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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

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.
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Figure 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.

Mentions: In response to stress such as hypoxia, cytokines, and infection, HO-1 is induced to have an anti-oxidant and anti-inflammatory function. HO-1 regulates cellular contents with suppression of LPS-induced inflammation.13 To determine the effects of HO-1 on LPS-induced COX-2 expression in RAW 264.7 macrophages, we examined the effect of the HO inhibitor ZnPP (20 µM) or the HO activator CoPP (10 µM) at 6 or 24 h post-treatment. As shown in Fig. 2, enhancement of HO-1 expression by CoPP treatment suppressed LPS-induced COX-2 expression at both time points. In contrast, inhibition of HO-1 expression by the HO inhibitor ZnPP augmented LPS-induced COX-2 expression. Notably, COX-2 expression was not influenced by HO-1 expression level in the absence of LPS. This suggests that induction of HO-1 can inhibit the intracellular signaling events associated with cellular expression of COX-2 enzyme in response to LPS.


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)

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

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

Figure 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.
Mentions: In response to stress such as hypoxia, cytokines, and infection, HO-1 is induced to have an anti-oxidant and anti-inflammatory function. HO-1 regulates cellular contents with suppression of LPS-induced inflammation.13 To determine the effects of HO-1 on LPS-induced COX-2 expression in RAW 264.7 macrophages, we examined the effect of the HO inhibitor ZnPP (20 µM) or the HO activator CoPP (10 µM) at 6 or 24 h post-treatment. As shown in Fig. 2, enhancement of HO-1 expression by CoPP treatment suppressed LPS-induced COX-2 expression at both time points. In contrast, inhibition of HO-1 expression by the HO inhibitor ZnPP augmented LPS-induced COX-2 expression. Notably, COX-2 expression was not influenced by HO-1 expression level in the absence of LPS. This suggests that induction of HO-1 can inhibit the intracellular signaling events associated with cellular expression of COX-2 enzyme in response to LPS.

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