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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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Expression profiling of wild-type and p100−/− MEFs.(A) Western blot analysis of cytoplasmic and nuclear protein extracts (20 µg/sample) were analyzed for the presence of NF-κB family members p100/p52, RelB, and RelA in wild-type (+/+) and in p100−/− (−/−) MEFs. As cytoplasmic loading control S6 ribosomal protein and as nuclear loading control RNA Pol II was assayed. (B) Increased κB DNA-binding activity in nuclear extracts from p100−/− (−/−) compared to wild-type MEFs (+/+). Five µg protein extract per cell line were incubated with a radioactively labeled Igκ oligo and analyzed by EMSA. Supershift analysis was performed using pre-immune serum (p.i.), anti-RelA (α-RelA), anti-RelB (α-RelB), and anti-p52 antibodies (α-p52). Super-shifted RelB and p52 complexes are indicated by arrow and arrowhead, respectively. (C) Heatmap displaying fold change values observed in p100−/− vs. wild-type cells. The color code indicates the fold change values between +6 fold up- (red) and −20 fold downregulation (green). Each horizontal line on the heatmap corresponds to one gene. Genes labeled with blue boxes on the left were verified by qRT-PCR. Gene symbols and abbreviations of GO terms are displayed on the right. CA, Cytokine activity; GPCRB, G-protein-coupled receptor binding; IGFB, insulin-like growth factor binding; ER, extracellular region; ESO, extracellular structure organization and biogenesis; D/M, developmental process; B/ND, forebrain development and nervous system development; CG/S, regulation of cell size; IR/RES, immune response and response to external stimulus; M/L/T, locomotory behavior. (D) Significantly regulated Gene Ontology terms with respective gene numbers.
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pone-0042741-g001: Expression profiling of wild-type and p100−/− MEFs.(A) Western blot analysis of cytoplasmic and nuclear protein extracts (20 µg/sample) were analyzed for the presence of NF-κB family members p100/p52, RelB, and RelA in wild-type (+/+) and in p100−/− (−/−) MEFs. As cytoplasmic loading control S6 ribosomal protein and as nuclear loading control RNA Pol II was assayed. (B) Increased κB DNA-binding activity in nuclear extracts from p100−/− (−/−) compared to wild-type MEFs (+/+). Five µg protein extract per cell line were incubated with a radioactively labeled Igκ oligo and analyzed by EMSA. Supershift analysis was performed using pre-immune serum (p.i.), anti-RelA (α-RelA), anti-RelB (α-RelB), and anti-p52 antibodies (α-p52). Super-shifted RelB and p52 complexes are indicated by arrow and arrowhead, respectively. (C) Heatmap displaying fold change values observed in p100−/− vs. wild-type cells. The color code indicates the fold change values between +6 fold up- (red) and −20 fold downregulation (green). Each horizontal line on the heatmap corresponds to one gene. Genes labeled with blue boxes on the left were verified by qRT-PCR. Gene symbols and abbreviations of GO terms are displayed on the right. CA, Cytokine activity; GPCRB, G-protein-coupled receptor binding; IGFB, insulin-like growth factor binding; ER, extracellular region; ESO, extracellular structure organization and biogenesis; D/M, developmental process; B/ND, forebrain development and nervous system development; CG/S, regulation of cell size; IR/RES, immune response and response to external stimulus; M/L/T, locomotory behavior. (D) Significantly regulated Gene Ontology terms with respective gene numbers.

Mentions: To explore the consequences of constitutively active alternative NF-κB signaling on genome-wide transcription, we investigated the gene expression profile of primary fibroblasts isolated from wild-type and p100−/− mouse embryos [20]. We have chosen this experimental system since the lack of p100 in mice results in multiple defects that affect organization and function of lymphoid tissues [20]–[22], thereby exerting secondary effects on gene expression. In contrast to Nfkb2−/− mouse embryonic fibroblasts (MEFs), which are deficient in both p100 and p52 [15], p100−/− MEFs lack only the p100 inhibitor but still express the p52 subunit [20]. Passage 3 MEFs were left untreated and protein extracts and total RNA were isolated. Western blots were performed to verify increased levels of p52 in nuclei of p100−/− MEFs. We also observed higher nuclear RelB levels in p100−/− compared to wild-type MEFs, whereas RelA levels remained unchanged (Figure 1A). Furthermore, electrophoretic mobility shift assays revealed strongly increased κB DNA-binding activity in p100−/− vs. wild-type MEFs (Figure 1B). Dissection of κB DNA-binding protein complexes revealed that the majority contained p52 and RelB (Figure 1B). Of note, RelA binding was not activated in p100−/− compared to wild-type MEFs (Figure 1A and B).


