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NIK is required for NF-κB-mediated induction of BAG3 upon inhibition of constitutive protein degradation pathways.

Rapino F, Abhari BA, Jung M, Fulda S - Cell Death Dis (2015)

Bottom Line: Furthermore, ST80/Bortezomib cotreatment stimulates NF-κB transcriptional activity and upregulates NF-κB target genes.Genetic inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) or by knockdown of p65 blocks the ST80/Bortezomib-stimulated upregulation of BAG3 mRNA and protein expression.Interestingly, inhibition of lysosomal activity by Bafilomycin A1 inhibits ST80/Bortezomib-stimulated IκBα degradation, NF-κB activation and BAG3 upregulation, indicating that IκBα is degraded via the lysosome in the presence of Bortezomib.

View Article: PubMed Central - PubMed

Affiliation: Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany.

ABSTRACT
Recently, we reported that induction of the co-chaperone Bcl-2-associated athanogene 3 (BAG3) is critical for recovery of rhabdomyosarcoma (RMS) cells after proteotoxic stress upon inhibition of the two constitutive protein degradation pathways, that is, the ubiquitin-proteasome system by Bortezomib and the aggresome-autophagy system by histone deacetylase 6 (HDAC6) inhibitor ST80. In the present study, we investigated the molecular mechanisms mediating BAG3 induction under these conditions. Here, we identify nuclear factor-kappa B (NF-κB)-inducing kinase (NIK) as a key mediator of ST80/Bortezomib-stimulated NF-κB activation and transcriptional upregulation of BAG3. ST80/Bortezomib cotreatment upregulates mRNA and protein expression of NIK, which is accompanied by an initial increase in histone H3 acetylation. Importantly, NIK silencing by siRNA abolishes NF-κB activation and BAG3 induction by ST80/Bortezomib. Furthermore, ST80/Bortezomib cotreatment stimulates NF-κB transcriptional activity and upregulates NF-κB target genes. Genetic inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) or by knockdown of p65 blocks the ST80/Bortezomib-stimulated upregulation of BAG3 mRNA and protein expression. Interestingly, inhibition of lysosomal activity by Bafilomycin A1 inhibits ST80/Bortezomib-stimulated IκBα degradation, NF-κB activation and BAG3 upregulation, indicating that IκBα is degraded via the lysosome in the presence of Bortezomib. Thus, by demonstrating a critical role of NIK in mediating NF-κB activation and BAG3 induction upon ST80/Bortezomib cotreatment, our study provides novel insights into mechanisms of resistance to proteotoxic stress in RMS.

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NF-κB is activated upon ST80/Bortezomib cotreatment. (a) RMS cells stably transfected with pTRH1- NF-κB EGFP plasmid were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and/or 50 μM ST80 at indicated time points. NF-κB activation was measured by flow cytometry. Data are shown as fold increase of GFP compared with the untreated cells. (b) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and/or 50 μM ST80 for 12 h. RelB and IκBα mRNA levels were quantified by RT-PCR. Mean+S.D. of three independent experiments performed in triplicate are shown; *P<0.05; **P<0.01
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fig1: NF-κB is activated upon ST80/Bortezomib cotreatment. (a) RMS cells stably transfected with pTRH1- NF-κB EGFP plasmid were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and/or 50 μM ST80 at indicated time points. NF-κB activation was measured by flow cytometry. Data are shown as fold increase of GFP compared with the untreated cells. (b) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and/or 50 μM ST80 for 12 h. RelB and IκBα mRNA levels were quantified by RT-PCR. Mean+S.D. of three independent experiments performed in triplicate are shown; *P<0.05; **P<0.01

Mentions: Recently, we showed that the co-chaperone BAG3 is transcriptionally upregulated in RMS cells that survive concomitant inhibition of the two major constitutive protein degradation pathways, that is, the UPS and the aggresome/autophagy pathway, by cotreatment with the proteasome inhibitor Bortezomib and the HDAC6 inhibitor ST80.10 Furthermore, we demonstrated that BAG3 has a pivotal role in mediating cell recovery upon ST80/Bortezomib cotreatment by promoting the clearance of cytotoxic protein aggregates via selective autophagy.10 Since NF-κB represents a key transcription factor that controls the cellular stress response,4 we asked whether NF-κB has a role in BAG3 induction upon ST80/Bortezomib cotreatment. To address this question we monitored NF-κB activation in RMS cell lines of both the embryonal and alveolar subtype, that is, RD and RMS13 cells, respectively. To this end, we created RMS cells with stable expression of a green fluorescent protein (GFP)-labeled NF-κB reporter construct and analyzed the kinetics of NF-κB activation. Intriguingly, ST80/Bortezomib cotreatment significantly enhanced NF-κB activity compared with cells treated with either agent alone (Figure 1a). Stimulation with the prototypic NF-κB stimulus TNFα was used as the positive control (Supplementary Figure S1). Furthermore, ST80/Bortezomib cotreatment significantly increased mRNA levels of IκBα and RelB, two known NF-κB target genes, compared with cells treated with Bortezomib alone (Figure 1b), confirming that ST80/Bortezomib cotreatment triggers NF-κB activation.


