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Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

Lee JH, Jung NH, Lee BH, Kim SH, Jun JH - Allergy Asthma Immunol Res (2013)

Bottom Line: This result suggests that the stimulatory effects of endogenous and exogenous PGE2 on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway.This induction was suppressed by exogenous PGE2 and enhanced by blockage of the endogenous PGE2 effect by the PKA inhibitor or AKAP disruptors.The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Eulji Hospital, Eulji University School of Medicine, Seoul, Korea.

ABSTRACT

Purpose: Prostaglandin (PG) E2 is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE2 on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE2-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress.

Methods: We explored the involvement of HO-1 on PGE2 regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages.

Results: LPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE2 or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE2 on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE2 and enhanced by blockage of the endogenous PGE2 effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE2. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.

Conclusions: AKAP plays an important role in PGE2 regulation of COX-2 expression, and the suppression of HO-1 by PGE2-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.

No MeSH data available.


Related in: MedlinePlus

Enhancement of HO-1 activity suppresses NF-κB signaling. (A) Cells were pretreated with vehicle control, or the HO activator CoPP (10 µM) for 10 min and then treated with PGE2 (1 µM) for 10 min before the addition of LPS (1 µg/mL). After 1 h, cells were harvested and the nuclear proteins were extracted as described in the Material and Methods. Immunoblotting of nuclear extracts was performed using anti-nuclear factor (NF)-κB p65. Results are representative of those from three experiments. *P<0.05. (B) Immunostaining for nuclear p65 localization in cells after 1 h exposure to: control, LPS only, LPS plus PGE2 (1 µM), LPS plus PGE2 plus CoPP, and LPS plus CoPP. Images depict nuclear translocation of NF-κB (arrows at green nuclei depict the translocation of NF-κB). (C) NF-κB immunofluorescence density determined by normalization with respect to translocation fluorescence. The nonfluorescent control cells were treated with second antibody only. Data are summarized and expressed as the means±SEM of three independent experiments. *P<0.05.
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Figure 6: Enhancement of HO-1 activity suppresses NF-κB signaling. (A) Cells were pretreated with vehicle control, or the HO activator CoPP (10 µM) for 10 min and then treated with PGE2 (1 µM) for 10 min before the addition of LPS (1 µg/mL). After 1 h, cells were harvested and the nuclear proteins were extracted as described in the Material and Methods. Immunoblotting of nuclear extracts was performed using anti-nuclear factor (NF)-κB p65. Results are representative of those from three experiments. *P<0.05. (B) Immunostaining for nuclear p65 localization in cells after 1 h exposure to: control, LPS only, LPS plus PGE2 (1 µM), LPS plus PGE2 plus CoPP, and LPS plus CoPP. Images depict nuclear translocation of NF-κB (arrows at green nuclei depict the translocation of NF-κB). (C) NF-κB immunofluorescence density determined by normalization with respect to translocation fluorescence. The nonfluorescent control cells were treated with second antibody only. Data are summarized and expressed as the means±SEM of three independent experiments. *P<0.05.

Mentions: TLR4 responses are mainly mediated via NF-κB activation.19,20 To evaluate whether induction of HO-1 involves LPS-induced NF-κB activation, we stimulated cells for 1 h with LPS, then observed NF-κB p65 expression in the nuclear fraction via Western blotting. As shown in Fig. 6A, NF-κB p65 proteins translocated to the nucleus only with LPS treatment, and PGE2 augmented the p65 activation via TLR4 ligation. In addition, the HO-1 activator CoPP suppressed NF-κB p65 expression, and exogenous PGE2 partially restored its effect. These findings were confirmed by assessing the nuclear translocation of NF-κB p65 subunit by immunofluorescence microscopy and by quantifying the nuclear colocalization of p65 with DAPI. LPS enhanced p65 nuclear translocation in macrophages; however, LPS-induced p65 translocation was attenuated with the HO-1 activator CoPP (Fig. 6B and C). Taken together, these results demonstrate that the induction of HO-1 has an inhibitory effect on LPS-induced COX-2 expression through the attenuation of LPS-induced p65 translocation.


Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

Lee JH, Jung NH, Lee BH, Kim SH, Jun JH - Allergy Asthma Immunol Res (2013)

Enhancement of HO-1 activity suppresses NF-κB signaling. (A) Cells were pretreated with vehicle control, or the HO activator CoPP (10 µM) for 10 min and then treated with PGE2 (1 µM) for 10 min before the addition of LPS (1 µg/mL). After 1 h, cells were harvested and the nuclear proteins were extracted as described in the Material and Methods. Immunoblotting of nuclear extracts was performed using anti-nuclear factor (NF)-κB p65. Results are representative of those from three experiments. *P<0.05. (B) Immunostaining for nuclear p65 localization in cells after 1 h exposure to: control, LPS only, LPS plus PGE2 (1 µM), LPS plus PGE2 plus CoPP, and LPS plus CoPP. Images depict nuclear translocation of NF-κB (arrows at green nuclei depict the translocation of NF-κB). (C) NF-κB immunofluorescence density determined by normalization with respect to translocation fluorescence. The nonfluorescent control cells were treated with second antibody only. Data are summarized and expressed as the means±SEM of three independent experiments. *P<0.05.
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Figure 6: Enhancement of HO-1 activity suppresses NF-κB signaling. (A) Cells were pretreated with vehicle control, or the HO activator CoPP (10 µM) for 10 min and then treated with PGE2 (1 µM) for 10 min before the addition of LPS (1 µg/mL). After 1 h, cells were harvested and the nuclear proteins were extracted as described in the Material and Methods. Immunoblotting of nuclear extracts was performed using anti-nuclear factor (NF)-κB p65. Results are representative of those from three experiments. *P<0.05. (B) Immunostaining for nuclear p65 localization in cells after 1 h exposure to: control, LPS only, LPS plus PGE2 (1 µM), LPS plus PGE2 plus CoPP, and LPS plus CoPP. Images depict nuclear translocation of NF-κB (arrows at green nuclei depict the translocation of NF-κB). (C) NF-κB immunofluorescence density determined by normalization with respect to translocation fluorescence. The nonfluorescent control cells were treated with second antibody only. Data are summarized and expressed as the means±SEM of three independent experiments. *P<0.05.
Mentions: TLR4 responses are mainly mediated via NF-κB activation.19,20 To evaluate whether induction of HO-1 involves LPS-induced NF-κB activation, we stimulated cells for 1 h with LPS, then observed NF-κB p65 expression in the nuclear fraction via Western blotting. As shown in Fig. 6A, NF-κB p65 proteins translocated to the nucleus only with LPS treatment, and PGE2 augmented the p65 activation via TLR4 ligation. In addition, the HO-1 activator CoPP suppressed NF-κB p65 expression, and exogenous PGE2 partially restored its effect. These findings were confirmed by assessing the nuclear translocation of NF-κB p65 subunit by immunofluorescence microscopy and by quantifying the nuclear colocalization of p65 with DAPI. LPS enhanced p65 nuclear translocation in macrophages; however, LPS-induced p65 translocation was attenuated with the HO-1 activator CoPP (Fig. 6B and C). Taken together, these results demonstrate that the induction of HO-1 has an inhibitory effect on LPS-induced COX-2 expression through the attenuation of LPS-induced p65 translocation.

Bottom Line: This result suggests that the stimulatory effects of endogenous and exogenous PGE2 on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway.This induction was suppressed by exogenous PGE2 and enhanced by blockage of the endogenous PGE2 effect by the PKA inhibitor or AKAP disruptors.The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Eulji Hospital, Eulji University School of Medicine, Seoul, Korea.

ABSTRACT

Purpose: Prostaglandin (PG) E2 is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE2 on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE2-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress.

Methods: We explored the involvement of HO-1 on PGE2 regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages.

Results: LPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE2 or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE2 on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE2 and enhanced by blockage of the endogenous PGE2 effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE2. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.

Conclusions: AKAP plays an important role in PGE2 regulation of COX-2 expression, and the suppression of HO-1 by PGE2-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.

No MeSH data available.


Related in: MedlinePlus