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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

The cyclic AMP/protein kinase A/A-kinase anchoring protein (cAMP/PKA/AKAP) axis is responsible for prostaglandin E2 (PGE2)-potentiated lipopolysaccharide (LPS)-induced COX-2 expression in RAW 264.7 macrophages. (A) Cells were pretreated with the COX inhibitor indomethacin (Indo, 5 µM) for 30 min followed by PGE2 (1 µM) for 10 min and incubated with LPS (1 µg/mL) for another 24 h. (B) Cells were incubated with dibutyryl cAMP (1 µM) for 10 min followed by LPS for 24 h. (C) Cells were pretreated with PKA inhibitor KT5720 (1 µM) for 30 min followed by PGE2 for 10 min before incubation for 24 h with LPS. (D) Cells were pretreated for 20 min with the AKAP/PKA RII-specific disruptor peptide Ht31 (25 µM) or the AKAP/PKA RI-specific disruptor RIAD (25 µM) followed by LPS for 24 h. In all experiments, cell lysates (30 µg protein) were subjected to Western blot analysis of COX-2 and β-actin, upper. Results from one experiment of three are shown. Relative expression of COX-2 was determined by densitometric analysis of immunoblots from three different experiments, normalized for β-actin expression, and expressed as percent of LPS alone, lower. The data are the means±S.E. values of three independent experiments, each performed in duplicate. *P<0.05.
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Figure 1: The cyclic AMP/protein kinase A/A-kinase anchoring protein (cAMP/PKA/AKAP) axis is responsible for prostaglandin E2 (PGE2)-potentiated lipopolysaccharide (LPS)-induced COX-2 expression in RAW 264.7 macrophages. (A) Cells were pretreated with the COX inhibitor indomethacin (Indo, 5 µM) for 30 min followed by PGE2 (1 µM) for 10 min and incubated with LPS (1 µg/mL) for another 24 h. (B) Cells were incubated with dibutyryl cAMP (1 µM) for 10 min followed by LPS for 24 h. (C) Cells were pretreated with PKA inhibitor KT5720 (1 µM) for 30 min followed by PGE2 for 10 min before incubation for 24 h with LPS. (D) Cells were pretreated for 20 min with the AKAP/PKA RII-specific disruptor peptide Ht31 (25 µM) or the AKAP/PKA RI-specific disruptor RIAD (25 µM) followed by LPS for 24 h. In all experiments, cell lysates (30 µg protein) were subjected to Western blot analysis of COX-2 and β-actin, upper. Results from one experiment of three are shown. Relative expression of COX-2 was determined by densitometric analysis of immunoblots from three different experiments, normalized for β-actin expression, and expressed as percent of LPS alone, lower. The data are the means±S.E. values of three independent experiments, each performed in duplicate. *P<0.05.

Mentions: To determine the role of endogenous prostanoids in LPS-induced COX-2 expression in RAW 264.7 macrophages, cells were challenged with LPS (1 µg/mL) in the absence or presence of the nonselective COX inhibitor indomethacin (5 µM). Indomethacin significantly reduced the LPS-induced expression of COX-2 protein (Fig. 1A), implicating that an endogenous prostanoid potentiates COX-2 expression. LPS-induced COX-2 expression was enhanced by pretreatment of macrophages with PGE2, which partially restored the inhibitory effects of indomethacin (Fig. 1A). This suggests that PGE2 involves the expression of COX-2 enzyme in response to LPS by modulating intracellular signaling events.


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)

The cyclic AMP/protein kinase A/A-kinase anchoring protein (cAMP/PKA/AKAP) axis is responsible for prostaglandin E2 (PGE2)-potentiated lipopolysaccharide (LPS)-induced COX-2 expression in RAW 264.7 macrophages. (A) Cells were pretreated with the COX inhibitor indomethacin (Indo, 5 µM) for 30 min followed by PGE2 (1 µM) for 10 min and incubated with LPS (1 µg/mL) for another 24 h. (B) Cells were incubated with dibutyryl cAMP (1 µM) for 10 min followed by LPS for 24 h. (C) Cells were pretreated with PKA inhibitor KT5720 (1 µM) for 30 min followed by PGE2 for 10 min before incubation for 24 h with LPS. (D) Cells were pretreated for 20 min with the AKAP/PKA RII-specific disruptor peptide Ht31 (25 µM) or the AKAP/PKA RI-specific disruptor RIAD (25 µM) followed by LPS for 24 h. In all experiments, cell lysates (30 µg protein) were subjected to Western blot analysis of COX-2 and β-actin, upper. Results from one experiment of three are shown. Relative expression of COX-2 was determined by densitometric analysis of immunoblots from three different experiments, normalized for β-actin expression, and expressed as percent of LPS alone, lower. The data are the means±S.E. values of three independent experiments, each performed in duplicate. *P<0.05.
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Figure 1: The cyclic AMP/protein kinase A/A-kinase anchoring protein (cAMP/PKA/AKAP) axis is responsible for prostaglandin E2 (PGE2)-potentiated lipopolysaccharide (LPS)-induced COX-2 expression in RAW 264.7 macrophages. (A) Cells were pretreated with the COX inhibitor indomethacin (Indo, 5 µM) for 30 min followed by PGE2 (1 µM) for 10 min and incubated with LPS (1 µg/mL) for another 24 h. (B) Cells were incubated with dibutyryl cAMP (1 µM) for 10 min followed by LPS for 24 h. (C) Cells were pretreated with PKA inhibitor KT5720 (1 µM) for 30 min followed by PGE2 for 10 min before incubation for 24 h with LPS. (D) Cells were pretreated for 20 min with the AKAP/PKA RII-specific disruptor peptide Ht31 (25 µM) or the AKAP/PKA RI-specific disruptor RIAD (25 µM) followed by LPS for 24 h. In all experiments, cell lysates (30 µg protein) were subjected to Western blot analysis of COX-2 and β-actin, upper. Results from one experiment of three are shown. Relative expression of COX-2 was determined by densitometric analysis of immunoblots from three different experiments, normalized for β-actin expression, and expressed as percent of LPS alone, lower. The data are the means±S.E. values of three independent experiments, each performed in duplicate. *P<0.05.
Mentions: To determine the role of endogenous prostanoids in LPS-induced COX-2 expression in RAW 264.7 macrophages, cells were challenged with LPS (1 µg/mL) in the absence or presence of the nonselective COX inhibitor indomethacin (5 µM). Indomethacin significantly reduced the LPS-induced expression of COX-2 protein (Fig. 1A), implicating that an endogenous prostanoid potentiates COX-2 expression. LPS-induced COX-2 expression was enhanced by pretreatment of macrophages with PGE2, which partially restored the inhibitory effects of indomethacin (Fig. 1A). This suggests that PGE2 involves the expression of COX-2 enzyme in response to LPS by modulating intracellular signaling events.

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