Cytokines decrease glutaminase expression in human fibroblasts

P. Sarantos, A. Abouhamze, S. Abcouwer, R. Chakrabarti, E. M. Copeland, W. W. Souba, A. Barbul, T. Billiar, J. E. Albina, David G Greenhalgh, R. A. Forse, S. I. Myers

Research output: Contribution to journalArticle

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Abstract

Background. Glutamine metabolism in fibroblasts is essential for energy production, nucleotide biosynthesis, and growth during wound healing. Because cytokines can impair fibroblast proliferation, we tested the hypothesis that cytokines impair glutamine metabolism. We studied the influence of several cytokines on the expression of glutaminase, the major enzyme of intracellular glutamine metabolism in fibroblasts. Methods. Human foreskin fibroblasts were incubated for 6 and 12 hours with varying doses (10, 100, or 1000 units/ml) of interleukin (IL)-1, IL-6, tumor necrosis factor-α, or γ-interferon. Cell lysates were assayed for glutaminase-specific activity, and glutaminase protein content was measured by Western blotting with a polyclonal antibody. Total cellular RNA was extracted, and relative glutaminase messenger RNA levels were determined by Northern blotting with a 32P-labeled glutaminase complement DNA-derived probe. These mRNA levels were normalized by blotting with a β-actin cDNA-derived probe as control. Cell nuclei were isolated, and nuclear run-ons were used to determine relative glutaminase mRNA transcription rates. Results. IL-1, IL-6, tumor necrosis factor-α, and γ- interferon decreased glutaminase activity and protein concentration after a 12-hour incubation in a dose-independent fashion. No difference was noted at 6 hours. Western blot analysis showed a 30% to 60% reduction in glutaminase protein in treated cells. These cytokines also decreased glutaminase mRNA levels, consistent with transcriptional regulation. This was confirmed by nuclear run-on assays that showed a decrease in the number of glutaminase transcripts. Conclusions. A variety of different pro-inflammatory cytokines decrease glutaminase expression in cultured human fibroblasts. This cytokine- mediated inhibition of glutamine metabolism may limit the availability of key glutamine-derived intermediates and impair fibroblast proliferation in certain patients.

Original languageEnglish (US)
Pages (from-to)276-284
Number of pages9
JournalSurgery
Volume116
Issue number2
StatePublished - 1994
Externally publishedYes

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Glutaminase
Fibroblasts
Cytokines
Glutamine
Messenger RNA
Interleukin-1
Interferons
Interleukin-6
Tumor Necrosis Factor-alpha
Western Blotting
Foreskin
Proteins
DNA Probes
Cell Nucleus
Northern Blotting
Wound Healing
Actins
Nucleotides
Complementary DNA

ASJC Scopus subject areas

  • Surgery

Cite this

Sarantos, P., Abouhamze, A., Abcouwer, S., Chakrabarti, R., Copeland, E. M., Souba, W. W., ... Myers, S. I. (1994). Cytokines decrease glutaminase expression in human fibroblasts. Surgery, 116(2), 276-284.

Cytokines decrease glutaminase expression in human fibroblasts. / Sarantos, P.; Abouhamze, A.; Abcouwer, S.; Chakrabarti, R.; Copeland, E. M.; Souba, W. W.; Barbul, A.; Billiar, T.; Albina, J. E.; Greenhalgh, David G; Forse, R. A.; Myers, S. I.

In: Surgery, Vol. 116, No. 2, 1994, p. 276-284.

Research output: Contribution to journalArticle

Sarantos, P, Abouhamze, A, Abcouwer, S, Chakrabarti, R, Copeland, EM, Souba, WW, Barbul, A, Billiar, T, Albina, JE, Greenhalgh, DG, Forse, RA & Myers, SI 1994, 'Cytokines decrease glutaminase expression in human fibroblasts', Surgery, vol. 116, no. 2, pp. 276-284.
Sarantos P, Abouhamze A, Abcouwer S, Chakrabarti R, Copeland EM, Souba WW et al. Cytokines decrease glutaminase expression in human fibroblasts. Surgery. 1994;116(2):276-284.
Sarantos, P. ; Abouhamze, A. ; Abcouwer, S. ; Chakrabarti, R. ; Copeland, E. M. ; Souba, W. W. ; Barbul, A. ; Billiar, T. ; Albina, J. E. ; Greenhalgh, David G ; Forse, R. A. ; Myers, S. I. / Cytokines decrease glutaminase expression in human fibroblasts. In: Surgery. 1994 ; Vol. 116, No. 2. pp. 276-284.
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title = "Cytokines decrease glutaminase expression in human fibroblasts",
abstract = "Background. Glutamine metabolism in fibroblasts is essential for energy production, nucleotide biosynthesis, and growth during wound healing. Because cytokines can impair fibroblast proliferation, we tested the hypothesis that cytokines impair glutamine metabolism. We studied the influence of several cytokines on the expression of glutaminase, the major enzyme of intracellular glutamine metabolism in fibroblasts. Methods. Human foreskin fibroblasts were incubated for 6 and 12 hours with varying doses (10, 100, or 1000 units/ml) of interleukin (IL)-1, IL-6, tumor necrosis factor-α, or γ-interferon. Cell lysates were assayed for glutaminase-specific activity, and glutaminase protein content was measured by Western blotting with a polyclonal antibody. Total cellular RNA was extracted, and relative glutaminase messenger RNA levels were determined by Northern blotting with a 32P-labeled glutaminase complement DNA-derived probe. These mRNA levels were normalized by blotting with a β-actin cDNA-derived probe as control. Cell nuclei were isolated, and nuclear run-ons were used to determine relative glutaminase mRNA transcription rates. Results. IL-1, IL-6, tumor necrosis factor-α, and γ- interferon decreased glutaminase activity and protein concentration after a 12-hour incubation in a dose-independent fashion. No difference was noted at 6 hours. Western blot analysis showed a 30{\%} to 60{\%} reduction in glutaminase protein in treated cells. These cytokines also decreased glutaminase mRNA levels, consistent with transcriptional regulation. This was confirmed by nuclear run-on assays that showed a decrease in the number of glutaminase transcripts. Conclusions. A variety of different pro-inflammatory cytokines decrease glutaminase expression in cultured human fibroblasts. This cytokine- mediated inhibition of glutamine metabolism may limit the availability of key glutamine-derived intermediates and impair fibroblast proliferation in certain patients.",
author = "P. Sarantos and A. Abouhamze and S. Abcouwer and R. Chakrabarti and Copeland, {E. M.} and Souba, {W. W.} and A. Barbul and T. Billiar and Albina, {J. E.} and Greenhalgh, {David G} and Forse, {R. A.} and Myers, {S. I.}",
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T1 - Cytokines decrease glutaminase expression in human fibroblasts

