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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 results in enhanced p52 and RelB binding to NF-κB target sites in the Enpp2/Atx promoter.(A) Schematic view of the Enpp2/Atx promoter. In silico analysis of the Enpp2/Atx promoter (−2485 bp upstream of the ATG codon) with MatInspector and TFSearch softwares revealed four potential NF-κB sites: ATX4 at position −1162 (CGGGGGCTTC), ATX3.2 at position −596 (GGAAGCTCCC), ATX3.1 at position −529 (AGGGTCATTCC), and ATX1 at position −375 (GGGAAATTCT). ATX3.2 and ATX1 κB target site have been previously described using the software Footer [30]. (B) In vitro binding of p52 and RelB to κB target sites in the Enpp2/Atx promoter was determined by the TransAM Flexi NF-κB Family Transcription Factor Assay (Active Motif). Three independent experiments were performed. Data are presented as mean values ± SD. Statistically significant differences are indicated by * (Student's t-test, P≤0.05). (C) In vivo binding of p52 and RelB to κB target sites in the Enpp2/Atx promoter. An unrelated site (ATXneg) served as a negative control. For ChIP experiments, the Express Magnetic Chromatin Immunoprecipitation Kit (Active Motif) was employed according to the manufacturer's instructions. ChIP assays for RNA polymerase II binding to the Gapdh promoter were used for normalization as input control. Data are presented as mean values ± SD, n = 3. Statistically significant differences are indicated by * (Student's t-test, P≤0.05).
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pone-0042741-g002: Lack of p100 results in enhanced p52 and RelB binding to NF-κB target sites in the Enpp2/Atx promoter.(A) Schematic view of the Enpp2/Atx promoter. In silico analysis of the Enpp2/Atx promoter (−2485 bp upstream of the ATG codon) with MatInspector and TFSearch softwares revealed four potential NF-κB sites: ATX4 at position −1162 (CGGGGGCTTC), ATX3.2 at position −596 (GGAAGCTCCC), ATX3.1 at position −529 (AGGGTCATTCC), and ATX1 at position −375 (GGGAAATTCT). ATX3.2 and ATX1 κB target site have been previously described using the software Footer [30]. (B) In vitro binding of p52 and RelB to κB target sites in the Enpp2/Atx promoter was determined by the TransAM Flexi NF-κB Family Transcription Factor Assay (Active Motif). Three independent experiments were performed. Data are presented as mean values ± SD. Statistically significant differences are indicated by * (Student's t-test, P≤0.05). (C) In vivo binding of p52 and RelB to κB target sites in the Enpp2/Atx promoter. An unrelated site (ATXneg) served as a negative control. For ChIP experiments, the Express Magnetic Chromatin Immunoprecipitation Kit (Active Motif) was employed according to the manufacturer's instructions. ChIP assays for RNA polymerase II binding to the Gapdh promoter were used for normalization as input control. Data are presented as mean values ± SD, n = 3. Statistically significant differences are indicated by * (Student's t-test, P≤0.05).

Mentions: As the alternative NF-κB pathway is a major factor in controlling secondary lymphoid organ development and since Enpp2 mRNA levels were increased in p100−/− vs. wild-type MEFs we investigated whether RelB and p52 directly regulate Enpp2 mRNA expression. Enpp2 (also known as autotaxin, ATX) is a recently described molecule involved in lymphocyte homing [31]. In addition to NFAT1 and HOXA13, NF-κB/RelA has been suggested to regulate Enpp2 expression in LPS-stimulated dendritic cells [32], [33], [34]. In silico analysis of the mouse Enpp2/Atx promoter identified four putative κB target sites: ATX4, ATX3.2, ATX3.1, and ATX1 (Figure 2A). In vitro assays showed that binding of p50 and RelA to any of the four κB sites was close to background and unaffected by the loss of p100 (Figure S1). In contrast, binding of both p52 and RelB to the putative κB sequences - in particular to sites ATX4, ATX3.2, and ATX1 - was strongly increased in p100−/− compared to wild-type MEFs. The κB sites ATX3.2 and ATX3.1 are separated by ca. 70 bp. However, we did not observe cooperative binding of RelB or p52 to these κB sequences (Figure 2B). Binding of NF-κB subunits to unrelated sequences (ATXunr1 and ATXunr2) in the Enpp2/Atx promoter was close to background and unchanged between wild-type and p100−/− MEFs (data not shown). Chromatin immunoprecipitation (ChIP) experiments verified direct binding of p52 and RelB to the ATX κB sites in vivo (Figure 2C). Thus, loss of p100 results in increased p52 and RelB binding to κB sites in the Enpp2/Atx promoter, indicating a direct transcriptional regulation of Enpp2/Atx by the alternative NF-κB pathway.


