Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes

David A. Geller, Andreas K. Nussler, Mauricio Di Silvio, Charles J. Lowenstein, Richard A. Shapiro, Stewart C. Wang, Richard L. Simmons, Timothy R. Billiar

Research output: Contribution to journalArticle

Abstract

Nitric oxide (NO·) is a short-lived mediator which can be induced in a variety of cell types and produces many physiologic and metabolic changes in target cells. The inducible or high-output NO· synthase (NOS) pathway was first characterized in macrophages activated by lipopolysaccharide (LPS) and interferon γ(IFN-γ). Hepatocytes also express an inducible NOS following exposure to the combination of endotoxin (LPS) and tumor necrosis factor (TNF), interleukin 1 (IL-1), and IFN-γ. In this study, to identify which of these cytokines, if any, was acting to induce the gene expression for hepatocyte NOS, we measured the levels of rat hepatocyte NOS mRNA by Northern blot analysis after stimulation by various combinations of endotoxin and cytokines in vitro. We found the mRNA for hepatocyte NOS to be a single band at ≈4.5 kilobases which was maximally up-regulated (≈70-fold) by the combination of TNF, IL-1, IFN-γ, and LPS. Abundance of NOS mRNA peaked 6-8 hr after stimulation and then declined by 25% at 24 hr. Unstimulated hepatocytes in vitro showed only a trace mRNA band after prolonged autoradiographic exposure. As single agents, TNF and IL-1 were the most effective inducers of hepatocyte NOS mRNA. Combinations of two or three stimuli revealed strong synergy between TNF, IL-1, and IFN-γ. The increased mRNA levels correlated with elevated nitrogen oxide release and cGMP levels in the culture supernatants. Dexamethasone and cycloheximide inhibited induction of mRNA for hepatocyte NOS in a dose-dependent fashion. The addition of NG-monomethyl-L-arginine had no effect on mRNA levels but effectively blocked NO· formation. The inducible hepatocyte NOS mRNA was also detected in rat hepatocytes following chronic hepatic inflammation triggered by Corynebacterium parvum injection in vivo. These data demonstrate that the inducible NOS is functional in rat hepatocytes both in vitro and in vivo and that this pathway is under complex control. Endotoxin and inflammatory cytokines act synergistically to up-regulate gene expression for hepatocyte NOS, whereas glucocorticoids down-regulate the mRNA.

Original languageEnglish (US)
Pages (from-to)522-526
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume90
Issue number2
StatePublished - Jan 15 1993

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Nitric Oxide Synthase Type II
Endotoxins
Nitric Oxide Synthase
Glucocorticoids
Hepatocytes
Cytokines
Messenger RNA
Interleukin-1
Interferons
Tumor Necrosis Factor-alpha
Lipopolysaccharides
Nitric Oxide
Gene Expression
omega-N-Methylarginine
Propionibacterium acnes
Cycloheximide
Northern Blotting
Dexamethasone
Up-Regulation
Down-Regulation

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

Geller, D. A., Nussler, A. K., Di Silvio, M., Lowenstein, C. J., Shapiro, R. A., Wang, S. C., ... Billiar, T. R. (1993). Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proceedings of the National Academy of Sciences of the United States of America, 90(2), 522-526.

Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. / Geller, David A.; Nussler, Andreas K.; Di Silvio, Mauricio; Lowenstein, Charles J.; Shapiro, Richard A.; Wang, Stewart C.; Simmons, Richard L.; Billiar, Timothy R.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 90, No. 2, 15.01.1993, p. 522-526.

Research output: Contribution to journalArticle

Geller, DA, Nussler, AK, Di Silvio, M, Lowenstein, CJ, Shapiro, RA, Wang, SC, Simmons, RL & Billiar, TR 1993, 'Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes', Proceedings of the National Academy of Sciences of the United States of America, vol. 90, no. 2, pp. 522-526.
Geller, David A. ; Nussler, Andreas K. ; Di Silvio, Mauricio ; Lowenstein, Charles J. ; Shapiro, Richard A. ; Wang, Stewart C. ; Simmons, Richard L. ; Billiar, Timothy R. / Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. In: Proceedings of the National Academy of Sciences of the United States of America. 1993 ; Vol. 90, No. 2. pp. 522-526.
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T1 - Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes

AU - Geller, David A.

AU - Nussler, Andreas K.

AU - Di Silvio, Mauricio

AU - Lowenstein, Charles J.

AU - Shapiro, Richard A.

AU - Wang, Stewart C.

AU - Simmons, Richard L.

AU - Billiar, Timothy R.

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N2 - Nitric oxide (NO·) is a short-lived mediator which can be induced in a variety of cell types and produces many physiologic and metabolic changes in target cells. The inducible or high-output NO· synthase (NOS) pathway was first characterized in macrophages activated by lipopolysaccharide (LPS) and interferon γ(IFN-γ). Hepatocytes also express an inducible NOS following exposure to the combination of endotoxin (LPS) and tumor necrosis factor (TNF), interleukin 1 (IL-1), and IFN-γ. In this study, to identify which of these cytokines, if any, was acting to induce the gene expression for hepatocyte NOS, we measured the levels of rat hepatocyte NOS mRNA by Northern blot analysis after stimulation by various combinations of endotoxin and cytokines in vitro. We found the mRNA for hepatocyte NOS to be a single band at ≈4.5 kilobases which was maximally up-regulated (≈70-fold) by the combination of TNF, IL-1, IFN-γ, and LPS. Abundance of NOS mRNA peaked 6-8 hr after stimulation and then declined by 25% at 24 hr. Unstimulated hepatocytes in vitro showed only a trace mRNA band after prolonged autoradiographic exposure. As single agents, TNF and IL-1 were the most effective inducers of hepatocyte NOS mRNA. Combinations of two or three stimuli revealed strong synergy between TNF, IL-1, and IFN-γ. The increased mRNA levels correlated with elevated nitrogen oxide release and cGMP levels in the culture supernatants. Dexamethasone and cycloheximide inhibited induction of mRNA for hepatocyte NOS in a dose-dependent fashion. The addition of NG-monomethyl-L-arginine had no effect on mRNA levels but effectively blocked NO· formation. The inducible hepatocyte NOS mRNA was also detected in rat hepatocytes following chronic hepatic inflammation triggered by Corynebacterium parvum injection in vivo. These data demonstrate that the inducible NOS is functional in rat hepatocytes both in vitro and in vivo and that this pathway is under complex control. Endotoxin and inflammatory cytokines act synergistically to up-regulate gene expression for hepatocyte NOS, whereas glucocorticoids down-regulate the mRNA.

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