CLCA2 as a p53-inducible senescence mediator

Abstract

p53 is a tumor suppressor gene that is frequently mutated in multiple cancer tissues. Activated p53 protein regulates its downstream genes and subsequently inhibits malignant transformation by inducing cell cycle arrest, apoptosis, DNA repair, and senescence. However, genes involved in the p53-mediated senescence pathway are not yet fully elucidated. Through the screening of two genome-wide expression profile data sets, one for cells in which exogenous p53 was introduced and the other for senescent fibroblasts, we have identified chloride channel accessory 2 (CLCA2) as a p53-inducible senescence-associated gene. CLCA2 was remarkably induced by replicative senescence as well as oxidative stress in a p53-dependent manner. We also found that ectopically expressed CLCA2 induced cellular senescence, and the down-regulation of CLCA2 by small interfering RNA caused inhibition of oxidative stress-induced senescence. Interestingly, the reduced expression of CLCA2 was frequently observed in various kinds of cancers including prostate cancer, whereas its expression was not affected in precancerous prostatic intraepithelial neoplasia. Thus, our findings suggest a crucial role of p53/CLCA2-mediated senescence induction as a barrier for malignant transformation.

Figure 1

CLCA2 expression is increased during replicative senescence. (A) Quantitative real-time PCR analysis of CLCA2 mRNA in NHDF cells at indicated passage levels. β2-Microglobulin was used for the normalization of expression levels. (B) Northern blot and quantitative real-time PCR analysis of CLCA2 transcript in U373MG cells at indicated times after infection with Ad-p53 or Ad-LacZ at 8 MOIs. β-Actin was used for the normalization of expression levels. p21^WAF1 served as a positive control. (C) Western blot analysis of CLCA2 protein in U373MG cells at indicated times after infection with Ad-p53 or Ad-LacZ at 40 MOIs. β-Actin was used for the normalization of expression levels. p53 and p21^WAF1 served as a positive control. (D) Quantitative real-time PCR analysis of CLCA2, p21^WAF1, and p16^Ink4a mRNA in IMR-90 cells at the indicated days after treatment with 400 µM of H₂O₂. β2-Microglobulin was used for the normalization of expression levels.

Figure 2

Clca5 (mouse homologue of human CLCA2) is a target of p53. (A, B) p53^+/+ and p53^-/- MEFs were treated with 100 µM (A) or 150 µM (B) of H₂O₂. (A) At 6 days after treatment, the cells were subjected to SA-β-gal staining. (B) Quantitative real-time PCR analysis of Clca5 mRNA at indicated days after treatment. β-Actin was used for the normalization of expression levels. (C) Genomic structure of the mouse Clca5 gene. Gray boxes indicate the locations and relative sizes of the two exons. Arrows indicate the locations of potential p53-binding sites (p53BS1-3) in a p53-binding region (p53BR). Identical nucleotides to the consensus sequence are written in capital letters. The underlined nucleotides were substituted for thymine to examine the specificity of each p53-binding site. (D) Results of luciferase assay of p53BR with or without substitutions at either of the p53BS fragments are shown. Luciferase activity is indicated relative to the activity of mock vector with SDs (n = 2).

Figure 3

CLCA2 is a key mediator of oxidative stress-induced senescence. (A, B) IMR-90 cells were transfected with siRNA oligonucleotides designed to suppress the expression of CLCA2 or p53 at 7 hours before treatment with 400 µM of H₂O₂. siEGFP was used as a control. At 8 days after treatment, the cells were subjected to quantitative real-time PCR analysis of CLCA2 mRNA (A) and SA-β-gal staining (B). β2-Microglobulin was used for the normalization of expression levels. The proportion of cells positive for SA-β-gal staining is indicated (B, upper panels). The representative images of cells are shown (B, lower panels). Asterisks indicate P value obtained by Student's t test: *P < .05 and **P < .01. (C, D) MCF7 cells were transfected with CLCA2 expression plasmid. (C) After 3 days, the cells were subjected to SA-β-gal staining. (D) The proportion of cells positive for SA-β-gal staining is indicated. Asterisk indicates P value obtained by Student's t test: **P < .01.

Figure 4

Down-regulation of CLCA2 in prostate cancer. (A) Relative CLCA2 expression in prostate cancer tissues compared with normal tissues was examined by quantitative real-time PCR analysis. β-Actin was used for the normalization of expression levels. (B) CLCA2 expressions in prostate cancer tissues, PIN, and normal tissues were examined by quantitative real-time PCR analysis. β-Actin was used for the normalization of expression levels. (C) CLCA2 expressions in prostate cancer cell lines were examined by quantitative real-time PCR analysis. β2-Microglobulin was used for the normalization of expression levels. (D) Three prostate cancer cell lines were treated with 5 µM of 5-Aza and/or 0.5 µM of TSA. CLCA2 expressions were examined by quantitative real-time PCR analysis. β2-Microglobulin was used for the normalization of expression levels. Asterisks indicate P value obtained by Student's t test: *P < .05, **P < .01, and N.S. (not statistically significant).