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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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TNF-α induces NOX/ROS generation and cPLA2 expression via PKCα. (a) Cells were incubated with 30 ng/mL of TNF-α for the indicated time intervals. Next, the cells were added with (5 μM) DHE and the number of DHE-labeled red-colored cells was counted under a fluorescence microscope. Open bars indicate statistical analysis of DHE staining. NADPH oxidase activity was determined (shaded bars). (b) RASFs were pretreated with Gö6976 (1 μM), DPI (5 μM), or APO (50 μM) for 1 h and then stimulated with TNF-α for 90 min (open bars) or 1 h (shaded bars). ROS generation and NADPH oxidase activity were determined. (c) Cells were pretreated with NAC, DPI, or APO for 1 h and then incubated with TNF-α for 16 h. The expression of cPLA2 was determined by Western blotting. (d) Cells were transfected with scrambled or p47phox siRNA and then incubated with TNF-α for 16 hours. The levels of p47phox and cPLA2 protein were determined by Western blotting. Cells were incubated with (e) TNF-α for the indicated time intervals or (f) pretreated with DPI (5 μM) or Gö6976 (Gö, 1 μM) for 1 h and then incubated with TNF-α for 60 min. The membrane (ME) and cytosolic (CE) fractions were prepared and subjected to Western blot using an anti-p47phox antibody. Values are the mean ± SEM of samples from 4 RA patients from 3 independent experiments. In (a) and (e), *P < 0.05; #P < 0.01 versus vehicle alone. In (b), (c), (d), and (f), *P < 0.05; #P < 0.01 versus TNF-α alone.
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fig3: TNF-α induces NOX/ROS generation and cPLA2 expression via PKCα. (a) Cells were incubated with 30 ng/mL of TNF-α for the indicated time intervals. Next, the cells were added with (5 μM) DHE and the number of DHE-labeled red-colored cells was counted under a fluorescence microscope. Open bars indicate statistical analysis of DHE staining. NADPH oxidase activity was determined (shaded bars). (b) RASFs were pretreated with Gö6976 (1 μM), DPI (5 μM), or APO (50 μM) for 1 h and then stimulated with TNF-α for 90 min (open bars) or 1 h (shaded bars). ROS generation and NADPH oxidase activity were determined. (c) Cells were pretreated with NAC, DPI, or APO for 1 h and then incubated with TNF-α for 16 h. The expression of cPLA2 was determined by Western blotting. (d) Cells were transfected with scrambled or p47phox siRNA and then incubated with TNF-α for 16 hours. The levels of p47phox and cPLA2 protein were determined by Western blotting. Cells were incubated with (e) TNF-α for the indicated time intervals or (f) pretreated with DPI (5 μM) or Gö6976 (Gö, 1 μM) for 1 h and then incubated with TNF-α for 60 min. The membrane (ME) and cytosolic (CE) fractions were prepared and subjected to Western blot using an anti-p47phox antibody. Values are the mean ± SEM of samples from 4 RA patients from 3 independent experiments. In (a) and (e), *P < 0.05; #P < 0.01 versus vehicle alone. In (b), (c), (d), and (f), *P < 0.05; #P < 0.01 versus TNF-α alone.

Mentions: ROS are released during the inflammatory responses of joint tissues and associated with cartilage degradation in RA [6, 25, 26]. TNF-α induces expression of several genes mediated through NOX-dependent ROS intermediaries including H2O2 and superoxide anion [11]. First, we measured whether TNF-α could induce intracellular ROS production. As shown in Figure 3(a), TNF-α induced a significant increase in NOX activity and ROS production. PKC isoforms, mainly, PKCα, βII, and δ, have been characterized as an important activator of NOX [27, 28]. Here, we also showed that pretreatment with the inhibitors of PKCα (Gö6976) and NOX (DPI and APO) markedly reduced TNF-α-induced NOX activity and ROS levels (Figure 3(b)), suggesting that TNF-α induced ROS generation via PKCα/NOX in RASFs. We further established that TNF-α induced cPLA2 expression via NOX and ROS by using NAC, DPI, APO, or, p47phox siRNA. As shown in Figures 3(c) and 3(d), pretreatment with NAC, DPI, or APO and transfection with p47phox siRNA significantly abrogated TNF-α-induced cPLA2 expression. On the other hand, we observed that TNF-α time-dependently stimulated p47phox translocation from the cytosol to the membrane, which was inhibited by pretreatment with DPI or Gö6976 (Figures 3(e) and 3(f)). Taken together, these data suggested that TNF-α induces cPLA2 expression via PKCα-dependent NOX activation and ROS generation in RASFs.


