Cytokine-induced nitric oxide synthase gene transcription is blocked by the heat shock response in human liver cells.
BACKGROUND
Previously we demonstrated that the heat shock response (HSR) inhibits cytokine-stimulated nitric oxide (NO) synthesis and inducible NO synthase (NOS2) expression in hepatocytes. In this study we sought to determine the molecular basis of this inhibition using a human liver cell line.


METHODS
After induction of the HSR by sodium arsenite or hyperthermia, the AKN-1 human liver cell line was treated with cytokines to stimulate NOS2 expression and NO production. Western blot analysis for hsp70 was performed, and NOS2 mRNA and 24-hour NO synthesis were quantitated. Cytokine-induced NOS2 promoter activity of AKN-1 cells transfected with a 7.0 kilobase NOS2 promoter luciferase construct and NO production of AKN-1 cells transduced with the NOS2 gene were measured.


RESULTS
Sodium arsenite or hyperthermia induced the synthesis of hsp70 protein in AKN-1 cells, indicating activation of the HSR. Cytokines stimulated high levels of NOS2 mRNA and NO production. However, prior induction of the HSR significantly inhibited NOS2 expression and NO synthesis. Cytokine-stimulated NOS2 promoter activity of transfected AKN-1 cells was decreased by 77%, but the HSR did not affect NOS2 enzyme activity in transduced AKN-1 cells.


CONCLUSIONS
These findings indicate that the HSR inhibits cytokine-induced NOS2 expression and NO synthesis in AKN-1 cells by preventing NOS2 promoter activation. Effects on NOS2 protein translation or stability were not observed. These data suggest that the HSR, which is expressed in the liver after trauma, shock, or ischemia-reperfusion, blocks NOS2 gene expression at the transcriptional level.
de Vera
M E
ME
Department of Surgery, University of Pittsburgh, Pa 15261, USA.
Wong
J M
JM
Zhou
J Y
JY
Tzeng
E
E
Wong
H R
HR
Billiar
T R
TR
Geller
D A
DA
D000210:Acute-Phase Reaction; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line / Q000187:drug effects / Q000201:enzymology; D016207:Cytokines / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000502:physiology*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000166:cytology*; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D006360:Heat-Shock Proteins; D007375:Interleukin-1; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase