Abstract
Purpose: :
Proinflammatory gene expression is induced in age-related macular degeneration (AMD). The bioactive phospholipid platelet-activating factor (PAF) is a potent inducer of pro-inflammatory gene expression. Therefore we have studied here the ability of low molecular weight PAF-receptor antagonists to block proinflamatory gene expression in RPE cells. The PAF receptor antagonists, LAU (Louisiana Alcala Universities) compounds, are potent inhibitors of apoptosis and mitigate inflammatory responses in different cell types. The purpose of this research is to evaluate the effect of structurally-related LAU compounds on interleukin-1β (IL-1β)-mediated upregulation of NF-ΚB and COX-2, in a human retinal pigment epithelial cell line (ARPE-19).
Methods: :
ARPE-19 cells, grown overnight, were transfected with an NF-ΚB-Luciferas (LUC) construct containing four tandem repeats of NF-ΚB enhancer sequences (Promega) linked to luciferase reporter gene by Fugene-6, according to the manufacturer’s protocol (Roche). A promoterless β-galactosidase construct was used to co-transfect as transfection control. Transfected cells were harvested, cell extracts were made, and luciferase assays were performed using luciferin as a substrate. In some experiments, an NF-ΚB-LUC stable transfectant cell line (NF-ΚB-LUC-ST), generated in this laboratory, was also used. Western blot analysis of COX-2 was performed using the COX-2 specific antibody (SantaCruz Biotech).
Results: :
Our results demonstrate that LAU compounds saliently inhibit IL-1β-induced NF-ΚB-LUC activity in a concentration-dependent manner in ARPE-19 and NF-ΚB-LUC stable transfectant (NF-ΚB-LUC-ST) cell lines. Utmost inhibition was achieved with 1μM, and the least inhibition with 10nM. Inhibitory capacity varied amongst the compounds assayed. Some were clearly efficacious (i.e. LAU-09030), while others were not as effective (i.e. LAU-09001). Additionally, IL-1β-induced COX-2 activity was attenuated by LAU compounds in both cell lines. In vitro LAU effects were complemented by in vivo cytoprotective activities in ARPE-19 cells.
Conclusions: :
LAU compounds were able to inhibit substantially the IL-1β-induced induction of NF-ΚB luciferase activity in ARPE-19 and NF-ΚB-LUC-ST cell lines and COX-2 induction. The inhibition of pro-inflammatory COX-2 and NF-ΚB by PAF receptor antagonists shows that the LAU compounds have therapeutic potential for inflammatory/degenerative diseases that display enhanced induction of proinflammtory signaling.
Keywords: retinal pigment epithelium • protective mechanisms • signal transduction