Korean J Physiol Pharmacol.  2014 Feb;18(1):73-78. 10.4196/kjpp.2014.18.1.73.

Bcl-2 Knockdown Accelerates T Cell Receptor-Triggered Activation-Induced Cell Death in Jurkat T Cells

• Lee YJ
• Won TJ
• Hyung KE
• Lee MJ
• Moon YH
• Lee IH
• Go BS
• Hwang KW

• KMID: 2285494
• DOI: http://doi.org/10.4196/kjpp.2014.18.1.73

Abstract

Cell death and survival are tightly controlled through the highly coordinated activation/inhibition of diverse signal transduction pathways to insure normal development and physiology. Imbalance between cell death and survival often leads to autoimmune diseases and cancer. Death receptors sense extracellular signals to induce caspase-mediated apoptosis. Acting upstream of CED-3 family proteases, such as caspase-3, Bcl-2 prevents apoptosis. Using short hairpin RNAs (shRNAs), we suppressed Bcl-2 expression in Jurkat T cells, and this increased TCR-triggered AICD and enhanced TNFR gene expression. Also, knockdown of Bcl-2 in Jurkat T cells suppressed the gene expression of FLIP, TNF receptor-associated factors 3 (TRAF3) and TRAF4. Furthermore, suppressed Bcl-2 expression increased caspase-3 and diminished nuclear factor kappa B (NF-kappaB) translocation.

Keyword

AICD; Apoptosis; Bcl-2; shRNA; T cell

MeSH Terms

Apoptosis
Autoimmune Diseases
Caspase 3
Cell Death*
Gene Expression
Humans
NF-kappa B
Peptide Hydrolases
Physiology
Receptors, Death Domain
RNA, Small Interfering
Signal Transduction
T-Lymphocytes*
TNF Receptor-Associated Factor 4
Tumor Necrosis Factor Receptor-Associated Peptides and Proteins
Caspase 3
NF-kappa B
Peptide Hydrolases
RNA, Small Interfering
Receptors, Death Domain
TNF Receptor-Associated Factor 4
Tumor Necrosis Factor Receptor-Associated Peptides and Proteins

Figure

• 

  Fig. 1 shRNAs knock down Bcl-2 in Jurkat T cells. The bcl-2 gene is knocked down using shRNA in Jurkat T cells. (A) Total RNA was extracted from Bcl-2-knockdown and control Jurkat T cells and bcl-2 gene expression was analyzed by real-time PCR using GAPDH as the internal control. (B) Total protein was extracted from Bcl2-knockdown and control Jurkat T cells. Bcl-2 protein expression was analyzed by western blotting using β-actin expression as a loading control. (C) A total of 1×105 Jurkat T cells were seeded in a 96-well plate and incubated for 24 h. After incubation, 0.4% Trypan Blue was added to the cell suspension, and cell numbers were estimated by counting under a microscope. Cells stained blue were considered non-viable. Data were presented as mean±SD for triplicate determinations. Student's t test; *p<0.05; **p<0.01; and ***p<0.001 vs. control sample. All data were representative of at least three individual experiments.

• 

  Fig. 2 Bcl-2 knockdown increases TCR-triggered AICD, downregulates FLIP gene expression, and upregulates caspase-3 cleavage. (A) Bcl-2-knockdown and control Jurkat T cells were incubated with 0.1 µg/ml or 1 µg/ml of plate bound anti-CD3 and anti-CD28 antibody for 24 h. Cells were washed with PBS, resuspended in PBS containing 5 µg/ml of PI, and analyzed by flow cytometry. (B~E) Bcl2-knockdown and control Jurkat T cells were incubated with 1 µg/ml of plate-bound anti-CD3 and anti-CD28 antibodies for 6 h. Total RNA was isolated, reverse transcribed, and gene expression was analyzed by real-time PCR. Relative gene expression levels were normalized with respect to those of GAPDH. (F) Bcl2-knockdown and control Jurkat T cells were incubated with 1 µg/ml of plate-bound anti-CD3 and anti-CD28 antibodies for 24 h. Total protein was extracted, and procaspase-3 and cleaved caspase-3 protein expression was analyzed by western blot. β-actin protein expression was used as a loading control. Data were presented as mean±SD for triplicate determinations. Student's t test; *p<0.05; **p<0.01; and ***p<0.001 vs. control sample. All data were representative of at least three individual experiments.

• 

  Fig. 3 Bcl-2 knockdown enhances the expression of TNFR and suppresses TRAF gene expression. Bcl-2-knockdown and control Jurkat T cells were incubated with 1 µg/ml of plate-bound anti-CD3 and anti-CD28 antibodies for 6 h. Total RNA was extracted and reverse transcribed, and gene expression was analyzed by real-time PCR. Relative gene expression levels of (A) TNFR1, (B) TNFR2, (C) TRAF3, (D) TRAF4 were normalized to those of GAPDH used as the internal control. Data were presented as mean±SD for triplicate determinations. Student's t test; *p<0.05; **p<0.01; and ***p<0.001 vs. control sample. All data were representative of at least three individual experiments.

• 

  Fig. 4 Bcl-2 shRNA suppresses the nuclear translocation of NF-κB. (A) Bcl-2-knockdown and control Jurkat T cells were incubated with 1 µg/ml of plate-bound anti-CD3 and anti-CD28 antibodies for 30 min. Nuclear extracts were prepared and analyzed by using an electrophoretic mobility shift assay (EMSA). (B, C) Bcl-2 knockdown and control Jurkat T cells were incubated with 1 µg/ml of plate-bound anti-CD3 and anti-CD28 antibodies. After 24 h, supernatants were collected and analyzed for cytokines by using the enzyme-linked immunosorbent assay (ELISA) method. After 6 h, total RNA was isolated and reverse transcribed, and gene expression was analyzed by real-time PCR. Relative gene expression levels were normalized to those of GAPDH used as the internal control.

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