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p100 Deficiency is insufficient for full activation of the alternative NF-κB pathway: TNF cooperates with p52-RelB in target gene transcription.

Lovas A, Weidemann A, Albrecht D, Wiechert L, Weih D, Weih F - PLoS ONE (2012)

Bottom Line: Here, we focused on the question how does the constitutive alternative NF-κB signaling exert its effects in these malignant processes.Our results show that p100 deficiency alone was insufficient for full induction of genes regulated by the alternative NF-κB pathway.Moreover, alternative NF-κB signaling strongly synergized both in vitro and in vivo with classical NF-κB activation, thereby extending the number of genes under the control of the p100 inhibitor of the alternative NF-κB signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Research Group Immunology, Leibniz-Institute for Age Research - Fritz-Lipmann-Institute, Jena, Germany.

ABSTRACT

Background: Constitutive activation of the alternative NF-κB pathway leads to marginal zone B cell expansion and disorganized spleen microarchitecture. Furthermore, uncontrolled alternative NF-κB signaling may result in the development and progression of cancer. Here, we focused on the question how does the constitutive alternative NF-κB signaling exert its effects in these malignant processes.

Methodology/principal findings: To explore the consequences of unrestricted alternative NF-κB activation on genome-wide transcription, we compared gene expression profiles of wild-type and NF-κB2/p100-deficient (p100(-/-)) primary mouse embryonic fibroblasts (MEFs) and spleens. Microarray experiments revealed only 73 differentially regulated genes in p100(-/-) vs. wild-type MEFs. Chromatin immunoprecipitation (ChIP) assays showed in p100(-/-) MEFs direct binding of p52 and RelB to the promoter of the Enpp2 gene encoding ENPP2/Autotaxin, a protein with an important role in lymphocyte homing and cell migration. Gene ontology analysis revealed upregulation of genes with anti-apoptotic/proliferative activity (Enpp2/Atx, Serpina3g, Traf1, Rrad), chemotactic/locomotory activity (Enpp2/Atx, Ccl8), and lymphocyte homing activity (Enpp2/Atx, Cd34). Most importantly, biochemical and gene expression analyses of MEFs and spleen, respectively, indicated a marked crosstalk between classical and alternative NF-κB pathways.

Conclusions/significance: Our results show that p100 deficiency alone was insufficient for full induction of genes regulated by the alternative NF-κB pathway. Moreover, alternative NF-κB signaling strongly synergized both in vitro and in vivo with classical NF-κB activation, thereby extending the number of genes under the control of the p100 inhibitor of the alternative NF-κB signaling pathway.

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Lack of p100 cooperates with TNF to increase binding of p52 and RelB to the κB elements in the Enpp2/Atx promoter.Wild-type and p100−/− primary MEFs were stimulated with recombinant murine TNF (20 ng/ml) for 6 and 24 h or were left untreated and subsequently nuclear proteins were isolated. In vitro binding of NF-κB subunits p52 (A) and RelB (B) to the κB target sites in the Enpp2/Atx promoter was determined as in Figure 2. Data are presented as mean values ± SD from n = 3 independent experiments. Significant differences (P≤0.05) in p52 and RelB binding were calculated by Student's t-test and are indicated (*).
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pone-0042741-g005: Lack of p100 cooperates with TNF to increase binding of p52 and RelB to the κB elements in the Enpp2/Atx promoter.Wild-type and p100−/− primary MEFs were stimulated with recombinant murine TNF (20 ng/ml) for 6 and 24 h or were left untreated and subsequently nuclear proteins were isolated. In vitro binding of NF-κB subunits p52 (A) and RelB (B) to the κB target sites in the Enpp2/Atx promoter was determined as in Figure 2. Data are presented as mean values ± SD from n = 3 independent experiments. Significant differences (P≤0.05) in p52 and RelB binding were calculated by Student's t-test and are indicated (*).

Mentions: To investigate how the alternative NF-κB transcription factors ensure synergistic regulation of Enpp2/Atx, we assayed p52 and RelB binding to the ATX κB elements in response to TNF stimulation in wild-type and p100−/− MEFs. TNF treatment resulted in stronger DNA binding activities by both p52 and RelB in p100−/− compared to wild-type MEFs at each ATX κB site. This difference of Enpp2/Atx promoter binding was further increased upon prolonged TNF stimulation (Figure 5A and 5B), which is in agreement with the gene expression pattern of Enpp2/Atx in these cells (Figure 4B).


p100 Deficiency is insufficient for full activation of the alternative NF-κB pathway: TNF cooperates with p52-RelB in target gene transcription.