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)

Expression profiling of wild-type and p100−/− MEFs.(A) Western blot analysis of cytoplasmic and nuclear protein extracts (20 µg/sample) were analyzed for the presence of NF-κB family members p100/p52, RelB, and RelA in wild-type (+/+) and in p100−/− (−/−) MEFs. As cytoplasmic loading control S6 ribosomal protein and as nuclear loading control RNA Pol II was assayed. (B) Increased κB DNA-binding activity in nuclear extracts from p100−/− (−/−) compared to wild-type MEFs (+/+). Five µg protein extract per cell line were incubated with a radioactively labeled Igκ oligo and analyzed by EMSA. Supershift analysis was performed using pre-immune serum (p.i.), anti-RelA (α-RelA), anti-RelB (α-RelB), and anti-p52 antibodies (α-p52). Super-shifted RelB and p52 complexes are indicated by arrow and arrowhead, respectively. (C) Heatmap displaying fold change values observed in p100−/− vs. wild-type cells. The color code indicates the fold change values between +6 fold up- (red) and −20 fold downregulation (green). Each horizontal line on the heatmap corresponds to one gene. Genes labeled with blue boxes on the left were verified by qRT-PCR. Gene symbols and abbreviations of GO terms are displayed on the right. CA, Cytokine activity; GPCRB, G-protein-coupled receptor binding; IGFB, insulin-like growth factor binding; ER, extracellular region; ESO, extracellular structure organization and biogenesis; D/M, developmental process; B/ND, forebrain development and nervous system development; CG/S, regulation of cell size; IR/RES, immune response and response to external stimulus; M/L/T, locomotory behavior. (D) Significantly regulated Gene Ontology terms with respective gene numbers.
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Related In: Results  -  Collection

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pone-0042741-g001: Expression profiling of wild-type and p100−/− MEFs.(A) Western blot analysis of cytoplasmic and nuclear protein extracts (20 µg/sample) were analyzed for the presence of NF-κB family members p100/p52, RelB, and RelA in wild-type (+/+) and in p100−/− (−/−) MEFs. As cytoplasmic loading control S6 ribosomal protein and as nuclear loading control RNA Pol II was assayed. (B) Increased κB DNA-binding activity in nuclear extracts from p100−/− (−/−) compared to wild-type MEFs (+/+). Five µg protein extract per cell line were incubated with a radioactively labeled Igκ oligo and analyzed by EMSA. Supershift analysis was performed using pre-immune serum (p.i.), anti-RelA (α-RelA), anti-RelB (α-RelB), and anti-p52 antibodies (α-p52). Super-shifted RelB and p52 complexes are indicated by arrow and arrowhead, respectively. (C) Heatmap displaying fold change values observed in p100−/− vs. wild-type cells. The color code indicates the fold change values between +6 fold up- (red) and −20 fold downregulation (green). Each horizontal line on the heatmap corresponds to one gene. Genes labeled with blue boxes on the left were verified by qRT-PCR. Gene symbols and abbreviations of GO terms are displayed on the right. CA, Cytokine activity; GPCRB, G-protein-coupled receptor binding; IGFB, insulin-like growth factor binding; ER, extracellular region; ESO, extracellular structure organization and biogenesis; D/M, developmental process; B/ND, forebrain development and nervous system development; CG/S, regulation of cell size; IR/RES, immune response and response to external stimulus; M/L/T, locomotory behavior. (D) Significantly regulated Gene Ontology terms with respective gene numbers.
Mentions: To explore the consequences of constitutively active alternative NF-κB signaling on genome-wide transcription, we investigated the gene expression profile of primary fibroblasts isolated from wild-type and p100−/− mouse embryos [20]. We have chosen this experimental system since the lack of p100 in mice results in multiple defects that affect organization and function of lymphoid tissues [20]–[22], thereby exerting secondary effects on gene expression. In contrast to Nfkb2−/− mouse embryonic fibroblasts (MEFs), which are deficient in both p100 and p52 [15], p100−/− MEFs lack only the p100 inhibitor but still express the p52 subunit [20]. Passage 3 MEFs were left untreated and protein extracts and total RNA were isolated. Western blots were performed to verify increased levels of p52 in nuclei of p100−/− MEFs. We also observed higher nuclear RelB levels in p100−/− compared to wild-type MEFs, whereas RelA levels remained unchanged (Figure 1A). Furthermore, electrophoretic mobility shift assays revealed strongly increased κB DNA-binding activity in p100−/− vs. wild-type MEFs (Figure 1B). Dissection of κB DNA-binding protein complexes revealed that the majority contained p52 and RelB (Figure 1B). Of note, RelA binding was not activated in p100−/− compared to wild-type MEFs (Figure 1A and B).

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