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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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Regulation of cPLA2 expression in TNF-α-treated mice. (a) Immunohistochemical staining for cPLA2 and vimentin and hematoxylin and eosin (H&E) staining in serial sections of ankle joints from PBS-treated mice (sham; (A)–(C)), TNF-α-injected mice (TNF-α; (D)–(F)), Gö6976-pretreated mice (Gö/TNF-α; (G)–(I)), NAC-pretreated mice (NAC/TNF-α; (J)–(L)), and helenalin-pretreated mice (HLN/TNF-α; (M)–(O)) are shown. Microscopic observation showed vimentin in the synovial membrane of ankle joints, and synovial fibroblasts overlapping with cPLA2 expression. Arrowheads indicate positive staining. (b) The diagrammatic representation of quantitative data of cPLA2-positive cells in the articular joints of mice injected with the indicated inhibitors. Results are representative of 3 mice per experimental group. In (b), values are the mean ± SEM. #P < 0.01, as compared with the cells exposed to TNF-α alone.
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fig6: Regulation of cPLA2 expression in TNF-α-treated mice. (a) Immunohistochemical staining for cPLA2 and vimentin and hematoxylin and eosin (H&E) staining in serial sections of ankle joints from PBS-treated mice (sham; (A)–(C)), TNF-α-injected mice (TNF-α; (D)–(F)), Gö6976-pretreated mice (Gö/TNF-α; (G)–(I)), NAC-pretreated mice (NAC/TNF-α; (J)–(L)), and helenalin-pretreated mice (HLN/TNF-α; (M)–(O)) are shown. Microscopic observation showed vimentin in the synovial membrane of ankle joints, and synovial fibroblasts overlapping with cPLA2 expression. Arrowheads indicate positive staining. (b) The diagrammatic representation of quantitative data of cPLA2-positive cells in the articular joints of mice injected with the indicated inhibitors. Results are representative of 3 mice per experimental group. In (b), values are the mean ± SEM. #P < 0.01, as compared with the cells exposed to TNF-α alone.

Mentions: To further confirm our in vitro results, we tested the effect of CORM-2 on the expression of cPLA2 and HO-1 in the ankle joints of mice challenged with TNF-α  in vivo. As shown in Figure 6(a)-(D), the synovial layer in TNF-α-treated ankle joints strongly expressed cPLA2, which was reduced by pretreatment with Gö6976, NAC, or helenalin (Figure 6(a)-(G), J, and M). The quantitative data of immunohistochemical staining (Figure 6(b)) demonstrated that TNF-α-induced cPLA2 expression occurs both in vitro and in vivo, which is mediated via PKCα-dependent NOX activation/ROS generation and NF-κB activation.


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)

Regulation of cPLA2 expression in TNF-α-treated mice. (a) Immunohistochemical staining for cPLA2 and vimentin and hematoxylin and eosin (H&E) staining in serial sections of ankle joints from PBS-treated mice (sham; (A)–(C)), TNF-α-injected mice (TNF-α; (D)–(F)), Gö6976-pretreated mice (Gö/TNF-α; (G)–(I)), NAC-pretreated mice (NAC/TNF-α; (J)–(L)), and helenalin-pretreated mice (HLN/TNF-α; (M)–(O)) are shown. Microscopic observation showed vimentin in the synovial membrane of ankle joints, and synovial fibroblasts overlapping with cPLA2 expression. Arrowheads indicate positive staining. (b) The diagrammatic representation of quantitative data of cPLA2-positive cells in the articular joints of mice injected with the indicated inhibitors. Results are representative of 3 mice per experimental group. In (b), values are the mean ± SEM. #P < 0.01, as compared with the cells exposed to TNF-α alone.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3927740&req=5

fig6: Regulation of cPLA2 expression in TNF-α-treated mice. (a) Immunohistochemical staining for cPLA2 and vimentin and hematoxylin and eosin (H&E) staining in serial sections of ankle joints from PBS-treated mice (sham; (A)–(C)), TNF-α-injected mice (TNF-α; (D)–(F)), Gö6976-pretreated mice (Gö/TNF-α; (G)–(I)), NAC-pretreated mice (NAC/TNF-α; (J)–(L)), and helenalin-pretreated mice (HLN/TNF-α; (M)–(O)) are shown. Microscopic observation showed vimentin in the synovial membrane of ankle joints, and synovial fibroblasts overlapping with cPLA2 expression. Arrowheads indicate positive staining. (b) The diagrammatic representation of quantitative data of cPLA2-positive cells in the articular joints of mice injected with the indicated inhibitors. Results are representative of 3 mice per experimental group. In (b), values are the mean ± SEM. #P < 0.01, as compared with the cells exposed to TNF-α alone.
Mentions: To further confirm our in vitro results, we tested the effect of CORM-2 on the expression of cPLA2 and HO-1 in the ankle joints of mice challenged with TNF-α  in vivo. As shown in Figure 6(a)-(D), the synovial layer in TNF-α-treated ankle joints strongly expressed cPLA2, which was reduced by pretreatment with Gö6976, NAC, or helenalin (Figure 6(a)-(G), J, and M). The quantitative data of immunohistochemical staining (Figure 6(b)) demonstrated that TNF-α-induced cPLA2 expression occurs both in vitro and in vivo, which is mediated via PKCα-dependent NOX activation/ROS generation and NF-κB activation.

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