-2 or iCORM-2 for 16 h and then incubated with TNF-α for 2 h. Chromatin immunoprecipitation (ChIP) assays were performed using an anti-p65 antibody. (b) Cells were transfected with NF-κB-luc reporter gene, pretreated with or without CORM-2 for 16 h, and then incubated with TNF-α for 4 h. NF-κB promoter activity was determined. ((c), (d), and (f)) Cells were pretreated with or without CORM-2 or iCORM-2 for 16 h and then treated with TNF-α for the indicated time intervals. The levels of phospho-p65, phospho-IKKα/β, phospho-JNK1/2, phospho-p38 MAPK, and HO-1 were determined by Western blotting. (e) Cells were pretreated with or without CORM-2 for 16 h and then incubated with TNF-α for 90 min. The cells were added with (5 μM) DHE and images acquired to determine the ROS generation using a fluorescence microscopy. All analyses were performed on samples from 4 RA patients. (f) Immunohistochemical staining for HO-1, cPLA2, or vimentin and hematoxylin and eosin (H&E) staining of serial sections of ankle joints from mice injected with phosphate-buffered saline (sham) ((A)–(E)), mice injected with TNF-α ((F)–(J)), and mice injected with CORM-2 for 16 h followed by TNF-α for 24 h ((K)–(O)) were performed. Arrowheads indicate positive staining. Results are representative of 3 mice per experimental group. Results are representative of 3 independent experiments. In (a), (b), (c), and (g), values are the mean ± SEM. *P < 0.05; #P < 0.01 versus TNF-α alone.
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fig7: Effect of CORM-2 on TNF-α-induced NF-κB activation and cPLA2 expression. (a) Cells were pretreated with CORM-2 or iCORM-2 for 16 h and then incubated with TNF-α for 2 h. Chromatin immunoprecipitation (ChIP) assays were performed using an anti-p65 antibody. (b) Cells were transfected with NF-κB-luc reporter gene, pretreated with or without CORM-2 for 16 h, and then incubated with TNF-α for 4 h. NF-κB promoter activity was determined. ((c), (d), and (f)) Cells were pretreated with or without CORM-2 or iCORM-2 for 16 h and then treated with TNF-α for the indicated time intervals. The levels of phospho-p65, phospho-IKKα/β, phospho-JNK1/2, phospho-p38 MAPK, and HO-1 were determined by Western blotting. (e) Cells were pretreated with or without CORM-2 for 16 h and then incubated with TNF-α for 90 min. The cells were added with (5 μM) DHE and images acquired to determine the ROS generation using a fluorescence microscopy. All analyses were performed on samples from 4 RA patients. (f) Immunohistochemical staining for HO-1, cPLA2, or vimentin and hematoxylin and eosin (H&E) staining of serial sections of ankle joints from mice injected with phosphate-buffered saline (sham) ((A)–(E)), mice injected with TNF-α ((F)–(J)), and mice injected with CORM-2 for 16 h followed by TNF-α for 24 h ((K)–(O)) were performed. Arrowheads indicate positive staining. Results are representative of 3 mice per experimental group. Results are representative of 3 independent experiments. In (a), (b), (c), and (g), values are the mean ± SEM. *P < 0.05; #P < 0.01 versus TNF-α alone.

Mentions: We have shown that CO-RM2 inhibited TNF-α-induced cPLA2 expression via HO-1 induction in RASFs (Figure 1). As upregulation of cPLA2 expression depends on activation of NF-κB, we next assessed whether CO-RM2 interfered with these processes. Chromatin was immunoprecipitated using an anti-p65 antibody, and the cPLA2 promoter region was amplified by PCR. As shown in Figure 7(a), TNF-α-induced p65 binding to the cPLA2 promoter was inhibited by pretreatment with CO-RM2, but not iCO-RM2. Moreover, exposure to TNF-α increased NF-κB promoter activity which was attenuated by pretreatment with CO-RM2 (Figure 7(b)). The classical NF-κB is activated by IκBα degradation, which occurs subsequent to IKKα/β phosphorylation. To investigate whether the inhibition of NF-κB promoter activity was due to the inhibition of IKKα/β and p65 phosphorylation, as shown in Figures 7(c) and 7(d), TNF-α-stimulated IKKα/β and p65 phosphorylation was attenuated by CO-RM2 but not iCO-RM2, during the period of observation. In addition, we demonstrated that TNF-α-stimulated generation of ROS was responsible for NF-κB transcriptional activity which was inhibited by pretreatment with CORM-2 (Figure 7(e)). On the other hand, CORM-2 had no effect on TNF-α-induced phosphorylation of p38 MAPK and JNK1/2 in RASFs (Figure 7(f)). These data demonstrated that HO-1 induction by CO-RM2 attenuates TNF-α-induced cPLA2 expression mediated through suppression of ROS and NF-κB. To confirm these results in in vivo studies, mice were intra-articularly administered with CO-RM2 for 16 h and then followed with TNF-α for 24 h. The images of immunohistochemical staining in the articular joints showed that the number of cPLA2-expressing cells was significantly higher in TNF-α-treated mice than those of PBS-treated mice (Figure 7(g)-(A, F)). Administration with CO-RM2 before TNF-α treatment resulted in decreased cPLA2 expression on synovial layer in the articular joints of mice (Figure 7(g)-(F, K)). The levels of cPLA2 expression normalized to vimentin are summarized in the bar graph (Figure 7(g), lower panel). These results suggested that CO-RM2 attenuates TNF-α-induced cPLA2 expression in the articular joints of mice.


