Increased transcription of IL-8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids.
Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) upregulates a spectrum of inflammatory cytokines and adhesion molecules different from those induced by classic inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide. Interestingly, Ox-PAPC also induces the expression of a set of proteins similar to those induced by TNF-alpha or lipopolysaccharide, which include the chemokines monocyte chemotactic protein-1 (MCP-1) and interleukin (IL)-8. To elucidate the molecular mechanisms of Ox-PAPC-induced gene expression and to determine whether Ox-PAPC and other inflammatory mediators such as TNF-alpha utilize common signaling pathways, we examined the transcriptional regulation of IL-8 by Ox-PAPC and TNF-alpha in human aortic endothelial cells. Both Ox-PAPC and TNF-alpha induced the expression of IL-8 mRNA in a dose-dependent fashion; however, the kinetics of IL-8 mRNA accumulation between the 2 ligands differed. Ox-PAPC-induced IL-8 mRNA was seen as early as 30 minutes, peaked between 4 and 8 hours, and decreased substantially by 24 hours. In contrast, TNF-alpha-induced IL-8 mRNA synthesis was elevated at 30 minutes, peaked at 2 hours, and reached basal/undetectable levels by 6 hours. Actinomycin D experiments suggested that both Ox-PAPC and TNF-alpha regulate the expression of IL-8 at the transcriptional level. Furthermore, the half-life of IL-8 mRNA for both ligands was similar (<30 minutes), suggesting that mRNA stability was not responsible for the differences in the kinetics of IL-8 accumulation between the 2 ligands. Transient transfection studies with reporter constructs containing 1.48 kb of the IL-8 promoter identified an Ox-PAPC-specific response region between -133 and -1481 bp of the IL-8 promoter. In contrast, TNF-alpha activation of the IL-8 promoter was mediated almost entirely through the nuclear factor-kappaB and activation protein-1 response elements present between -70 and -133 bp of the IL-8 promoter. Thus, although Ox-PAPC and TNF-alpha both induced IL-8 synthesis, our data suggest that the 2 ligands utilize different mechanisms in the regulation of IL-8 transcription.
Yeh
M
M
Department of Pathology, Division of Cardiology, University of California, Los Angeles, USA.
Leitinger
N
N
de Martin
R
R
Onai
N
N
Matsushima
K
K
Vora
D K
DK
Berliner
J A
JA
Reddy
S T
ST
D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D017930:Genes, Reporter; D006367:HeLa Cells; D051863:Host Cell Factor C1; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism; D050811:Octamer Transcription Factor-1; D010084:Oxidation-Reduction; D010742:Phospholipid Ethers / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D020218:Response Elements; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D016209:Interleukin-8; D008077:Lipoproteins, LDL; D016328:NF-kappa B; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D010742:Phospholipid Ethers; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C099252:oxidized low density lipoprotein; C043051:1-O-hexadecyl-2-arachidonyl-sn-glycero-3-phosphocholine