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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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TNF synergizes with the lack of p100 in the induction of target gene expression.(A) Western blot of NF-κB family members in cytoplasmic and nuclear protein extracts (15 µg/sample) from unstimulated (0 h) and TNF-stimulated (6 and 24 h, 20 ng/ml recombinant murine TNF) wild-type and p100−/− MEFs. As cytoplasmic and nuclear loading controls β-actin and RNA Pol II were assayed, respectively. (B) Changes in mRNA levels of selected genes were analyzed by qRT-PCR. From 16 analyzed genes, 12 responded synergistically to TNF and the lack of p100 whereas only four genes responded similarly to TNF treatment of wild-type (grey squares) and p100−/− MEFs (black circles; see also Figure S4). qRT-PCR data represent n = 3 independent TNF stimulation experiments and are expressed as mean values ± SD. Differences between wild-type and p100−/− MEFs at each time-point were analyzed by Welch tests. P≤0.05 was considered significant (*).
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pone-0042741-g004: TNF synergizes with the lack of p100 in the induction of target gene expression.(A) Western blot of NF-κB family members in cytoplasmic and nuclear protein extracts (15 µg/sample) from unstimulated (0 h) and TNF-stimulated (6 and 24 h, 20 ng/ml recombinant murine TNF) wild-type and p100−/− MEFs. As cytoplasmic and nuclear loading controls β-actin and RNA Pol II were assayed, respectively. (B) Changes in mRNA levels of selected genes were analyzed by qRT-PCR. From 16 analyzed genes, 12 responded synergistically to TNF and the lack of p100 whereas only four genes responded similarly to TNF treatment of wild-type (grey squares) and p100−/− MEFs (black circles; see also Figure S4). qRT-PCR data represent n = 3 independent TNF stimulation experiments and are expressed as mean values ± SD. Differences between wild-type and p100−/− MEFs at each time-point were analyzed by Welch tests. P≤0.05 was considered significant (*).

Mentions: In agreement with a previous report [21], we observed increased Ccl21, Baff, Vcam1, Icam1, Madcam1, and Glycam1 mRNA expression in spleens of p100−/− mice compared to wild-type controls (data not shown). In addition, mutant spleens had increased mRNA expression of the proinflammatory chemokines encoding genes Cx3cl1 and Cxcl10 (Figure S3). Surprisingly, none of these genes was regulated in p100−/− vs. wild-type MEFs (data not shown). Therefore, we reasoned that in contrast to p100−/− MEFs kept under non-stimulating cell culture conditions, splenocytes of p100−/− mice may provide additional signals that sensitize alternative NF-κB signaling. To test whether signals from the classical pathway cross-talk to alternative NF-κB activation we chose TNF, a well-known and selective inducer of the classical pathway [16]. As shown in Figure 4A, TNF treatment resulted in increased cytoplasmic p100 in wild-type and increased nuclear p52 levels in p100−/− MEFs. Nuclear accumulation of p52 was restricted to p100−/− cells and accompanied by a marked increase in nuclear RelB. This result reflects enhanced nuclear translocation of p52-RelB heterodimers due to the TNF-induced increase of the cytoplasmic pools of p52 and RelB in combination with the lack of the p100 inhibitor [16] (data not shown). In contrast to p52 and RelB, TNF-induced nuclear translocation of RelA was similar in both wild-type and mutant MEFs. Collectively, these data show that in the absence of the p100 inhibitor TNF stimulates nuclear translocation of alternative p52-RelB complexes (Figure 4A).


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)

TNF synergizes with the lack of p100 in the induction of target gene expression.(A) Western blot of NF-κB family members in cytoplasmic and nuclear protein extracts (15 µg/sample) from unstimulated (0 h) and TNF-stimulated (6 and 24 h, 20 ng/ml recombinant murine TNF) wild-type and p100−/− MEFs. As cytoplasmic and nuclear loading controls β-actin and RNA Pol II were assayed, respectively. (B) Changes in mRNA levels of selected genes were analyzed by qRT-PCR. From 16 analyzed genes, 12 responded synergistically to TNF and the lack of p100 whereas only four genes responded similarly to TNF treatment of wild-type (grey squares) and p100−/− MEFs (black circles; see also Figure S4). qRT-PCR data represent n = 3 independent TNF stimulation experiments and are expressed as mean values ± SD. Differences between wild-type and p100−/− MEFs at each time-point were analyzed by Welch tests. P≤0.05 was considered significant (*).
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Related In: Results  -  Collection

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pone-0042741-g004: TNF synergizes with the lack of p100 in the induction of target gene expression.(A) Western blot of NF-κB family members in cytoplasmic and nuclear protein extracts (15 µg/sample) from unstimulated (0 h) and TNF-stimulated (6 and 24 h, 20 ng/ml recombinant murine TNF) wild-type and p100−/− MEFs. As cytoplasmic and nuclear loading controls β-actin and RNA Pol II were assayed, respectively. (B) Changes in mRNA levels of selected genes were analyzed by qRT-PCR. From 16 analyzed genes, 12 responded synergistically to TNF and the lack of p100 whereas only four genes responded similarly to TNF treatment of wild-type (grey squares) and p100−/− MEFs (black circles; see also Figure S4). qRT-PCR data represent n = 3 independent TNF stimulation experiments and are expressed as mean values ± SD. Differences between wild-type and p100−/− MEFs at each time-point were analyzed by Welch tests. P≤0.05 was considered significant (*).
Mentions: In agreement with a previous report [21], we observed increased Ccl21, Baff, Vcam1, Icam1, Madcam1, and Glycam1 mRNA expression in spleens of p100−/− mice compared to wild-type controls (data not shown). In addition, mutant spleens had increased mRNA expression of the proinflammatory chemokines encoding genes Cx3cl1 and Cxcl10 (Figure S3). Surprisingly, none of these genes was regulated in p100−/− vs. wild-type MEFs (data not shown). Therefore, we reasoned that in contrast to p100−/− MEFs kept under non-stimulating cell culture conditions, splenocytes of p100−/− mice may provide additional signals that sensitize alternative NF-κB signaling. To test whether signals from the classical pathway cross-talk to alternative NF-κB activation we chose TNF, a well-known and selective inducer of the classical pathway [16]. As shown in Figure 4A, TNF treatment resulted in increased cytoplasmic p100 in wild-type and increased nuclear p52 levels in p100−/− MEFs. Nuclear accumulation of p52 was restricted to p100−/− cells and accompanied by a marked increase in nuclear RelB. This result reflects enhanced nuclear translocation of p52-RelB heterodimers due to the TNF-induced increase of the cytoplasmic pools of p52 and RelB in combination with the lack of the p100 inhibitor [16] (data not shown). In contrast to p52 and RelB, TNF-induced nuclear translocation of RelA was similar in both wild-type and mutant MEFs. Collectively, these data show that in the absence of the p100 inhibitor TNF stimulates nuclear translocation of alternative p52-RelB complexes (Figure 4A).

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