TY - JOUR
T1 - Cytokine-induced nitric oxide synthase gene transcription is blocked by the heat shock response in human liver cells
AU - De Vera, M. E.
AU - Wong, J. M.
AU - Zhou, J. Y.
AU - Tzeng, E.
AU - Wong, H. R.
AU - Billiar, T. R.
AU - Geller, D. A.
N1 - Funding Information:
THE SMALL, INTERMITTENTB URSTS of nitric oxide (NO) synthesized by the constitutive forms of NO synthases (NOS) (types I and III) mediate basic physiologic processes such as vasodilation and neurotransmission. In contrast, the larger, sustained amounts of NO produced by the inducible form ofNOS (iNOS or NOS2) can exhibit either cytoprotective or cytotoxic actions depending on the setting. NO has been shown to be beneficial in sepsis and inflammation by preventing platelet and leukocyte adhesion (thus maintaining perfusion in the microcirculation), by scavenging oxygen radicals, and by exerting antimicrobial actions) However, detrimental effects such as pressor-resistant hypotension during septic shock and increased liver injury during endotox- Supported by National Institutes of Health grants GM-52021 and GM-44100. Presented at the Fifty-seventhA nnual Meeting of the Societyo f University Surgeons, Washington, D.C., Feb. 8-10, 1996. Reprint requests: Michael E. de Vera, MD, Department of Surgery, University of Pittsburgh, 497 Scaife Hall, Pittsburgh, PA 15261. Copyright 9 1996 by Mosby-YearB ook. Inc. 0039-6060/96/$5.00 + 0 11/6/73521
PY - 1996
Y1 - 1996
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0029819656&partnerID=8YFLogxK
U2 - 10.1016/S0039-6060(96)80281-9
DO - 10.1016/S0039-6060(96)80281-9
M3 - Article
C2 - 8751576
AN - SCOPUS:0029819656
SN - 0039-6060
VL - 120
SP - 144
EP - 149
JO - Surgery
JF - Surgery
IS - 2
ER -