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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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Synergistic regulation of gene expression by the classical (TNF) and the alternative (p100−/−) NF-κB pathway in vivo.Changes in mRNA levels of selected genes contributing to chemotaxis, lymphocyte homing, and cell adhesion were analyzed by qRT-PCR using RNA samples isolated from spleens of control (wild-type for the Nfkb2 locus and heterozygous for the TnfLta locus), p100−/− (deficient for the p100/Nfkb2 and wild-type for the TnfLta locus), and p100−/− Tnf−/− (deficient for both the p100/Nfkb2 and the TnfLta locus) animals. Four independent experiments were performed representing n = 6 control mice, n = 5 p100−/− mice, and n = 4 p100−/− Tnf−/− mice. Data are presented as mean values ± SD. Significant differences (P≤0.05) were calculated by Student's t-test and are indicated (*). * P≤0.05; ** P≤0.01; n.s. = not significant.
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pone-0042741-g006: Synergistic regulation of gene expression by the classical (TNF) and the alternative (p100−/−) NF-κB pathway in vivo.Changes in mRNA levels of selected genes contributing to chemotaxis, lymphocyte homing, and cell adhesion were analyzed by qRT-PCR using RNA samples isolated from spleens of control (wild-type for the Nfkb2 locus and heterozygous for the TnfLta locus), p100−/− (deficient for the p100/Nfkb2 and wild-type for the TnfLta locus), and p100−/− Tnf−/− (deficient for both the p100/Nfkb2 and the TnfLta locus) animals. Four independent experiments were performed representing n = 6 control mice, n = 5 p100−/− mice, and n = 4 p100−/− Tnf−/− mice. Data are presented as mean values ± SD. Significant differences (P≤0.05) were calculated by Student's t-test and are indicated (*). * P≤0.05; ** P≤0.01; n.s. = not significant.

Mentions: To further investigate synergistic regulation exerted by TNF signaling in combination with the constitutively active alternative NF-κB pathway, we analyzed gene expression in spleens from control, p100−/−, and p100−/− TnfLta−/− mutant animals. To allow a direct comparison with the data obtained from TNF-treated MEFs (see Figure 4), we focused our analysis on genes regulated by LTβR signaling in stromal cells [27], [28], [29], [30]. Expression of Ccl21, Cxcl13, Madcam1, Glycam1, and Enpp2/Atx was markedly upregulated in p100−/− compared to control spleen. This upregulation was strongly reduced in p100−/− TnfLta−/− mutant spleen. In contrast, expression of Ccl8 was not affected by TNF in p100−/− spleen, similar to the result from MEFs (Figure 6). Thus, the observed in vitro synergy between the lack of the p100 inhibitor and TNF signaling could largely be reproduced in vivo.


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)

Synergistic regulation of gene expression by the classical (TNF) and the alternative (p100−/−) NF-κB pathway in vivo.Changes in mRNA levels of selected genes contributing to chemotaxis, lymphocyte homing, and cell adhesion were analyzed by qRT-PCR using RNA samples isolated from spleens of control (wild-type for the Nfkb2 locus and heterozygous for the TnfLta locus), p100−/− (deficient for the p100/Nfkb2 and wild-type for the TnfLta locus), and p100−/− Tnf−/− (deficient for both the p100/Nfkb2 and the TnfLta locus) animals. Four independent experiments were performed representing n = 6 control mice, n = 5 p100−/− mice, and n = 4 p100−/− Tnf−/− mice. Data are presented as mean values ± SD. Significant differences (P≤0.05) were calculated by Student's t-test and are indicated (*). * P≤0.05; ** P≤0.01; n.s. = not significant.
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Related In: Results  -  Collection

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pone-0042741-g006: Synergistic regulation of gene expression by the classical (TNF) and the alternative (p100−/−) NF-κB pathway in vivo.Changes in mRNA levels of selected genes contributing to chemotaxis, lymphocyte homing, and cell adhesion were analyzed by qRT-PCR using RNA samples isolated from spleens of control (wild-type for the Nfkb2 locus and heterozygous for the TnfLta locus), p100−/− (deficient for the p100/Nfkb2 and wild-type for the TnfLta locus), and p100−/− Tnf−/− (deficient for both the p100/Nfkb2 and the TnfLta locus) animals. Four independent experiments were performed representing n = 6 control mice, n = 5 p100−/− mice, and n = 4 p100−/− Tnf−/− mice. Data are presented as mean values ± SD. Significant differences (P≤0.05) were calculated by Student's t-test and are indicated (*). * P≤0.05; ** P≤0.01; n.s. = not significant.
Mentions: To further investigate synergistic regulation exerted by TNF signaling in combination with the constitutively active alternative NF-κB pathway, we analyzed gene expression in spleens from control, p100−/−, and p100−/− TnfLta−/− mutant animals. To allow a direct comparison with the data obtained from TNF-treated MEFs (see Figure 4), we focused our analysis on genes regulated by LTβR signaling in stromal cells [27], [28], [29], [30]. Expression of Ccl21, Cxcl13, Madcam1, Glycam1, and Enpp2/Atx was markedly upregulated in p100−/− compared to control spleen. This upregulation was strongly reduced in p100−/− TnfLta−/− mutant spleen. In contrast, expression of Ccl8 was not affected by TNF in p100−/− spleen, similar to the result from MEFs (Figure 6). Thus, the observed in vitro synergy between the lack of the p100 inhibitor and TNF signaling could largely be reproduced in vivo.

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