FoxO3a and BCR-ABL regulate cyclin D2 transcription through a STAT5/BCL6-dependent mechanism

Abstract

Cell cycle arrest by FoxO transcription factors involves transcriptional repression of cyclin D, although the exact mechanism remains unclear. In this study, we used the BCR-ABL-expressing cell line BV173 as a model system to investigate the mechanisms whereby FoxO3a regulates cyclin D2 expression. Inhibition of BCR-ABL by STI571 results in down-regulation of cyclin D2 expression, activation of FoxO3a activity, and up-regulation of BCL6 expression. Using reporter gene assays, we demonstrate that STI571, FoxO3a, and BCL6 can repress cyclin D2 transcription through a STAT5/BCL6 site located within the cyclin D2 promoter. We propose that BCR-ABL inhibition leads to FoxO3a activation, which in turn induces the expression of BCL6, culminating in the repression of cyclin D2 transcription through this STAT5/BCL6 site. This process was verified by mobility shift and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to accumulation of BCL6 and down-regulation of cyclin D2 at protein and mRNA levels. Furthermore, silencing of FoxO3a and BCL6 in BCR-ABL-expressing cells abolishes STI571-mediated effects on cyclin D2. This report establishes the signaling events whereby BCR-ABL signals are relayed to cyclin D2 to mediate cell cycle progression and defines a potential mechanism by which FoxO proteins regulate cyclin D2 expression.

FIG. 1.

Expression of signaling proteins and cell cycle regulators in BV173 cells after STI571 treatment. BV173 cells were treated for the indicated times with STI571. Cell lysates were prepared at the times indicated, separated on polyacrylamide gels, and immunoblotted with specific antibodies. The expressions of cyclin D2, FoxO3a, cdk4, and tubulin were analyzed by Western blotting.

FIG. 2.

Effect of STI571 treatment on the mRNA levels of cyclin D2 in BV173 cells. (A) BV173 cells were treated with STI571 for the indicated times, and total RNA was extracted and analyzed for expression of cyclin D2. Also shown are the expressions of GAPDH mRNA and 28S rRNA as a control for RNA loading. (B) Expression of cyclin D2 RNA was also analyzed in parallel by real-time PCR and normalized to the level of L19. The results shown are the average of triplicate results from two independent sets of samples.

FIG. 3.

Effect of treatment with STI571 and expression of FoxO3a and BCL6 on cyclin D2 promoter activity in BV173 cells. (A) Schematic representation of the 5′ deletion mutants of the human cyclin D2 promoter used in this study. CRE, cAMP-responsive element. (B) BV173 cells were transiently transfected with pGL3cyclinD2 promoter constructs (10 μg) and incubated the day after with STI571 for 24 h. Cells were harvested and assayed for luciferase activity. Values are corrected for cotransfected Renilla activity and expressed as the percentage of inhibition by STI571 compared with the nontreated conditions. (C and D) BV173 cells were transiently transfected with pGL3cyclinD2 promoter constructs (10 μg) together with 5 μg of pLPC-FoxO3a(wt) or pLPC-FoxO3a(A3) (C), 5 μg of pcDNA3BCL6 (D), or empty vector. Cells were harvested 24 h after transfection and assayed for luciferase activity. Values are corrected for cotransfected Renilla activity, and the relative luciferase activity is standardized to that observed with the empty vector and the full-length promoter. All data shown represent the averages of results from three independent experiments, and the error bars show the standard deviation.

FIG. 4.

Expression of BCL6 and STAT5 in BV173 cells after STI571 treatment and effect of FoxO3a expression on BCL6 promoter activity. (A) BV173 cells were treated for the indicated times with STI571. Cell lysates were prepared at the indicated times, separated on polyacrylamide gels, and subjected to immunoblotting with specific antibodies for BCL6 and STAT5. (B) BV173 cells were treated with STI571 for the times indicated, and total RNA was extracted and analyzed for expression of BCL6. Also shown is the expression of GAPDH as a control for RNA loading and blotting. (C) BV173 cells were transiently transfected with pGL3.1296 or pGL3.1296.mutBCL6 promoter reporter constructs (10 μg) along with pLPC-FoxO3a(wt), pLPC-FoxO3a(A3), or the empty vector (5 μg). Cells were harvested 24 h after transfection and assayed for luciferase activity. Values are corrected for cotransfected Renilla activity and expressed as arbitrary units. The data shown represent the averages of results from two independent experiments, and the error bars show the standard deviation.

