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Neisseria gonorrhoeae epithelial cell interaction leads to the activation of the transcription factors nuclear factor kappaB and activator protein 1 and the induction of inflammatory cytokines.

Naumann M, Wessler S, Bartsch C, Wieland B, Meyer TF - J. Exp. Med. (1997)

Bottom Line: In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer.Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation.Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, 10117 Berlin. naumann@mpiib-berlin.mpg.de

ABSTRACT
We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-kappaB and the transcriptional activation of inflammatory cytokine genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-kappaB DNA-protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer. The NF-kappaB complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-kappaB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection. Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes. Activation of NF-kappaB and cytokine mRNA upregulation also occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.

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Ngo infection induces expression of IL-6 promoter–hGH  constructs transfected in HeLa cells. (A, left) Schematic representation of  the deleted IL-6 promoter constructs. The transcription start site is designated with a black arrow. (A, right, and B) HeLa cells were transfected by  cationic liposomes with 3 μg of plasmids. Cells were maintained in medium containing 10% FCS for 24 h. 1 h before the infection, the medium  was exchanged and the cells either untreated or infected with the Ngo P+  strain at a MOI of 100. hGH activity was assessed in culture supernatants  by ELISA, and the results of three independent experiments expressed as  fold induction against noninfected cells.
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Figure 5: Ngo infection induces expression of IL-6 promoter–hGH constructs transfected in HeLa cells. (A, left) Schematic representation of the deleted IL-6 promoter constructs. The transcription start site is designated with a black arrow. (A, right, and B) HeLa cells were transfected by cationic liposomes with 3 μg of plasmids. Cells were maintained in medium containing 10% FCS for 24 h. 1 h before the infection, the medium was exchanged and the cells either untreated or infected with the Ngo P+ strain at a MOI of 100. hGH activity was assessed in culture supernatants by ELISA, and the results of three independent experiments expressed as fold induction against noninfected cells.

Mentions: As shown in Fig. 1 A, IL-6 mRNA is upregulated in response to Ngo infection. Therefore, the following experiment was aimed to investigate the molecular mechanism mediating this effect. To this end, deleted forms of IL-6 promoter fragments linked to the hGH gene as a reporter gene were used (Fig. 5 A), and plasmids were transiently transfected into HeLa cells. Promoter activity was assayed after Ngo infection by analysing the activity of the hGH in cell-free culture supernatants. Infection experiments were performed for 3 h in the presence of 10% FCS. Transcriptional induction by infection with P+ Ngo was observed in all promoter constructs with the exception of the promoter construct deleted upstream of position −49, containing no enhancer elements. As much as approximately threefold hGH activity was observed from the longest promoter construct deleted upstream of position −524 containing the NF-κB, NF–IL-6, and AP-1 enhancer elements (Fig. 5 A). With this construct, the exchange of the medium before the infection already led to high hGH levels (Fig. 5 B). Transfection of the promoter construct upstream of position −219 lacking the binding site for the transcription factor AP-1 (position −283 to −277) caused a drastic reduction of the residual hGH release, but did not affect the inducibility of hGH release by Ngo infection. Although the NF–IL-6–binding site at position −158 to −145 has been demonstrated to be involved in transcriptional activation of the IL-6 promoter (25), we observed no activation of NF– IL-6 at the C/EBP DNA-binding site in response to Ngo infection by EMSA (Fig. 2 D). Thus, NF–IL-6 does not appear to contribute in IL-6 promoter activation. These data rather suggest that NF-κB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection.


Neisseria gonorrhoeae epithelial cell interaction leads to the activation of the transcription factors nuclear factor kappaB and activator protein 1 and the induction of inflammatory cytokines.

Naumann M, Wessler S, Bartsch C, Wieland B, Meyer TF - J. Exp. Med. (1997)

Ngo infection induces expression of IL-6 promoter–hGH  constructs transfected in HeLa cells. (A, left) Schematic representation of  the deleted IL-6 promoter constructs. The transcription start site is designated with a black arrow. (A, right, and B) HeLa cells were transfected by  cationic liposomes with 3 μg of plasmids. Cells were maintained in medium containing 10% FCS for 24 h. 1 h before the infection, the medium  was exchanged and the cells either untreated or infected with the Ngo P+  strain at a MOI of 100. hGH activity was assessed in culture supernatants  by ELISA, and the results of three independent experiments expressed as  fold induction against noninfected cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198971&req=5

Figure 5: Ngo infection induces expression of IL-6 promoter–hGH constructs transfected in HeLa cells. (A, left) Schematic representation of the deleted IL-6 promoter constructs. The transcription start site is designated with a black arrow. (A, right, and B) HeLa cells were transfected by cationic liposomes with 3 μg of plasmids. Cells were maintained in medium containing 10% FCS for 24 h. 1 h before the infection, the medium was exchanged and the cells either untreated or infected with the Ngo P+ strain at a MOI of 100. hGH activity was assessed in culture supernatants by ELISA, and the results of three independent experiments expressed as fold induction against noninfected cells.
Mentions: As shown in Fig. 1 A, IL-6 mRNA is upregulated in response to Ngo infection. Therefore, the following experiment was aimed to investigate the molecular mechanism mediating this effect. To this end, deleted forms of IL-6 promoter fragments linked to the hGH gene as a reporter gene were used (Fig. 5 A), and plasmids were transiently transfected into HeLa cells. Promoter activity was assayed after Ngo infection by analysing the activity of the hGH in cell-free culture supernatants. Infection experiments were performed for 3 h in the presence of 10% FCS. Transcriptional induction by infection with P+ Ngo was observed in all promoter constructs with the exception of the promoter construct deleted upstream of position −49, containing no enhancer elements. As much as approximately threefold hGH activity was observed from the longest promoter construct deleted upstream of position −524 containing the NF-κB, NF–IL-6, and AP-1 enhancer elements (Fig. 5 A). With this construct, the exchange of the medium before the infection already led to high hGH levels (Fig. 5 B). Transfection of the promoter construct upstream of position −219 lacking the binding site for the transcription factor AP-1 (position −283 to −277) caused a drastic reduction of the residual hGH release, but did not affect the inducibility of hGH release by Ngo infection. Although the NF–IL-6–binding site at position −158 to −145 has been demonstrated to be involved in transcriptional activation of the IL-6 promoter (25), we observed no activation of NF– IL-6 at the C/EBP DNA-binding site in response to Ngo infection by EMSA (Fig. 2 D). Thus, NF–IL-6 does not appear to contribute in IL-6 promoter activation. These data rather suggest that NF-κB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection.

Bottom Line: In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer.Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation.Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Infektionsbiologie, Abteilung Molekulare Biologie, 10117 Berlin. naumann@mpiib-berlin.mpg.de

ABSTRACT
We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-kappaB and the transcriptional activation of inflammatory cytokine genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-kappaB DNA-protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer. The NF-kappaB complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-kappaB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection. Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes. Activation of NF-kappaB and cytokine mRNA upregulation also occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.

Show MeSH
Related in: MedlinePlus