HO-1 induction by CO-RM2 attenuates TNF-α-induced cytosolic phospholipase A2 expression via inhibition of PKCα-dependent NADPH oxidase/ROS and NF-κB.

Chi PL, Liu CJ, Lee IT, Chen YW, Hsiao LD, Yang CM - Mediators Inflamm. (2014)

Effect of CORM-2 on TNF-α-induced NF-κB activation and cPLA2 expression. (a) Cells were pretreated with CORM-2 or iCORM-2 for 16 h and then incubated with TNF-α for 2 h. Chromatin immunoprecipitation (ChIP) assays were performed using an anti-p65 antibody. (b) Cells were transfected with NF-κB-luc reporter gene, pretreated with or without CORM-2 for 16 h, and then incubated with TNF-α for 4 h. NF-κB promoter activity was determined. ((c), (d), and (f)) Cells were pretreated with or without CORM-2 or iCORM-2 for 16 h and then treated with TNF-α for the indicated time intervals. The levels of phospho-p65, phospho-IKKα/β, phospho-JNK1/2, phospho-p38 MAPK, and HO-1 were determined by Western blotting. (e) Cells were pretreated with or without CORM-2 for 16 h and then incubated with TNF-α for 90 min. The cells were added with (5 μM) DHE and images acquired to determine the ROS generation using a fluorescence microscopy. All analyses were performed on samples from 4 RA patients. (f) Immunohistochemical staining for HO-1, cPLA2, or vimentin and hematoxylin and eosin (H&E) staining of serial sections of ankle joints from mice injected with phosphate-buffered saline (sham) ((A)–(E)), mice injected with TNF-α ((F)–(J)), and mice injected with CORM-2 for 16 h followed by TNF-α for 24 h ((K)–(O)) were performed. Arrowheads indicate positive staining. Results are representative of 3 mice per experimental group. Results are representative of 3 independent experiments. In (a), (b), (c), and (g), values are the mean ± SEM. *P < 0.05; #P < 0.01 versus TNF-α alone.
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Related In: Results  -  Collection

