Limits...
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: Importantly, NIK silencing by siRNA abolishes NF-κB activation and BAG3 induction by ST80/Bortezomib.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

NIK mediates NF-κB activity and BAG3 transcription. RMS cells stably transfected with pTRH1- NF-κB EGFP plasmid were transiently transfected with siRNA control sequence (siCtrl) or siRNA sequence against NIK (siNIK). (a) Cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 8 h. NF-κB key regulatory proteins levels were assessed by western blot analysis. GAPDH was used as loading control. (b) Cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 16 h (RD) or 24 h (RMS13). NF-κB activation was measured by flow cytometry. Data are shown as fold increase of GFP compared with the untreated cells. (c and d) Cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 48 h. BAG1 and BAG3 mRNA levels were assessed by RT-PCR (c). BAG3 protein levels were assessed by western blot analysis. GAPDH was used as loading control (d). In (b) and (c), mean+S.D. of three independent experiments performed in triplicate are shown; **P<0.01
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: NIK mediates NF-κB activity and BAG3 transcription. RMS cells stably transfected with pTRH1- NF-κB EGFP plasmid were transiently transfected with siRNA control sequence (siCtrl) or siRNA sequence against NIK (siNIK). (a) Cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 8 h. NF-κB key regulatory proteins levels were assessed by western blot analysis. GAPDH was used as loading control. (b) Cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 16 h (RD) or 24 h (RMS13). NF-κB activation was measured by flow cytometry. Data are shown as fold increase of GFP compared with the untreated cells. (c and d) Cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 48 h. BAG1 and BAG3 mRNA levels were assessed by RT-PCR (c). BAG3 protein levels were assessed by western blot analysis. GAPDH was used as loading control (d). In (b) and (c), mean+S.D. of three independent experiments performed in triplicate are shown; **P<0.01

Mentions: To determine the functional requirement of NIK, we transiently knocked down NIK by siRNA. NIK silencing attenuated phosphorylation of p65 and IκBα as well as degradation of IκBα upon ST80/Bortezomib cotreatment, while it did not interfere with acetylation of H3 (Figure 4a and Supplementary Figure S3), suggesting that NIK is involved in the activation of the canonical NF-κB pathway. In addition, knockdown of NIK significantly reduced ST80/Bortezomib-stimulated NF-κB transcriptional activation in RMS cells expressing a GFP-labeled NF-κB reporter plasmid compared with cells transfected with control siRNA (Figure 4b).


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)

NIK mediates NF-κB activity and BAG3 transcription. RMS cells stably transfected with pTRH1- NF-κB EGFP plasmid were transiently transfected with siRNA control sequence (siCtrl) or siRNA sequence against NIK (siNIK). (a) Cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 8 h. NF-κB key regulatory proteins levels were assessed by western blot analysis. GAPDH was used as loading control. (b) Cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 16 h (RD) or 24 h (RMS13). NF-κB activation was measured by flow cytometry. Data are shown as fold increase of GFP compared with the untreated cells. (c and d) Cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 48 h. BAG1 and BAG3 mRNA levels were assessed by RT-PCR (c). BAG3 protein levels were assessed by western blot analysis. GAPDH was used as loading control (d). In (b) and (c), mean+S.D. of three independent experiments performed in triplicate are shown; **P<0.01
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: NIK mediates NF-κB activity and BAG3 transcription. RMS cells stably transfected with pTRH1- NF-κB EGFP plasmid were transiently transfected with siRNA control sequence (siCtrl) or siRNA sequence against NIK (siNIK). (a) Cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 8 h. NF-κB key regulatory proteins levels were assessed by western blot analysis. GAPDH was used as loading control. (b) Cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 16 h (RD) or 24 h (RMS13). NF-κB activation was measured by flow cytometry. Data are shown as fold increase of GFP compared with the untreated cells. (c and d) Cells were treated with 20 nM (RD) or 50 nM (RMS13) Bortezomib and 50 μM ST80 for 48 h. BAG1 and BAG3 mRNA levels were assessed by RT-PCR (c). BAG3 protein levels were assessed by western blot analysis. GAPDH was used as loading control (d). In (b) and (c), mean+S.D. of three independent experiments performed in triplicate are shown; **P<0.01
Mentions: To determine the functional requirement of NIK, we transiently knocked down NIK by siRNA. NIK silencing attenuated phosphorylation of p65 and IκBα as well as degradation of IκBα upon ST80/Bortezomib cotreatment, while it did not interfere with acetylation of H3 (Figure 4a and Supplementary Figure S3), suggesting that NIK is involved in the activation of the canonical NF-κB pathway. In addition, knockdown of NIK significantly reduced ST80/Bortezomib-stimulated NF-κB transcriptional activation in RMS cells expressing a GFP-labeled NF-κB reporter plasmid compared with cells transfected with control siRNA (Figure 4b).

Bottom Line: Importantly, NIK silencing by siRNA abolishes NF-κB activation and BAG3 induction by ST80/Bortezomib.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