Rapino F, Abhari B A, Jung M, Fulda S
Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany.
1] Institute of Pharmaceutical Sciences, Albert-Ludwigs-University, Freiburg, Germany [2] German Cancer Consortium (DKTK), Heidelberg, Germany [3] German Cancer Research Center (DKFZ), Heidelberg, Germany.
Cell Death Dis. 2015 Mar 12;6(3):e1692. doi: 10.1038/cddis.2014.584.
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.
最近，我们报道了伴侣蛋白Bcl-2相关抗凋亡基因3（BAG3）的诱导对于横纹肌肉瘤（RMS）细胞在两种组成型蛋白质降解途径受到抑制后的蛋白毒性应激恢复至关重要，这两种途径分别是硼替佐米抑制的泛素-蛋白酶体系统和组蛋白去乙酰化酶6（HDAC6）抑制剂ST80抑制的聚集体自噬系统。在本研究中，我们调查了在这些条件下介导BAG3诱导的分子机制。在此，我们确定核因子-κB（NF-κB）诱导激酶（NIK）是ST80/硼替佐米刺激的NF-κB激活和BAG3转录上调的关键介质。ST80/硼替佐米联合处理上调了NIK的mRNA和蛋白表达，同时伴随着组蛋白H3乙酰化的初步增加。重要的是，通过小干扰RNA（siRNA）沉默NIK可消除ST80/硼替佐米诱导的NF-κB激活和BAG3诱导。此外，ST80/硼替佐米联合处理刺激了NF-κB转录活性并上调了NF-κB靶基因。通过过表达显性负性IκBα超阻遏物（IκBα-SR）或敲低p65对NF-κB进行基因抑制，可阻断ST80/硼替佐米刺激的BAG3 mRNA和蛋白表达上调。有趣的是，巴弗洛霉素A1抑制溶酶体活性可抑制ST80/硼替佐米刺激的IκBα降解、NF-κB激活和BAG3上调，表明在存在硼替佐米的情况下IκBα通过溶酶体降解。因此，通过证明NIK在介导ST80/硼替佐米联合处理后的NF-κB激活和BAG3诱导中的关键作用，我们的研究为RMS中对蛋白毒性应激的抗性机制提供了新的见解。