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fig7: Effect of CORM-2 on TNF-α-induced NF-κB activation and cPLA2 expression. (a) Cells were pretreated with CORM-2 or iCORM-2 for 16 h and then incubated with TNF-α for 2 h. Chromatin immunoprecipitation (ChIP) assays were performed using an anti-p65 antibody. (b) Cells were transfected with NF-κB-luc reporter gene, pretreated with or without CORM-2 for 16 h, and then incubated with TNF-α for 4 h. NF-κB promoter activity was determined. ((c), (d), and (f)) Cells were pretreated with or without CORM-2 or iCORM-2 for 16 h and then treated with TNF-α for the indicated time intervals. The levels of phospho-p65, phospho-IKKα/β, phospho-JNK1/2, phospho-p38 MAPK, and HO-1 were determined by Western blotting. (e) Cells were pretreated with or without CORM-2 for 16 h and then incubated with TNF-α for 90 min. The cells were added with (5 μM) DHE and images acquired to determine the ROS generation using a fluorescence microscopy. All analyses were performed on samples from 4 RA patients. (f) Immunohistochemical staining for HO-1, cPLA2, or vimentin and hematoxylin and eosin (H&E) staining of serial sections of ankle joints from mice injected with phosphate-buffered saline (sham) ((A)–(E)), mice injected with TNF-α ((F)–(J)), and mice injected with CORM-2 for 16 h followed by TNF-α for 24 h ((K)–(O)) were performed. Arrowheads indicate positive staining. Results are representative of 3 mice per experimental group. Results are representative of 3 independent experiments. In (a), (b), (c), and (g), values are the mean ± SEM. *P < 0.05; #P < 0.01 versus TNF-α alone.
Mentions: We have shown that CO-RM2 inhibited TNF-α-induced cPLA2 expression via HO-1 induction in RASFs (Figure 1). As upregulation of cPLA2 expression depends on activation of NF-κB, we next assessed whether CO-RM2 interfered with these processes. Chromatin was immunoprecipitated using an anti-p65 antibody, and the cPLA2 promoter region was amplified by PCR. As shown in Figure 7(a), TNF-α-induced p65 binding to the cPLA2 promoter was inhibited by pretreatment with CO-RM2, but not iCO-RM2. Moreover, exposure to TNF-α increased NF-κB promoter activity which was attenuated by pretreatment with CO-RM2 (Figure 7(b)). The classical NF-κB is activated by IκBα degradation, which occurs subsequent to IKKα/β phosphorylation. To investigate whether the inhibition of NF-κB promoter activity was due to the inhibition of IKKα/β and p65 phosphorylation, as shown in Figures 7(c) and 7(d), TNF-α-stimulated IKKα/β and p65 phosphorylation was attenuated by CO-RM2 but not iCO-RM2, during the period of observation. In addition, we demonstrated that TNF-α-stimulated generation of ROS was responsible for NF-κB transcriptional activity which was inhibited by pretreatment with CORM-2 (Figure 7(e)). On the other hand, CORM-2 had no effect on TNF-α-induced phosphorylation of p38 MAPK and JNK1/2 in RASFs (Figure 7(f)). These data demonstrated that HO-1 induction by CO-RM2 attenuates TNF-α-induced cPLA2 expression mediated through suppression of ROS and NF-κB. To confirm these results in in vivo studies, mice were intra-articularly administered with CO-RM2 for 16 h and then followed with TNF-α for 24 h. The images of immunohistochemical staining in the articular joints showed that the number of cPLA2-expressing cells was significantly higher in TNF-α-treated mice than those of PBS-treated mice (Figure 7(g)-(A, F)). Administration with CO-RM2 before TNF-α treatment resulted in decreased cPLA2 expression on synovial layer in the articular joints of mice (Figure 7(g)-(F, K)). The levels of cPLA2 expression normalized to vimentin are summarized in the bar graph (Figure 7(g), lower panel). These results suggested that CO-RM2 attenuates TNF-α-induced cPLA2 expression in the articular joints of mice.

Bottom Line: Here, we reported that TNF-α-induced cPLA2 expression was mediated through TNFR1/PKCα-dependent signaling pathways, including NADPH oxidase (NOX) activation/ROS production and NF-κB activation.CO-RM2 significantly suppressed TNF-α-induced cPLA2 expression by inhibiting the ROS generation and the phosphorylation of NF-κB p65 and IKK α/β, but not the phosphorylation of p38 MAPK and JNK1/2.These results were further confirmed by a ChIP assay to detect the NF-κB DNA-binding activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 333, Taiwan.

ABSTRACT
Rheumatoid arthritis (RA) is characterized by chronic inflammatory infiltration of the synovium and elevation of proinflammatory cytokines. Cytosolic phospholipase A2 (cPLA2) is involved in the development of inflammatory diseases. Heme oxygenase-1 (HO-1) has been shown to possess anti-inflammatory properties. The objective of the study was to investigate the detailed mechanisms of TNF-α-induced cPLA2 expression and to determine whether carbon monoxide releasing molecule-2 (CO-RM2) suppresses TNF-α-induced expression of NF-κB-related proinflammatory genes, including cPLA2, via HO-1 induction in RA synovial fibroblasts (RASFs). Here, we reported that TNF-α-induced cPLA2 expression was mediated through TNFR1/PKCα-dependent signaling pathways, including NADPH oxidase (NOX) activation/ROS production and NF-κB activation. CO-RM2 significantly suppressed TNF-α-induced cPLA2 expression by inhibiting the ROS generation and the phosphorylation of NF-κB p65 and IKK α/β, but not the phosphorylation of p38 MAPK and JNK1/2. These results were further confirmed by a ChIP assay to detect the NF-κB DNA-binding activity. Our results demonstrated that induction of HO-1 by CO-RM2 exerted anti-inflammatory and antioxidant effects which were required in concert to prevent the activation of NF-κB leading to induction of various inflammatory genes implicated in the pathogenesis of RA.

Show MeSH
Related in: MedlinePlus