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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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IκBα degradation is mediated by lysosomes upon ST80/Bortezomib cotreatment. (a) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 8 h. Lysosomal acidification was quantified by FACS measurement of Lysotracker Red stained cells. (b) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 in the presence and absence of 10 nM BafA1 for 8 h. NF-κB key regulatory proteins levels were assessed by western blot analysis. GAPDH was used as loading control. (c) RMS cells stably transfected with pTRH1- NF-κB EGFP plasmid were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 in the presence or absence of 10 nM BafA1 at indicated time points. NF-κB activation was measured by FACS analysis of FITC florescence. Data are shown as fold increase of GFP compared with untreated cells. (d and e) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 in the presence and absence of 10 nM BafA1 for 48 h. BAG1 and BAG3 mRNA levels were assessed by RT-PCR (d). BAG3 protein levels were assessed by western blot analysis. GAPDH was used as loading control (e). In (a, c and d), mean+S.D. of three independent experiments performed in triplicate are shown; *P<0.05; **P<0.01
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fig5: IκBα degradation is mediated by lysosomes upon ST80/Bortezomib cotreatment. (a) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 8 h. Lysosomal acidification was quantified by FACS measurement of Lysotracker Red stained cells. (b) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 in the presence and absence of 10 nM BafA1 for 8 h. NF-κB key regulatory proteins levels were assessed by western blot analysis. GAPDH was used as loading control. (c) RMS cells stably transfected with pTRH1- NF-κB EGFP plasmid were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 in the presence or absence of 10 nM BafA1 at indicated time points. NF-κB activation was measured by FACS analysis of FITC florescence. Data are shown as fold increase of GFP compared with untreated cells. (d and e) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 in the presence and absence of 10 nM BafA1 for 48 h. BAG1 and BAG3 mRNA levels were assessed by RT-PCR (d). BAG3 protein levels were assessed by western blot analysis. GAPDH was used as loading control (e). In (a, c and d), mean+S.D. of three independent experiments performed in triplicate are shown; *P<0.05; **P<0.01

Mentions: Since we observed that ST80/Bortezomib cotreatment triggers the degradation of IκBα (Figure 3a), we next asked how IκBα is degraded when the proteasome is inhibited by Bortezomib. Since the lysosomal compartment has been implicated in the degradation of key components of the NF-κB signaling pathway,13 we hypothesized that IκBα degradation occurs via the lysosomal route. To test this hypothesis, we quantified lysosomal activity by Lysotracker Red staining. Of note, ST80/Bortezomib cotreatment significantly increased lysosomal activity compared to either compound alone (Figure 5a). To explore whether lysosomal degradation is responsible for IκBα degradation and subsequent NF-κB activation following ST80/Bortezomib cotreatment, we monitored NF-κB activation in the presence and absence of Bafilomycin A1 (BafA1), an inhibitor of vacuolar H+ ATPases that inhibits lysosomal degradation.14 Intriguingly, addition of BafA1 attenuated the ST80/Bortezomib-triggered degradation of IκBα protein, whereas it did not block NIK accumulation, phosphorylation of IκBα and p65 or acetylation of histone H3 (Figure 5b). Furthermore, addition of BafA1 significantly impaired ST80/Bortezomib-stimulated NF-κB activation at all tested time points, whereas BafA1 did not alter constitutive NF-κB activity in untreated cells (Figure 5c and Supplementary Figure S4a). Importantly, addition of BafA1 prevented the ST80/Bortezomib-stimulated upregulation of BAG3 mRNA and protein levels, whereas it had no effect on BAG1 mRNA levels (Figures 5d and e). Also, BafA1 blocked the ST80/Bortezomib-stimulated transcriptional induction of other prototypic NF-κB target genes such as IκBα and RelB (Supplementary Figure S4b), confirming that inhibition of lysosomal degradation by BafA1 blocks the ST80/Bortezomib-mediated transcriptional activation of NF-κB.


