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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

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.
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Figure 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.

Mentions: HO-1 is induced by TLR4 ligation.18 Because induction of HO-1 inhibits LPS-induced COX-2 expression, we hypothesized that PGE2-PKA-AKAP signaling could suppress LPS-induced HO-1 expression and cause positive feedback augmentation of COX-2 expression. As shown in Fig. 3, the HO-1 activator CoPP significantly reduced LPS-induced COX-2 expression, and exogenous PGE2 restored CoPP-inhibited responses to LPS at 6 h. Exogenous PGE2 attenuated LPS-induced HO-1 expression and modestly blocked the potentiating effects of exogenous CoPP on LPS-induced HO-1. This suggests that suppression of LPS-induced HO-1 expression by the addition of PGE2 is responsible for the amplification of COX-2 in response to ligation of TLR4. To delineate the role of endogenous PGE2-PKA-AKAP signaling on LPS-induced HO-1 expression, we challenged cells with LPS in the presence or absence of a PKA inhibitor (KT5720) or AKAP disruptors (Ht31 or RIAD). The PKA inhibitor enhanced LPS-induced HO-1 expression, and these potentiating effects were blocked by exogenous PGE2 (Fig. 4A). To determine whether AKAPs are involved in the PKA-dependent suppression of LPS-induced HO-1, we disrupted the potential PKA RI-AKAP interactions with RIAD peptide and RII-AKAP interactions with Ht31 peptide. Both AKAP disruptors augmented LPS-induced HO-1 expression to an extent similar to that observed with the PKA inhibitor KT5720 (Fig. 4B). To confirm that PKA is indeed responsible for blocking HO-1 expression, we measured bilirubin generated from HO activity. In the presence of KT5720, bilirubin increased markedly, and this effect was restored by exogenous PGE2 (Fig. 5). Taken together, these data clearly demonstrate that suppression of HO-1 expression via PGE2-PKA-AKAP signaling potentiates LPS-induced COX2 expression in RAW 264.7 macrophages.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3756181&req=5

Figure 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.
Mentions: HO-1 is induced by TLR4 ligation.18 Because induction of HO-1 inhibits LPS-induced COX-2 expression, we hypothesized that PGE2-PKA-AKAP signaling could suppress LPS-induced HO-1 expression and cause positive feedback augmentation of COX-2 expression. As shown in Fig. 3, the HO-1 activator CoPP significantly reduced LPS-induced COX-2 expression, and exogenous PGE2 restored CoPP-inhibited responses to LPS at 6 h. Exogenous PGE2 attenuated LPS-induced HO-1 expression and modestly blocked the potentiating effects of exogenous CoPP on LPS-induced HO-1. This suggests that suppression of LPS-induced HO-1 expression by the addition of PGE2 is responsible for the amplification of COX-2 in response to ligation of TLR4. To delineate the role of endogenous PGE2-PKA-AKAP signaling on LPS-induced HO-1 expression, we challenged cells with LPS in the presence or absence of a PKA inhibitor (KT5720) or AKAP disruptors (Ht31 or RIAD). The PKA inhibitor enhanced LPS-induced HO-1 expression, and these potentiating effects were blocked by exogenous PGE2 (Fig. 4A). To determine whether AKAPs are involved in the PKA-dependent suppression of LPS-induced HO-1, we disrupted the potential PKA RI-AKAP interactions with RIAD peptide and RII-AKAP interactions with Ht31 peptide. Both AKAP disruptors augmented LPS-induced HO-1 expression to an extent similar to that observed with the PKA inhibitor KT5720 (Fig. 4B). To confirm that PKA is indeed responsible for blocking HO-1 expression, we measured bilirubin generated from HO activity. In the presence of KT5720, bilirubin increased markedly, and this effect was restored by exogenous PGE2 (Fig. 5). Taken together, these data clearly demonstrate that suppression of HO-1 expression via PGE2-PKA-AKAP signaling potentiates LPS-induced COX2 expression in RAW 264.7 macrophages.

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