Lovas A, Weidemann A, Albrecht D, Wiechert L, Weih D, Weih F - PLoS ONE (2012)

Lack of p100 cooperates with TNF to increase binding of p52 and RelB to the κB elements in the Enpp2/Atx promoter.Wild-type and p100−/− primary MEFs were stimulated with recombinant murine TNF (20 ng/ml) for 6 and 24 h or were left untreated and subsequently nuclear proteins were isolated. In vitro binding of NF-κB subunits p52 (A) and RelB (B) to the κB target sites in the Enpp2/Atx promoter was determined as in Figure 2. Data are presented as mean values ± SD from n = 3 independent experiments. Significant differences (P≤0.05) in p52 and RelB binding were calculated by Student's t-test and are indicated (*).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412832&req=5

pone-0042741-g005: Lack of p100 cooperates with TNF to increase binding of p52 and RelB to the κB elements in the Enpp2/Atx promoter.Wild-type and p100−/− primary MEFs were stimulated with recombinant murine TNF (20 ng/ml) for 6 and 24 h or were left untreated and subsequently nuclear proteins were isolated. In vitro binding of NF-κB subunits p52 (A) and RelB (B) to the κB target sites in the Enpp2/Atx promoter was determined as in Figure 2. Data are presented as mean values ± SD from n = 3 independent experiments. Significant differences (P≤0.05) in p52 and RelB binding were calculated by Student's t-test and are indicated (*).
Mentions: To investigate how the alternative NF-κB transcription factors ensure synergistic regulation of Enpp2/Atx, we assayed p52 and RelB binding to the ATX κB elements in response to TNF stimulation in wild-type and p100−/− MEFs. TNF treatment resulted in stronger DNA binding activities by both p52 and RelB in p100−/− compared to wild-type MEFs at each ATX κB site. This difference of Enpp2/Atx promoter binding was further increased upon prolonged TNF stimulation (Figure 5A and 5B), which is in agreement with the gene expression pattern of Enpp2/Atx in these cells (Figure 4B).

Bottom Line: Here, we focused on the question how does the constitutive alternative NF-κB signaling exert its effects in these malignant processes.Our results show that p100 deficiency alone was insufficient for full induction of genes regulated by the alternative NF-κB pathway.Moreover, alternative NF-κB signaling strongly synergized both in vitro and in vivo with classical NF-κB activation, thereby extending the number of genes under the control of the p100 inhibitor of the alternative NF-κB signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Research Group Immunology, Leibniz-Institute for Age Research - Fritz-Lipmann-Institute, Jena, Germany.

ABSTRACT

Background: Constitutive activation of the alternative NF-κB pathway leads to marginal zone B cell expansion and disorganized spleen microarchitecture. Furthermore, uncontrolled alternative NF-κB signaling may result in the development and progression of cancer. Here, we focused on the question how does the constitutive alternative NF-κB signaling exert its effects in these malignant processes.

Methodology/principal findings: To explore the consequences of unrestricted alternative NF-κB activation on genome-wide transcription, we compared gene expression profiles of wild-type and NF-κB2/p100-deficient (p100(-/-)) primary mouse embryonic fibroblasts (MEFs) and spleens. Microarray experiments revealed only 73 differentially regulated genes in p100(-/-) vs. wild-type MEFs. Chromatin immunoprecipitation (ChIP) assays showed in p100(-/-) MEFs direct binding of p52 and RelB to the promoter of the Enpp2 gene encoding ENPP2/Autotaxin, a protein with an important role in lymphocyte homing and cell migration. Gene ontology analysis revealed upregulation of genes with anti-apoptotic/proliferative activity (Enpp2/Atx, Serpina3g, Traf1, Rrad), chemotactic/locomotory activity (Enpp2/Atx, Ccl8), and lymphocyte homing activity (Enpp2/Atx, Cd34). Most importantly, biochemical and gene expression analyses of MEFs and spleen, respectively, indicated a marked crosstalk between classical and alternative NF-κB pathways.

Conclusions/significance: Our results show that p100 deficiency alone was insufficient for full induction of genes regulated by the alternative NF-κB pathway. Moreover, alternative NF-κB signaling strongly synergized both in vitro and in vivo with classical NF-κB activation, thereby extending the number of genes under the control of the p100 inhibitor of the alternative NF-κB signaling pathway.

Show MeSH
Related in: MedlinePlus