Oxidized phospholipids increase interleukin 8 (IL-8) synthesis by activation of the c-src/signal transducers and activators of transcription (STAT)3 pathway.
Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) and its component phospholipids 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine induce endothelial cells to synthesize chemotactic factors, such as interleukin 8 (IL-8). We have shown recently that Ox-PAPC-mediated induction of IL-8 transcription is independent of NF-kappaB activation, a major transcription factor utilized by cytokines and lipopolysaccharide for the induction of IL-8 transcription. In this study, we provide evidence for the role of c-src in Ox-PAPC and, specifically, PEIPC-mediated IL-8 induction. Ox-PAPC and its component phospholipids induced a rapid and transient phosphorylation of c-src Tyr418, a hallmark of c-src activation, in human aortic endothelial cells (HAEC). Ox-PAPC-mediated IL-8 protein synthesis in HAEC was inhibited by Src family kinase inhibitors, PP1 and PP2, but not by an inactive analog, PP3. Transient expression of plasmids containing C-terminal Src kinase or kinase-deficient dominant-negative c-src resulted in a 72 and 50% reduction in Ox-PAPC-induced IL-8 promoter activation in human microvascular endothelial cells, respectively. In contrast, overexpression of v-src kinase resulted in a 4-fold increase in IL-8 promoter activation, without inducing NF-kappaB promoter activation. Furthermore, treatment of HAEC with Ox-PAPC and its component PEIPC induced the activation of STAT3 by phosphorylating Tyr705, a feature of STAT3 activation. STAT3 is a known downstream effector of c-src. Ox-PAPC-induced activation of STAT3 resulted in the translocation of STAT3 from the cytoplasm of HAEC into their nuclear compartment. Transient expression of a dominant-negative STAT3beta construct in HMEC strongly inhibited IL-8 induction by Ox-PAPC. Taken together, these data demonstrate the role of the c-src kinase/STAT3 pathway in Ox-PAPC-mediated IL-8 expression in endothelial cells.
Yeh
Michael
M
Division of Cardiology, Department of Medicine, University of California, Los Angeles, California 90095, USA. myeh@mednet.ucla.edu
Gharavi
Nima M
NM
Choi
Jenny
J
Hsieh
Xavier
X
Reed
Erin
E
Mouillesseaux
Kevin P
KP
Cole
Amy L
AL
Reddy
Srinivasa T
ST
Berliner
Judith A
JA
D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005799:Genes, Dominant; D006801:Humans; D007249:Inflammation; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010713:Phosphatidylcholines / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases
D004268:DNA-Binding Proteins; D016209:Interleukin-8; D016328:NF-kappa B; D010713:Phosphatidylcholines; D010743:Phospholipids; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; C472349:oxidized-L-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; D014443:Tyrosine; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D010100:Oxygen