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 results in enhanced p52 and RelB binding to NF-κB target sites in the Enpp2/Atx promoter.(A) Schematic view of the Enpp2/Atx promoter. In silico analysis of the Enpp2/Atx promoter (−2485 bp upstream of the ATG codon) with MatInspector and TFSearch softwares revealed four potential NF-κB sites: ATX4 at position −1162 (CGGGGGCTTC), ATX3.2 at position −596 (GGAAGCTCCC), ATX3.1 at position −529 (AGGGTCATTCC), and ATX1 at position −375 (GGGAAATTCT). ATX3.2 and ATX1 κB target site have been previously described using the software Footer [30]. (B) In vitro binding of p52 and RelB to κB target sites in the Enpp2/Atx promoter was determined by the TransAM Flexi NF-κB Family Transcription Factor Assay (Active Motif). Three independent experiments were performed. Data are presented as mean values ± SD. Statistically significant differences are indicated by * (Student's t-test, P≤0.05). (C) In vivo binding of p52 and RelB to κB target sites in the Enpp2/Atx promoter. An unrelated site (ATXneg) served as a negative control. For ChIP experiments, the Express Magnetic Chromatin Immunoprecipitation Kit (Active Motif) was employed according to the manufacturer's instructions. ChIP assays for RNA polymerase II binding to the Gapdh promoter were used for normalization as input control. Data are presented as mean values ± SD, n = 3. Statistically significant differences are indicated by * (Student's t-test, P≤0.05).
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Related In: Results  -  Collection

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pone-0042741-g002: Lack of p100 results in enhanced p52 and RelB binding to NF-κB target sites in the Enpp2/Atx promoter.(A) Schematic view of the Enpp2/Atx promoter. In silico analysis of the Enpp2/Atx promoter (−2485 bp upstream of the ATG codon) with MatInspector and TFSearch softwares revealed four potential NF-κB sites: ATX4 at position −1162 (CGGGGGCTTC), ATX3.2 at position −596 (GGAAGCTCCC), ATX3.1 at position −529 (AGGGTCATTCC), and ATX1 at position −375 (GGGAAATTCT). ATX3.2 and ATX1 κB target site have been previously described using the software Footer [30]. (B) In vitro binding of p52 and RelB to κB target sites in the Enpp2/Atx promoter was determined by the TransAM Flexi NF-κB Family Transcription Factor Assay (Active Motif). Three independent experiments were performed. Data are presented as mean values ± SD. Statistically significant differences are indicated by * (Student's t-test, P≤0.05). (C) In vivo binding of p52 and RelB to κB target sites in the Enpp2/Atx promoter. An unrelated site (ATXneg) served as a negative control. For ChIP experiments, the Express Magnetic Chromatin Immunoprecipitation Kit (Active Motif) was employed according to the manufacturer's instructions. ChIP assays for RNA polymerase II binding to the Gapdh promoter were used for normalization as input control. Data are presented as mean values ± SD, n = 3. Statistically significant differences are indicated by * (Student's t-test, P≤0.05).
Mentions: As the alternative NF-κB pathway is a major factor in controlling secondary lymphoid organ development and since Enpp2 mRNA levels were increased in p100−/− vs. wild-type MEFs we investigated whether RelB and p52 directly regulate Enpp2 mRNA expression. Enpp2 (also known as autotaxin, ATX) is a recently described molecule involved in lymphocyte homing [31]. In addition to NFAT1 and HOXA13, NF-κB/RelA has been suggested to regulate Enpp2 expression in LPS-stimulated dendritic cells [32], [33], [34]. In silico analysis of the mouse Enpp2/Atx promoter identified four putative κB target sites: ATX4, ATX3.2, ATX3.1, and ATX1 (Figure 2A). In vitro assays showed that binding of p50 and RelA to any of the four κB sites was close to background and unaffected by the loss of p100 (Figure S1). In contrast, binding of both p52 and RelB to the putative κB sequences - in particular to sites ATX4, ATX3.2, and ATX1 - was strongly increased in p100−/− compared to wild-type MEFs. The κB sites ATX3.2 and ATX3.1 are separated by ca. 70 bp. However, we did not observe cooperative binding of RelB or p52 to these κB sequences (Figure 2B). Binding of NF-κB subunits to unrelated sequences (ATXunr1 and ATXunr2) in the Enpp2/Atx promoter was close to background and unchanged between wild-type and p100−/− MEFs (data not shown). Chromatin immunoprecipitation (ChIP) experiments verified direct binding of p52 and RelB to the ATX κB sites in vivo (Figure 2C). Thus, loss of p100 results in increased p52 and RelB binding to κB sites in the Enpp2/Atx promoter, indicating a direct transcriptional regulation of Enpp2/Atx by the alternative NF-κB pathway.

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