Complement component C3 production in IL-1β-stimulated human intestinal epithelial cells is blocked by NF-κB inhibitors and by transfection with Ser 32/36 mutant IκBα

M. Ryan Moon, Alexander A. Parikh, Timothy A. Pritts, Josef E. Fischer, Sarah Cottongim, Csaba Szabo, Andrew L. Salzman, Per Olof Hasselgren

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Background. Recent studies suggest that interleukin-1β (IL-1β) stimulates the production of the acute phase protein complement component C3 in human intestinal epithelial cells. The transcription factor NF-κB activates different genes involved in the response to cytokines. It is not known if IL-1β-induced C3 production in the enterocyte is regulated by NF- κB. Materials and methods. Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with one of the NF-κB inhibitors, tosyl- lys-chloromethylketone (TLCK), genistein, or pyrrolidine dithiocarbamate (PDTC), or with N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor known to block the degradation of IκB, the cytosolic inhibitor of NF-κB. Following this treatment, the Caco-2 cells were stimulated with IL-1β, and C3 levels in the culture medium were measured after 24 h by ELISA. C3 mRNA levels were determined after 4 h by Northern blot analysis. In other experiments, Caco-2 cells were transfected with a mutant IκBα in which serines 32 and 36 were substituted by alanine. This mutation prevents IkBα phosphorylation and subsequent NF-κB nuclear translocation. After transfection, the cells were stimulated with IL-1β, and C3 levels in the culture medium were measured after 24 h. Cytosolic IκBα was determined by Western blot analysis. Results. TLCK, genistein, and LLnL each inhibited IL- 1β-induced C3 production in a dose-dependent fashion. These responses were associated with decreased C3 mRNA levels. In contrast, PDTC did not influence C3 production or C3 mRNA in the Caco-2 cells. Transfection of the Caco-2 cells with the Ser 32/36 mutant IkBα resulted in maintained IκBα levels and decreased IL-β-induced C3 production. Conclusions. IL-1β-stimulated C3 production in the enterocyte may be regulated by NF-κB.

Original languageEnglish (US)
Pages (from-to)48-55
Number of pages8
JournalJournal of Surgical Research
Volume82
Issue number1
DOIs
StatePublished - Mar 1999
Externally publishedYes

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Complement C3
Interleukin-1
Caco-2 Cells
Transfection
Epithelial Cells
leucylleucine
Enterocytes
Genistein
Messenger RNA
Culture Media
Proteasome Inhibitors
Acute-Phase Proteins
Alanine
Northern Blotting
Serine
Cultured Cells
Transcription Factors
Western Blotting
Enzyme-Linked Immunosorbent Assay
Phosphorylation

Keywords

  • Complement component C3
  • Enterocyte
  • Interleukin-1
  • NF-κB

ASJC Scopus subject areas

  • Surgery

Cite this

Complement component C3 production in IL-1β-stimulated human intestinal epithelial cells is blocked by NF-κB inhibitors and by transfection with Ser 32/36 mutant IκBα. / Moon, M. Ryan; Parikh, Alexander A.; Pritts, Timothy A.; Fischer, Josef E.; Cottongim, Sarah; Szabo, Csaba; Salzman, Andrew L.; Hasselgren, Per Olof.

In: Journal of Surgical Research, Vol. 82, No. 1, 03.1999, p. 48-55.

Research output: Contribution to journalArticle

Moon, M. Ryan ; Parikh, Alexander A. ; Pritts, Timothy A. ; Fischer, Josef E. ; Cottongim, Sarah ; Szabo, Csaba ; Salzman, Andrew L. ; Hasselgren, Per Olof. / Complement component C3 production in IL-1β-stimulated human intestinal epithelial cells is blocked by NF-κB inhibitors and by transfection with Ser 32/36 mutant IκBα. In: Journal of Surgical Research. 1999 ; Vol. 82, No. 1. pp. 48-55.
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title = "Complement component C3 production in IL-1β-stimulated human intestinal epithelial cells is blocked by NF-κB inhibitors and by transfection with Ser 32/36 mutant IκBα",
abstract = "Background. Recent studies suggest that interleukin-1β (IL-1β) stimulates the production of the acute phase protein complement component C3 in human intestinal epithelial cells. The transcription factor NF-κB activates different genes involved in the response to cytokines. It is not known if IL-1β-induced C3 production in the enterocyte is regulated by NF- κB. Materials and methods. Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with one of the NF-κB inhibitors, tosyl- lys-chloromethylketone (TLCK), genistein, or pyrrolidine dithiocarbamate (PDTC), or with N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor known to block the degradation of IκB, the cytosolic inhibitor of NF-κB. Following this treatment, the Caco-2 cells were stimulated with IL-1β, and C3 levels in the culture medium were measured after 24 h by ELISA. C3 mRNA levels were determined after 4 h by Northern blot analysis. In other experiments, Caco-2 cells were transfected with a mutant IκBα in which serines 32 and 36 were substituted by alanine. This mutation prevents IkBα phosphorylation and subsequent NF-κB nuclear translocation. After transfection, the cells were stimulated with IL-1β, and C3 levels in the culture medium were measured after 24 h. Cytosolic IκBα was determined by Western blot analysis. Results. TLCK, genistein, and LLnL each inhibited IL- 1β-induced C3 production in a dose-dependent fashion. These responses were associated with decreased C3 mRNA levels. In contrast, PDTC did not influence C3 production or C3 mRNA in the Caco-2 cells. Transfection of the Caco-2 cells with the Ser 32/36 mutant IkBα resulted in maintained IκBα levels and decreased IL-β-induced C3 production. Conclusions. IL-1β-stimulated C3 production in the enterocyte may be regulated by NF-κB.",
keywords = "Complement component C3, Enterocyte, Interleukin-1, NF-κB",
author = "Moon, {M. Ryan} and Parikh, {Alexander A.} and Pritts, {Timothy A.} and Fischer, {Josef E.} and Sarah Cottongim and Csaba Szabo and Salzman, {Andrew L.} and Hasselgren, {Per Olof}",
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T1 - Complement component C3 production in IL-1β-stimulated human intestinal epithelial cells is blocked by NF-κB inhibitors and by transfection with Ser 32/36 mutant IκBα