NIK is required for NF-κB-mediated induction of BAG3 upon inhibition of constitutive protein degradation pathways.

Rapino F, Abhari BA, Jung M, Fulda S - Cell Death Dis (2015)

NF-κB is activated upon ST80/Bortezomib cotreatment. (a) RMS cells stably transfected with pTRH1- NF-κB EGFP plasmid were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and/or 50 μM ST80 at indicated time points. NF-κB activation was measured by flow cytometry. Data are shown as fold increase of GFP compared with the untreated cells. (b) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and/or 50 μM ST80 for 12 h. RelB and IκBα mRNA levels were quantified by RT-PCR. Mean+S.D. of three independent experiments performed in triplicate are shown; *P<0.05; **P<0.01
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4385908&req=5

fig1: NF-κB is activated upon ST80/Bortezomib cotreatment. (a) RMS cells stably transfected with pTRH1- NF-κB EGFP plasmid were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and/or 50 μM ST80 at indicated time points. NF-κB activation was measured by flow cytometry. Data are shown as fold increase of GFP compared with the untreated cells. (b) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and/or 50 μM ST80 for 12 h. RelB and IκBα mRNA levels were quantified by RT-PCR. Mean+S.D. of three independent experiments performed in triplicate are shown; *P<0.05; **P<0.01
Mentions: Recently, we showed that the co-chaperone BAG3 is transcriptionally upregulated in RMS cells that survive concomitant inhibition of the two major constitutive protein degradation pathways, that is, the UPS and the aggresome/autophagy pathway, by cotreatment with the proteasome inhibitor Bortezomib and the HDAC6 inhibitor ST80.10 Furthermore, we demonstrated that BAG3 has a pivotal role in mediating cell recovery upon ST80/Bortezomib cotreatment by promoting the clearance of cytotoxic protein aggregates via selective autophagy.10 Since NF-κB represents a key transcription factor that controls the cellular stress response,4 we asked whether NF-κB has a role in BAG3 induction upon ST80/Bortezomib cotreatment. To address this question we monitored NF-κB activation in RMS cell lines of both the embryonal and alveolar subtype, that is, RD and RMS13 cells, respectively. To this end, we created RMS cells with stable expression of a green fluorescent protein (GFP)-labeled NF-κB reporter construct and analyzed the kinetics of NF-κB activation. Intriguingly, ST80/Bortezomib cotreatment significantly enhanced NF-κB activity compared with cells treated with either agent alone (Figure 1a). Stimulation with the prototypic NF-κB stimulus TNFα was used as the positive control (Supplementary Figure S1). Furthermore, ST80/Bortezomib cotreatment significantly increased mRNA levels of IκBα and RelB, two known NF-κB target genes, compared with cells treated with Bortezomib alone (Figure 1b), confirming that ST80/Bortezomib cotreatment triggers NF-κB activation.

Bottom Line: Furthermore, ST80/Bortezomib cotreatment stimulates NF-κB transcriptional activity and upregulates NF-κB target genes.Genetic inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) or by knockdown of p65 blocks the ST80/Bortezomib-stimulated upregulation of BAG3 mRNA and protein expression.Interestingly, inhibition of lysosomal activity by Bafilomycin A1 inhibits ST80/Bortezomib-stimulated IκBα degradation, NF-κB activation and BAG3 upregulation, indicating that IκBα is degraded via the lysosome in the presence of Bortezomib.

View Article: PubMed Central - PubMed

Affiliation: Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany.

ABSTRACT
Recently, we reported that induction of the co-chaperone Bcl-2-associated athanogene 3 (BAG3) is critical for recovery of rhabdomyosarcoma (RMS) cells after proteotoxic stress upon inhibition of the two constitutive protein degradation pathways, that is, the ubiquitin-proteasome system by Bortezomib and the aggresome-autophagy system by histone deacetylase 6 (HDAC6) inhibitor ST80. In the present study, we investigated the molecular mechanisms mediating BAG3 induction under these conditions. Here, we identify nuclear factor-kappa B (NF-κB)-inducing kinase (NIK) as a key mediator of ST80/Bortezomib-stimulated NF-κB activation and transcriptional upregulation of BAG3. ST80/Bortezomib cotreatment upregulates mRNA and protein expression of NIK, which is accompanied by an initial increase in histone H3 acetylation. Importantly, NIK silencing by siRNA abolishes NF-κB activation and BAG3 induction by ST80/Bortezomib. Furthermore, ST80/Bortezomib cotreatment stimulates NF-κB transcriptional activity and upregulates NF-κB target genes. Genetic inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) or by knockdown of p65 blocks the ST80/Bortezomib-stimulated upregulation of BAG3 mRNA and protein expression. Interestingly, inhibition of lysosomal activity by Bafilomycin A1 inhibits ST80/Bortezomib-stimulated IκBα degradation, NF-κB activation and BAG3 upregulation, indicating that IκBα is degraded via the lysosome in the presence of Bortezomib. Thus, by demonstrating a critical role of NIK in mediating NF-κB activation and BAG3 induction upon ST80/Bortezomib cotreatment, our study provides novel insights into mechanisms of resistance to proteotoxic stress in RMS.

Show MeSH
Related in: MedlinePlus