FIG. 5.

Electrophoretic mobility shift analysis of STAT5 DNA-binding complexes after STI571 treatment of BV173 cells. (A) DNA-binding protein extracts were prepared from BV173 cells at the indicated times after STI571 treatment. The extracts were then used for gel mobility shift experiments with a ³²P-labeled oligonucleotide probe containing the STAT5 binding site in the human cyclin D2 promoter. (B) Antibody supershift analysis of components of STAT5 complexes at different times after STI571 treatment. Supershifts were performed using specific antibodies against STAT5 and BCL6, as indicated, on whole-cell extracts prepared from cells at 0, 1, and 4 h after STI571 treatment.

FIG. 6.

ChIP assay of BV173 cells after STI571 treatment. (A) BV173 cells were treated with STI571 for 4 h. Protein-DNA complexes were formaldehyde cross-linked in vivo. Chromatin fragments from these cells were subjected to immunoprecipitation with antibodies to STAT5 or BCL6 as indicated. After cross-link reversal, the coimmunoprecipitated DNA was amplified by PCR using the A (−1446/−1149), B (−713/−499), and C (−548/−323) primers and resolved in 2% agarose gel. The PCR products obtained with A primers were also detected by Southern blotting, and shown is the quantitation by phosphorimaging of the results obtained. (B) BV173 cells were treated with STI571 for 4 or 8 h. Protein-DNA complexes were formaldehyde cross-linked in vivo. Chromatin fragments from these cells were subjected to immunoprecipitation with a specific antibody to FoxO3a. After cross-link reversal, the coimmunoprecipitated DNA was amplified by PCR using the A (−1446/−1149), D (−839/−501), and E (+38/+349) primers and resolved in 2% agarose gel.

FIG. 7.

Effect of 4-OHT treatment on BaF3 and BaF3 · FoxO3a(A3):ER cells. BaF3 and BaF3 · FoxO3a(A3):ER cells were treated with 4-OHT for the indicated times. (A) Cell lysates were prepared at the times indicated, separated on polyacrylamide gels, and subjected to immunoblotting with specific antibodies. The expressions of cyclin D2, BCL6, FoxO3a, and tubulin were analyzed by Western blotting. (B) Total RNA was extracted and analyzed for expression of cyclin D2 and BCL6. Also shown is the expression of GAPDH mRNA as a control for RNA loading and blotting. (C) The expression of BCL6 mRNA was also analyzed in parallel by real-time PCR and normalized to the level of L19 RNA. The results shown are averages of triplicate results from two independent sets of samples.

FIG. 8.

Effect of FoxO3a gene silencing on the effects of STI571 treatment of BaF3/BCR-ABL cells. The pSUPER vector coding for FoxO3a RNAi sequence was introduced into BaF3/BCR-ABL cells in order to silence FoxO3a expression. BaF3/BCR-ABL and BaF3/BCR-ABL FoxO3a-negative cells were treated in parallel with STI571 for the indicated times. (A) Cell cultures were fixed in ethanol, and DNA content was analyzed by flow cytometry following propidium iodide staining. The percentage of cells in each phase of the cell cycle (<2N, G₁, S, and G₂/M) is indicated. (B) Cell lysates were prepared at the times indicated, separated on polyacrylamide gels, and subjected to immunoblotting with specific antibodies. The expressions of FoxO3a, cyclin D2, BCL6, and BCR-ABL were analyzed by Western blotting. (C) Total RNA was extracted and analyzed for expression of cyclin D2 and BCL6. Also shown is the expression of 28S rRNA as a control for RNA loading and blotting. (D) Expressions of cyclin D2 and BCL6 mRNA were analyzed in parallel by real-time PCR and normalized to the level of L19 RNA. The results shown are the averages of triplicate results from two independent sets of samples.

FIG. 9.

Effect of BCL6 gene silencing on STI571 treatment of BV173 cells. BV173 cells were transfected by nucleofection with either a control oligonucleotide or a combination of two different BCL6 siRNAs and then treated with STI571 for 4 h. (A) Cell lysates were prepared, separated on polyacrylamide gels, and subjected to immunoblotting with specific antibodies. The expressions of BCL6, cyclin D2, FoxO3a, and tubulin were analyzed by Western blotting. (B) The expression of cyclin D2 RNA was analyzed in parallel by real-time PCR and normalized to the level of L19.