Korean J Physiol Pharmacol.  2018 Jul;22(4):457-465. 10.4196/kjpp.2018.22.4.457.

Effect of BIS depletion on HSF1-dependent transcriptional activation in A549 non-small cell lung cancer cells

• Yun HH
• Baek JY
• Seo G
• Kim YS
• Ko JH
• Lee JH

• KMID: 2414270
• DOI: http://doi.org/10.4196/kjpp.2018.22.4.457

Abstract

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.

Keyword

A549; BCL-2 interacting cell death suppressor; Heat shock factor1; Heat shock protein

MeSH Terms

Carcinoma, Non-Small-Cell Lung*
Cell Death
Down-Regulation
Glioblastoma
Heat-Shock Proteins
Homeostasis
Hot Temperature
Oxidative Stress
RNA Stability
RNA, Messenger
RNA, Small Interfering
Shock
Transcriptional Activation*
Heat-Shock Proteins
RNA, Messenger
RNA, Small Interfering

Figure

• 

  Fig. 1 The induction of HSP70 and HSP27 mRNA was not affected by BIS depletion under stress conditions.(A) Wild type (WT) A549 cells and BIS-knockout (KO) A549 cells were exposed to heat shock (43℃) for 30 min and subsequent recovery for up to 6 h, to MG132 for 6 h, or to glucose free and serum free conditions for 3 h followed by 3 h of recovery. The mRNA expression levels at the indicated times or indicated treatment concentrations were measured by qRT-PCR analyses. The fold induction was determined as the relative value of each mRNA level compared to the untreated WT A549 cells, which was designated arbitrarily as 1.0. Data are represented as the mean±SEM from three independent experiments. *p≤0.05, **p≤0.01, ***p≤0.001 vs. control in each group; #p≤0.05, ##p≤0.01, ###p≤0.001 vs. WT at the indicated times or concentrations. ns, statistically non-significant. (B) The protein levels of BIS, HSP70, and HSP27 in the WT and BIS-KO A549 were determined by western blotting of samples after 3 h of recovery (+R3) following heat shock and control cell samples. Actin expression was used as a loading control.

• 

  Fig. 2 BIS knockdown did not affect HSP70 and HSP27 induction by various stress conditions.(A) BIS expression was transiently suppressed by 200 nM of control siRNA (siCON) and BIS specific siRNA (siBIS) for 48 h in A549 cells, and the fold induction was compared for BIS, HSP70, and HSP27 mRNA expression in response to heat shock, MG132, or oxidative stress as described in Fig. 1. (B) U87 cells were transfected with HSF1 siRNA (siHSF1) or BIS siRNA (siBIS) for 48 h and exposed to heat shock. The expression levels of HSF1, BIS, HSP70, and HSP27 mRNA were determined as in Fig. 1. *p≤0.05, **p≤0.01, ***p≤0.001 vs. untreated control cells in each group; #p≤0.05, ##p≤0.01, ###p≤0.001 vs. siCON at the indicated times or treatment concentrations. ns, statistically non-significant.

• 

  Fig. 3 The promoter activities of BIS and HSP70 were not affected by BIS depletion.(A) Schematic diagram for the relative position of heat shock element (HSE) to ATG in the promoter region of the HSP70 and BIS genes. (B) WT A549 and BIS-KO A549 cells were transfected with BIS or HSP70 promoter constructs for 24 h and then exposed to heat shock (HS) at 43℃ for 30 min, and then further at 37℃ for 1 h. Transcriptional activation of the BIS (P3) and HSP70 promoters are presented as fold changes compared to the value of the pGL3 basic construct in untreated conditions in WT and BIS-KO A549 cells. (C) Western blotting shows that BIS protein expression was sufficiently repressed by siRNA. (D) Following suppression of BIS in A549 cells with BIS siRNA (siBIS), the reporter constructs were expressed and compared to the control (siCON). After normalization with renilla activity, the results are presented as fold change compared to the activity from the pGL3 basic vector (mean±SE, n=3). #p≤0.05 vs. siCON for P1. ns, statistically non-significant.

• 

  Fig. 4 Autoregulation of BIS was not mediated by the alteration of mRNA stability.(A) The sequencing results of genotyping in BIS-KO A549 cells show the deletion in exon 1 of the BIS gene and consequent generation of a premature stop codon. (B) WT and BIS-KO A549 cells were treated with actinomycin D for up to 24 h. At the indicated times, mRNA was extracted, and the remaining BIS mRNA was measured and compared to the initial value with qRT-PCR analysis (mean±SE, n=3). (C) The coding region of the WT BIS and the deletion mutant of the BIS gene, in which 14 bp were deleted (Δ14), were cloned into the pEGFP-C1 construct (left). After transfection for 48 h, the GFP mRNA levels representing WT and Δ14-BIS were determined with PCR amplification and agarose electrophoresis by loading different quantities of PCR products (right). (D) The BIS pre-mRNA levels were determined in WT A549 and BIS-KO A549 cells using qRT-PCR analysis with specific primers for intron 2 (solid arrows). The BIS mature mRNA was levels were also determined by the primers covering exon 2 and 3 (dotted arrows). Reverse transcription was performed as described in Method section. *p≤0.05 vs. WT.

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