AU - Sarantos, P.

AU - Abouhamze, A.

AU - Abcouwer, S.

AU - Chakrabarti, R.

AU - Copeland, E. M.

AU - Souba, W. W.

AU - Barbul, A.

AU - Billiar, T.

AU - Albina, J. E.

AU - Greenhalgh, David G

AU - Forse, R. A.

AU - Myers, S. I.

PY - 1994

Y1 - 1994

N2 - Background. Glutamine metabolism in fibroblasts is essential for energy production, nucleotide biosynthesis, and growth during wound healing. Because cytokines can impair fibroblast proliferation, we tested the hypothesis that cytokines impair glutamine metabolism. We studied the influence of several cytokines on the expression of glutaminase, the major enzyme of intracellular glutamine metabolism in fibroblasts. Methods. Human foreskin fibroblasts were incubated for 6 and 12 hours with varying doses (10, 100, or 1000 units/ml) of interleukin (IL)-1, IL-6, tumor necrosis factor-α, or γ-interferon. Cell lysates were assayed for glutaminase-specific activity, and glutaminase protein content was measured by Western blotting with a polyclonal antibody. Total cellular RNA was extracted, and relative glutaminase messenger RNA levels were determined by Northern blotting with a 32P-labeled glutaminase complement DNA-derived probe. These mRNA levels were normalized by blotting with a β-actin cDNA-derived probe as control. Cell nuclei were isolated, and nuclear run-ons were used to determine relative glutaminase mRNA transcription rates. Results. IL-1, IL-6, tumor necrosis factor-α, and γ- interferon decreased glutaminase activity and protein concentration after a 12-hour incubation in a dose-independent fashion. No difference was noted at 6 hours. Western blot analysis showed a 30% to 60% reduction in glutaminase protein in treated cells. These cytokines also decreased glutaminase mRNA levels, consistent with transcriptional regulation. This was confirmed by nuclear run-on assays that showed a decrease in the number of glutaminase transcripts. Conclusions. A variety of different pro-inflammatory cytokines decrease glutaminase expression in cultured human fibroblasts. This cytokine- mediated inhibition of glutamine metabolism may limit the availability of key glutamine-derived intermediates and impair fibroblast proliferation in certain patients.

AB - Background. Glutamine metabolism in fibroblasts is essential for energy production, nucleotide biosynthesis, and growth during wound healing. Because cytokines can impair fibroblast proliferation, we tested the hypothesis that cytokines impair glutamine metabolism. We studied the influence of several cytokines on the expression of glutaminase, the major enzyme of intracellular glutamine metabolism in fibroblasts. Methods. Human foreskin fibroblasts were incubated for 6 and 12 hours with varying doses (10, 100, or 1000 units/ml) of interleukin (IL)-1, IL-6, tumor necrosis factor-α, or γ-interferon. Cell lysates were assayed for glutaminase-specific activity, and glutaminase protein content was measured by Western blotting with a polyclonal antibody. Total cellular RNA was extracted, and relative glutaminase messenger RNA levels were determined by Northern blotting with a 32P-labeled glutaminase complement DNA-derived probe. These mRNA levels were normalized by blotting with a β-actin cDNA-derived probe as control. Cell nuclei were isolated, and nuclear run-ons were used to determine relative glutaminase mRNA transcription rates. Results. IL-1, IL-6, tumor necrosis factor-α, and γ- interferon decreased glutaminase activity and protein concentration after a 12-hour incubation in a dose-independent fashion. No difference was noted at 6 hours. Western blot analysis showed a 30% to 60% reduction in glutaminase protein in treated cells. These cytokines also decreased glutaminase mRNA levels, consistent with transcriptional regulation. This was confirmed by nuclear run-on assays that showed a decrease in the number of glutaminase transcripts. Conclusions. A variety of different pro-inflammatory cytokines decrease glutaminase expression in cultured human fibroblasts. This cytokine- mediated inhibition of glutamine metabolism may limit the availability of key glutamine-derived intermediates and impair fibroblast proliferation in certain patients.

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