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)

IκBα degradation is mediated by lysosomes upon ST80/Bortezomib cotreatment. (a) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 8 h. Lysosomal acidification was quantified by FACS measurement of Lysotracker Red stained cells. (b) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 in the presence and absence of 10 nM BafA1 for 8 h. NF-κB key regulatory proteins levels were assessed by western blot analysis. GAPDH was used as loading control. (c) RMS cells stably transfected with pTRH1- NF-κB EGFP plasmid were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 in the presence or absence of 10 nM BafA1 at indicated time points. NF-κB activation was measured by FACS analysis of FITC florescence. Data are shown as fold increase of GFP compared with untreated cells. (d and e) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 in the presence and absence of 10 nM BafA1 for 48 h. BAG1 and BAG3 mRNA levels were assessed by RT-PCR (d). BAG3 protein levels were assessed by western blot analysis. GAPDH was used as loading control (e). In (a, c and d), 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

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Show All Figures
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fig5: IκBα degradation is mediated by lysosomes upon ST80/Bortezomib cotreatment. (a) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 8 h. Lysosomal acidification was quantified by FACS measurement of Lysotracker Red stained cells. (b) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 in the presence and absence of 10 nM BafA1 for 8 h. NF-κB key regulatory proteins levels were assessed by western blot analysis. GAPDH was used as loading control. (c) RMS cells stably transfected with pTRH1- NF-κB EGFP plasmid were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 in the presence or absence of 10 nM BafA1 at indicated time points. NF-κB activation was measured by FACS analysis of FITC florescence. Data are shown as fold increase of GFP compared with untreated cells. (d and e) RMS cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 in the presence and absence of 10 nM BafA1 for 48 h. BAG1 and BAG3 mRNA levels were assessed by RT-PCR (d). BAG3 protein levels were assessed by western blot analysis. GAPDH was used as loading control (e). In (a, c and d), mean+S.D. of three independent experiments performed in triplicate are shown; *P<0.05; **P<0.01
Mentions: Since we observed that ST80/Bortezomib cotreatment triggers the degradation of IκBα (Figure 3a), we next asked how IκBα is degraded when the proteasome is inhibited by Bortezomib. Since the lysosomal compartment has been implicated in the degradation of key components of the NF-κB signaling pathway,13 we hypothesized that IκBα degradation occurs via the lysosomal route. To test this hypothesis, we quantified lysosomal activity by Lysotracker Red staining. Of note, ST80/Bortezomib cotreatment significantly increased lysosomal activity compared to either compound alone (Figure 5a). To explore whether lysosomal degradation is responsible for IκBα degradation and subsequent NF-κB activation following ST80/Bortezomib cotreatment, we monitored NF-κB activation in the presence and absence of Bafilomycin A1 (BafA1), an inhibitor of vacuolar H+ ATPases that inhibits lysosomal degradation.14 Intriguingly, addition of BafA1 attenuated the ST80/Bortezomib-triggered degradation of IκBα protein, whereas it did not block NIK accumulation, phosphorylation of IκBα and p65 or acetylation of histone H3 (Figure 5b). Furthermore, addition of BafA1 significantly impaired ST80/Bortezomib-stimulated NF-κB activation at all tested time points, whereas BafA1 did not alter constitutive NF-κB activity in untreated cells (Figure 5c and Supplementary Figure S4a). Importantly, addition of BafA1 prevented the ST80/Bortezomib-stimulated upregulation of BAG3 mRNA and protein levels, whereas it had no effect on BAG1 mRNA levels (Figures 5d and e). Also, BafA1 blocked the ST80/Bortezomib-stimulated transcriptional induction of other prototypic NF-κB target genes such as IκBα and RelB (Supplementary Figure S4b), confirming that inhibition of lysosomal degradation by BafA1 blocks the ST80/Bortezomib-mediated transcriptional activation of NF-κB.

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