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)

TNF-α induces NOX/ROS generation and cPLA2 expression via PKCα. (a) Cells were incubated with 30 ng/mL of TNF-α for the indicated time intervals. Next, the cells were added with (5 μM) DHE and the number of DHE-labeled red-colored cells was counted under a fluorescence microscope. Open bars indicate statistical analysis of DHE staining. NADPH oxidase activity was determined (shaded bars). (b) RASFs were pretreated with Gö6976 (1 μM), DPI (5 μM), or APO (50 μM) for 1 h and then stimulated with TNF-α for 90 min (open bars) or 1 h (shaded bars). ROS generation and NADPH oxidase activity were determined. (c) Cells were pretreated with NAC, DPI, or APO for 1 h and then incubated with TNF-α for 16 h. The expression of cPLA2 was determined by Western blotting. (d) Cells were transfected with scrambled or p47phox siRNA and then incubated with TNF-α for 16 hours. The levels of p47phox and cPLA2 protein were determined by Western blotting. Cells were incubated with (e) TNF-α for the indicated time intervals or (f) pretreated with DPI (5 μM) or Gö6976 (Gö, 1 μM) for 1 h and then incubated with TNF-α for 60 min. The membrane (ME) and cytosolic (CE) fractions were prepared and subjected to Western blot using an anti-p47phox antibody. Values are the mean ± SEM of samples from 4 RA patients from 3 independent experiments. In (a) and (e), *P < 0.05; #P < 0.01 versus vehicle alone. In (b), (c), (d), and (f), *P < 0.05; #P < 0.01 versus TNF-α alone.
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Related In: Results  -  Collection

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fig3: TNF-α induces NOX/ROS generation and cPLA2 expression via PKCα. (a) Cells were incubated with 30 ng/mL of TNF-α for the indicated time intervals. Next, the cells were added with (5 μM) DHE and the number of DHE-labeled red-colored cells was counted under a fluorescence microscope. Open bars indicate statistical analysis of DHE staining. NADPH oxidase activity was determined (shaded bars). (b) RASFs were pretreated with Gö6976 (1 μM), DPI (5 μM), or APO (50 μM) for 1 h and then stimulated with TNF-α for 90 min (open bars) or 1 h (shaded bars). ROS generation and NADPH oxidase activity were determined. (c) Cells were pretreated with NAC, DPI, or APO for 1 h and then incubated with TNF-α for 16 h. The expression of cPLA2 was determined by Western blotting. (d) Cells were transfected with scrambled or p47phox siRNA and then incubated with TNF-α for 16 hours. The levels of p47phox and cPLA2 protein were determined by Western blotting. Cells were incubated with (e) TNF-α for the indicated time intervals or (f) pretreated with DPI (5 μM) or Gö6976 (Gö, 1 μM) for 1 h and then incubated with TNF-α for 60 min. The membrane (ME) and cytosolic (CE) fractions were prepared and subjected to Western blot using an anti-p47phox antibody. Values are the mean ± SEM of samples from 4 RA patients from 3 independent experiments. In (a) and (e), *P < 0.05; #P < 0.01 versus vehicle alone. In (b), (c), (d), and (f), *P < 0.05; #P < 0.01 versus TNF-α alone.
Mentions: ROS are released during the inflammatory responses of joint tissues and associated with cartilage degradation in RA [6, 25, 26]. TNF-α induces expression of several genes mediated through NOX-dependent ROS intermediaries including H2O2 and superoxide anion [11]. First, we measured whether TNF-α could induce intracellular ROS production. As shown in Figure 3(a), TNF-α induced a significant increase in NOX activity and ROS production. PKC isoforms, mainly, PKCα, βII, and δ, have been characterized as an important activator of NOX [27, 28]. Here, we also showed that pretreatment with the inhibitors of PKCα (Gö6976) and NOX (DPI and APO) markedly reduced TNF-α-induced NOX activity and ROS levels (Figure 3(b)), suggesting that TNF-α induced ROS generation via PKCα/NOX in RASFs. We further established that TNF-α induced cPLA2 expression via NOX and ROS by using NAC, DPI, APO, or, p47phox siRNA. As shown in Figures 3(c) and 3(d), pretreatment with NAC, DPI, or APO and transfection with p47phox siRNA significantly abrogated TNF-α-induced cPLA2 expression. On the other hand, we observed that TNF-α time-dependently stimulated p47phox translocation from the cytosol to the membrane, which was inhibited by pretreatment with DPI or Gö6976 (Figures 3(e) and 3(f)). Taken together, these data suggested that TNF-α induces cPLA2 expression via PKCα-dependent NOX activation and ROS generation in RASFs.

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