Hypoxia induces macrophage inflammatory protein-2 (MIP-2) gene expression in murine macrophages via NF-kappaB: the prominent role of p42/ p44 and PI3 kinase pathways.
We have previously reported that hypoxia induces a pronounced inflammatory response in the mouse lung associated with elevated levels of specific chemokines. To further explore the mechanisms involved in lung inflammation, we exposed RAW 264.7 cells as well as mouse primary macrophages to hypoxia and analyzed chemokine gene expression. Among the genes examined, macrophage inflammatory protein-2 (MIP-2) expression was prominently induced by hypoxia both at the mRNA and the protein level. When RAW 264.7 cells were transfected with a panel of plasmids harboring a luciferase marker gene under the control of wild-type or mutant variants of the MIP-2 gene promoter, a strong hypoxic induction of expression (9- to 17-fold) was observed. This induction was abolished by a mutation targeted to an NF-kappaB binding site in the MIP-2 promoter. Concordantly, specific NF-kappaB binding to the cognate sequence was enriched in nuclear extracts from hypoxic but not normoxic RAW 264.7 cells. The mechanism of MIP-2 gene induction by hypoxia was further characterized using inhibitors of signaling kinases. Inhibition of the p42/p44 and PI3 kinases but not p38 MAPK abolished the NF-kappaB-driven upregulation of MIP-2 gene expression by hypoxia. This attenuation of the NF-kappaB response to hypoxia did not involve decreased nuclear NF-kappaB abundance but correlated with diminished transactivation potential of the p65 subunit. Our results indicate that the hypoxic signal for induction of MIP-2 gene expression is implemented through enhanced NF-kappaB activity and transmitted along the p42/44 and PI3 kinase pathways.
Zampetaki
A
A
Department of Medicine, Division of Newborn Medicine, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Mitsialis
S A
SA
Pfeilschifter
J
J
Kourembanas
Stella
S
D000818:Animals; D015687:Cell Hypoxia / Q000235:genetics*; D002460:Cell Line; D054426:Chemokine CXCL2; D018925:Chemokines / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology*; D048052:Mitogen-Activated Protein Kinase 3 / Q000502:physiology*; D008954:Models, Biological; D016328:NF-kappa B / Q000502:physiology*; D010100:Oxygen / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D015533:Transcriptional Activation
D054426:Chemokine CXCL2; D018925:Chemokines; C404915:Cxcl2 protein, mouse; D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D010100:Oxygen