Induction of interleukin 6 (IL-6) by hypoxia in vascular cells. Central role of the binding site for nuclear factor-IL-6

Fang Yan Shi Fang Yan, I. Tritto, D. Pinsky, H. Liao, J. Huang, G. Fuller, J. Brett, L. May, David Stern

Research output: Contribution to journalArticle

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Abstract

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 (pO 2 ≃ 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 acetyltransferase (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 β (C/EBP-β), but antibody to C/EBP-α or -δ was without effect. Transfection of ECs with a construct comprising thymidine kinase promoter, -225/-111 in either the 5' to 3' or 3' to 5' orientation, and the reporter CAT showed this region to be an enhancer (≃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.

Original languageEnglish (US)
Pages (from-to)11463-11471
Number of pages9
JournalJournal of Biological Chemistry
Volume270
Issue number19
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

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CCAAT-Enhancer-Binding Protein-beta
Blood Vessels
Interleukin-6
Endothelial cells
Binding Sites
Endothelial Cells
Electrophoretic mobility
Chloramphenicol O-Acetyltransferase
Electrophoretic Mobility Shift Assay
Lung
Assays
Genes
Transfection
Tumor Necrosis Factor-alpha
Glycyrrhetinic Acid
CCAAT-Enhancer-Binding Proteins
Cytokines
Antigens
Messenger RNA
Thymidine Kinase

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Induction of interleukin 6 (IL-6) by hypoxia in vascular cells. Central role of the binding site for nuclear factor-IL-6. / Shi Fang Yan, Fang Yan; Tritto, I.; Pinsky, D.; Liao, H.; Huang, J.; Fuller, G.; Brett, J.; May, L.; Stern, David.

In: Journal of Biological Chemistry, Vol. 270, No. 19, 01.01.1995, p. 11463-11471.

Research output: Contribution to journalArticle

Shi Fang Yan, Fang Yan ; Tritto, I. ; Pinsky, D. ; Liao, H. ; Huang, J. ; Fuller, G. ; Brett, J. ; May, L. ; Stern, David. / Induction of interleukin 6 (IL-6) by hypoxia in vascular cells. Central role of the binding site for nuclear factor-IL-6. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 19. pp. 11463-11471.
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abstract = "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 (pO 2 ≃ 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 acetyltransferase (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 β (C/EBP-β), but antibody to C/EBP-α or -δ was without effect. Transfection of ECs with a construct comprising thymidine kinase promoter, -225/-111 in either the 5' to 3' or 3' to 5' orientation, and the reporter CAT showed this region to be an enhancer (≃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.",
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T1 - Induction of interleukin 6 (IL-6) by hypoxia in vascular cells. Central role of the binding site for nuclear factor-IL-6

AU - Shi Fang Yan, Fang Yan

AU - Tritto, I.

AU - Pinsky, D.

AU - Liao, H.

AU - Huang, J.

AU - Fuller, G.

AU - Brett, J.

AU - May, L.

AU - Stern, David

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N2 - 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 (pO 2 ≃ 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 acetyltransferase (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 β (C/EBP-β), but antibody to C/EBP-α or -δ was without effect. Transfection of ECs with a construct comprising thymidine kinase promoter, -225/-111 in either the 5' to 3' or 3' to 5' orientation, and the reporter CAT showed this region to be an enhancer (≃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.

AB - 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 (pO 2 ≃ 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 acetyltransferase (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 β (C/EBP-β), but antibody to C/EBP-α or -δ was without effect. Transfection of ECs with a construct comprising thymidine kinase promoter, -225/-111 in either the 5' to 3' or 3' to 5' orientation, and the reporter CAT showed this region to be an enhancer (≃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.

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