Korean J Physiol Pharmacol.  2014 Oct;18(5):403-409. 10.4196/kjpp.2014.18.5.403.

Bis is Induced by Oxidative Stress via Activation of HSF1

Affiliations
  • 1Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea. leejh@catholic.ac.kr
  • 2Cancer Research Institute, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea.
  • 3Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea.

Abstract

The Bis protein is known to be involved in a variety of cellular processes including apoptosis, migration, autophagy as well as protein quality control. Bis expression is induced in response to a number of types of stress, such as heat shock or a proteasome inhibitor via the activation of heat shock factor (HSF)1. We report herein that Bis expression is increased at the transcriptional level in HK-2 kidney tubular cells and A172 glioma cells by exposure to oxidative stress such as H2O2 treatment and oxygen-glucose deprivation, respectively. The pretreatment of HK-2 cells with N-acetyl cysteine, suppressed Bis induction. Furthermore, HSF1 silencing attenuated Bis expression that was induced by H2O2, accompaniedby increase in reactive oxygen species (ROS) accumulation. Using a series of deletion constructs of the bis gene promoter, two putative heat shock elements located in the proximal region of the bis gene promoter were found to be essential for the constitutive expression is as well as the inducible expression of Bis. Taken together, our results indicate that oxidative stress induces Bis expression at the transcriptional levels via activation of HSF1, which might confer an expansion of antioxidant capacity against pro-oxidant milieu. However, the possible role of the other cis-element in the induction of Bis remains to be determined.

Keyword

Bis; HSF1; Oxidative stress; ROS

MeSH Terms

Apoptosis
Autophagy
Cysteine
Glioma
Hot Temperature
Kidney
Oxidative Stress*
Proteasome Inhibitors
Quality Control
Reactive Oxygen Species
Shock
Cysteine
Proteasome Inhibitors
Reactive Oxygen Species

Figure

  • Fig. 1 Induction of Bis expression by oxidative stress in HK-2 and A172 cells. (A) Western blot assay for Bis expression in HK-2 (left column) and A172 (right column) cells after exposure to H2O2 and OGD, respectively, and then incubated in the normal medium for indicated times. Beta-actin expression was used as a loading control. (B) The relative levels of Bis mRNA from HK-2 (left column) or A172 (right column) cells at the indicated conditions were determined by quantitative real time RT-PCR analysis after normalizing with beta-actin mRNA level in the same sample. The mean values from three independent experiments are shown with SD. The value from the control cells before exposure to oxidative stress is designated as 1.0. *p<0.05 and **p<0.01 vs. the value from control cells. (C) HK-2 (left column) or A172 (right column) cells were transfected with control or Bis specific siRNA with indicated doses for 48 h and then treated with 100 µM of H2O2 for 3 h followed by additional 6 h in normal medium. The relative cell viability was determined using MTT assay as described in METHODS section. Values from triplicate experiments are provided as mean±SE. *p<0.05 and **p<0.01 vs. the value from control cells treated only with H2O2. (D) ROS accumulation was determined in HK-2 cells after treatment of H2O2 by measuring DCF-DA fluorescence intensity using flow cytometric analysis. 1 mM of NAC was pretreated before exposure to H2O2. Fold changes in the mean from three experiments are provided as mean±SE. *p<0.05 and **p<0.01 vs. the value from control cells, ##p<0.01 vs. the value in the absence of NAC. (E) Effect of NAC on the induction of Bis mRNA was determined by pretreatment of NAC prior to H2O2 treatment and then Bis mRNA level was determined as in (B). The mean values from four independent experiments are present with SD. ***p<0.001 vs. the value from control cells, ##p<0.01 vs. the value in the absence of NAC.

  • Fig. 2 HSF1 suppressed induction of Bis mRNA upon oxidative stress. HK-2 cells were transfected with control or HSF1-specific siRNA and incubated with H2O2 for 3 h followed by additional 6 h in normal medium. HSF1 and Bis expressions were evaluated by Western assay (A) and by real-time RT-PCR (B) as in Fig. 1. (C) Effect of downregulation of HSF1 on ROS accumulation upon H2O2 treatment was examined by DCF-DA staining and FACS analysis. Data are presented with fold changes in the mean intensities from three independent experiments with SE. *p<0.05 vs. the value from control cells, #p<0.05 vs. the value from the control siRNA-treated cells.

  • Fig. 3 Identification of oxidative stress-responsive region in the Bis promoter (A) Schematic diagram of the deletion mutants for proximal region of Bis promoter, which were cloned into pGL3 basic vector.The relative position to the transcription site (+1) and the locations of putative HSE (H) are shown. (B) Transcriptional activation of pBis-1 was determined at the indicated time points following treatment of H2O2 after normalization with renilla activity. Luciferase activity before exposure to H2O2 is designated at 1.0. The mean value from triplicate experiments are present with SD. **p<0.01 and ***p<0.001 vs. the value from control cells. (C) Fold activation of various deletion mutants of Bis promoter in response to H2O2 treatment was shown. The luciferase activityof pBis-1 before exposure to H2O2 is designated at 1.0. The values for pBis-3 and pBis-4 were provided as a magnified graph (inlet). The mean values from triplicate experiments are present with SD. *p<0.05 and **p<0.01 vs. the value from H2O2-untreated cells in each construct. (D) Effect of HSF1 knockdown on the expression of pBis-2 was shown. Expression of HSF1 was suppressed by transfection of HSF1 specific siRNA for 24 h and then HK-2 cells were transfected with pBis-2 construct as described in Materials and Methods section. Fold change in luciferase activity compared with that of control HK-2 cells are presented as mean value with SD. *p<0.05 vs. the value from H2O2-untreated control cells, #p<0.05 vs. the value from the control siRNA-treated cells.


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