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BAG3 protects against hyperthermic stress by modulating NF-κB and ERK activities in human retinoblastoma cells

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Abstract

Purpose

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.

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Acknowledgments

This study was supported in part by a Grant-in-Aid for Scientific Research B (24310046) from the Japan Society for the Promotion of Science.

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Authors and Affiliations

  1. Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan

    Tatsuya Yunoki & Takashi Kondo

  2. Department of Ophthalmology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan

    Tatsuya Yunoki & Atsushi Hayashi

  3. Division of Molecular Genetics Research, Life Science Research Center, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan

    Yoshiaki Tabuchi

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  1. Tatsuya Yunoki
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  2. Yoshiaki Tabuchi
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  4. Takashi Kondo
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Correspondence to Tatsuya Yunoki.

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Yunoki, T., Tabuchi, Y., Hayashi, A. et al. BAG3 protects against hyperthermic stress by modulating NF-κB and ERK activities in human retinoblastoma cells. Graefes Arch Clin Exp Ophthalmol 253, 399–407 (2015). https://doi.org/10.1007/s00417-014-2874-1

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  • DOI: https://doi.org/10.1007/s00417-014-2874-1

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