Role of PPAR and PKC isoforms on NOSII activity induced by lipopolysaccharide in murine macrophages

Maria Jose Zarzuelo1,2, Rosario Jimenez2, Juan Duarte2, Hime Gashaw1, Lucy K Bailey1, Mark Paul-Clark1, Laura Moreno1, Jane A Mitchell1. 1Cardiothoracic Pharmacology, National Heart and Lung Institute, Imperial College, London, United Kingdom, 2Pharmacology Department, School of Pharmacy, University of Granada, Granada, Spain.

The peroxisome proliferator-activated receptors (PPARs) are a group of ligand-activated nuclear receptors. There are three PPAR isoforms: α, β/δ and γ. PPAR ligands have anti-inflammatory actions in some vascular and inflammatory cells suggesting that these types of drugs may represent a promising therapeutic strategy for a variety of inflammation-related disorders (Moraes et al., 2006). Our group has shown that PPARβ/δ agonists inhibit NOSII activity in murine macrophages (Bailey et al., 2007). Recently, we have also shown that PPARβ/δ agonists inhibit platelet aggregation by repressing PKC α activation (Ali et al., 2009). In the current study we aimed to analyse the effects of PPAR activation and PKC inhibition on lipopolysaccharide (LPS) induced nitric oxide synthase (NOS) II activity in murine macrophages (J774). J774 macrophages were cultured using standard techniques in a humidified incubator at 37°C, 5% CO2 and 95% air. Cells were stimulated with LPS (1μg/ml) for 24 hours before NOSII activity was assessed by the accumulation of nitrite using the Griess reaction.

Figure 1: Effect of different PPAR agonists (A) and PKC inhibitors (B) on NOSII activity induced by LPS (1 μg/ml). GW0742 (PPARβ/δ agonist), rosiglitazone (PPARγ agonist), bezafibrate (PPARα agonist), Gö6976 (blocks PKC α, β, and μ), Gö6983 (blocks PKC α, β, γ, δ, ζ and λ) or vehicule (Control; 0.1 % dimethyl sulfoxide) were added 90 minutes prior to LPS. Data is the mean±S.E.M. of n = 8-9. Drug induced responses were compared with control using one-sample t-test. *GW0742, +Rosiglitazone, #Gö6976 denotes P <0.05.

Murine J774.2 macrophages evoked increased NOSII activity when challenged with LPS (a TLR4 agonist). Pre-treatment of cells with rosiglitazone or bezafibrate had no significant effect on NOSII activity in LPS-stimulated macrophages. However, the PPARβ/δ agonist GW0742 caused concentration dependent inhibition of NO release induced by LPS.

The data confirm our previous observations that GW0742 inhibits NOSII activity in J774 cells. The finding that Gö6976, but not Gö6983, also inhibits NOSII does not support a role for PKC α in the effects of GW0742 in this system, but does reveal a potentially novel role for PKC μ in NOSII induction in response to LPS.

1. Ali, FY, Hall, MG, Desvergne, B, Warner, TD, Mitchell, JA (2009). Arterioscler Thromb Vasc Biol 29(11): 1871-1873.]
2. Bailey, L, Moreno, L, Paul-Clark, M, Mitchell JA (2007) Proceedings of the British Pharmacological Society at http://www.pA2online.org/abstracts/Vol5Issue2abst036P.pdf.
3. Moraes, LA, Piqueras, L, Bishop-Bailey, D (2006). Pharmacol Ther 110(3): 371-385.