title: BAG3 protects against hyperthermic stress by modulating NF-κB and ERK activities in human retinoblastoma cells.
abstract: OBJECTIVE
  BCL2-associated athanogene 3 (BAG3), a co-chaperone of HSP70, is a cytoprotective and anti-apoptotic protein that acts against various stresses, including heat stress. Here, we examined the effect of BAG3 on the sensitivity of human retinoblastoma cells to hyperthermia (HT).
  
  
  METHODS
  We examined the effects of BAG3 knockdown on the sensitivity of Y79 and WERI-Rb-1cells to HT (44 °C, 1 h) by evaluating apoptosis and cell proliferation using western blotting, real-time quantitative PCR (qPCR), flow cytometry, and a WST-8 assay kit. Furthermore, we examined the effects of activating nuclear factor-kappa B (NF-κB) and extracellular signal-regulated kinase (ERK) using western blotting and real time qPCR.
  
  
  RESULTS
  HT induced considerable apoptosis along with the activation of caspase-3 and chromatin condensation. The sensitivity of Y79 and WERI-Rb-1 cells to HT was significantly enhanced by BAG3 knockdown. Compared to HT alone, the combination of BAG3 knockdown and HT reduced phosphorylation of the inhibitors of kappa B α (IκBα) and p65, a subunit of NF-κB, and degraded IκB kinase γ (IKKγ) during the recovery period after HT. Furthermore, BAG3 knockdown increased the HT-induced phosphorylation of ERK after HT treatment, and the ERK inhibitor U0126 significantly improved the viability of the cells treated with a combination of BAG3 knockdown and HT.
  
  
  CONCLUSIONS
  The silencing of BAG3 seems to enhance the effects of HT, at least in part, by maintaining HT-induced inactivity of NF-κB and the phosphorylation of ERK. These findings indicate that BAG3 may be a potential molecular target for modifying the outcomes of HT in retinoblastoma.
authors.affiliation: Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan, yunokiki@med.u-toyama.ac.jp.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Yunoki
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Tabuchi
authors.forename: Atsushi
authors.initials: A
authors.lastname: Hayashi
authors.forename: Takashi
authors.initials: T
authors.lastname: Kondo
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology*; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000502:physiology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D006801:Humans; D006979:Hyperthermia, Induced; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D019572:Retinal Neoplasms / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D012175:Retinoblastoma / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C414431:BAG3 protein, human; D016328:NF-kappa B; D034741:RNA, Small Interfering; D048049:Extracellular Signal-Regulated MAP Kinases