Induction of interleukin 6 (IL-6) by hypoxia in vascular cells. Central role of the binding site for nuclear factor-IL-6.
The pathologic picture in ischemic tissue injury shares features with the inflammatory response, including production of proinflammatory cytokines. Hypoxia-mediated induction of interleukin-6 (IL-6), a cytokine with anti-inflammatory properties, could set in motion mechanisms limiting inflammation in ischemia. Exposure of cultured endothelial cells (ECs) to H (pO2 approximately 12-16 torr) increased transcription of IL-6, elevated levels of IL-6 mRNA, and induced elaboration of IL-6 antigen in a time-dependent manner. Exposure of mice to hypoxia increased IL-6 transcripts in the lung, and immunostaining revealed a striking increase in IL-6 antigen in pulmonary vasculature. Transfection of ECs with deletion chimeric IL-6 promoter-chloramphenicol acetyl-transferase (CAT) constructs showed hypoxia-mediated 9-11-fold induction with -1200/+13, -596/+13, and -225/+13 but no induction with -111/+13. Electrophoretic mobility shift assays (EMSAs) using -225/-111 as the labeled probe demonstrated enhanced binding activity in nuclear extracts of hypoxic ECs and lung; the appearance of the gel shift band was prevented by excess unlabeled probe (-225/-111), and hypoxia-mediated enhancement of the band was blocked by a probe corresponding to the nuclear factor (NF)-IL-6 site (-158/-145). The hypoxia-enhanced band on EMSA displayed a supershift with antibody to CCAAT-enhancer-binding protein beta (C/EBP-beta), but antibody to C/EBP-alpha or -delta was without effect. Transfection of ECs with a construct comprising thymidine kinase promoter, -225/-111 in either the 5' to 3' to 5' orientation, and the reporter CAT showed this region to be an enhancer (approximately 8-fold) under hypoxia. EMSA with the NF-IL-6 probe revealed a prominent induction of binding activity with nuclear extracts from hypoxic ECs and whole lung. Constructs with -158/-145 and the CAT reporter gene showed induction when transfected into hypoxic ECs, whereas a similar construct with the NF-IL-6 motif mutationally inactivated failed to display hypoxia-induced expression. Furthermore, the tumor necrosis factor (TNF) gene, whose product contributes to ischemic pathology and contains a putative regulatory NF-IL-6 site, demonstrated enhanced binding activity for its NF-IL-6 motif and induction of TNF mRNA based on analysis of hypoxic lung. These data indicate that hypoxia induces expression of IL-6, most likely a result of hypoxic activation at the NF-IL-6 site, and suggest that other genes with regulatory NF-IL-6 sites may also be induced by a similar mechanism.
Yan
S F
SF
Department of Physiology, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA.
Tritto
I
I
Pinsky
D
D
Liao
H
H
Huang
J
J
Fuller
G
G
Brett
J
J
May
L
L
Stern
D
D
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D015687:Cell Hypoxia / Q000276:immunology; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000276:immunology* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis*; D007700:Kinetics; D008168:Lung / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D013937:Thymidine Kinase / Q000096:biosynthesis; D013997:Time Factors; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D014471:Umbilical Veins
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase