Korean J Physiol Pharmacol.  2009 Dec;13(6):483-489. 10.4196/kjpp.2009.13.6.483.

Knockdown of RCAN1.4 Increases Susceptibility to FAS-mediated and DNA-damage-induced Apoptosis by Upregulation of p53 Expression

• Kim YS
• Lee HJ
• Jang C
• Kim HS
• Cho YJ

• KMID: 2285385
• DOI: http://doi.org/10.4196/kjpp.2009.13.6.483

Abstract

Despite the potential importance of the human regulator of calcineurin 1 (RCAN-1) gene in the modulation of cell survival under stress, little is known about its role in death-inducing signal pathways. In this study, we addressed the effects of RCAN1.4 knockdown on cellular susceptibility to apoptosis and the activation of death pathway proteins. Transfection of siRNAs against RCAN1.4 resulted in enhanced Fas- and etoposide-induced apoptosis, which was associated with increased expression and translocation of Bax to mitochondria. Our results suggest that enhanced expression and activation of p53 was responsible for the upregulation of Bax and the increased sensitivity to apoptosis, which could be reversed by p53 knockdown. To explain the observed upregulation of p53, we propose a downregulation of the ubiquitin ligase HDM2, probably translationally. These findings show the importance of appropriate RCAN1.4 expression in the modulation of cell survival and reveal a link between RCAN1.4 and p53.

Keyword

RCAN1; Apoptosis; p53; Bax; HDM2

MeSH Terms

Aluminum Hydroxide
Apoptosis
Calcineurin
Carbonates
Cell Survival
Down-Regulation
Humans
Mitochondria
Proteins
RNA, Small Interfering
Signal Transduction
Transfection
Ubiquitin
Up-Regulation
Aluminum Hydroxide
Calcineurin
Carbonates
Proteins
RNA, Small Interfering
Ubiquitin

Figure

• 

  Fig. 1. RCAN1.4 knockdown enhances Fas-antibody- or etoposide-mediated apoptosis in U87MG cells. (A) Cells were transfected with siRNA against RCAN1.4 (siRCAN259) or negative control siRNA (siCon). After 48 h, cells were treated with an activating anti-Fas antibody (CH11) for 5 h to induce apoptosis. Cells were then washed, stained with Annexin V, and analyzed by FACS. (B) Cells transfected with two doses of siRCAN259 (20 and 40 pmol/well in a 6-well plate) were treated with activating anti-Fas antibody and then subjected to immunoblot analysis for the indicated proteins. (C) Cells transfected with the indicated siRNAs were treated with activating anti-Fas antibody for 6 h or etoposide for 16 h. The cells were then subjected to a caspase activity assay (Promega) according to the manufacturer's instructions.

• 

  Fig. 2. RCAN1.4 knockdown enhances mitochondria-mediated apoptosis. Forty-eight hours after transfection with siRCAN259, siRCAN1348, or siCon, cells were treated with activating anti-Fas antibody for 6 h or etoposide for 16 h. After cells were disrupted as indicated in the methods section, the cytosol fractions (A) or the mitochondrial fractions (B) were subjected to immunoblot analysis for the indicated proteins.

• 

  Fig. 3. RCAN1.4 knockdown upregulates Bax and p53 expression. (A) Forty-eight hours after transfection with the indicated siRNAs, cells were treated with activating anti-Fas antibody for 6 h or etoposide for 16 h. Whole lysates from the cells were subjected to immunoblot analysis for the indicated proteins. (B) Twenty-four hours after transfection with the indicated siRNAs, cells were transfected with a vector containing p53-Luc. Twenty-four hours after the final transfection, luciferase activities were measured. The results are presented as the average±SD (Student's t test; ∗∗p<0.001). (C) Forty-eight hours after transfection with the indicated siRNAs, whole lysates from the cells were subjected to immunoblot analysis for the indicated proteins. (D) Forty-eight hours after transfection with the indicated siRNAs, cells were treated with etoposide for 16 h. Whole lysates from the cells were subjected to immunoblot analysis for the indicated proteins.

• 

  Fig. 4. RCAN1.4 knockdown increases p53 expression while decreasing HDM2 expression. (A) Forty-eight hours after transfection with three doses of siRCAN259 (5, 20, or 40 pmol/well in a 6-well plate), whole lysates from the cells were subjected to immunoblot analysis for the indicated proteins. (B, C) Forty-eight hours after transfection with siRCAN259, p53 and HDM2 transcript levels were measured by real-time RT-PCR. (D) Twenty-four hours after transfection with siRCAN259 or siCon, cells were transfected with a vector containing wild-type (wt-HDM2) or mutants (C464A and 440Δ) of HDM2. Twenty-four hours after the final transfection, whole lysates from the cells were subjected to immunoblot analysis with an antibody specific to HDM2.

Reference

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