AU - Moon, M. Ryan

AU - Parikh, Alexander A.

AU - Pritts, Timothy A.

AU - Fischer, Josef E.

AU - Cottongim, Sarah

AU - Szabo, Csaba

AU - Salzman, Andrew L.

AU - Hasselgren, Per Olof

PY - 1999/3

Y1 - 1999/3

N2 - Background. Recent studies suggest that interleukin-1β (IL-1β) stimulates the production of the acute phase protein complement component C3 in human intestinal epithelial cells. The transcription factor NF-κB activates different genes involved in the response to cytokines. It is not known if IL-1β-induced C3 production in the enterocyte is regulated by NF- κB. Materials and methods. Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with one of the NF-κB inhibitors, tosyl- lys-chloromethylketone (TLCK), genistein, or pyrrolidine dithiocarbamate (PDTC), or with N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor known to block the degradation of IκB, the cytosolic inhibitor of NF-κB. Following this treatment, the Caco-2 cells were stimulated with IL-1β, and C3 levels in the culture medium were measured after 24 h by ELISA. C3 mRNA levels were determined after 4 h by Northern blot analysis. In other experiments, Caco-2 cells were transfected with a mutant IκBα in which serines 32 and 36 were substituted by alanine. This mutation prevents IkBα phosphorylation and subsequent NF-κB nuclear translocation. After transfection, the cells were stimulated with IL-1β, and C3 levels in the culture medium were measured after 24 h. Cytosolic IκBα was determined by Western blot analysis. Results. TLCK, genistein, and LLnL each inhibited IL- 1β-induced C3 production in a dose-dependent fashion. These responses were associated with decreased C3 mRNA levels. In contrast, PDTC did not influence C3 production or C3 mRNA in the Caco-2 cells. Transfection of the Caco-2 cells with the Ser 32/36 mutant IkBα resulted in maintained IκBα levels and decreased IL-β-induced C3 production. Conclusions. IL-1β-stimulated C3 production in the enterocyte may be regulated by NF-κB.

AB - Background. Recent studies suggest that interleukin-1β (IL-1β) stimulates the production of the acute phase protein complement component C3 in human intestinal epithelial cells. The transcription factor NF-κB activates different genes involved in the response to cytokines. It is not known if IL-1β-induced C3 production in the enterocyte is regulated by NF- κB. Materials and methods. Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with one of the NF-κB inhibitors, tosyl- lys-chloromethylketone (TLCK), genistein, or pyrrolidine dithiocarbamate (PDTC), or with N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor known to block the degradation of IκB, the cytosolic inhibitor of NF-κB. Following this treatment, the Caco-2 cells were stimulated with IL-1β, and C3 levels in the culture medium were measured after 24 h by ELISA. C3 mRNA levels were determined after 4 h by Northern blot analysis. In other experiments, Caco-2 cells were transfected with a mutant IκBα in which serines 32 and 36 were substituted by alanine. This mutation prevents IkBα phosphorylation and subsequent NF-κB nuclear translocation. After transfection, the cells were stimulated with IL-1β, and C3 levels in the culture medium were measured after 24 h. Cytosolic IκBα was determined by Western blot analysis. Results. TLCK, genistein, and LLnL each inhibited IL- 1β-induced C3 production in a dose-dependent fashion. These responses were associated with decreased C3 mRNA levels. In contrast, PDTC did not influence C3 production or C3 mRNA in the Caco-2 cells. Transfection of the Caco-2 cells with the Ser 32/36 mutant IkBα resulted in maintained IκBα levels and decreased IL-β-induced C3 production. Conclusions. IL-1β-stimulated C3 production in the enterocyte may be regulated by NF-κB.

KW - Complement component C3

KW - Enterocyte

KW - Interleukin-1

KW - NF-κB

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