An ARF/CtBP2 complex regulates BH3-only gene expression and p53-independent apoptosis

Abstract

The alternative reading frame (ARF) tumor suppressor exerts both p53-dependent and p53-independent functions. The corepressor C-terminal binding protein (CtBP) interacts with ARF, resulting in proteasome-mediated degradation of CtBP. ARF can induce apoptosis in p53-null colon cancer cells, in a manner dependent on ARF interaction with CtBP. Bik was uniquely identified in an apoptotic gene array as coordinately upregulated in colon cancer cells after either CtBP2 knockdown or ARF overexpression. Validating the array findings, ARF induced Bik mRNA and protein expression, and this activity required an intact CtBP binding domain. Apoptosis induced by CtBP deficiency was substantially impaired when Bik expression was simultaneously silenced. An analysis of the Bik promoter revealed binding sites for the CtBP-interacting basic Kruppel-like factor (BKLF). A Bik promoter luciferase reporter was repressed by BKLF and CtBP2, and ARF reversed CtBP-associated repression. Chromatin immunoprecipitation analyses showed that CtBP was recruited to the Bik promoter largely by BKLF. Expression profiling of BH3-only gene expression in ARF-expressing or CtBP-deficient cells revealed that Bik was uniquely regulated by ARF/CtBP in colon cancer cells, whereas additional BH3-only proteins (Bim, Bmf) showed CtBP-dependent repression in osteosarcoma cells. ARF antagonism of CtBP repression of Bik and other BH3-only genes may have a critical role in ARF-induced p53-independent apoptosis and tumor suppression.

Figure 1

Bik is upregulated upon ARF overexpression or CtBP depletion in p53-null human colon cancer cells. (a) Total RNA isolated from HCT116 p53−/− cells after either ARF overexpression or CtBP knockdown was subjected to an apoptotic gene array (SABiosciences, Frederick, MD, USA) analysis. The relative expression level of genes relevant to apoptosis was estimated by comparing signal intensities of spots of cDNA for each relevant gene with the intensity of spots of housekeeping genes. The fold changes in gene expression level after ARF overexpression or CtBP knockdown was calculated by using GEArray Expression Analysis Suite (SABiosciences). (b) ARF/CtBP regulates Bik at the mRNA level. Quantitative real-time PCR using RNA prepared from HCT116 p53−/− cells infected with either vector (Ev) or hARF retrovirus, or transfected with either control (siCon) or CtBP2 (siCtBP2) siRNA, was carried out using Bik- and GAPDH-specific primers. The bars represent GAPDH-normalized average fold change of Bik in the treated cells. The error bars represent ±1 S.D. (c) CtBP2 regulates Bik expression. HCT116 p53^−/− cells were treated with control (siCon) or CtBP2 (siCtBP2) siRNA duplexes, and CtBP2, Bik, and GAPDH levels were determined by immunoblotting 24 h after transfection. (d) CtBP2 interaction with ARF is required for regulation of Bik expression. HCT116 p53 −/− cells were infected with vector, hARF, or hARF (L50D) mutant lentiviruses for 24 h. Cell lysates were analyzed for Bik, GAPDH, and ARF levels using immunoblotting

Figure 2

p53-independent stress-induced apoptosis through an ARF/CtBP/Bik pathway in colon cancer cells. (a) Apoptosis assay: HCT 116: p53−/− cells were stably infected with control (shCon), shBik1, or shBik2 lentiviruses and transiently transfected with either control (siCon) or CtBP2 (siC2) siRNA. All cells were exposed to 20 J/m² UV-C. After 24 h of transfection and UV treatment, apoptosis was determined by annexin V-PE/7-AAD staining. (b) Cell viability assay: the percentage of non-viable cells was determined after treatment as in (a) by staining cells with 0.4% Trypan blue. (c) ARF/CtBP2 regulation of stress-induced apoptosis. HCT116 p53−/− cells infected with empty, ARF, or L50D retroviruses were exposed to 20 J/m² UV-C or hypoxia (0.5% O₂), and the level of apoptosis determined after 24 h using annexin V-PE/7-AAD staining. All experiments were performed in triplicate and the results were expressed as Mean±1 S.D., *P<0.05 for (a, b) siC2/shCon versus siCon/shCon; (c) ARF versus Ev. ** P<0.05 for (a, b) siC2/shBik1 versus siC2/shCon; ^†P = 0.07 for (a, b) siC2/shBik2 versus siC2/shCon

Figure 3

ARF/CtBP regulates the Bik promoter through BKLF recognition elements. (a) BH3-only genes contain BKLF recognition elements. (Left) Diagram of BKLF recognition elements (BK) in the Bik promoter and (right) alignment and BKLF element localization in the Noxa, Puma, Bim, Bmf, and Bik promoters. (b) ARF antagonizes CtBP/BKLF repression of the Bik promoter. A Bik promoter luciferase reporter plasmid (pGL3-Bikluc) was co-transfected with or without expression constructs for ARF, BKLF, or CtBP2 into U2OS cells along with a control reporter plasmid expressing Renilla luciferase (pRL-TK). Normalized firefly luciferase activity from three independent experiments was averaged, and error bars indicate ± 1 S.D. *P<0.05 for CtBP2 +BKLF versus BKLF; **P<0.05 for ARF + CtBP2 +BKLF versus CtBP2 + BKLF

Figure 4

BKLF-mediated recruitment of CtBP to the Bik promoter. (a) ChIP assay of the Bik promoter was performed using chromatin prepared from H1299 cells and CtBP2 or control IgG antibody. Immunoprecipitated and input DNAs were amplified by PCR using PS1 and PS2 primer sets specific for fragments of the Bik promoter indicated in the graphic, and a negative control primer set (NS) that amplifies a fragment 10 kb upstream of the Bik promoter. PCR products were electrophoresed in an agarose gel and stained with ethidium bromide. (b) ChIP assay for CtBP2. H1299 chromatin was immunoprecipitated with or without CtBP2 antibody. Immunoprecipitated and input DNAs were amplified by PCR using NS, specific Bik PS2 primers, or positive control E-cadherin promoter primers. (c) Efficacy of BKLF shRNA. shRNA targeting BKLF or GFP was stably expressed in H1299 cells. RT-PCR was performed to determine the knockdown of BKLF mRNA with GAPDH mRNA level as an internal control. RT-PCR products were electrophoresed in an agarose gel and stained with ethidium bromide. (d) BKLF regulation of Bik expression. shBKLF or shGFP were stably expressed in HCT116 p53−/− cells, and protein levels of GAPDH and Bik were determined using immunoblotting. (e) CtBP2 recruitment to the Bik promoter requires BKLF. CtBP2 or control (no antibody) ChIP was performed with chromatin obtained from shGFP or shBKLF-expressing cells. Immunoprecipitated DNA was analyzed by PCR using Bik PS2, NS, and E-cadherin primers as in (a). (f) A model of BKLF/CtBP2-mediated transcription regulation of Bik expression

Figure 5

Regulation of BH3-only genes by ARF and CtBP. RNA isolated after CtBP2 or control siRNA treatment of HCT116 p53−/− (a), or U2OS (b) cells was subjected to RQ-PCR using GAPDH, β-actin and Bik, Bim, Bmf, Puma, and Noxa primers. Cell lysates from HCT116 p53−/− (c), or U2OS (d) cells treated with control or CtBP2 siRNA and HCT116 p53−/− cells infected with either empty, ARF, or ARF^L50D retroviruses (e) were analyzed for GAPDH, Bik, Bim, Bmf, Puma, and Noxa protein levels using immunoblotting. siCon, control siRNA; siCtBP2, CtBP2 siRNA