Allergy Asthma Immunol Res.  2013 Sep;5(5):329-336. 10.4168/aair.2013.5.5.329.

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

• KMID: 2260815
• DOI: http://doi.org/10.4168/aair.2013.5.5.329

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-kappaB 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.

Keyword

Cyclooxygenase-2; heme oxygenase-1; lipopolysaccharide; prostaglandin E2; macrophages

MeSH Terms

Arachidonic Acid
Copper
Cyclic AMP
Cyclic AMP-Dependent Protein Kinases
Cyclooxygenase 2
Dinoprostone
Heme
Heme Oxygenase-1
Homeostasis
Inflammation
Intracellular Signaling Peptides and Proteins
Macrophages
NF-kappa B
Phosphotransferases
Toll-Like Receptors
Arachidonic Acid
Copper
Cyclic AMP
Cyclic AMP-Dependent Protein Kinases
Cyclooxygenase 2
Dinoprostone
Heme
Heme Oxygenase-1
Intracellular Signaling Peptides and Proteins
NF-kappa B
Phosphotransferases
Toll-Like Receptors

Figure

• 

  Fig. 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.

• 

  Fig. 2 Heme oxygenase-1 (HO-1) activation results in decreased LPS-induced COX-2 expression. Cells were pretreated with the HO activator copper protoporphyrin (CoPP, 10 µM) or the HO inhibitor zinc protoporphyrin (ZnPP, 20 µM) for 10 min followed by LPS (1 µg/mL) or vehicle for 6 h (left) and 24 h (right). Cell lysates at the indicated time points were subject to Western blot analysis of COX-2, HO-1, and β-actin. The blots are representative of three independently performed experiments.

• 

  Fig. 3 Exogenous PGE2 suppresses LPS-induced HO-1 expression. Cells were pretreated with CoPP (10 µM) for 10 min followed by PGE2 (1 µM) for 10 min and then incubated with LPS (1 µg/mL) for 6 h. Lysates were subjected to Western blot analysis for COX-2, HO-1, and β-actin. Results from one experiment of three are shown. Relative expression levels of COX-2 and HO-1 were determined by densitometry analysis of immunoblots from three different experiments, normalized for β-actin expression, and expressed as percent of LPS alone. Data are the means±SEM of three independent experiments. *P<0.05.

• 

  Fig. 4 PKA/AKAP interaction is required for PGE2-suppressed effects on LPS-induced HO-1 expression. (A) Cells were pretreated with the PKA inhibitor KT5720 (1 µM) for 30 min followed by PGE2 for 10 min before incubation for 6 h with LPS. (B) Cells were pretreated with Ht31 (25 µM) or RIAD (25 µM) for 20 min and then incubated with LPS (1 µg/mL) for 6 h. In all experiments, lysates were subjected to Western blot analysis for HO-1 and β-actin. Results from one experiment of three are shown (upper). Relative expression of COX-2 and HO-1 were determined by densitometry analysis of immunoblots from three different experiments, normalized for β-actin expression, and expressed as percent of LPS alone (lower). Data represent the means±SEM of three separate experiments. *P<0.05.

• 

  Fig. 5 Inhibition of PKA induces an increase in HO-1 activity. Cells were pretreated with the PKA inhibitor KT5720 (1 µM) for 20 min followed by PGE2 (1 µM) or vehicle for 10 min and then incubated with LPS for 6 h. Supernatants were harvested, and HO-1 activity was measured as described in the Materials and Methods. Data represent the means±SEM of three separate experiments. *P< 0.05.

• 

  Fig. 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.

• 

  Fig. 7 Schematic representation of the proposed mechanism by which PGE2 signaling amplifies COX-2 expression through an autocrine manner in LPS-stimulated macrophages. Upon LPS binding to the toll-like receptor 4 (TLR4), the NF-κB signaling pathway becomes activated, leading to an increase in COX-2 expression, which in turn activates cAMP/PKA/AKAP through the production of PGE2. The PKA-AKAP complex blocks HO-1 activity, which results in enhanced translocation of NF-κB p65 to the nucleus.

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