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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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Gene expression analysis of wild-type and p100−/− spleens.(A) Changes in mRNA levels of selected genes were analyzed by qRT-PCR using RNA samples isolated from spleens of four wild-type and four p100−/− animals. Data are expressed as mean values ± SD. Differences were analyzed by Welch tests. P≤0.05 was considered significant. All genes shown were significantly differentially regulated in wild-type compared to mutant mice. (B) Western blot analysis of total protein extracts from wild-type and p100−/− spleens (30 µg/sample; protein extracts from three independent spleens are shown for each genotype) for the presence of p100/p52, ENPP2/ATX, and TRAF1. p52 wild-type protein and p52 protein resulting from the knock-in of the stop codon migrated with different speed due to a few amino acids difference in length. The asterisk indicates an unspecific signal. The membrane was probed for β-actin as a loading control.
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pone-0042741-g003: Gene expression analysis of wild-type and p100−/− spleens.(A) Changes in mRNA levels of selected genes were analyzed by qRT-PCR using RNA samples isolated from spleens of four wild-type and four p100−/− animals. Data are expressed as mean values ± SD. Differences were analyzed by Welch tests. P≤0.05 was considered significant. All genes shown were significantly differentially regulated in wild-type compared to mutant mice. (B) Western blot analysis of total protein extracts from wild-type and p100−/− spleens (30 µg/sample; protein extracts from three independent spleens are shown for each genotype) for the presence of p100/p52, ENPP2/ATX, and TRAF1. p52 wild-type protein and p52 protein resulting from the knock-in of the stop codon migrated with different speed due to a few amino acids difference in length. The asterisk indicates an unspecific signal. The membrane was probed for β-actin as a loading control.

Mentions: Collectively, gene ontology analysis revealed enrichment of terms related to development/structure morphogenesis, extracellular region, but also to cell growth, inflammatory response, taxis/locomotory behavior, suggesting structural rearrangements in organs of p100−/− mice, which is in agreement with earlier studies [20], [21]. As the alternative NF-κB signaling pathway has a pivotal role in the development and maintenance of secondary lymphoid organs, we examined selected genes annotated to the above GO terms. Expression analysis of p100−/− vs. wild-type spleens showed significant 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). Gene expression of Nfkb2, as assayed with 3′-end primers, was diminished in p100−/− spleen as this mutant lacks the 3′-end of the gene. Expression of the remaining 5′-part, encoding p52, was increased since p52 exerts a positive feedback loop on its own regulation [20] (Figure 3A). ENPP2/ATX was also tested in Western blots. As expected, ENPP2/ATX protein levels were increased in spleen of p100−/− vs. wild-type mice. Expression of the anti-apoptotic protein TRAF1 was also elevated in spleens of p100−/− mutant mice (Figure 3B).


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)

Gene expression analysis of wild-type and p100−/− spleens.(A) Changes in mRNA levels of selected genes were analyzed by qRT-PCR using RNA samples isolated from spleens of four wild-type and four p100−/− animals. Data are expressed as mean values ± SD. Differences were analyzed by Welch tests. P≤0.05 was considered significant. All genes shown were significantly differentially regulated in wild-type compared to mutant mice. (B) Western blot analysis of total protein extracts from wild-type and p100−/− spleens (30 µg/sample; protein extracts from three independent spleens are shown for each genotype) for the presence of p100/p52, ENPP2/ATX, and TRAF1. p52 wild-type protein and p52 protein resulting from the knock-in of the stop codon migrated with different speed due to a few amino acids difference in length. The asterisk indicates an unspecific signal. The membrane was probed for β-actin as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042741-g003: Gene expression analysis of wild-type and p100−/− spleens.(A) Changes in mRNA levels of selected genes were analyzed by qRT-PCR using RNA samples isolated from spleens of four wild-type and four p100−/− animals. Data are expressed as mean values ± SD. Differences were analyzed by Welch tests. P≤0.05 was considered significant. All genes shown were significantly differentially regulated in wild-type compared to mutant mice. (B) Western blot analysis of total protein extracts from wild-type and p100−/− spleens (30 µg/sample; protein extracts from three independent spleens are shown for each genotype) for the presence of p100/p52, ENPP2/ATX, and TRAF1. p52 wild-type protein and p52 protein resulting from the knock-in of the stop codon migrated with different speed due to a few amino acids difference in length. The asterisk indicates an unspecific signal. The membrane was probed for β-actin as a loading control.
Mentions: Collectively, gene ontology analysis revealed enrichment of terms related to development/structure morphogenesis, extracellular region, but also to cell growth, inflammatory response, taxis/locomotory behavior, suggesting structural rearrangements in organs of p100−/− mice, which is in agreement with earlier studies [20], [21]. As the alternative NF-κB signaling pathway has a pivotal role in the development and maintenance of secondary lymphoid organs, we examined selected genes annotated to the above GO terms. Expression analysis of p100−/− vs. wild-type spleens showed significant 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). Gene expression of Nfkb2, as assayed with 3′-end primers, was diminished in p100−/− spleen as this mutant lacks the 3′-end of the gene. Expression of the remaining 5′-part, encoding p52, was increased since p52 exerts a positive feedback loop on its own regulation [20] (Figure 3A). ENPP2/ATX was also tested in Western blots. As expected, ENPP2/ATX protein levels were increased in spleen of p100−/− vs. wild-type mice. Expression of the anti-apoptotic protein TRAF1 was also elevated in spleens of p100−/− mutant mice (Figure 3B).

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